Patent Publication Number: US-2023158174-A1

Title: Chemically modified guide rnas for genome editing with cas9

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims benefit under 35 U.S.C. § 119 from Provisional Application Ser. No. 63/007,803, filed Apr. 9, 2020; Provisional Application Ser. No. 63/007,797, filed Apr. 9, 2020; Provisional Application Ser. No. 63/136,087, filed Jan. 11, 2021; Provisional Application Ser. No. 63/045,032, filed Jun. 26, 2020; Provisional Application Ser. No. 63/045,033, filed Jun. 26, 2020, the disclosures of which are incorporated herein by reference in their entirety. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 8, 2021, is named 53989-708_601_SL.txt and is 1,105,762 bytes in size. 
     FIELD OF DISCLOSURE 
     Provided herein are compositions for gene modification or editing, and methods of using same that are capable of treating or preventing certain conditions, such as cardiovascular disease and conditions or diseases associated therewith such as diabetes. 
     BACKGROUND 
     All publications, patents, and patent applications mentioned in this specification are herein incorporated by references to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. It is not an admission that any publication or information specifically or implicitly referenced herein is prior art or necessarily relevant to the claimed subject matter. To the extent publications or patents or patent applications incorporated by reference contradict or are inconsistent with the disclosure contained in the specification, such cited or incorporated references should be considered supplementary to this disclosure with the understanding that the specification is intended to supersede and/or take precedence over any irreconcilable inconsistencies or contradictory material. 
     SUMMARY 
     The inventive subject matter disclosed in this application is directed to compositions capable of editing a polynucleotide or target gene and methods of use of those compositions. The compositions and the constituent components thereof, individiualy and in combination, are considered separate aspects of the inventive subject matter, which may be defined and claimed by their respective physical and/or functional attributes described herein in any combination without limitation. The breadth, specificity, and variation of the inventive subject matter are further illustrated by specific aspects summarized here. 
     One significant aspect of the inventive subject matter described herein is directed to the treatment of cardiovascular disease (CVD), which is the leading cause of death worldwide, responsible for nearly one in three deaths according to the World Health Organization. CVD is also a leading contributor to reductions in life expectancy and is one of the most expensive health conditions to care for. According to the Centers for Disease Control and Prevention (CDC), CVD is a significant economic burden, costing the U.S. healthcare system more than $320 billion per year in annual costs and lost productivity. CVD collectively refers to diseases of the heart and blood vessels, which are diagnosed as either atherosclerotic cardiovascular disease (ASCVD) or cardiomyopathy, among others. ASCVD is a large subset of CVD for which cholesterol drives the development of atherosclerotic plaque, a mixture of cholesterol, cells and cellular debris in the wall of a blood vessel that results in the hardening of the arteries. The current cornerstone of the treatment and prevention of ASCVD is to lower cumulative exposure to blood lipids aiming to maintain low-density lipoprotein cholesterol (LDL-C, commonly known as “bad” cholesterol) and/or triglycerides as low as possible for as long as possible. There is significant evidence, for example, demonstrating that individuals who maintain LDL-C at sufficiently low levels over a sufficiently long period of time are substantially less likely to develop ASCVD. The relationship between the lowering of LDL-C and reduction in ASCVD is amongst the best understood of all relationships in medicine. It has been shown that lowering LDL-C by 39 mg/dL for five years in a patient with established ASCVD reduces ASCVD risk by 21%, whereas that same 39 mg/dL degree of LDL-C reduction over a lifetime reduces risk of a first ASCVD event by 88%. The current standard of care is a chronic care model that typically requires numerous daily pills and/or intermittent injections and often insufficiently controls cumulative exposure to LDL-C. Despite the availability of such chronic care therapies, cumulative exposure to LDL-C is often insufficiently controlled in many patients with ASCVD, and a large fraction of individuals with established ASCVD have LDL-C levels above the recommend goal. Higher cumulative LDL-C exposure leads to accelerated cholesterol plaque build-up in the heart or neck arteries and the rupture of which can result in heart attack, cardiac death, stroke and the need for invasive medical procedures, such as intracoronary stenting and coronary artery bypass surgery. 
     One aspect, disclosed herein, are compositions and methods that are capable of safely and effectively editing gene targets expressed in the liver to durably lower LDL-C and/or triglycerides thereby treating CVD such as ASCVD. 
     Another aspect, disclosed herein, is the efficacy and safety of the gene editing compositions described herein when administered as a single-course or dose (once-and-done) therapy, in repeat or successive doses, and combination gene editing therapy doses. The efficacy and safety of the compositions described herein are shown in in vitro and in vivo studies described herein involving various cell and animal experiments including mouse and non-human primate experiments, and the results or performance of the compositions disclosed herein represented in those studies each constitute aspects of the invention. 
     Another aspect of the composition and methods disclosed herein relates to the location where the edit in the gene is made, including for example compositions and methods directed at editing a gene in the splice site. 
     Another aspect of the compositions and methods disclosed herein are the constituent guide RNAs (gRNAs) and base editors that comprise the compositions that are capable of precisely editing a gene at a single base pair without imparting double-stranded breaks in the target gene. The compositions and method of use of those gRNAs and base editors including nucleotide or mRNA sequences that express and encode the base editor constitute yet another aspect. 
     Another aspect, disclosed herein, are the lipid nanoparticle (LNPs) formulations that encapsulate the gRNA and base editor drug substances, the selection of an LNP, and the relative ratios between the various components of the drug substance compositions alone and as part of the LNP. 
     Another aspect, disclosed herein, is directed at the dosing of gene editing compositions, such as those described herein, and the impact of dosing and repeat dosing on efficacy and safety profile indicia. 
     Another aspect, disclosed herein, are that the compositions and methods of use include implementations that are designed to target or edit specific genes such PCSK9, ANGPTL3, APOC3, and/or Lp(a) and/or have an impact on the protein levels of proteins expressed by those genes. 
     In one aspect, provided herein is a single guide RNA that comprises: (a) a spacer sequence, wherein the spacer sequence comprises (i) one or more chemical modification(s) and (ii) one or more unmodified nucleotide(s) at select position(s), and (b) a tracr sequence having at least 70% identity to SEQ ID NO: 61, wherein the tracr sequence serve as a binding scaffold for a Type II Cas protein, and wherein the tracr sequence comprises (i) one or more chemical modification(s) and (ii) one or more unmodified nucleotide(s) at select position(s). In some embodiments, the spacer sequence hybridizes with a target polynucleotide sequence in a gene of interest when contacted with the target polynucleotide sequence, wherein the single guide RNA directs the Cas protein to effect alteration in the gene when administered to a mammalian subject. In some embodiments, the tracr sequence comprises unmodified nucleotides at positions 2 to 7, 23 to 25, 27, 29, 31, 38, 39, 42 to 45 48, 49 and 62 as numbered in SEQ ID NO: 61 or a corresponding position thereof. In some embodiments, the tracr sequence comprises unmodified nucleotides at positions 2 to 7, 13, 23 to 25, 27, 29, 31, 38, 39, 42 to 45, 48, 49, 53 and 62 as numbered in SEQ ID NO: 61 or a corresponding position thereof. In some embodiments, the tracr sequence comprises unmodified nucleotides at positions 2 to 7, 13, 23 to 25, 27, 29, 31, 38, 39, 42 to 45, 48, 49, 53, 61, 68, 70 and 11 as numbered in SEQ ID NO: 61 or a corresponding position thereof. In some embodiments, the tracr sequence comprises modified nucleotides at positions 1, 8 to 22, 26, 28, 30, 32 to 37, 40, 41, 46, 47, 50 to 61, and 63 to 80 as numbered in SEQ ID NO: 61 or a corresponding position thereof. In some embodiments, the tracr sequence comprises modified nucleotides at positions 1, 8 to 12, 14 to 22, 26, 28, 30, 32 to 37, 40, 41, 46, 47, 50 to 52, 54 to 61, and 63 to 80 as numbered in SEQ ID NO: 61 or a corresponding position thereof. In some embodiments, the tracr sequence comprises modified nucleotides at positions 1, 8 to 12, 14 to 22, 26, 28, 30, 32 to 34, 37, 41, 46, 47, 50 to 52, 54 to 60, 62 to 67, 69 and 72 to 80 as numbered in SEQ ID NO: 61 or a corresponding position thereof. In some embodiments, more than 60% of the nucleotides in the tracr sequence with SEQ ID NO: 61 are modified. In some embodiments, more than 70% of the nucleotides in the tracr sequence with SEQ ID NO: 61 are modified. In some embodiments, the tracr sequence comprises unmodified nucleotides at positions 13, 49, 53 and 62 as numbered in SEQ ID NO: 61 or a corresponding position thereof. In some embodiments, the tracr sequence comprises unmodified nucleotides at positions 49 and 62 as numbered in SEQ ID NO: 61 or a corresponding position thereof. In some embodiments, the tracr sequence comprises unmodified nucleotides at positions 13, 40, 49, 53, 61, 68, 70 and 71 as numbered in SEQ ID NO: 61 or a corresponding position thereof. In some embodiments, the tracr sequence comprises unmodified nucleotides at positions 13, 49 and 53 as numbered in SEQ ID NO: 61 or a corresponding position thereof. In some embodiments, the tracr sequence comprises modified nucleotides at positions 1, 8, 21, 22, 26, 28, 30, 32 to 37, 40, 41, 46 and 47 as numbered in SEQ ID NO: 61 or a corresponding position thereof. In some embodiments, the tracr sequence comprises at least 80% identity to SEQ ID NO: 61.In some embodiments, the tracr sequence comprises at least 90% identity to SEQ ID NO: 61. In some embodiments, the chemical modification comprises a 2′-OMe modification. In some embodiments, the chemical modification comprises a nebularin or a deoxynebularin. In some embodiments, the chemical modification comprises a phosphorothioate linkage. In some embodiments, the tracr sequence comprises SEQ ID No. 61, and the single guide RNA further comprises one or more phosphorothioate linkage at a 5′ end, at a 3′ end, at select internal positions or any combinations thereof. In some embodiments, the single guide RNA further comprises two and no more than two contiguous phosphorothioate linkages at the 5′ end, at the 3′ end or both. In some embodiments, the single guide RNA further comprises three contiguous phosphorothioate linkages at the 5′ end, at the 3′ end or both. In some embodiments, the single guide RNA comprises the sequence 5′-ususuNNN-3′ at the 3′ end, wherein N independently indicates a unmodified ribonucleotide, and wherein each u indicates 2′-O-methyluridine and each s indicates phosphorothioate linkage. In some embodiments, each N is uridine. In some embodiments, the single guide RNA comprises the sequence 5′-ususuNNn-3′ at the 3′ end, wherein each N independently indicates a unmodified ribonucleotide, wherein the n indicates a modified nucleotide, wherein each u indicates 2′-O-methyluridine and wherein each s indicates phosphorothioate linkage. In some embodiments, each N is uridine and the n is 2′-O-methyluridine. 
     In another aspect, provided herein is a single guide RNA that comprises (i) a spacer sequence and (ii) a tracr sequence, wherein the spacer sequence hybridizes with a target polynucleotide sequence in a PCSK9 gene or an ANGPTL3 gene when contacted with the target polynucleotide sequence, wherein the tracr sequence binds a Type II Cas protein when contacted with the Type II Cas protein, and wherein the single guide RNA comprises a nebularine, a deoxynebularine, or a 2′-O-methylnebularine. In another aspect, provided herein is a single guide RNA that comprises (i) a spacer sequence and (ii) a tracr sequence, wherein the spacer sequence hybridizes with a target polynucleotide sequence in a PCSK9 gene or an ANGPTL3 gene when contacted with the target polynucleotide sequence, wherein the tracr sequence binds a Type II Cas protein when contacted with the Type II Cas protein, and wherein the single guide RNA comprises two and no more than two phosphorothioate linkages at a 5′ end or at a 3′ end. In some embodiments, the single guide RNA comprises two and no more than two phosphorothioate linkages at the 5′ end and at the 3′ end. In some embodiments, the single guide RNA comprises three phosphorothioate linkages at the 5′ end. In some embodiments, the single guide RNA comprises three phosphorothioate linkages at the 3′ end. In some embodiments, the two phosphorothioate linkages at the 5′ end are two contiguous phosphorothioate linkages at the first two nucleotide positions of the 5′ end. In some embodiments, the two phosphorothioate linkages at the 5′ end are within the first 3-10 nucleotides of the 5′ end. In some embodiments, the two phosphorothioate linkages at the 3′ end are two contiguous phosphorothioate linkages at the last two nucleotide positions of the 3′ end. In some embodiments, the two phosphorothioate linkages at the 3′ end are within the last 3-10 nucleotides of the 3′ end. In some embodiments, the single guide RNA as provided herein comprises the sequence 5′-UsUsUs-3′ at the 3′ end, wherein U indicates a uridine and s indicates a phosphorothioate linkage. In some embodiments, the single guide RNA as provided herein comprises the sequence 5′-UUU-3′ at the 3′end. In some embodiments, the tracr sequence binds the Type II Cas protein with increased binding affinity compared to a tracr sequence in an unmodified single guide RNA. In some embodiments, the Type II Cas protein is a Cas9 protein. In some embodiments, the Cas9 protein is a  Streptococcus pyogenes  Cas9. 
     In another aspect, provided herein is a single guide RNA that comprises a guide RNA sequence selected from Table 1, wherein a, u, g, and c indicate 2′-OMe modified adenine, uridine, guanine, and cytidine, wherein s indicates a phosphorothioate linkage, wherein X indicates a nebularine, wherein x indicates a 2′-O-methylnebularine and wherein dX indicates a 2′-deoxynebularine. 
     In another aspect, provided herein is a pharmaceutical composition for gene modification comprising the single guide RNA as provided herein and a Type II Cas protein or a nucleic acid sequence encoding the Type II Cas protein. In some embodiments, the pharmaceutical composition as provided herein further comprises a vector that comprises the nucleic acid sequence encoding the Type II Cas protein. In some embodiments, the Type II Cas protein is a Cas9. In some embodiments, the pharmaceutical composition as provided herein further comprises a pharmaceutically acceptable carrier. 
     In another aspect, provided herein is a lipid nanoparticle comprising the pharmaceutical composition as provided herein. 
     In another aspect, provided herein is a method for modifying a target polynucleotide sequence in a cell comprising introducing into the cell the pharmaceutical composition as provided herein, wherein the single guide RNA directs the Type II Cas protein to effect a modification in the target polynucleotide sequence in the cell. In some embodiments, the target polynucleotide sequence is in a PCSK9 gene. In some embodiments, the modification reduces expression of functional PCSK9 protein encoded by the PCSK9 gene in the cell. In some embodiments, the target polynucleotide sequence is in an ANGPTL3 gene. In some embodiments, the modification reduces expression of functional ANGPTL3 protein encoded by the ANGPTL3 gene in the cell. In some embodiments, the introduction is performed via a lipid nanoparticle that comprises the composition. 
     In another aspect, provided herein is a method for treating or preventing a condition in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition as provided herein or the lipid nanoparticle as provided herein, wherein the single guide RNA directs the Type II Cas protein to effect a modification in a target polynucleotide sequence in a cell of the subject, thereby treating or preventing the condition. In some embodiments, the target polynucleotide sequence is in a PCSK9 gene. In some embodiments, the target polynucleotide sequence is in an ANGPTL3 gene. In some embodiments, the modification reduces expression of functional PCSK9 protein encoded by the PCSK9 gene in the subject. In some embodiments, the modification reduces expression of functional ANGPTL3 protein encoded by the ANGPTL3 gene in the subject. In some embodiments, the condition is an atherosclerotic vascular disease. In some embodiments, the condition is an atherosclerotic vascular disease, hypertriglyceridemia, or diabetes. In some embodiments, the subject exhibits a reduced blood LDL cholesterol level, and/or a reduced blood triglycerides level as compared to before the administration. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The features, principles, advantages, and illustrative embodiments, implementations, analysis, and examples of the subject matter of this application are set forth herein including in the appended claims with aspects of which being illustrated in the accompanying drawings of which: 
         FIGS.  1 A -IC illustrate the modes of operation of Cas9, cytidine base editors (CBE), and adenine base editors (ABE) respectively, along with relevant terminology used in this application including “protospacer”, “PAM”, “spacer” ( Mali , P. et al 2013  Nat Methods  10, 957-963; Anzalone, A V. 2020  Nat Biotechnol  38, 824-844).  FIG.  1 D  is a schematic representation of how mcPCSK9 guide RNA (gRNA)-Tracr disclosed herein were designed. Shown is a ribonucleoprotein (RNP)-single guide RNA (sgRNA) alignment with stem-loop guide intramolecular interactions (W—C base pairing at the stem). The tracrRNA sequence of the gRNA serves as a binding scaffold for the cas protein. When designing a gRNA, loop nucleotides can be aligned with a protein and the interaction of base (H-bond), 2′-hydroxyl (2′-OH), 4′-oxygen (ring-oxygen) of the sugar moiety, phosphate linkage of each nucleotide to amino acid side chains of the protein can be considered in the design of a gRNA-Tracr. Spatial arrangement of nucleotide within RNP-steric interaction, room to accommodate bulky substitution like 2′-O-Methyl (2′-OMe) and phosphorothioate (PS) can be also taken into consideration.  FIG.  1 D  discloses SEQ ID NOS 70-71, respectively, in order of appearance. 
         FIG.  2    are two tables that describe how SpCas9 crystal structures were chosen in connection with designing a gRNAs disclosed herein based on structure. The top panel is a table summarizing different components of CRISPR/Cas system with Protein Data Bank (PDB) IDs and the state. The bottom panel shows the root mean square deviation (RMSD) values after alignment between the protein chains of each structure. The RNP state (4ZT0) and pre-catalytic ternary complex (5F9R) are the most relevant to determine contacts related to RNP formation and contacts related to catalysis. Some rearrangement of the protein occurs between these two states. Massive rearrangements may occur in the protein between the apo state and the RNP. 
         FIG.  3    depicts a gRNA secondary structure of a sgRNA based on the 4ZT0 and 5F9R crystal structures, in which the sgRNA is bound to SpCas9 either as an RNP alone or as part of a ternary complex with DNA. Secondary structure relationships are shown in the Leontis and Westhof nomenclature (RNA, 2001, 7, 499-512) (SEQ ID NO: 72). 
         FIG.  4    depicts a gRNA secondary structure with a PCSK9 spacer showing predicted contacts based on RNP PDB 4ZT0 (sgRNA+SpCas9 RNP). Positions 1-10 and positions 83-100 were not modeled in this crystal structure due to a lack of clear electron density (SEQ ID NO: 73). Black circle with white letter labels: protein contact, light gray with black letter: steric clash if 2′-O-Me incorporated or distant contact, dark grey with black letter: RNA contact. As a person of skill in the art would understand, the term “clash” as used herein refers to physically-unlikely overlapping atomic volumes in a structure. Incorporation of a 2′O-Me modification in these situations would potentially result in structural rearrangement(s) that could be detrimental to RNP function. As a person of skill in the art would understand, the term “contact” as used herein means a stable, non-covalent interaction between two functional groups, e.g. a hydrogen bond. 
         FIG.  5    depicts a gRNA secondary structure with a PCSK9 spacer showing predicted contacts based on PDB 5F9R (precatalytic ternary complex) (SEQ ID NO: 74). Black circle with white letter labels: protein contact, light gray with black letter: steric clash if 2′-O-Me incorporated or distant contact, dark grey with black letter: RNA contact. 
         FIG.  6    depicts plausible positions of 2′-OMe substitutions determined by structure-based designs (SEQ ID NO: 73). Black circle with white letter labels: 2′-OMe and phosphorothioate substitution, light gray circle with black letter labels: 2′-OMe substitution only, white circle with black letter labels: unmodified nucleotide. 
         FIG.  7 A  depicts three patterns of 2′-OMe position identified in the tracr region of the single guide RNA from structure-guided incorporation of 2′-O-methylribosugar modification that produced robust editing in vivo: (1) mice:  FIG.  16   , GA054 (tracr1) and GA055 (tracr2) and (2) NHP:  FIG.  28   , GA096 (tracr1), GA097 (tracr2) and GA346 (tracr3). Black circle with white letter labels: 2′-OMe and phosphorothioate substitution, light gray circle with black letter labels: 2′-OMe substitution only, white circle with black letter labels: unmodified nucleotide. (SEQ ID NO: 73)  FIG.  7 B  depicts the sequence alignment of unmodified SEQ ID NO: 61, a reference tracrRNA sequence (“Lit Tracr”), tracr1, tracr2, and tracr3. (SEQ ID NO: 73) 
         FIG.  8    shows base editing of the target splice site by an adenosine base editor system in modifying PCSK9 in primary human hepatocytes. The dark line represents the percent splice site editing obtained using gRNA identified a GA066. 
         FIG.  9    depicts a Sanger sequencing chromatogram demonstrating editing of adenine base in the antisense strand at the splice donor at the end of PCSK9 exon 1 (PCR amplification from the genomic DNA of the cells transfected with the 2500-ng/mL dose), portraying how splice-site disruption results in an in-frame stop codon. Heterozygosity for a naturally occurring single nucleotide polymorphism (SNP) is evident downstream of the editing site. The scheme shows A-to-G base editing by an adenosine base editor system to knock-out PCSK9 in primary human hepatocytes.  FIG.  9    discloses SEQ ID NO: 75. 
         FIG.  10    depicts a Sanger sequencing chromatogram demonstrating editing of adenine base in the antisense strand at the splice donor at the end of ANGPTL3 exon 6 (PCR amplification from the genomic DNA of the cells transfected with the 2500-ng/mL dose), portraying how splice-site disruption results in an in-frame stop codon. A-to-G base editing effected by an adenosine base editor system in modifying ANGPTL3 in primary human hepatocytes.  FIG.  10    discloses SEQ ID NO: 76. 
         FIG.  11    shows three sanger sequencing chromatograms from PCR amplified genomic DNA isolated from: 1) untreated primary hepatocytes (top panel); 2) SpCas9 mRNA/PCSK9 gRNA GA097 treated primary hepatocytes (middle panel); 3) ABE8.8 mRNA/PCSK9 gRNA treated primary hepatocytes (bottom panel). The PCSK9-gRNA GA097 protospacer sequence is highlighted in grey. The arrow points to the position 6 of the protospacer that is targeted for A-to-G base editing by the ABE8.8 editor. The scissors depict the general site of double stranded break that occurs upon cutting by the SpCas9 nuclease at this target site. SpCas9 mRNA and GA097 delivery in cells resulted in significant gene editing at the site of the double strand break (as denoted with the scissors) but this was not seen in the ABE8.8 and GA097 delivery in cells. Instead, the same gRNA GA097 in combination with ABE8.8 mRNA resulted in robust A-to-G base editing.  FIG.  11    discloses SEQ ID NO 77-79, respectively in order of appearance. 
         FIG.  12    depicts a schematic showing potential splicing outcomes with disruption of splice donor or splice acceptor sequences. Other outcomes are possible, such as inclusion of part of intron 1 in the splicing product. Alteration of the splice donor or splice acceptor sites are shown (top panel). Alternative splice donor sites within PCSK9 intron 1 resulting from editing of PCSK9 exon 1 splice-donor adenine base in primary human hepatocytes is shown in the bottom panel. *Four different primer pairs (Table 9) were used for RT-PCR of the control/treated samples. 
         FIG.  13    shows lack of guide RNA-dependent DNA off-target editing in primary human hepatocytes. Human primary hepatocytes were incubated with gRNA (GA097 or GA346) and ABE8.8 mRNA as described in Example 4. The off-target reading was calculated as net adenine editing (proportion of sequencing reads with alteration of one or more adenine bases in LNP-treated cells versus untreated cells) at the on-target PCSK9 site and more than 50 candidate off-target PCSK9 sites in primary human hepatocytes from four individual donors. 
         FIG.  14    depicts guide RNA-independent RNA editing, assessed in SpCas9-treated or ABE8.8-treated hepatocytes after 2 days (n=4 biological replicates). Each replicate was compared against each of four untreated hepatocyte samples to eliminate any positions with editing that were common to both conditions. The jitter plots portray transcriptomic loci with editing in the treated sample (number indicates total edited loci identified in the treated sample, boxplot indicates median ±interquartile range of proportion of edited reads across all edited loci in the sample). gRNA GA097, SpCas9 mRNA MS010, and ABE8.8 mRNA MA004 were used in this study as described in example 4. 
         FIG.  15    shows PCSK9 and ANGPTL3 base editing with lipid nanoparticles (LNPs) formulated with an embodiment of base editor system, ABE mRNA and a guide RNA, in primary human hepatocytes and primary non-human primates (NHP) hepatocytes. The human-specific ANGPTL3 guide RNA showed low editing efficiency in NHP hepatocytes within the concentration evaluated, whereas the PCSK9 guide RNA cross-reactive to both human and NHP showed high editing efficiency in both cell lines. 
         FIG.  16    shows gene editing of Pcsk9 in mice (n=2-5 mice) via LNPs containing SpCas9 mRNA MS002 (TriLink Biotechnologies) and mouse Pcsk9-targeting gRNA with different tracr designs (GA052, GA053, GA054, and GA055) at a 1:1 weight ratio. Wild-type C57BL/6 mice were dosed with 2 mg/kg of the LNP test article. The mg/kg dose was calculated based on total RNA and the total RNA is the quantified sum of mRNA and gRNA present in the LNP after formulation. Seven days after dosing, the mice were euthanized and genomic DNA was harvested from mouse liver, and then assessed for base editing of the target site with next-generation sequencing. All four guide RNAs evaluated carry same spacer and same pattern of chemical modification within the first 20 nucleotides from the 5′-end, but all four differ in chemical modification pattern within the tracr between nucleotides at position 21 and at position 100 of the 100-mer guide RNA. 
         FIG.  17    shows base editing of PCSK9 in mice (n=5) via LNPs with an embodiment of base editor system, ABE mRNA MA004 and PCSK9 guide RNA GA256 (targeting mouse intron 1 splice donor). Wild-type C57BL/6 mice were dosed with 2 mg/kg total RNA of the LNP test article. Seven days after dosing, the mice were euthanized and genomic DNA was harvested from mouse liver, and then assessed for base editing of the target site with next-generation sequencing. 
         FIG.  18    depicts editing of the Pcsk9 exon 1 splice-donor adenine base in wild-type mouse liver, assessed 1 week following treatment with different doses of same LNP formulation with ABE8.8 mRNA MA004 and Pcsk9 gRNA GA256 (n=4 to 5 mice per dosing group, bar indicates mean editing in group). 
         FIG.  19    depicts editing of the Pcsk9 exon 1 splice-donor adenine base in wild-type mouse liver, after dosed with LNPs at 0.05 mg/kg total RNA dose containing different ratios of gRNA GA256 and mRNA MA002. Additional guides GA255 and GA257 with different chemical modifications were also assessed for base editing efficiency, at mRNA to gRNA 1:1 wt ratio. 
         FIG.  20    shows the results from dosing LNPs containing ABE8.8 mRNA and mouse Angptl3-targeting gRNAs (GA258, GA259, GA260, GA349, GA353) at 0.05 mg/kg total RNA dose at a 1:1 weight ratio into mice. GA258, GA259 and GA260 contain three different structure-guided tracr design. GA349 and GA353 tracr designs were from published literature (Cell Reports, 2018 22, 2227-2235). The mice were later euthanized and genomic DNA was harvested from mouse liver, and then assessed for base editing of the target splice site with next-generation sequencing. 
         FIG.  21    is a schematic showing the general dosing strategy for introducing adenine base editing of a target gene in NHP via LNPs formulated with an embodiment of base editor system, ABE mRNA and a guide RNA, and subsequent analysis after 2 weeks. 
         FIG.  22    shows that administration of LNPs formulated with an embodiment of base editor system, ABE mRNA MA002 and PCSK9 guide RNA GA066, at 1 mg/kg and 3 mg/kg total RNA dose to cynomolgus monkeys via intravenous infusion, induced adenine base editing at the PCSK9 target splice site in the liver of cynomolgus monkeys. 
         FIG.  23    shows that administration of LNPs formulated with an embodiment of base editor system, ABE mRNA MA004 and PCSK9 guide RNA GA066, to cynomolgus monkeys via intravenous infusion resulted in reduction in the blood PCSK9 protein level compared to pre-dosing levels at 2 weeks after dosing. 
         FIG.  24    shows that administration of LNPs at 3 mg/kg formulated with an embodiment of base editor system, ABE mRNA MA002 and PCSK9 guide RNA GA066, to cynomolgus monkeys via intravenous infusion resulted in reduction of the blood low-density lipoprotein cholesterol (LDL-C) level at 1 and 2 weeks after dosing compared to pre-dosing levels. 
         FIGS.  25 A- 25 C  show short-term adenine base editing of PCSK9 in non-human primates.  FIG.  25 A  depicts editing of the PCSK9 exon 1 splice-donor adenine base in the livers of cynomolgus monkeys receiving an intravenous infusion of a 1 mg/kg dose of an LNP formulation with ABE8.8 mRNA MA004 and PCSK9 gRNA GA097 with necropsy at either 2 weeks (three animals) or 24 hours (two animals) following treatment. For each animal, editing was assessed in samples collected from sites distributed throughout the liver (n=8 samples; bar indicates mean editing in animal). Reduction of the blood PCSK9 protein level ( FIG.  25 B ) or blood LDL-C level ( FIG.  25 C ) in the three animals that underwent necropsy at 2 weeks following treatment are shown, comparing the level at 2 weeks versus the baseline pre-treatment level (n=1 blood sample per animal). 
         FIGS.  26 A- 26 C  shows adenine base editing of PCSK9 in non-human primates.  FIG.  26 A  depicts editing of the PCSK9 exon 1 splice-donor adenine base in the livers of cynomolgus monkeys receiving an intravenous infusion of 0.5, 1.0, or 1.5 mg/kg dose of an LNP formulation with ABE8.8 mRNA MA004 and PCSK9 gRNA GA346. Reduction of the blood PCSK9 protein level ( FIG.  26 B ) or blood LDL-C level ( FIG.  26 C ) for the animals are shown. 
         FIG.  27    depicts tissue distribution of editing of the PCSK9 exon 1 splice donor adenine base in the three animals that underwent necropsy at 2 weeks following treatment (n=1 sample per animal for each indicated organ except liver; the liver data represent the means shown in a calculated from eight liver samples each). The LNP constituted with ABE mRNA MA004 and guide RNA GA346 was used in this study, and the dose administered was 0.5 mg/kg. 
         FIG.  28    illustrates editing of the PCSK9 exon 1 splice-donor adenine base editing in the livers of cynomolgus monkeys following intravenous infusions of individual lipid nanoparticles (LNPs) constituted with ABE mRNA MA004 and different guide RNAs with same spacer but with different tracer modifications. The guide RNAs used in this study were GA066, GA096, GA097 and GA346. The gRNA GA097 from two different sources were used in the same study and the sources are identified as (1) and (2). For tracr comparison studies the LNPS were dosed at 1 mg/kg total RNA dose where the guide RNA and mRNA were mixed at 1:1 weight ratio. The GA066 with published tracr design (Cell Reports, 2018 22, 2227-2235) produced lower base editing in monkey compared to all other tracr designs ( FIG.  7   )—GA095, GA097, and GA346—under same experimental conditions. 
         FIG.  29    shows SpCas9 nuclease versus adenine base editing of PCSK9 in non-human primates. LNPs containing either SpCas9 mRNA and PCSK9 gRNA, (MS010/GA097) or ABE8.8 mRNA and PCSK9 gRNA (MA004/GA097) were infused intravenously in cynomolgus monkeys. The MS010/GA097 LNP was dosed at 0.75 and 1.5 mg/kg, and the MA004/GA097 LNP was dosed at 1 mg/kg total RNA dose. The MS010/GA097 LNP test article at 1.5 mg/kg produced low single digit gene editing in NHP whereas the ABE/GA097 test article produced about 40% adenine base editing at 1 mg/kg, showing the robustness of ABE base editor over SpCas9 system. All LNPs used in this study were prepared using same excipients and compositions. 
         FIG.  30    shows adenine base editing of PCSK9 in non-human primates. This graph depicts editing of the PCSK9 exon 1 splice-donor adenine base in the livers of cynomolgus monkeys (each bar is an individual animal, with editing recorded for multiple sampling areas) receiving an intravenous infusion of 0.5, 1.0, 1.5, or 3 mg/kg total RNA dose of LNP #1. LNP #2 at 3 mg/kg total RNA was dosed as a benchmark from a previous study. Both LNP formulations contain ABE8.8 mRNA MA004 and PCSK9 gRNA GA346. 
         FIG.  31    shows the reduction of PCSK9 protein levels from basal on Day 15, from the same experiment described in  FIG.  30   . 
         FIG.  32    depicts editing of the PCSK9 exon 1 splice-donor adenine base in the livers of four cynomolgus monkeys received an intravenous infusion of a 3 mg/kg total RNA dose of an LNP formulation with ABE8.8 mRNA MA004 and PCSK9 gRNA GA066. For each animal, editing was assessed in a liver biopsy sample at 2 weeks following treatment (n=1 sample per animal). 
         FIG.  33    depicts reduction of the blood PCSK9 protein levels in the four animals from  FIG.  32    (animals that received 3 mg/kg total RNA dose of an LNP formulation with ABE8.8 mRNA MA004 and PCSK9 gRNA GA066) and in two contemporaneous control animals that received phosphate-buffered saline, comparing levels at various time points following treatment versus the baseline pre-treatment level (mean±standard deviation for each group, n=4 or n=2, at each time point). The dotted lines indicate 100% and 10% of baseline levels, respectively. 
         FIG.  34    depicts reduction of the blood LDL-C level in the four animals from  FIG.  32    (animals that received 3 mg/kg total RNA dose of an LNP formulation with ABE8.8 mRNA MA004 and PCSK9 gRNA GA066) and in two contemporaneous control animals that received phosphate-buffered saline, comparing levels at various time points following treatment versus the baseline pre-treatment level (mean±standard deviation for each group, n=4 or n=2, at each time point). The dotted lines indicate 100% and 40% of baseline levels, respectively (top panel). The absolute values of individual animals are shown in the bottom panel. 
         FIG.  35    depicts reduction of Lipoprotein(a) in the four animals from  FIG.  32    and in two contemporaneous control animals that received phosphate-buffered saline, comparing levels at various time points following treatment versus the baseline pre-treatment level (mean±standard deviation for each group, n=4 or n=2, at each time point). 
         FIG.  36    shows long-term phenotypic effects of liver PCSK9 base editing in non-human primates. Absolute values of aspartate aminotransferase (AST) (top panel), and alanine aminotransferase (ALT) (bottom panel) in the individual animals portrayed in  FIG.  32    (n=4 animals treated with 3 mg/kg dose of an LNP formulation with ABE8.8 mRNA and PCSK9-gRNA, and n=2 animals treated with phosphate-buffered saline) at various time points following treatment. 
         FIGS.  37 A- 37 G  show liver function markers of individual animals. AST ( FIG.  37 A ,  FIG.  37 B ), ALT ( FIG.  37 A ,  FIG.  37 C ), Alkaline phosphate ( FIG.  37 A ,  FIG.  37 D ), gamma-glutamyltransferase ( FIG.  37 A ,  FIG.  37 E ), total bilirubin ( FIG.  37 A ,  FIG.  37 F ), and albumin ( FIG.  37 A ,  FIG.  37 G ), up to 15 days post dose, from cynomolgus monkeys that received an intravenous infusion of a 0.5, 1.0, or 1.5 mg/kg dose of an LNP formulation with ABE8.8 mRNA and PCSK9 gRNA. 
         FIG.  38    is a schematic of the representative candidate ONE-seq sites using a specific library designed against the cynomolgus genome. The PCSK9 protospacer is depicted at the top with a ONE-seq score of 1.00. All sites listed are ranked by decreasing ONE-seq score, with mismatches to the protospacer sequence identified.  FIG.  38    discloses SEQ ID NO: 80, 2193-2196, 566, 2197-2205, 675, 2206-2220, 564, 2221-224, 638, 644, 506, 621, 2225-2226, 681, and 2227-2230, respectively, in order of appearance. 
         FIG.  39    shows gRNA-dependent, DNA off-target analysis of the sites identified in  FIG.  38   . Samples from cynomolgus primary hepatocytes (top panel) or cynomolgus monkey livers (bottom panel) were assessed for net A&gt;G base editing (n=3 treated, n=3 untreated samples). 
         FIG.  40    shows net A&gt;G % base editing at one off-target site (C5), identified in  FIG.  39   , from livers of NHPs that received either 0.5, 1.0, or 1.5 mg/kg LNP. 
         FIG.  41    shows base editing of ANGPTL3 in NHPs via LNPs formulated with an embodiment of base editor system, ABE mRNA MA004 and ANGPTL3 guide RNA GA067. Liver editing (left panel), ANGPTL3 protein levels (middle panel), and triglyceride levels (right panel) are shown for three NHPs. 
         FIG.  42    shows simultaneous ANGPTL3 and PCSK9 base editing with lipid nanoparticles (LNPs) formulated with an embodiment of base editor system, ABE mRNA MA002 and dual guide RNAs GA095 (hcPCSK9) and GA098 (hANGPTL3), in human primary hepatocytes at concentrations ranging from 0-2500 ng/test article/mL. 
         FIG.  43    shows Day 15 and Day 44 liver biopsy adenosine base editing results. NHPs were dosed with LNPs formulated with ABE8.8 mRNA MA004 and either a gRNA targeting PCSK9 (GA346) or a gRNA targeting ANGPTL3 (GA347) via intravenous infusion at a total RNA doses ranging from 0.5-2 mg/kg. Two weeks after administration of test article, biopsies were performed to assess base editing. After 30 days from the initiation of the study, the opposite LNP was administered. Following a second biopsy after an additional 2 weeks, the gDNA was extracted, and base editing was assessed using next generation sequencing. Results for PCSK9 base editing (top panel) and ANGPTL3 base editing (bottom panel) are shown. 2 mg/kg total RNA dose of LNP encapsulating ABE8.8 mRNA, PCSK9 gRNA GA346 and ANGPTL3 gRNA GA347 at 1:0.5:0.5 weight ratio produced robust synchronized PCSK9 and ANGPTL3 gene editing (Example 10). 
         FIG.  44    illustrates the corresponding % change in PCSK9 (top panel) and ANGPTL3 (bottom panel) protein levels from NHPs described in  FIG.  43   . 
         FIG.  45    illustrates that repeat LNP dosing in NHPs causes additive adenosine base editing in the liver, post Day 14, Day 46, and Day75 liver biopsies. NHPs were dosed with either LNP #1 or LNP #2 formulated with ABE8.8 mRNA MA004 and a gRNA targeting PCSK9 (GA097) via intravenous infusion at a total RNA doses of 0.5 mg/kg (see Example 10 for details on dosing intervals and related details). 
         FIG.  46    illustrates that repeat LNP dosing in NHPs causes additive base editing in the liver and translates to dose-dependent additive decrease in plasma PCSK9 protein levels over 90 days. As described in  FIG.  45   , NHPs were repeat dosed with LNPs formulated with ABE8.8 mRNA MA004 and a gRNA targeting PCSK9 (GA097). NHPs were dosed via intravenous infusion at a total RNA doses of 0.5 mg/kg at days 0, 30, and 60 (arrow is illustrated on graph to depict dosing). For description of analysis of PCSK9 protein levels, see detailed methods section. 
         FIG.  47    illustrates that repeat LNP dosing in NHPs causes additive base editing in the liver with only transient liver marker increase, and the transient liver marker increase correlates well with the day of each dose administered. The data shows 71 days of the liver marker levels of ALT, AST, total bilirubin, and creatine kinase post first dose (see Example 10,  FIG.  45    for details). 
         FIG.  48    illustrates that repeat LNP dosing in NHPs causes additive base editing in the liver, with only transient liver marker increase showing up to 71 days post dose of the liver enzyme levels of LDH, GLDH, GGT, and ALP, in NHPs (See Example 10,  FIG.  45    for details). 
         FIGS.  49 A and  49 B  illustrate that base editing of ANGPTL3 results in long-term decreased ANGPTL3 protein and triglyceride levels after a single dose of LNP constituted with ABE8.8 mRNA MA004 and ANGPTL3 gRNA GA067. The results illustrates the effect of long-term adenine base editing of ANGPTL3 on ANGPTL3 protein ( FIG.  49 A ) and triglycerides ( FIG.  49 B ) in non-human primates over 6 months. ANGPTL3 protein (96% reduction) and triglyceride levels were substantially decreased upon single dose administration of the LNP, and remain stably reduced for more than 170 days (see Example 10 for details). 
         FIGS.  50 A- 50 E  illustrate the effect of LNP dosing in NHPs on cytokine activation and immune response. Cynomolgus monkeys received intravenous infusions of 0.5 mg/kg doses at specified time points ( FIG.  50 A  and  FIG.  50 B ) of an LNP formulation with ABE8.8 mRNA MA004 and PCSK9 gRNA GA346. Blood was collected at timepoints specified and the graph, and IP-10 and MCP-1 were analyzed. In additional studies, IL-6, MCP-1, and SC5b-9 ( FIG.  50 C ,  FIG.  50 D , and  FIG.  50 E , respectively) were analyzed at different time points from blood collected from NHPs that received an intravenous infusion of 1.0 mg/kg total RNA dose of LNP formulated with MA004 and PCSK9 gRNA GA346. 
         FIG.  51    illustrates liver editing in NHPs at 15 days after treatment with an LNP containing SpCas9 mRNA/gRNA.  FIG.  51    shows the results from gene editing of ANGPTL3 or PCSK9 in non-human primates. Cynomolgus monkeys received an intravenous infusion of a 1.5 mg/kg dose of an LNP formulation with SpCas9 mRNA MS004 and one gRNA targeting either ANGPTL3 (GA261-GA263) or PCSK9 (GA266-GA271). Upon necropsy after 2 weeks, two pieces from each liver lobe (8 pieces total) were isolated and gDNA was extracted. Samples were processed as described in the detailed methods section. Indel % was analyzed for each separate piece and are graphed as individual points. High editing efficiency was observed in most NHP livers. 
         FIG.  52    illustrates LDL-C levels in NHPs at 15 days after treatment with SpCas9/gRNA.  FIG.  52    shows the reduction of LDL-C from gene editing of ANGPTL3 or PCSK9 in non-human primates. Cynomolgus monkeys received an intravenous infusion of a 1.5 mg/kg dose of an LNP formulation with SpCas9 mRNA MS004 and one gRNA targeting either ANGPTL3 (GA261-GA263) or PCSK9 (GA266-GA271. Samples were processed as described in the detailed methods section. All NHPs that received LNPs with SpCas9 mRNA/PCSK9 gRNA had at least 35% reduction in circulating LDL-C levels. Although more modest, LNPs with SpCas9 mRNA/ANGPTL3 gRNA had 10-25% reduction in circulating LDL-C levels. 
         FIG.  53    illustrates triglyceride levels in NHPs at 15 days after treatment with SpCas9/gRNA.  FIG.  53    shows the triglyceride levels from gene editing of ANGPTL3 or PCSK9 in non-human primates. Cynomolgus monkeys received an intravenous infusion of a 1.5 mg/kg dose of an LNP formulation with SpCas9 mRNA MS004 and one gRNA targeting either ANGPTL3 (GA261-GA263) or PCSK9 (GA266-GA271. Samples were processed as described in the detailed methods section. NHPs that received LNPs with SpCas9 mRNA/ANGPTL3 gRNA had around 10-50% reduction in triglyceride levels. NHPs that received LNPs with SpCas9 mRNA/PCSK9 gRNA did not show a significant reduction in triglyceride levels. 
         FIGS.  54 A and  54 B  illustrate that PACE-modifications to the gRNA decrease off-target editing efficiency. Human primary hepatocytes were transfected at 2500, 1250, 500, and 250 ng/test article/mL with SpCas9 mRNA (commercially purchased from Trilink) and a gRNA targeting PCSK9 with modifications to the tracr. Genomic DNA was processed, sequenced, and analyzed as described in the detailed methods section. GA156 was transfected to serve as a positive control. GA248 and GA249 contain PACE-modifications to the gRNA that have previously been demonstrated to decrease off-target editing efficiency. Indeed, although GA248 and GA249 had lower on-target editing compared to the unmodified gRNA, GA156 ( FIG.  54 A ), GA248 and GA249 showed decreased off-target editing at an identified off-target site ( FIG.  54 B ). 
         FIG.  55 A  illustrates the GC comparison of ABE-encoding nucleotides, MA004, MA019, MA020, MA021, and ABE8.8 m (Table 23), as well as a more detailed look at MA004 ( FIG.  55 A , bottom panel).  FIG.  55 B  illustrates the editing % obtained using ABE-encoding nucleotides, MA004, MA019, MA020, and MA021 (Table 23). 
     
    
    
     DETAILED DESCRIPTION 
     Certain specific details of this description are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the present disclosure may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods, and materials are described below. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure. 
     Definitions 
     To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below. 
     As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. 
     It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. The terms “and/or” and “any combination thereof” and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use. 
     The term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. 
     As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. 
     As used herein, “some embodiments,” “an embodiment,” “one embodiment,” “embodiments” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. 
     The term “nucleic acid” as used herein refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form and includes DNA and RNA. “Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. “Bases” include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides. A nucleic acid includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally termed oligonucleotides, and longer fragments termed polynucleotides. A deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5′ and 3′ carbons of this sugar to form an alternating, unbranched polymer. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, pre-condensed DNA, a PCR product, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. A ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose. Accordingly, the terms “polynucleotide” and “oligonucleotide” can refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally-occurring bases, sugars and intersugar (backbone) linkages. Additionally, nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, nonstandard, and/or non-naturally occurring, and which have similar binding properties as the reference nucleic acid. The nucleic acid may be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety, or phosphate backbone. Backbone modifications can include, but are not limited to, a phosphorothioate, a phosphorodithioate, a phosphoroselenoate, a phosphorodiselenoate, a phosphoroanilothioate, a phosphoraniladate, a phosphoramidate, and a phosphorodiamidate linkage. A phosphorothioate linkage substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone and delays nuclease degradation of oligonucleotides. A phosphorodiamidate linkage (N3′→P5′) allows preventing nuclease recognition and degradation. Backbone modifications can also include having peptide bonds instead of phosphorous in the backbone structure (e.g., N-(2-aminoethyl)-glycine units linked by peptide bonds in a peptide nucleic acid), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups. Oligonucleotides with modified backbones are reviewed in Micklefield, Backbone modification of nucleic acids: synthesis, structure and therapeutic applications, Curr. Med. Chem., 8 (10): 1157-79, 2001 and Lyer et al., Modified oligonucleotides-synthesis, properties and applications, Curr. Opin. Mol. Ther., 1 (3): 344-358, 1999. Nucleic acid molecules described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog. The examples of modified sugar moieties include, but are not limited to, 2′-O-methyl, 2′-O-methoxyethyl, 2′-O-aminoethyl, 2′-Flouro, N3′→P5′ phosphoramidate, 2′dimethylaminooxyethoxy, 2′ 2′dimethylaminoethoxyethoxy, 2′-guanidinidium, 2′-O-guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. 2′-O-methyl or 2′-O-methoxyethyl modifications promote the A-form or RNA-like conformation in oligonucleotides, increase binding affinity to RNA, and have enhanced nuclease resistance. Modified sugar moieties can also include having an extra bridge bond (e.g., a methylene bridge joining the 2′-0 and 4′-C atoms of the ribose in a locked nucleic acid) or sugar analog such as a morpholine ring (e.g., as in a phosphorodiamidate morpholino). Examples of such analogs and/or modified residues include, but are not limited to diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, 5-methyl-2-thiouracil, 3-(3-amino-3- N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine, methyl phosphonates, chiral-methyl phosphonates, 2′-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. In some cases, nucleotides may include modifications in their phosphate moieties, including modifications to a triphosphate moiety. Non-limiting examples of such modifications include phosphate chains of greater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphates). Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases. Thus, the terms “polynucleotide” and “oligonucleotide” can also include polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. 
     Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). 
     The present disclosure encompasses isolated or substantially purified nucleic acid molecules and compositions containing those molecules. As used herein, an “isolated” or “purified” DNA molecule or RNA molecule is a DNA molecule or RNA molecule that exists apart from its native environment. An isolated DNA molecule or RNA molecule may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an “isolated” or “purified” nucleic acid molecule or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in some embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. 
     The term “vector,” as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some examples, a vector is an expression vector that is capable of directing the expression of nucleic acids to which they are operatively linked. The term “operably linked,” as used herein, means that the nucleotide sequence of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. The term “regulatory sequence,” as used herein, includes, but is not limited to promoters, enhancers and other expression control elements. Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Examples of expression vectors include, but are not limited to, plasmid vectors, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors. 
     As used herein, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably and refer to a polymer of amino acid residues linked via peptide bonds and which may be composed of two or more polypeptide chains. The terms “polypeptide,” “protein,” and “peptide” refer to a polymer of at least two amino acid monomers joined together through amide bonds. An amino acid may be the L-optical isomer or the D-optical isomer. More specifically, the terms “polypeptide,” “protein,” and “peptide” refer to a molecule composed of two or more amino acids in a specific order; for example, the order as determined by the base sequence of nucleotides in the gene or RNA coding for the protein. Proteins are essential for the structure, function, and regulation of the body&#39;s cells, tissues, and organs, and each protein has unique functions. Examples are hormones, enzymes, antibodies, and any fragments thereof. In some cases, a protein can be a portion of the protein, for example, a domain, a subdomain, or a motif of the protein. In some cases, a protein can be a variant (or mutation) of the protein, wherein one or more amino acid residues are inserted into, deleted from, and/or substituted into the naturally occurring (or at least a known) amino acid sequence of the protein. A protein or a variant thereof can be naturally occurring or recombinant. Methods for detection and/or measurement of polypeptides in biological material are well known in the art and include, but are not limited to, Western-blotting, flow cytometry, ELISAs, RIAs, and various proteomics techniques. An exemplary method to measure or detect a polypeptide is an immunoassay, such as an ELISA. This type of protein quantitation can be based on an antibody capable of capturing a specific antigen, and a second antibody capable of detecting the captured antigen. Exemplary assays for detection and/or measurement of polypeptides are described in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press. 
     The term “sequence identity,” as used herein, refers to the amount of nucleotide which match exactly between two different sequences. When comparing RNA and DNA sequences Uracil and Thymine bases are considered to be the same base. Gaps are not counted and the measurement is typically in relation to the shorter of the two sequences. For example: 
                            A: AAGGCTT                       B: AAGGC                       C: AAGGCAT            
Here identity (A,B)=100% (5 identical nucleotides/min(length(A),length(B))). Identity(B,C)=100%, but identity(A,C)=85% ((6 identical nucleotides/7). So 100% identity does not mean two sequences are the same.
 
     The term “sequence similarity,” as used herein, can be described as an optimal matching problem that finds the minimal number of edit operations (inserts, deletes, and substitutions) in order to transform the one sequence into an exact copy of the other sequence being aligned (edit distance). Using this, the percentage sequence similarity of the examples above are sim(A,B)=60%, sim(B,C)=60%, sim(A,C)=86% (semi-global, sim=1-(edit distance/unaligned length of the shorter sequence)). 
     A “subject” in need thereof, refers to an individual who has a disease, a symptom of the disease, or a predisposition toward the disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptom of the disease, or the predisposition toward the disease. In some embodiments, the subject has hypercholesterolemia. In some embodiments, the subject has atherosclerotic vascular disease. In some embodiments, the subject has hypertriglyceridemia. In some embodiments, the subject has diabetes. The term “subject” or “patient” encompasses mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. 
     The term “condition,” as used herein, includes diseases, disorders, and susceptibilities. In some embodiments, the condition is an atherosclerotic vascular disease. In some embodiments, the condition is a hypertriglyceridemia. In some embodiments, the condition is a diabetes. 
     The term “atherosclerosis” or “atherosclerotic vascular disease,” as used herein, refers to a disease in which the inside of an artery narrows due to the buildup of plaque. In some embodiments, it may result in coronary artery disease, stroke, peripheral artery disease, or kidney problems. 
     The term “hypertriglyceridemia,” as used herein, refers to high (hyper-) blood levels (-emia) of triglycerides, the most abundant fatty molecule in most organisms. In some embodiments, elevated levels of triglycerides are associated with atherosclerosis, even in the absence of hypercholesterolemia (high cholesterol levels), and predispose to cardiovascular disease. In some embodiments, very high triglyceride levels increase the risk of acute pancreatitis. In some embodiments, hypertriglyceridemia is associated with overeating, obesity, diabetes mellitus and insulin resistance, excess alcohol consumption, kidney failure, nephrotic syndrome, genetic predisposition (e.g., familial combined hyperlipidemia, i.e., Type II hyperlipidemia), lipoprotein lipase deficiency, lysosomal acid lipase deficiency, cholesteryl ester storage disease, certain medications (e.g., isotretinoin, hydrochlorothiazide diuretics, beta blockers, protease inhibitors), hypothyroidism (underactive thyroid), systemic lupus erythematosus and associated autoimmune responses, glycogen storage disease type 1, propofol, or HIV medications. 
     The term “diabetes,” as used herein, refers to a group of metabolic disorders characterized by a high blood sugar level over a prolonged period of time. In some embodiments, diabetes is type 1 diabetes that results from the pancreas&#39;s failure to produce enough insulin due to loss of beta cells. In some embodiments, diabetes is type 2 diabetes characterized by insulin resistance, a condition in which cells fail to respond to insulin properly. In some embodiments, diabetes is gestational diabetes that occurs when pregnant women without a previous history of diabetes develop high blood sugar levels. 
     The term “low-density lipoprotein (LDL),” as used herein, refers to a microscopic blob made up of an outer rim of lipoprotein and a cholesterol center. In some embodiments, LDL has a highly hydrophobic core composed of a polyunsaturated fatty acid known as linoleate and hundreds to thousands esterified and unesterified cholesterol molecules. In some embodiments, the core of LDL also carries triglycerides and other fats and is surrounded by a shell of phospholipids and unesterified cholesterol. 
     The term “high-density lipoprotein (HDL),” as used herein, refers to the smallest lipoprotein particles. In embodiments, plasma enzyme lecithin-cholesterol acyltransferase (LCAT) converts the free cholesterol into cholesteryl, which is then sequestered into the core of the lipoprotein particle, eventually causing the newly synthesized HDL to assume a spherical shape. In embodiments, HDL particles increase in size as they circulate through the bloodstream and incorporate more cholesterol and phospholipid molecules from cells and other lipoproteins. 
     The term “cholesterol,” as used herein, refers to a lipid with a unique structure composed of four linked hydrocarbon rings forming the bulky steroid structure. The term “triglyceride,” as used herein, refers to a tri-ester composed of a glycerol bound to three fatty acid molecules. In some embodiments, the fatty acids are saturated or unsaturated fatty acids. 
     The terms “treat,” “treating,” or “treatment,” and its grammatical equivalents as used herein, can include alleviating, abating, or ameliorating at least one symptom of a disease or a condition, preventing additional symptoms, inhibiting the disease or the condition, e.g., delaying, decreasing, suppressing, attenuating, diminishing, arresting, or stabilizing the development or progression of a disease or the condition, relieving the disease or the condition, causing regression of the disease or the condition, relieving a condition caused by the disease or the condition, reducing disease severity, or stopping the symptoms of the disease or the condition either prophylactically and/or therapeutically. “Treating” also includes lessening the frequency of occurrence or recurrence, or the severity, of any symptoms or other ill effects related to a disease or condition and/or the side effects associated with the disease or condition. “Treating” does not necessarily require curative results. It is appreciated that, although not precluded, treating a disorder or condition also does not require that the disorder, condition, or symptoms associated therewith be completely eliminated. The term “treating” encompasses the concept of “managing” which refers to reducing the severity of a particular disease or disorder in a patient or delaying its recurrence, e.g., lengthening the period of remission in a patient who had suffered from the disease. “Treating” may refer to the application or administration or a composition to a subject after the onset, or suspected onset, of a disease or condition. 
     The term “treating” further encompasses the concept of “prevent,” “preventing,” and “prevention.” The terms “prevent,” “preventing,” and “prevention,” as used herein, refer to a decrease in the occurrence of pathology of a condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition. The prevention may be complete, e.g., the total absence of pathology of a condition in a subject. The prevention may also be partial, such that the occurrence of pathology of a condition in a subject is less than that which would have occurred without the present disclosure. 
     By “treating or preventing a condition,” for example, as compared with an equivalent untreated control, alleviating a symptom of a disorder may involve reduction or degree of prevention at least 3%, 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% as measured by any standard technique. In some embodiments, alleviating a symptom of a disorder may involve reduction or degree of prevention by at least 2, 3, 4, 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 fold as compared with an equivalent untreated control. 
     As used therein, “delaying” the development of a disease means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result. 
     “Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. 
     As used herein “onset” or “occurrence” of a disease includes initial onset and/or recurrence. 
     “Administering” and its grammatical equivalents as used herein can refer to providing pharmaceutical compositions described herein to a subject or a patient. Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the composition to the subject, depending upon the type of disease to be treated or the site of the disease. For example, the composition can be administered, e.g., orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, or via infusion. One or more such routes can be employed. 
     The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraperitoneal, intradermal, intraarterial, intrasynovial, intrastemal, intrathecal, intravascular, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods. 
     By “co-administering” is meant administering one or more additional therapeutic regimens or agents or treatments and the composition of the disclosure sufficiently close in time to enhance the effect of one or more additional therapeutic agents, or vice versa. In this regard, the composition of the disclosure described herein can be administered simultaneously with one or more additional therapeutic regimens or agents or treatments, at a different time, or on an entirely different therapeutic schedule (e.g., the first treatment can be daily, while the additional treatment is weekly). For example, in embodiments, the secondary therapeutic regimens or agents or treatments are administered simultaneously, prior to, or subsequent to the composition of the disclosure. 
     The terms “pharmaceutical composition” and its grammatical equivalents as used herein can refer to a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients, carriers, and/or a therapeutic agent to be administered to a subject, e.g., a human in need thereof. 
     The term “pharmaceutically acceptable” and its grammatical equivalents as used herein can refer to an attribute of a material which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and is acceptable for veterinary as well as human pharmaceutical use. “Pharmaceutically acceptable” can refer a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the pharmaceutical composition in which it is contained. 
     A “pharmaceutically acceptable excipient, carrier, or diluent” refers to an excipient, carrier, or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent. 
     A “pharmaceutically acceptable salt” may be an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication. Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2-hydroxyethyl sulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC—(CH 2 ) n —COOH where n is 0-4, and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize from this disclosure and the knowledge in the art that further pharmaceutically acceptable salts include those listed by Remington&#39;s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985). In general, a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in an appropriate solvent. 
     The term “therapeutic agent” can refer to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. Therapeutic agents can also be referred to as “actives” or “active agents.” Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids. 
     “A therapeutically effective amount” as used herein refers to the amount of each composition of the present disclosure required to confer therapeutic effect on the subject, either alone or in combination with one or more other therapeutic agents. Hence, as used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, composition, therapeutic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition. In terms of treatment, a “therapeutically effective amount” is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of a disease or a condition, e.g., an atherosclerotic vascular disease, hypertriglyceridemia, or diabetes. A “therapeutically effective amount” varies, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual subject parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a subject may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons. Additionally, other medication the patient may be receiving will affect the determination of the therapeutically effective amount of the therapeutic agent to administer. Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. A “therapeutically effective amount” may be of any of the compositions of the disclosure used alone or in conjunction with one or more agents used to treat a condition. A therapeutically effective amount can be administered in one or more administrations. 
     An effective initial method to determine a “therapeutically effective amount” may be by carrying out cell culture assays (for example, using neuronal cells) or using animal models (for example, mice, rats, rabbits, dogs or pigs). A dose may be formulated in animal models to achieve a concentration range that includes the IC50 (i.e., the concentration of the composition which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. In addition to determining the appropriate concentration range for an disclosure composition to be therapeutically effective, animal models may also yield other relevant information such as preferable routes of administration that will give maximum effectiveness. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan. 
     Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction. 
     The term “protospacer,” or “target sequence” and their grammatical equivalents as used herein can refer to a DNA sequence of a target gene. In the native state, a protospacer is adjacent to a PAM (protospacer adjacent motif). The term “spacer” can be the RNA version of the protospacer that binds to the complementary strand of the protospacer. A spacer can be within a guide RNA (gRNA). The site of cleavage by an RNA-guided nuclease is within a protospacer sequence. Please see  FIG.  1 A  for an illustration. 
     The term “base editing,” “gene editing,” or “gene modification” and its grammatical equivalents as used herein can refer to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a genome. Gene editing can be performed using a nuclease (e.g., a natural-existing nuclease or an artificially engineered nuclease). Gene modification can include introducing a double stranded break, a non-sense mutation, a frameshift mutation, a splice site alteration, or an inversion in a polynucleotide sequence, e.g., a target polynucleotide sequence. 
     The term “base editor (BE)” or “nucleobase editor (NBE)” as used herein can refer to an agent that binds a polynucleotide and has nucleobase modifying activity. In various embodiments, the base editor comprises a nucleobase modifying polypeptide (e.g., a deaminase) and a nucleic acid programmable nucleotide binding domain in conjunction with a guide polynucleotide (e.g., guide RNA), or nucleic acids encoding the programmable nucleotide binding domain and the deaminase. In various embodiments, the agent is a biomolecular complex comprising a protein domain having base editing activity, i.e., a domain capable of modifying a base (e.g., A, T, C, G, or U) within a nucleic acid molecule (e.g., DNA), or a nucleic acid encoding the same. In some embodiments, the polynucleotide programmable DNA binding domain is fused or linked to a deaminase domain, resulting in a base editor fusion protein. In some embodiments, the base editor comprises a nucleic acid encoding the base editor fusion protein, e.g., a mRNA encoding the base editor fusion protein. The base editor fusion protein may comprise one or more linkers, for example, peptide linkers. In one embodiment, the agent is a fusion protein comprising a domain having base editing activity. In another embodiment, the protein domain having base editing activity is linked to the guide RNA (e.g., via an RNA binding motif on the guide RNA and an RNA binding domain fused to the deaminase). In some embodiments, the domain having base editing activity is capable of deaminating a base within a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating one or more bases within a DNA molecule. In some embodiments, the base editor is capable of deaminating an adenosine (A) within DNA. In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the base editor is capable of deaminating an cytosine (C) within DNA. In some embodiments, the base editor is a cytosine base editor (CBE). 
     The term “base editor system” refers to a system for editing a nucleobase of a target nucleotide sequence. In various embodiments, the base editor system comprises (1) a polynucleotide programmable nucleotide binding domain (e.g., Cas9); (2) a deaminase domain (e.g., an adenosine deaminase or a cytidine deaminase) for deaminating said nucleobase; and (3) one or more guide polynucleotide (e.g., guide RNA). In some embodiments, the base editor system comprises a base editor fusion protein comprising (1) and (2). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is a cytosine base editor (CBE). 
     Nucleobase Editor Systems 
     In some aspects, provided herein are base editor systems capable of nucleobase modifications. In some embodiments, the base editor system comprises (i) a guide polynucleotide or a nucleic acid encoding same, and (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same. In some embodiments, the base editor system comprises a guide polynucleotide. In some embodiments, the base editor system comprises a nucleic acid encoding a guide polynucleotide. In some embodiments, the base editor system comprises a base editor fusion protein comprising a programmable DNA binding domain and a deaminase. In some embodiments, the base editor system comprises a nucleic acid encoding a base editor fusion protein comprising a programmable DNA binding domain and a deaminase. 
     In some embodiments, the guide polynucleotide directs the base editor system to effect a nucleobase alteration in a PCSK9 or ANGPTL3 gene in vivo when administered to a subject. 
     In some embodiments, the base alteration occurs in at least 35% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. 
     In some embodiments, the base alteration occurs in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 10%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 1%-90%, 5%-85%, 10%-80%, 15%-75%, 20%-70%, 25%-65%, 30%-60%, 35%-55%, or 40%-50% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 100% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. 
     In some embodiments, the base alteration occurs in hepatocytes in the subject. In some embodiments, the base alteration occurs in at least 30% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in hepatocytes in the subject. In some embodiments, the base alteration occurs in at least % of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in at most 1%, 2%, 3%, 4%, 5%0, 10%, 15%, 20%, 25%, 30%, 3500 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 100%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 1%-90%, 5%-85%, 10%-80%, 15%-75%, 20%-70%, 25%-65%, 30%-60%, 35%-55%, or 40%-50% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 100% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. 
     In some embodiments, the base alteration occurred in whole liver cells in the subject is measured by next generation sequencing. In some embodiments, the base alteration occurred in whole liver cells in the subject is measured by Sanger sequencing. In some embodiments, the base alteration occurred in hepatocytes in the subject is measured by next generation sequencing. In some embodiments, the base alteration occurred in hepatocytes in the subject is measured by Sanger sequencing. 
     In some embodiments, the nucleobase alteration results in a reduction of at least 35% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of at least 35% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS 
     In some embodiments, the nucleobase alteration results in a reduction of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 100%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 31%-99.9%, 32%-99.9%, 33%-99.9%, 34%-99.9%, 35%-99.9%, 36%-99.9%, 37%-99.9%, 38%-99.9%, 39%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-79%, 1%-78%, 1%-77%, 1%-76%, 1%-75%, 1%-74%, 1%-73%, 1%-72%, 1%-71%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-39%, 1%-38%, 1%-37%, 1%-36%, 1%-35%, 1%-34%, 1%-33%, 1%-32%, 1%-31%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%, 25%-90%, 30%-85%, 31%-80%, 32%-79%, 33%-78%, 34%-77%, 35%-76%, 36%-76%, 37%-75%, 38%-74%, 39%-73%, 40%-72%, 45%-71%, 50%-70%, or 55%-65% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 100% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. 
     In some embodiments, the nucleobase alteration results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, 1000% less blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. 
     In some embodiments, the reduction of blood PCSK9 protein level or the blood PCSK9 protein level in the subject as compared to prior to the administration is measured by ELISA (enzyme-linked immunosorbent assay). In some embodiments, the reduction of blood PCSK9 protein level or the blood PCSK9 protein level in the subject as compared to prior to the administration is measured by Western blot analysis. In some embodiments, the reduction of blood PCSK9 protein level or the blood PCSK9 protein level in the subject as compared to prior to the administration is measured by LC-MS/MS (liquid chromatography-tandem mass spectrometry). 
     In some embodiments, the nucleobase alteration results in a reduction of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 100%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 31%-99.9%, 32%-99.9%, 33%-99.9%, 34%-99.9%, 35%-99.9%, 36%-99.9%, 37%-99.9%, 38%-99.9%, 39%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-79%, 1%-78%, 1%-77%, 1%-76%, 1%-75%, 1%-74%, 1%-73%, 1%-72%, 1%-7%, 1%0-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-39%, 1%-38%, 1%-37%, 1%-36%, 1%-35%, 1%-34%, 1%-33%, 1%-32%, 1%-31%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%, 25%-90%, 30%-85%, 31%-80%, 32%-79%, 33%-78%, 34%-77%, 35%-76%, 36%-76%, 37%-75%, 38%-74%, 39%-73%, 40%-72%, 45%-71%, 50%-70%, or 55%-65% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 100% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. 
     In some embodiments, the nucleobase alteration results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, 1000% less blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. 
     In some embodiments, the reduction of blood ANGPTL3 protein level or the blood ANGPTL3 protein level in the subject as compared to prior to the administration is measured by ELISA (enzyme-linked immunosorbent assay). In some embodiments, the reduction of blood ANGPTL3 protein level or the blood ANGPTL3 protein level in the subject as compared to prior to the administration is measured by Western blot analysis. In some embodiments, the reduction of blood ANGPTL3 protein level or the blood ANGPTL3 protein level in the subject as compared to prior to the administration is measured by LC-MS/MS (liquid chromatography-tandem mass spectrometry). 
     In some embodiments, the nucleobase alteration results in a reduction of at least 35% in blood or low-density lipoprotein cholesterol (LDL-C) levels in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of at least 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 1%-990.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 100%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%, 25%-90%, 30%-85%, 35%-80%, 40%-75%, 45%-70%, 50%-65%, or 55%-60% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 100% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10%, 11%, 2%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, 1000% less blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. 
     In some embodiments, the nucleobase alteration results in a reduction of at least 35% in blood triglyceride levels in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of at least 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of at least 1%, 2%, 3%, 4%, 5%, 6%7, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 10%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%, 25%-90%, 30%-85%, 35%-80%, 40%-75%, 45%-70%, 50%-65%, or 55%-60% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 100% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, 1000% less blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less blood triglyceride level in the subject as compared to prior to the administration. 
     In some embodiments, the blood triglyceride level or the reduction of blood triglyceride level in the subject as compared to prior to the administration is measured by any standard technique. In some embodiments, the blood low-density lipoprotein cholesterol (LDL-C) level or the reduction of blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration is measured by any standard technique. For example, a clinical analyzer instrument may be used to measure a ‘lipid panel’ in serum samples which entails the direct measurement of cholesterol (total C), triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C) enzymatically. Reagent kits specific for each analyte contain buffers, calibrators, blanks and controls. As used in the present disclosure, cholesterol, triglycerides and HDL-C may be quantified using absorbance measurements of specific enzymatic reaction products. LDL-C may be determined indirectly. In some instances, most of circulating cholesterol can be found in three major lipoprotein fractions: very low-density lipoproteins (VLDL), LDL and HDL. In some embodiments, total circulating cholesterol may be estimated with the formula [Total C]=[VLDL-C]+[LDL-C]+[HDL-C]. Thus the LDL-C can be calculated from measured values of total cholesterol, triglycerides and HDL-C according to the relationship: [LDL-C]=[total C]-[HDL-C]-[TG]/5, where [TG]/5 is an estimate of VLDL-cholesterol. A reagent kit specific for triglycerides containing buffers, calibrators, blanks and controls may be used. As used herein, serum samples from the study may be analyzed and triglycerides may be measured using a series of coupled enzymatic reactions. In some embodiments, H 2 O 2  may be used to quantify the analyte.as the end product of the last one and its absorbance at 500 nm, and the color intensity is proportional to triglyceride concentrations. 
     In some embodiments, the guide polynucleotide is a guide RNA, wherein the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with 0, 1, or 2 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with no mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with 1 mismatch. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with 2 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with 3 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with 4 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with 5 mismatches. 
     In some embodiments, the guide polynucleotide is a guide RNA, wherein the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with 0, 1, or 2 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with no mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with 1 mismatch. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with 2 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with 3 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with 4 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with 5 mismatches. 
     In some embodiments, the nucleobase alteration is outside of the protospacer sequence in less than 1% of whole liver cells in the subject as measured by net nucleobase editing. In some embodiments, the nucleobase alteration is outside of the protospacer sequence in less than 1% of hepatocytes in the subject as measured by net nucleobase editing. In some embodiments, the nucleobase alteration is only within the protospacer sequence as measured by net nucleobase editing. 
     In some embodiments, the nucleobase alteration is outside of the protospacer sequence in less than 0.01%. 0.02%, 0.03% 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 65%, 80%, 85%, 90% of whole liver cells in the subject as measured by net nucleobase editing. In some embodiments, the nucleobase alteration is outside of the protospacer sequence in less than 0.01%. 0.02%, 0.03% 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 65%, 80%, 85%, 90% of hepatocytes in the subject as measured by net nucleobase editing. In some embodiments, the nucleobase alteration is outside of the protospacer sequence in less than 0.01%. 0.02%, 0.03% 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 65%, 80%, 85%, 90% of cells in the subject as measured by net nucleobase editing. 
     In some embodiments, the deaminase is an adenine deaminase. In some embodiments, the nucleobase alteration is a A•T to G•C alteration. In some embodiments, the deaminase is an adenine deaminase and the nucleobase alteration is a A•T to G•C alteration. In some embodiments, the programmable DNA binding domain comprises a nuclease inactive Cas9 or a Cas9 nickase. In some embodiments, the programmable DNA binding domain comprises a Cas9. 
     In some embodiments, the nucleobase alteration is at a splice site of the PCSK9 gene. In some embodiments, the nucleobase alteration is at a splice donor site of the PCSK9 gene. In some embodiments, the splice donor site is at 5′ end of PCSK9 intron 1 as referenced in SEQ ID NO: 5. In some embodiments, the nucleobase alteration is at a splice acceptor site of the PCSK9 gene. In some embodiments, the nucleobase alteration results in a frame shift, a premature stop codon, a insertion or deletion in a transcript encoded by the PCSK9 gene. In some embodiments, the nucleobase alteration results in an aberrant transcript encoded by the PCSK9 gene. In some embodiments, the guide polynucleotide is a guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the guide RNA comprises a tracrRNA sequence. In some embodiments, the guide RNA comprises a chemical modification as set forth in Table 1 or Table 24. 
     In some embodiments, the nucleobase alteration is at a splice site of the ANGPTL3 gene. In some embodiments, the nucleobase alteration is at a splice donor site of the ANGPTL3 gene. In some embodiments, the splice donor site is at 5′ end of ANGPTL3 intron 6 as referenced in SEQ ID NO: 7. In some embodiments, the nucleobase alteration is at a splice acceptor site of the ANGPTL3 gene. In some embodiments, the nucleobase alteration results in a frame shift, a premature stop codon, a insertion or deletion in a transcript encoded by the ANGPTL3 gene. In some embodiments, the nucleobase alteration results in an aberrant transcript encoded by the ANGPTL3 gene. In some embodiments, the guide polynucleotide is a guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the guide RNA comprises a tracrRNA sequence. In some embodiments, the guide RNA comprises a chemical modification as set forth in Table 1 or Table 24. 
     In some embodiments, the guide RNA comprises a guide RNA sequence set forth in Table 1 or Table 24. In some embodiments, the guide RNA comprises the sequence 5′-5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUC cG UUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu-3′ (SEQ ID NO: 9), 5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUcc GU UAucAAcuugaaaaagugGcaccgagucggugcusususu-3′ (SEQ ID NO: 9), 5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUcc GUUAucAAcuuGaaaaagugGcaccgagucggugcusususu-3′ (SEQ ID NO: 9) (GA346), 5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUcc GUUAacAAcuugaaaaagugGcaccgagucggugcusususu-3′ (SEQ ID NO: 10) (GA374), 5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUcc GUUAucAAcuugaaaaagugGcaccgagucggugcusususuuuu-3′ (SEQ ID NO: 11) (GA385), 5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUcc GUUAucAAcuugaaaaagugGcaccgagucggugcusususuuUu-3′ (SEQ ID NO: 11) (GA386) or 5′-cscscsGCACCUUGGCGCAGCGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccG UUAucAAcuuGaaaaagugGcaccgagucggugcuususuuuu-3′ (SEQ ID NO: 12) (GA387). 
     In some embodiments, the protospacer sequence comprises a protospacer sequence set forth in Table 1 or Table 24. In some embodiments, the protospacer comprises the sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 13) 
               
               
                   
                 5′-CCCGCACCTTGGCGCAGCGG-3′, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 14) 
               
               
                   
                 AAGATACCTGAATAACTCTC-3′, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 15) 
               
               
                   
                 5′-AAGATACCTGAATAACCCTC-3′. 
               
            
           
         
       
     
     In some embodiments, the base editor fusion protein comprises the sequence of SEQ ID NO: 3. In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 3 or to any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identity plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to the amino acid sequence set forth in SEQ ID NO: 3 or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NO: 3 or any of the adenosine deaminases provided herein. 
     In some embodiments, the nucleic acid encoding the base editor fusion protein is a mRNA. The mRNA may comprise modifications, for example, modifications at 3′ or 5′ end of the mRNA. In some embodiments, the mRNA comprises a cap analog. 
     In some embodiments, the mRNA comprises at least 1, 2, or 3 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 1, 2, or 3 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 1 nucleotide at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 2 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 3 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 4 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 5 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 6 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 7 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 8 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 9 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 10 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. 
     In some embodiments, the mRNA comprises a poly A tail. The poly A tail may be at the 3′ end of the mRNA. 
     In some embodiments, the GC % content of the mRNA sequence is greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, the GC % content of the mRNA sequence is greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%. 
     In some embodiments, the mRNA sequence comprises an adenine tTNA deaminase (TadA) region. In some embodiments, the GC % of the TadA region is greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, the GC % content of the TadA region is greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%. 
     In some embodiments, the mRNA sequence comprises a Cas9 region. In some embodiments, the GC % of the Cas9 region is greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, the GC % content of the Cas9 region is greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%. 
     In some embodiments, the mRNA sequence comprises a NLS region. In some embodiments, the GC % of the NLS region is greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, the GC % content of the NLS region is greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%. 
     In some embodiments, the mRNA sequence comprises a first linker region that connects the TadA region and the Cas9 region. In some embodiments, the GC % of the first linker region is greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, the GC % content of the first linker region is greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%. 
     In some embodiments, the mRNA sequence comprises a second linker region that connects the Cas9 region and the NLS region. In some embodiments, the GC % of the second linker region is greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, the GC % content of the second linker region is greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%. 
     In some embodiments, the base editor system as provided herein further comprises a lipid nanoparticle (LNP) enclosing a guide polynucleotide or a nucleic acid encoding the guide polynucleotide (i). In some embodiments, the LNP further encloses a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same (ii). In some embodiments, the base editor system further comprises a second LNP enclosing a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same (ii). 
     A base editor system as provided herein can include one or more LNPs. For example, a base editor system may comprise a LNP enclosing both a guide polynucleotide and a nucleic acid encoding the base editor fusion protein, e.g. an mRNA encoding the base editor fusion protein. In another example, a base editor system may comprise a LNP enclosing a guide polynucleotide, e.g. a guide RNA, and a LNP enclosing a nucleic acid, e.g. an mRNA, encoding the base editor fusion protein. LNPs separately enclosing the guide polynucleotide and the base editor fusion protein or mRNA encoding the base editor fusion protein may allow for flexible dosing and administration of the base editor system. For example, a LNP enclosing a guide RNA can be administered first, followed by administration of a LNP enclosing a mRNA encoding the base editor fusion protein. In some embodiments, a LNP enclosing a guide RNA and a second LNP enclosing a mRNA encoding the base editor fusion protein are administered to a subject at the same time. In some embodiments, a LNP enclosing a guide RNA and a LNP enclosing a mRNA encoding the base editor protein are administered to a subject sequentially. In some embodiments, a LNP enclosing mRNA encoding the base editor fusion protein is administered to a subject, followed by multiple administration or doses of a second LNP enclosing a guide RNA after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or more. The multiple doses of the second LNP may be administered with intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or more. 
     In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1:10 to about 10:1 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1:1, 1.5:1, 2:1, 3:1, 4:1, 1:1.5, 1:2, 1:3, 1:4 or any ratio between 4:1 or 1:4 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein can be determined by titration of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein. 
     In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 500:1 to about 1:500. 
     In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1000:1 to about 1:1000 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2; 1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2; 1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2; 1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. 
     In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 10000:1 to about 1:10000. In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. 
     In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 10:1 to about 1:10 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 4:1, 3;1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, or 1:4 by weight. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 500:1 to about 1:500. 
     In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1000:1 to about 1:1000 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. 
     In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 10000:1 to about 1:10000. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. 
     Precision genome editing is a growing field with industrial, agricultural, and biomedical applications. One of the dominant genome-editing systems available today is clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9). Through the use of a guide RNA (gRNA) with a sequence homologous to that of a sequence of DNA in the target genome (known as the protospacer) adjacent to a specific protospacer-adjacent motif (PAM) comprising the sequence NGG (N is any standard base) in the DNA, Cas9 can be used to create a double-strand break (DSB) at the targeted sequence. Non-homologous end joining (NHEJ) at DSBs can be used to create indels and knock out genes at genetic loci; likewise, homology-directed repair (HDR) can be used, with an introduced template DNA, to insert genes or modify the targeted sequence. A variety of Cas9-based tools have been developed in recent years, including tools that methylate DNA, recognize broader sequence space, or create single-strand nicks. In 2016, Komor et al. described the use of CRISPR-Cas9 to convert a cytosine base to a thymine base without the introduction of a template DNA strand and without the need for DSBs (Komor A C, Kim Y B, Packer MS, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.  Nature,  2016, 533: 420-4, incorporated herein by reference in its entirety). After the cytidine deaminase domain of rat APOBEC1 was fused to the N-terminus of catalytically-dead Cas9 (dCas9) using the linker XTEN (resulting in a fusion protein called base editor 1, or BE1), conversion of cytosine to uracil was observed between position 4 and position 8 within the 20-nt protospacer region of DNA (or, to express it a different way, 13 to 17 nucleotides upstream of the PAM). Of note, any cytosine base within this “window” was amenable to editing, resulting in varied outcomes depending on how many and which cytosines were edited. After DNA replication or repair, each uracil was replaced by a thymine, completing the C to T base editing.3 The next version of base editor (BE2) incorporated a uracil glycosylase inhibitor fused to the C-terminus of dCas9 to help inhibit base excision repair of the uracil bases resulting from the cytidine deaminase activity (which otherwise would act to restore the original cytosine bases); this improved the efficiency of C to T base editing. The final version, BE3, used a Cas9 nickase rather than dCas9; the nickase cut the unedited strand opposite the edited C to T bases, stimulating the removal of the opposing guanidine through eukaryotic mismatch repair. BE2 and BE3 base editing was observed in both human and murine cell lines. The specificity of base editing has been further improved through the addition of mutations to the Cas9 nickase; in similar fashion, Cas9 has been mutated to narrow the width of the editing window from approximately 5 nucleotides to as little as 1-2 nucleotides (Rees H A, Komor A C, Yeh W H, et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun, 2017, 8: 15790, Kim Y B, Komor A C, Levy J M, et al. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol, 2017, 35: 371-6, each of which is incorporated herein by reference in its entirety). 
     An alternative cytosine base editing platform is by linking the activation-induced cytosine deaminase domain PmCDA1 to dCas9 (Target-AID), they were able to demonstrate targeted C to T base editing in yeast. Furthermore, an alternative C to T editing strategy was also demonstrated without fusing a deaminase domain to Cas9; instead, a SH3 (Src 3 homology) domain was added to the C-terminus of dCas9 while a SHL (SH3 interaction ligand) was added to PmCDA1.6 Optimization of efficiency was achieved through the use of a Cas9 nickase rather than dCas9. Further, an uracil DNA glycosylase inhibitor was added to enhance base editing in the mammalian CHO cell line. The resulting platform was able to consistently edit bases within 3 to 5 bases of the 18th nucleotide upstream of the PAM sequence (Nishida K, Arazoe T, Yachie N, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science, 2016, 353: aaf8729, incorporated herein by reference in its entirety). 
     A distinct cytosine base editing platform used Cpf1 (also known as Cas12a) instead of Cas9 as the RNA-guided endonuclease. Catalytically-inactive Cpf1 was fused to APOBEC1 (dLbCpfl-BE0), leading to C to T conversion in a human cell line. While the Cas9 base editor variants BE3 and Target-AID recognize the PAM sequence NGG, dLbCpfl-BE0 recognizes the T-rich PAM sequence TTTV. Although base editing was observed between positions 8 and 13 of the protospacer sequence with dLbCpfl-BE0, the introduction of additional mutations into Cpf1 was able to reduce the window to positions 10 to 12. However, narrowing of the base editing window correlated with a decrease in editing efficiency (Li X, Wang Y, Liu Y, et al. Base editing with a Cpf1-cytidine deaminase fusion. Nat Biotechnol, 2018, 36: 324-7, incorporated herein by reference in its entirety). 
     Cytosine base editing is not wholly predictable; indels can occur at the target site, albeit at lower frequencies that those observed for C to T editing editors. Furthermore, cytosine base editors can occasionally cause C to A or C to G edits rather than the expected C to T edits. Adding linker lengths between Cas9 nickase and the rat APOBEC1 cytosine deaminase domain from 16 amino acids to 32 amino acids, the linker between the Cas9 nickase and the uracil glycosylase inhibitor from 4 amino acids to 9 amino acids, and using a second uracil glycosylase inhibitor was appended to the C-terminus of the new cytosine base editor using another 9 amino acid linker improved cytosine base editor termed “BE4” (Komor A C, Zhao K T, Packer M S, et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv, 2017, 3: eaao4774. Incorporated herein by reference in its entirety). 
     By fusing  Escherichia coli  adenine tTNA deaminase TadA (ecTadA) to dCas9 and mutagenesis of the ecTadA domain in conjunction with selection for editing activity revealed that A106V and D108N mutations yielded a base editor capable of editing adenine to guanine in DNA, termed ABE7.10 (Gaudelli N M, Komor A C, Rees H A, et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature, 2017, 551: 464-71. Incorporated herein by reference in its entirety). Koblan et al. improved the efficiency of ABE7.10 through modification of nuclear localization signals and codon optimization, yielding a version called ABEmax; a similar approach improved the efficiency of the cytosine base editor BE4.10 Huang et al. performed further development of both adenine and cytosine base editors to use alternative PAMs and to expand their editing windows, thereby increasing their targeting range (Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol, 2018, 36: 843-6, Incorporated herein by reference in its entirety). 
     The same has proven to be true of base editors, 12-14 although comparisons of Cas9, cytosine base editors, and adenine base editors using the same gRNAs have shown distinct off-target profiles. 
     A variety of studies have raised concern about gRNA-independent off-target base editing, caused by the deaminase domain acting in isolation (without the need for engagement of DNA by the Cas9-gRNA complex). Additional studies showed that the gRNA-independent off-target effects of base editors are not limited to DNA. RNA sequencing of cells treated with either cytosine base editors or adenine base editors revealed transcriptome-wide off-target editing of RNA, and that introduction of amino acid substitution in the deaminase domain of adenine base (e.g. R106W) editors reduced off-target editing of RNA without substantially reducing on-target DNA base-editing efficiency. Zuo E, Sun Y, Wei W, et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science, 2019, 364: 289-92; Jin S, Zong Y, Gao Q, et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science, 2019, 364: 292-5; Grunewald J, Zhou R, Garcia SP, et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature, 2019, 569: 433-7; Grünewald J, Zhou R, Iyer S, et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat Biotechnol, 2019, 37: 1041-8; Zhou C, Sun Y, Yan R, et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature, 2019, 571: 275-8; Rees H A, Wilson C, Doman J L, et al. Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci Adv, 2019, 5: eaax5717, each of which is incorporated herein by reference in its entirety). 
     Correction of disease-causing mutations via precision editing with standard Cas9 genome editing has largely required HDR. Since HDR is limited to cells in S or G2 phase of mitosis, precision editing of non-mitotic cells is difficult. However, base editors are not reliant on HDR; the editing of postmitotic cochlear cells in mice is feasible with cytosine base editor BE3. By injecting BE3 and a gRNA in the form of a preassembled ribonucleoprotein via cationic liposomes, serine-33 in beta-catenin was edited to phenylalanine (TCT codon edited to TTT), allowing for the transdifferentiation of supporting cells into hair cells. Base editing of cochlear tissue was confirmed via sequencing, showing an editing rate between 0.7% and 3.0% depending on the region of the cochlea. In contrast, standard Cas9 editing via HDR showed negligible signs of efficacy in cochlear cells. A variant of SaBE3, delivered into the liver via adeno-associated viral (AAV) vectors, was reported to directly correct a pathogenic T to C mutation in the Pah gene with an editing rate as high as 29% and thereby treat the disease phenylketonuria in adult mice. Adenine base editing has also been demonstrated to generate mutations in mice. Yeh W H, Chiang H, Rees H A, et al. In vivo base editing of post-mitotic sensory cells. Nat Commun, 2018, 9: 2184; Villiger L, Grisch-Chan H M, Lindsay H, et al. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Nat Med, 2018, 24: 1519-25; Ryu S M, Koo T, Kim K, et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat Biotechnol, 2018, 36: 536-9; Song C Q, Jiang T, Richter M, et al. Adenine base editing in an adult mouse model of tyrosinaemia. Nat Biomed Eng, 2020, 4: 125-30; Pisciotta L, Favari E, Magnolo L, et al. Characterization of three kindreds with familial combined hypolipidemia caused by loss-of-function mutations of ANGPTL3. Circ Cardiovasc Genet, 2012, 5: 42-50. each of which is incorporated herein by reference in its entirety. 
     Provided herein are compositions of nucleobase editor systems that comprises nucleobase editor proteins, complexes, or compounds that is capable of making a modification or conversion to a nucleobase (e.g., A, T, C, G, or U) within a target nucleotide sequence. 
     A nucleobase editor or a base editor (BE) refers to an agent comprising a polypeptide that is capable of making a modification to a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid. In some embodiments, the base editor is capable of deaminating a base within a DNA molecule. In some embodiments, the base editor is capable of deaminating an adenine (A) in DNA. 
     In some embodiments, the base editor comprises a fusion protein comprising a programmable DNA binding protein fused to an adenosine deaminase. In some embodiments, the base editor comprises a fusion protein comprising a Cas9 protein and an adenosine deaminase. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to an adenosine deaminase. In some embodiments, the base editor is a nuclease-inactive Cas9 (dCas9) fused to an adenosine deaminase. In some embodiments, the base editor comprises a fusion protein comprising a programmable DNA binding protein fused to an cytidine deaminase. In some embodiments, the base editor comprises a fusion protein comprising a Cas9 protein and an cytidine deaminase. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to an cytidine deaminase. In some embodiments, the base editor is a nuclease-inactive Cas9 (dCas9) fused to an cytidine deaminase. In some embodiments, the base editor further comprises, an inhibitor of base excision repair, for example, a UGI domain. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair, such as a UGI or dISN domain. In some embodiments, the dCas9 domain of the fusion protein comprises a D10A and a H840A mutation as numbered in the wild type SpCas9 amino acid sequence. In some embodiments, the UGI comprises the following amino acid sequence: 
     
       
         
           
               
            
               
                 &gt;sp|P14739IUNGI_BPPB2 Uracil-DNA glycosylase 
               
               
                 inhibitor 
               
               
                 (SEQ ID NO: 16) 
               
               
                 MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDES 
               
               
                   
               
               
                 TDENVMLLTS D APE YKPW ALVIQDS NGENKIKML 
               
            
           
         
       
     
     In some embodiments, a base editor system provided herein comprises a base editor fusion protein. For example, a base editor fusion protein may comprise a programmable DNA binding protein and a deaminase, e.g. an adenosine deaminase. In some embodiments, any of the fusion proteins provided herein are base editors. In some embodiments, the programmable DNA binding protein is a Cas9 domain, a Cpf1 domain, a CasX domain, a CasY domain, a Cas12b domain, a C2c2 domain, aC2c3 domain, or an Argonaute domain. In some embodiments, the programmable DNA binding protein is a Cas9 domain. The Cas9 domain may be any of the Cas9 domains or Cas9 proteins (e.g., nuclease inactive Cas9 or Cas9 nickase, or a Cas9 variant from any species) provided herein. In some embodiments, any of the Cas9 domains or Cas9 proteins provided herein may be fused with any of the deaminases provided herein. In some embodiments, the base editor comprises a deaminase, e.g., an adenosine deaminase and a programmable DNA binding protein, e.g., a Cas9 domain joined via a linker. In some embodiments, the base editor comprises a fusion protein comprising a deaminase, e.g., an adenosine deaminase and a programmable DNA binding protein, e.g., a Cas9 domain joined via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, a linker is present between the deaminase domain and the Cas9 domain. In some embodiments, an deaminase and a programmable DNA binding domain are fused via any of the peptide linkers provided herein. For example, an adenosine deaminase and a Cas9 domain may be fused via a linker that comprises between 1 and 200 amino acids. In some embodiments, the adenosine deaminase and the programmable DNA binding protein are fused via a linker that comprises from 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 80, 1 to 100, 1 to 150, 1 to 200, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 60, 5 to 80, 5 to 100, 5 to 150, 5 to 200, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 80, 10 to 100, 10 to 150, 10 to 200, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 80, 20 to 100, 20 to 150, 20 to 200, 30 to 40, 30 to 50, 30 to 60, 30 to 80, 30 to 100, 30 to 150, 30 to 200, 40 to 50, 40 to 60, 40 to 80, 40 to 100, 40 to 150, 40 to 200, 50 to 60 50 to 80, 50 to 100, 50 to 150, 50 to 200, 60 to 80, 60 to 100, 60 to 150, 60 to 200, 80 to 100, 80 to 150, 80 to 200, 100 to 150, 100 to 200, or 150 to 200 amino acids in length. In some embodiments, the adenosine deaminase and the programmable DNA binding protein are fused via a linker that comprises 4, 16, 32, or 104 amino acids in length. In some embodiments, the adenosine deaminase and the programmable DNA binding protein are fused via a linker that comprises the amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 17), SGGS (SEQ ID NO: 18), SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 19), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 20), or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 21). In some embodiments, the adenosine deaminase and the programmable DNA binding protein are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 17), which may also be referred to as the XTEN linker. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 23). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 24). 
     In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS (SEQ ID NO: 25). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 26). 
     In some embodiments, a base editor system provided herein comprises a base editor comprising a fusion protein comprising an inhibitor of base repair. In some embodiments, a base editor comprises a fusion protein comprising a cytidine deaminase and a programmable DNA binding domain, e.g. a Cas9 domain. In some embodiments, a base editor comprises a fusion protein comprising an adenosine deaminase and a programmable DNA binding domain, e.g. a Cas9 domain. In some embodiments, the base editor or the fusion protein further comprises an inhibitor of base repair (IBR). In some embodiments, the IBR comprises an inhibitor of inosine base repair. In some embodiments, the IBR is an inhibitor of inosine base excision repair. In some embodiments, the inhibitor of inosine base excision repair is a catalytically inactive inosine specific nuclease (dISN). In some embodiments, a dISN may inhibit (e.g., by steric hindrance) inosine removing enzymes from excising the inosine residue from DNA. For example, catalytically dead inosine glycosylases (e.g., alkyl adenine glycosylase [AAG]) will bind inosine but will not create an abasic site or remove the inosine, thereby sterically blocking the newly-formed inosine moiety from potential DNA damage/repair mechanisms. Thus, this disclosure contemplates a fusion protein comprising a programmable DNA binding protein and an adenosine deaminase further fused to a dISN. This disclosure contemplates a fusion protein comprising any Cas9 domain, for example, a Cas9 nickase (nCas9) domain, a catalytically inactive Cas9 (dCas9) domain, a high fidelity Cas9 domain, or a Cas9 domain with reduced PAM exclusivity. It should be understood that the use of a dISN may increase the editing efficiency of a adenosine deaminase that is capable of catalyzing a A to I change. For example, fusion proteins comprising a dISN domain may be more efficient in deaminating A residues. 
     In some embodiments, the base editors provided herein comprise fusion proteins that further comprise one or more nuclear targeting sequences, for example, a nuclear localization sequence (NLS). In some embodiments, the fusion protein comprises multiple NLSs. In some embodiments, the fusion protein comprises a NLS at the N-terminus and the C-terminus of the fusion protein. In some embodiments, a NLS comprises an amino acid sequence that facilitates the importation of a protein, that comprises an NLS, into the cell nucleus. In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the programmable DNA binding protein, e.g. the Cas9. In some embodiments, the NLS is fused to the C-terminus of the programmable DNA binding protein. In some embodiments, the NLS is fused to the N-terminus of the adenosine deaminase. In some embodiments, the NLS is fused to the C-terminus of the adenosine deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises an amino acid sequence of any one of the NLS sequences provided or referenced herein. In some embodiments, a NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 27) or MD SLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 28). Additional nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et ah, PCT/EP2000/011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. 
     In some embodiments, the fusion proteins provided herein do not comprise a linker. In some embodiments, a linker is present between one or more of the domains or proteins (e.g., adenosine deaminase, napDNAbp, NLS, and/or IBR). In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker. 
     Some aspects of the disclosure provide base editors or fusion proteins that comprise a programmable DNA binding protein and at least two adenosine deaminase domains. Without wishing to be bound by any particular theory, dimerization of adenosine deaminases (e.g., in cis or in trans) may improve the ability (e.g., efficiency) of the fusion protein to modify a nucleic acid base, for example to deaminate adenine. In some embodiments, any of the fusion proteins may comprise 2, 3, 4 or 5 adenosine deaminase domains. In some embodiments, any of the fusion proteins provided herein comprise two adenosine deaminases. In some embodiments, any of the fusion proteins provided herein contain only two adenosine deaminases. In some embodiments, the adenosine deaminases are the same. In some embodiments, the adenosine deaminases are any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminases are different. In some embodiments, the first adenosine deaminase is any of the adenosine deaminases provided herein, and the second adenosine is any of the adenosine deaminases provided herein, but is not identical to the first adenosine deaminase. In some embodiments, the first adenosine deaminase comprises any one of the mutations provided herein as numbered in SEQ ID NO: 1. In some embodiments, the second adenosine deaminase comprises any one of the mutations provided herein as numbered in SEQ ID NO: 1. In some embodiments, the first adenosine deaminase comprises any one of the mutations provided herein as numbered in SEQ ID NO: 1, and the second adenosine deaminase comprises a wild type adenosine deaminase sequence. In some embodiments, the second adenosine deaminase comprises any one of the mutations provided herein as numbered in SEQ ID NO: 1, and the first adenosine deaminase comprises a wild type adenosine deaminase sequence. As one example, the fusion protein may comprise a first adenosine deaminase and a second adenosine deaminase that both comprise a A106V, D108N, D147Y, and E155V mutation from ecTadA (SEQ ID NO: 1). As another example, the fusion protein may comprise a first adenosine deaminase domain that comprises a A106V, D108N, D147Y, and E155V mutation from ecTadA (SEQ ID NO: 1), and a second adenosine deaminase that comprises a L84F, A106V, D108N, H123Y, D147Y, E155V, and I156F mutation from ecTadA (SEQ ID NO: 1). 
     In some embodiments, the adenosine deaminase comprises the amino acid sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 1) 
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG 
               
               
                   
               
               
                 RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR 
               
               
                   
               
               
                 MRRQEIKAQKKAQSSTD 
               
            
           
         
       
     
     In some embodiments, the fusion protein comprises two adenosine deaminases (e.g., a first adenosine deaminase and a second adenosine deaminase). In some embodiments, the first adenosine deaminase is N-terminal to the second adenosine deaminase in the fusion protein. In some embodiments, the first adenosine deaminase is C-terminal to the second adenosine deaminase in the fusion protein. In some embodiments, the first adenosine deaminase and the second deaminase are fused directly or via a linker. In some embodiments, the linker is any of the linkers provided herein, for example, any of the linkers described in the “Linkers” section. In some embodiments, the first adenosine deaminase is the same as the second adenosine deaminase. In some embodiments, the first adenosine deaminase and the second adenosine deaminase are any of the adenosine deaminases described herein. In some embodiments, the first adenosine deaminase and the second adenosine deaminase are different. In some embodiments, the first adenosine deaminase is any of the adenosine deaminases provided herein. In some embodiments, the second adenosine deaminase is any of the adenosine deaminases provided herein but is not identical to the first adenosine deaminase. In some embodiments, the first adenosine deaminase is an ecTadA adenosine deaminase. In some embodiments, the first adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence set forth SEQ ID NO: 1 or to any of the adenosine deaminases provided herein. In some embodiments, the second adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence set forth SEQ ID NO: 1 or to any of the adenosine deaminases provided herein. In some embodiments, the second adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 1. 
     In some embodiments, the fusion proteins provided herein do not comprise a linker. In some embodiments, a linker is present between one or more of the domains or proteins (e.g., first adenosine deaminase, second adenosine deaminase, programmable DNA binding protein, and/or NLS). 
     It should be appreciated that the fusion proteins of the present disclosure may comprise one or more additional features. For example, in some embodiments, the fusion protein may comprise cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more His tags. 
     In some embodiments, the fusion protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the amino acid sequences listed in Table 23. In some embodiments, the fusion protein comprises any one of the amino acid sequences listed in Table 23. In some embodiments, the sequence of the fusion protein is any one of the amino acid sequences listed in Table 23. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the amino acid sequences of SEQ ID NOs: 2137, 2149, 2154, 2158, 2188, 2140, 40, 2146, 2152, 2156, and 2160. In some embodiments, the fusion protein comprises any one of the amino acid sequences of SEQ ID NOs: 2137, 2149, 2154, 2158, 2188, 2140, 40, 2146, 2152, 2156, and 2160. In some embodiments, the sequence of the fusion protein is any one of the amino acid sequences of SEQ ID NOs: 2137, 2149, 2154, 2158, and 2188. 
     In some embodiments, the fusion protein is encoded by the polynucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences listed in Table 23. In some embodiments, the fusion protein is encoded by any one of the polynucleotide sequences listed in Table 23. In some embodiments, the fusion protein is expressed by the polynucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences listed in Table 23. In some embodiments, the fusion protein is expressed by any one of the polynucleotide sequences listed in Table 23. In some embodiments, the fusion protein is encoded by the polynucleotide sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. In some embodiments, the fusion protein is encoded by the polynucleotide sequence that comprises any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. In some embodiments, the fusion protein is encoded by any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, and 2189. In some embodiments, the fusion protein is expressed by the polynucleotide sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2190, 2138, 2147, 2158, or a combination thereof: In some embodiments, the fusion protein is encoded by the polynucleotide sequence that comprises any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2190, 2138, 2147, 2158, or a combination thereof. In some embodiments, the fusion protein is expressed by any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, and 2189. In some embodiments, the polynucleotide sequence further comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences of SEQ ID NOs: 2138, 2147, 2158, or a combination thereof. In some embodiments, the polynucleotide sequence further comprises any one of the polynucleotide sequences of SEQ ID NOs: 2138, 2147, 2158, or a combination thereof. 
     In some embodiments, the nucleobase editor ABE8.8 comprises a fusion protein comprising the sequence as provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 3) 
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
               
               
                   
               
               
                 LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFR 
               
               
                   
               
               
                 MPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
               
               
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL 
               
               
                   
               
               
                 FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE 
               
               
                   
               
               
                 ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR 
               
               
                   
               
               
                 LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
               
               
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF 
               
               
                   
               
               
                 KSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL 
               
               
                   
               
               
                 SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF 
               
               
                   
               
               
                 DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
               
               
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV 
               
               
                   
               
               
                 GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNL 
               
               
                   
               
               
                 PNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL 
               
               
                   
               
               
                 FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
               
               
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK 
               
               
                   
               
               
                 RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL 
               
               
                   
               
               
                 TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMG 
               
               
                   
               
               
                 RHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
               
               
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSI 
               
               
                   
               
               
                 DNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK 
               
               
                   
               
               
                 AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE 
               
               
                   
               
               
                 VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
               
               
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL 
               
               
                   
               
               
                 ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT 
               
               
                   
               
               
                 GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG 
               
               
                   
               
               
                 KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
               
               
                 LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN 
               
               
                   
               
               
                 EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
               
               
                   
               
               
                 REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI 
               
               
                   
               
               
                 TGLYETRIDLSQLGGD 
               
            
           
         
       
     
     In some embodiments, the nucleobase editor comprises a fusion protein comprising a polypeptide encoded by the polynucleotide (herein also referenced as MA002) sequence as provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 4) 
               
               
                 ATGAGCGAGGTCGAGTTCTCTCACGAATATTGGATGAGACACGCTCTCAC 
               
               
                   
               
               
                 CCTGGCTAAGAGAGCCAGGGACGAAAGAGAGGTGCCAGTTGGCGCTGTCC 
               
               
                   
               
               
                 TGGTGTTGAACAATCGCGTCATCGGAGAAGGATGGAATCGCGCCATTGGC 
               
               
                   
               
               
                 CTGCACGATCCAACCGCACATGCCGAAATTATGGCTCTGCGGCAAGGCGG 
               
               
                   
               
               
                 CCTCGTGATGCAAAATTACAGACTGATCGATGCTACCCTCTACGTCACCT 
               
               
                   
               
               
                 TCGAGCCCTGTGTCATGTGTGCTGGGGCAATGATTCACTCCCGGATTGGC 
               
               
                   
               
               
                 CGCGTGGTGTTTGGAGTGCGGAATGCCAAGACTGGCGCCGCTGGATCTCT 
               
               
                   
               
               
                 GATGGACGTCCTGCACcatCCTGGGATGAACCACCGGGTCGAGATCACAG 
               
               
                   
               
               
                 AGGGAATTCTGGCTGACGAGTGCGCTGCCCTGCTGTGCaggTTCTTTAGA 
               
               
                   
               
               
                 ATGCCtAGAaggGTGTTCAACGCCCAGAAAAAAGCTCAGAGCAGCACCGA 
               
               
                   
               
               
                 TTCCGGCGGAAGCAGCGGAGGATCTTCTGGAAGCGAAACCCCAGGCACCA 
               
               
                   
               
               
                 GCGAGTCTGCCACACCAGAATCATCTGGCGGTAGCTCCGGCGGCAGCGAC 
               
               
                   
               
               
                 AAGAAGTATTCTATCGGACTGGCCATCGGCACCAACTCTGTTGGATGGGC 
               
               
                   
               
               
                 CGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGG 
               
               
                   
               
               
                 GCAACACCGACAGGCACAGCATCAAGAAGAACCTGATCGGCGCACTGCTG 
               
               
                   
               
               
                 TTCGACTCTGGCGAAACAGCCGAGGCCACCAGACTGAAGAGAACAGCCCG 
               
               
                   
               
               
                 CAGACGGTACACCAGAAGAAAGAACCGGATCTGCTACCTCCAAGAGATCT 
               
               
                   
               
               
                 TCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAA 
               
               
                   
               
               
                 GAGTCCTTCCTGGTGGAAGAGGACAAGAAGCACGAGAGACACCCCATCTT 
               
               
                   
               
               
                 CGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCT 
               
               
                   
               
               
                 ACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGAGA 
               
               
                   
               
               
                 CTGATCTATCTGGCCCTGGCTCACATGATCAAGTTCCGGGGCCACTTCCT 
               
               
                   
               
               
                 GATCGAGGGCGACCTGAATCCTGACAACAGCGACGTGGACAAGCTGTTCA 
               
               
                   
               
               
                 TCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAAC 
               
               
                   
               
               
                 GCCAGCGGAGTGGATGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAG 
               
               
                   
               
               
                 CAGACGGCTGGAAAATCTGATCGCCCAGCTGCCTGGCGAGAAGAAGAATG 
               
               
                   
               
               
                 GCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACACCTAACTTC 
               
               
                   
               
               
                 AAGAGCAACTTCGACCTGGCCGAGGACGCCAAACTGCAGCTGAGCAAGGA 
               
               
                   
               
               
                 CACCTACGACGACGACCTGGACAATCTGCTGGCCCAGATCGGCGATCAGT 
               
               
                   
               
               
                 ACGCCGACTTGTTTCTGGCCGCCAAGAATCTGAGCGACGCCATCCTGCTG 
               
               
                   
               
               
                 TCCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCACCTCTGAGCGC 
               
               
                   
               
               
                 CTCTATGATCAAGAGATACGACGAGCACCACCAGGATCTGACCCTGCTGA 
               
               
                   
               
               
                 AGGCCCTCGTTAGACAGCAGCTGCCAGAGAAGTACAAAGAGATTTTCTTC 
               
               
                   
               
               
                 GACCAGAGCAAGAACGGCTACGCCGGCTACATTGATGGCGGAGCCAGCCA 
               
               
                   
               
               
                 AGAGGAATTCTACAAGTTCATCAAGCCCATCCTCGAGAAGATGGACGGCA 
               
               
                   
               
               
                 CCGAGGAACTGCTGGTCAAGCTGAACAGAGAGGACCTGCTGAGAAAGCAG 
               
               
                   
               
               
                 AGAACCTTCGACAACGGCAGCATCCCTCACCAGATCCACCTGGGAGAACT 
               
               
                   
               
               
                 GCACGCCATTCTGCGGAGACAAGAGGACTTTTACCCATTCCTGAAGGACA 
               
               
                   
               
               
                 ACCGGGAAAAGATCGAGAAAATCCTGACCTTCAGGATCCCCTACTACGTG 
               
               
                   
               
               
                 GGACCACTGGCCAGAGGCAATAGCAGATTCGCCTGGATGACCAGAAAGAG 
               
               
                   
               
               
                 CGAGGAAACCATCACTCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCG 
               
               
                   
               
               
                 CCAGCGCTCAGTCCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTG 
               
               
                   
               
               
                 CCTAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCAC 
               
               
                   
               
               
                 CGTGTACAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAA 
               
               
                   
               
               
                 AGCCCGCCTTTCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGATCTGCTG 
               
               
                   
               
               
                 TTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTT 
               
               
                   
               
               
                 CAAGAAAATCGAGTGCTTCGACAGCGTCGAGATCTCCGGCGTGGAAGATC 
               
               
                   
               
               
                 GGTTCAATGCCAGCCTGGGCACATACCACGATCTGCTGAAAATTATCAAG 
               
               
                   
               
               
                 GACAAGGACTTCCTGGACAACGAAGAGAACGAGGACATCCTTGAGGACAT 
               
               
                   
               
               
                 CGTGCTGACACTGACCCTGTTTGAGGACAGAGAGATGATCGAGGAACGGC 
               
               
                   
               
               
                 TGAAAACATACGCCCACCTGTTCGACGACAAAGTGATGAAGCAACTGAAG 
               
               
                   
               
               
                 CGGCGGAGATACACCGGCTGGGGCAGACTGTCTCGGAAGCTGATCAACGG 
               
               
                   
               
               
                 CATCCGGGATAAGCAGTCCGGCAAGACCATCCTGGACTTTCTGAAGTCCG 
               
               
                   
               
               
                 ACGGCTTCGCCAACAGAAACTTCATGCAGCTGATTCACGACGACAGCCTC 
               
               
                   
               
               
                 ACCTTCAAAGAGGATATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATTC 
               
               
                   
               
               
                 TCTGCATGAGCACATTGCCAACCTGGCCGGCTCTCCCGCCATTAAGAAAG 
               
               
                   
               
               
                 GCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTTGTGAAAGTGATGGGC 
               
               
                   
               
               
                 AGACACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGAC 
               
               
                   
               
               
                 CACACAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAG 
               
               
                   
               
               
                 AGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAA 
               
               
                   
               
               
                 AACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGG 
               
               
                   
               
               
                 ACGGGATATGTACGTGGACCAAGAGCTGGACATCAACAGACTGTCCGACT 
               
               
                   
               
               
                 ACGATGTGGACCATATCGTGCCCCAGTCTTTTCTGAAGGACGACTCCATC 
               
               
                   
               
               
                 GACAACAAGGTCCTGACCAGATCCGACAAGAATCGGGGCAAGAGCGACAA 
               
               
                   
               
               
                 CGTGCCCTCCGAAGAGGTGGTCAAGAAGATGAAGAACTACTGGCGACAGC 
               
               
                   
               
               
                 TGCTGAACGCCAAGCTGATTACCCAGCGGAAGTTCGACAATCTGACCAAG 
               
               
                   
               
               
                 GCCGAAAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAG 
               
               
                   
               
               
                 ACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATTCTGG 
               
               
                   
               
               
                 ACTCTCGGATGAACACTAAGTACGACGAGAACGACAAACTGATCCGCGAA 
               
               
                   
               
               
                 GTGAAAGTCATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGA 
               
               
                   
               
               
                 TTTCCAGTTCTACAAAGTGCGCGAGATCAACAACTACCATCACGCCCACG 
               
               
                   
               
               
                 ACGCCTACCTGAATGCCGTTGTTGGAACAGCCCTGATCAAAAAGTACCCT 
               
               
                   
               
               
                 AAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCG 
               
               
                   
               
               
                 GAAGATGATCGCCAAGAGCGAGCAAGAGATTGGCAAGGCAACCGCCAAGT 
               
               
                   
               
               
                 ACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACAGAGATCACCCTC 
               
               
                   
               
               
                 GCCAACGGCGAGATCAGAAAGCGGCCTCTGATCGAGACAAACGGCGAAAC 
               
               
                   
               
               
                 CGGCGAGATTGTGTGGGATAAGGGCAGAGACTTTGCCACAGTGCGGAAAG 
               
               
                   
               
               
                 TGCTGAGCATGCCCCAAGTGAATATCGTGAAGAAAACCGAGGTGCAGACA 
               
               
                   
               
               
                 GGCGGCTTCAGCAAAGAGTCTATCCTGCCTAAGCGGAACTCCGACAAGCT 
               
               
                   
               
               
                 GATCGCCAGAAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGATT 
               
               
                   
               
               
                 CTCCTACCGTGGCCTATAGCGTGCTGGTGGTGGCCAAAGTGGAAAAGGGC 
               
               
                   
               
               
                 AAGTCCAAGAAACTCAAGAGCGTGAAAGAGCTGCTGGGGATCACCATCAT 
               
               
                   
               
               
                 GGAAAGAAGCAGCTTCGAGAAGAATCCGATCGATTTCCTCGAGGCCAAGG 
               
               
                   
               
               
                 GCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTCCCCAAGTACTCC 
               
               
                   
               
               
                 CTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCTGGCGA 
               
               
                   
               
               
                 ACTGCAGAAGGGAAACGAACTGGCCCTGCCTAGCAAATATGTGAACTTCC 
               
               
                   
               
               
                 TGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCAGCCCCGAGGACAAT 
               
               
                   
               
               
                 GAGCAAAAGCAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGAT 
               
               
                   
               
               
                 CATCGAGCAGATCAGCGAGTTTAGCAAGAGAGTGATTCTGGCCGACGCCA 
               
               
                   
               
               
                 ATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGACAAGCCTATC 
               
               
                   
               
               
                 AGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAACCTGGG 
               
               
                   
               
               
                 AGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGCGGT 
               
               
                   
               
               
                 ACACCTCCACCAAAGAGGTGCTGGACGCCACTCTGATCCACCAGTCTATC 
               
               
                   
               
               
                 ACCGGCCTGTACGAGACACGGATCGACCTGTCTCAACTCGGAGGCGACGA 
               
               
                   
               
               
                 AGGCGCCGATAAGAGAACCGCCGATGGCTCTGAGTTCGAGAGCCCCAAGA 
               
               
                   
               
               
                 AAAAGCGCAAAGTG 
               
            
           
         
       
     
     In an aspect, a nucleobase editor system provided herein comprises a guide polynucleotide. In some embodiments, the guide polynucleotide binds and forms a complex with the base editor fusion protein. In some embodiments, the guide polynucleotide directs the base editor fusion protein to effect a modification at a target sequence. The guide polynucleotide may comprise a single nucleic acid sequence or two separate nucleic acid sequences. In some embodiments, the guide polynucleotide is a single guide, e.g. a single guide RNA. In some embodiments, the single guide RNA comprises a spacer sequence that is capable of hybridizing with a target sequence. In some embodiments, the single guide RNA comprises a tracrRNA sequence that binds the programmable DNA binding protein, e.g. the Cas9 protein of the base editor fusion protein. In some embodiments, the single guide RNA comprises a stem loop structure, a tracrRNA sequence, a crRNA sequence, a direct repeat, and/or an anti-repeat. In some embodiments, the single guide RNA comprises a chemical modification. In some embodiments, the guide polynucleotide is any one of the guide polynucleotides provided herein, as described in the “Guide polynucleotide” section. 
     Deaminase Domains 
     Disclosed herein are base editor systems for editing, modifying or altering a target nucleotide sequence of a polynucleotide. 
     A base editor system provided herein may comprise a programmable DNA binding protein and a deaminase. As used herein, a deaminase may refer to an enzyme that catalyzes the removal of an amine group from a molecule, or deamination, for example through hydrolysis. In some embodiments, the deaminase is a cytidine deaminase, catalyzing the deamination of cytidine (C) to uridine (U), deoxycytidine (dC) to deoxyuridine (dU), or 5-methyl-cytidine to thymidine (T, 5-methyl-U), respectively. Subsequent DNA repair mechanisms ensure that a dU is replaced by T, as described in Komor et al, Nature, Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, 533, 420-424 (2016), which is incorporated herein by reference in its entirety. In some embodiments, the deaminase is a cytosine deaminase, catalyzing and promoting the conversion of cytosine to uracil (e.g., in RNA) or thymine (e.g., in DNA). In some embodiments, the deaminase is an adenosine deaminase, catalyzing and promoting the conversion of adenine to guanine. In some embodiments, the deaminase is a naturally-occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is a variant of a naturally-occurring deaminase from an organism, and the variants do not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase from an organism 
     A cytidine deaminase (or cytosine deaminase) comprises an enzyme that catalyzes the chemical reaction “cytosine+H20—&gt;uracil+NH3” or “5-methyl-cytosine+H20—&gt;thymine+NH3”. In the context of a gene, such nucleotide change, or mutation, may in turn lead to an amino acid change in the protein, which may affect the protein&#39;s function, e.g., loss-of-function or gain-of-function. Subsequent DNA repair mechanisms ensure that uracil bases in DNA are replaced by T, as described in Komor et al. (Nature, Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, 533, 420-424 (2016), which is incorporated herein by reference in its entirety). 
     One exemplary suitable class of cytosine deaminases is the apolipoprotein B mRNA-editing complex (APOBEC) family of cytosine deaminases encompassing eleven proteins that serve to initiate mutagenesis in a controlled and beneficial manner. The apolipoprotein B editing complex 3 (APOBEC3) enzyme provides protection to human cells against a certain HIV-1 strain via the deamination of cytosines in reverse-transcribed viral ssDNA. These cytosine deaminases all require a Zn—coordinating motif (His-X-Glu-X23_26-Pro-Cys-X2_4-Cys (SEQ ID NO: 29)) and bound water molecule for catalytic activity. The glutamic acid residue acts to activate the water molecule to a zinc hydroxide for nucleophilic attack in the deamination reaction. Each family member preferentially deaminates at its own particular “hotspot,” for example, WRC (W is A or T, R is A or G) for hAID, or TTC for hAPOBEC3F. A recent crystal structure of the catalytic domain of APOBEC3G revealed a secondary structure comprising a five-stranded β-sheet core flanked by six α-helices, which is believed to be conserved across the entire family. The active center loops have been shown to be responsible for both ssDNA binding and in determining “hotspot” identity. Overexpression of these enzymes has been linked to genomic instability and cancer, thus highlighting the importance of sequence-specific targeting. Another suitable cytosine deaminase is the activation-induced cytidine deaminase (AID), which is responsible for the maturation of antibodies by converting cytosines in ssDNA to uracils in a transcription-dependent, strand-biased fashion. 
     An adenosine deaminase (or adenine deaminase) comprises an enzyme that catalyzes the hydrolytic deamination of adenosine or deoxy adenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases (e.g. engineered adenosine deaminases, evolved adenosine deaminases) provided herein may be from any organism, such as a bacterium. In some embodiments, the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase. In some embodiments, the adenosine deaminase is from a bacterium, such as,  E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae , or  C. crescentus . In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is an  E. coli  TadA deaminase. In some embodiments, the TadA deaminase is a truncated  E. coli  TadA deaminase. For example, the truncated ecTadA may be missing one or more N-terminal amino acids relative to a full-length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the ecTadA deaminase does not comprise an N-terminal methionine. 
     In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO:1 or to any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identity plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to the amino acid sequence set forth in SEQ ID NO:1 or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NO:1 or any of the adenosine deaminases provided herein. 
     In some embodiments, the adenosine deaminase comprises a D108X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108G, D108N, D108V, D108A, or D108Y mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. It should be appreciated, however, that additional deaminases may similarly be aligned to identify homologous amino acid residues that can be mutated as provided herein. 
     In some embodiments, the adenosine deaminase comprises an A106X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A106V mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises a E155X mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a E155D, E155G, or E155V mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises a D147X mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D147Y, mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     It should be appreciated that any of the mutations provided herein (e.g., based on the ecTadA amino acid sequence of SEQ ID NO: 1) may be introduced into other adenosine deaminases, such as  S. aureus  TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). It would be apparent to the skilled artisan how to are homologous to the mutated residues in ecTadA. Thus, any of the mutations identified in ecTadA may be made in other adenosine deaminases that have homologous amino acid residues. It should also be appreciated that any of the mutations provided herein may be made individually or in any combination in ecTadA or another adenosine deaminase. For example, an adenosine deaminase may contain a D108N, a A106V, a E155V, and/or a D147Y mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, an adenosine deaminase comprises the following group of mutations (groups of mutations are separated by a “;”) in ecTadA SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase: D108N and A106V; D108N and E155V; D108N and D147Y; A106V and E155V; A106V and D147Y; E155V and D147Y; D108N, A106V, and E55V; D108N, A106V, and D147Y; D108N, E55V, and D147Y; A106V, E55V, and D 147Y; and D108N, A106V, E55V, and D147Y. It should be appreciated, however, that any combination of corresponding mutations provided herein may be made in an adenosine deaminase (e.g., ecTadA) 
     In some embodiments, the adenosine deaminase comprises one or more of a H8X, T17X, L18X, W23X, L34X, W45X, R51X, A56X, E59X, E85X, M94X, I95X, V102X, F104X, A106X, R107X, D108X, K110X, M118X, N127X, A138X, F149X, M151X, R153X, Q154X, I156X, and/or K157X mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8Y, T17S, L18E, W23L, L34S, W45L, R51H, A56E, or A56S, E59G, E85K, or E85G, M94L, 1951, V102A, F104L, A106V, R107C, or R107H, or R107P, D108G, or D108N, or D108V, or D108A, or D108Y, K1101, M118K, N127S, A138V, F149Y, M151V, R153C, Q154L, 1156D, and/or K157R mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the mutations corresponding to SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the mutation or mutations of in any constructs shown in Table 23 corresponding to SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of a H8X, D108X, and/or N127X mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase, where X indicates the presence of any amino acid. In some embodiments, the adenosine deaminase comprises one or more of a H8Y, D108N, and/or N127S mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises one or more of H8X, R26X, M61X, L68X, M70X, A106X, D108X, A109X, N127X, D147X, R152X, Q154X, E155X, K161X, Q163X, and/or T166X mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8Y, R26W, M61I, L68Q, M70V, A106T, D108N, A109T, N127S, D147Y, R152C, Q154H or Q154R, E155G or E155V or E155D, K161Q, Q163H, and/or T166P mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8X, D108X, N127X, D147X, R152X, and Q154X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, M61X, M70X, D108X, N127X, Q154X, E155X, and Q163X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8X, D108X, N127X, E155X, and T166X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8X, A106X, D108X, mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, R126X, L68X, D108X, N127X, D147X, and E155X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8X, D108X, A109X, N127X, and E155X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, D108N, N127S, D147Y, R152C, and Q154H in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, M61I, M70V, D108N, N127S, Q154R, E155G and Q163H in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8Y, D108N, N127S, E155V, and T166P in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, A106T, D108N, N127S, E155D, and K161Q in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, R126W, L68Q, D108N, N127S, D147Y, and E155V in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8Y, D108N, A109T, N127S, and E155G in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises a D108N, D108G, or D108V mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a A106V and D108N mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises R107C and D108N mutations in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a H8Y, D108N, N127S, D147Y, and Q154H mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a H8Y, R24W, D108N, N127S, D147Y, and E155V mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108N, D147Y, and E155V mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a H8Y, D108N, and S 127S mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a A106V, D108N, D147Y and E155V mutation in SEQ ID NO: 1, or corresponding mutations in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises one or more of a S2X, H8X, I49X, L84X, H123X, N127X, I156X and/or K160X mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of S2A, H8Y, I49F, L84F, H123Y, N127S, I156F and/or K160S mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the mutations in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the mutation or mutations of any one of clones corresponding to SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an L84X mutation adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an L84F mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an H123X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H123Y mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an I157X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an I157F mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of L84X, A106X, D108X, H123X, D147X, E155X, and I156X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2X, I49X, A106X, D108X, D147X, and E155X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8X, A106X, D108X, N127X, and K160X in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of L84F, A106V, D108N, H123Y, D147Y, E155V, and 1156F in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2A, I49F, A106V, D108N, D147Y, and E155V in SEQ ID NO: 1, or a corresponding amino acid in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8Y, A106T, D108N, N127S, and K160S in SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of a E25X, R26X, R107X, A142X, and/or A143X mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of E25M, E25D, E25A, E25R, E25V, E25S, E25Y, R26G, R26N, R26Q, R26C, R26L, R26K, R107P, R07K, R107A, R107N, R107W, R107H, R107S, A142N, A142D, A142G, A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q and/or A143R mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the mutations provided in Table 23 corresponding to SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the mutation or mutations of any one shown in Table 23 corresponding to SEQ ID NO: 1, or a corresponding mutation or mutations in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an E25X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E25M, E25D, E25A, E25R, E25V, E25S, or E25Y mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an R26X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an, R26G, R26N, R26Q, R26C, R26L, or R26K mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an R107X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R107P, R07K, R107A, R107N, R107W, R107H, or R107S mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an A142X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A142N, A142D, A142G, mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an A143X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q and/or A143R mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises one or more of a H36X, N37X, P48X, I49X, R51X, M70X, N72X, D77X, E134X, S 146X, Q154X, K157X, and/or K161X mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H36L, N37T, N37S, P48T, P48L, I49V, R51H, R51L, M70L, N72S, D77G, E134G, S 146R, S 146C, Q154H, K157N, and/or K161T mutation in SEQ ID NO: 1, or one or more corresponding mutations in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an H36X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H36L mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises an N37X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an N37T, or N37S mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an P48X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an P48T, or P48L mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an R51X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R51H, or R51L mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an S 146X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an S 146R, or S 146C mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an K157X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a K157N mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an P48X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48S, P48T, or P48A mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an A142X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a A142N mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an W23X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a W23R, or W23L mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an R152X mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R152P, or R52H mutation in SEQ ID NO: 1, or a corresponding mutation in another adenosine deaminase. 
     Additional adenosine deaminase mutations and variants are described in Patent Application WO2018119354, which is incorporated herein by reference in its entirety. 
     Additional adenosine deaminases useful in the present application would be apparent to the skilled artisan and are within the scope of this disclosure. For example, the adenosine deaminase may be a homolog of an AD AT. Exemplary AD AT homologs include, without limitation: 
     
       
         
           
               
            
               
                   Staphylococcus aureus  TadA: 
               
               
                 (SEQ ID NO: 30) 
               
               
                 MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAITKDDEVIARAHNLRETL 
               
               
                   
               
               
                 QQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPR 
               
               
                   
               
               
                 VVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKN 
               
               
                   
               
               
                 LRANKKSTN 
               
               
                   
               
               
                   Bacillus subtilis  TadA: 
               
               
                 (SEQ ID NO: 31) 
               
               
                 MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRS 
               
               
                   
               
               
                 IAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVF 
               
               
                   
               
               
                 GAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRK 
               
               
                   
               
               
                 KKKAARKNLSE 
               
               
                   
               
               
                   Salmonella typhimurium  ( S. typhimurium ) 
               
               
                 TadA: 
               
               
                 (SEQ ID NO: 32) 
               
               
                 MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHR 
               
               
                   
               
               
                 VIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVM 
               
               
                   
               
               
                 CAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRD 
               
               
                   
               
               
                 ECATLLSDFFRMRRQEIKALKKADRAEGAGPAV 
               
               
                   
               
               
                   Shewanella putrefaciens  ( S. putrefaciens ) 
               
               
                 TadA: 
               
               
                 (SEQ ID NO: 33) 
               
               
                 MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTA 
               
               
                   
               
               
                 HAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGA 
               
               
                   
               
               
                 RDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEK 
               
               
                   
               
               
                 KALKLAQRAQQGIE 
               
               
                   
               
               
                   Haemophilus influenzae  F3031 ( H. influenzae ) 
               
               
                 TadA: 
               
               
                 (SEQ ID NO: 34) 
               
               
                 MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWN 
               
               
                   
               
               
                 LSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILH 
               
               
                   
               
               
                 SRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLS 
               
               
                   
               
               
                 TFFQKRREEKKIEKALLKSLSDK 
               
               
                   
               
               
                   Caulobacter crescentus  ( C. crescentus ) 
               
               
                 TadA: 
               
               
                 (SEQ ID NO: 35) 
               
               
                 MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGN 
               
               
                   
               
               
                 GPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISH 
               
               
                   
               
               
                 ARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLR 
               
               
                   
               
               
                 GFFRARRKAKI 
               
               
                   
               
               
                   Geobacter sulfurreducens  ( G. sulfurreducens ) 
               
               
                 TadA: 
               
               
                 (SEQ ID NO: 36) 
               
               
                 MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHN 
               
               
                   
               
               
                 LREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIIL 
               
               
                   
               
               
                 ARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLS 
               
               
                   
               
               
                 DFFRDLRRRKKAKATPALFIDERKVPPEP 
               
            
           
         
       
     
     In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the amino acid sequences listed in Table 23. In some embodiments, the adenosine deaminase comprises any one of the amino acid sequences listed in Table 23. In some embodiments, the sequence of the adenosine deaminase is any one of the amino acid sequences listed in Table 23. In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the amino acid sequences of SEQ ID NOs: SEQ ID NOs: 2137, 2149, 2154, 2158, 2188, 2140, 40, 2146, 2152, 2156, and 2160. In some embodiments, the adenosine deaminase comprises any one of the amino acid sequences of SEQ ID NOs: 2137, 2149, 2154, 2158, 2188, 2140, 40, 2146, 2152, 2156, and 2160. In some embodiments, the sequence of the adenosine deaminase is any one of the amino acid sequences of SEQ ID NOs: 2137, 2149, 2154, 2158, and 2188. 
     In some embodiments, the adenosine deaminase is encoded by the polynucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences listed in Table 23. In some embodiments, the adenosine deaminase is encoded by any one of the polynucleotide sequences listed in Table 23. In some embodiments, the adenosine deaminase is expressed by the polynucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences listed in Table 23. In some embodiments, the adenosine deaminase is expressed by any one of the polynucleotide sequences listed in Table 23. In some embodiments, the adenosine deaminase is encoded by the polynucleotide sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. In some embodiments, the adenosine deaminase is encoded by the polynucleotide sequence that comprises any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. In some embodiments, the adenosine deaminase is encoded by any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, and 2189. In some embodiments, the adenosine deaminase is expressed by the polynucleotide sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2190, 2138, 2147, 2158, or a combination thereof: In some embodiments, the adenosine deaminase is encoded by the polynucleotide sequence that comprises any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2190, 2138, 2147, 2158, or a combination thereof. In some embodiments, the adenosine deaminase is expressed by any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, and 2189. In some embodiments, the polynucleotide sequence further comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences of SEQ ID NOs: 2138, 2147, 2158, or a combination thereof. In some embodiments, the polynucleotide sequence further comprises any one of the polynucleotide sequences of SEQ ID NOs: 2138, 2147, 2158, or a combination thereof. 
     Programmable DNA Binding Proteins 
     Provided herein are programmable DNA-binding proteins that can be programmed to target to bind DNA sequences in any desired nucleotide sequence within a genome. To program the DNA-binding protein to bind a desired nucleotide sequence, the DNA binding protein may be modified to change its binding specificity, e.g., zinc finger DNA-binding domain, zinc finger nuclease (ZFN), a CRISPR-Cas9 protein, or a transcription activator—like effector proteins (TALE). ZFNs are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-fingers to bind unique sequences within complex genomes. Transcription activator-like effector nucleases (TALEN) are engineered restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a nuclease domain (e.g. Fokl). Transcription activator-like effectors (TALEs) can be engineered to bind practically any desired DNA sequence. Methods for programming ZFNs and TALEs are familiar to one skilled in the art. For example, such methods are described in Maeder, et al, Mol. Cell 31 (2): 294-301, 2008; Carroll et al, Genetics Society of America, 188 (4): 773-782, 2011; Miller et al., Nature Biotechnology 25 (7): 778-785, 2007; Christian et al, Genetics 186 (2): 757-61, 2008; Li et al, Nucleic Acids Res. 39 (1): 359-372, 2010; and Moscou et al, Science 326 (5959): 1501, 2009, each of which are incorporated herein by reference. 
     A CRISPR/Cas system or a Cas protein in a base editor system provided herein may comprise Class 1 or Class 2 system components, including ribonucleic acid protein complexes. The Class 2 Cas nuclease families of proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as described further herein. A Class 2 CRISPR/Cas system component may be from a Type II, Type IIA, Type IIB, Type IIC, Type V, or Type VI system. Class 2 Cas nucleases include, for example, Cas9 (also known as Csn1 or Csx12), Csn2, Cas4, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas13a (C2c2), Cas13b, Cas13c, and Cas13d proteins. In some embodiments, the Cas protein is from a Type II CRISPR/Cas system, i.e., a Cas9 protein from a CRISPR/Cas9 system, or a Type V CRISPR/Cas system, e.g., a Cas12a protein. In some embodiments, the Cas protein is from a Class 2 CRISPR/Cas system, i.e., a single-protein Cas nuclease such as a Cas9 protein or a Cas12a protein. 
     Other non-limiting examples of Cas proteins can include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, Cas9HiFi, homologues thereof, or modified versions thereof. 
     In some embodiments, provided herein are guide nucleotide sequence-programmable DNA-binding protein or RNA guided programable DNA binding proteins that are able to bind DNA, and the binding to its target DNA sequence is mediated by a guide nucleotide sequence. Thus, it is appreciated that the guide nucleotide sequence-programmable DNA-binding protein binds to a guide nucleotide sequence. The guide nucleotide may be an RNA or DNA molecule (e.g., a single-stranded DNA or ssDNA molecule) that is complementary to the target sequence and can guide the DNA binding protein to the target sequence. As such, a guide nucleotide sequence-programmable DNA-binding protein may be a RNA-programmable DNA-binding protein (e.g., a Cas9 protein), or an ssDNA-programmable DNA-binding protein (e.g., an Argonaute protein). “Programmable” means the DNA-binding protein may be programmed to bind any DNA sequence that the guide nucleotide targets. Exemplary guide nucleotide sequence-programmable DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), saCas9 (e.g., saCas9d, saCas9d, saKKH Cas9) CasX, CasY, Cpf1, C2c1, C2c2, C2c3, Argonaute, and any other suitable protein described herein, or variants thereof. 
     In some embodiments, the guide nucleotide sequence exists as a single nucleotide molecule and comprises two domains: (1) a domain that shares homology to a target nucleic acid (e.g., and directs binding of a guide nucleotide sequence-programmable DNA-binding protein to the target); and (2) a domain that binds a guide nucleotide sequence-programmable DNA-binding protein. In some embodiments, domain (1) comprises a spacer sequence. In some embodiments, domain (2) is referred to as a tracrRNA sequence. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA, and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821(2012), which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. Patent Application Publication US20160208288 and U.S. Patent Application Publication US20160200779 each of which is herein incorporated by reference in their entirety. 
     Methods of using guide nucleotide sequence-programmable DNA-binding protein, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013);  Mali , P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W. Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, J. E. et al. Genome engineering in  Saccharomyces cerevisiae  using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013); each of which are incorporated herein by reference). 
     CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. A CRISPR/Cas system comprises a non-coding RNA molecule (e.g., guide RNA) that binds to DNA (e.g., target DNA sequence) and Cas proteins (e.g., Cas9) with nuclease functionality (e.g., two nuclease domains). See, e.g., Sander, et al., Nature Biotechnology, 32:347-355 (2014); see also e.g., Hsu, et al., Cell 157(6):1262-1278 (2014). The general mechanism and recent advances of CRISPR system is discussed in Cong, et al., Science, 339(6121): 819-823 (2013); Fu, et al., Nature Biotechnology, 31, 822-826 (2013); Chu, et al., Nature Biotechnology 33, 543-548 (2015); Shmakov, et al., Molecular Cell, 60, 1-13 (2015); Makarova, et al., Nature Reviews Microbiology, 13, 1-15 (2015). CRISPR/Cas systems can be used to introduce site-specific cleavage of a target DNA. The locations for site-specific cleavage are determined by both 1) base-pairing complementarity between the guide RNA (gRNA) and the target DNA (a protospacer) and 2) a short motif in the target DNA referred to as the protospacer adjacent motif (PAM). CRISPR/Cas systems (e.g., Type II CRISPR/Cas system) can be used to generate, e.g., an engineered cell in which a target gene is disrupted or mutated. A Cas enzyme (e.g., Cas9) can be used to catalyze DNA cleavage. A Cas9 protein (e.g., a  Streptococcus pyogenes  Cas9 or any closely related Cas9) can derive an enzymatic action to generate double stranded breaks at target site sequences which hybridize to about 20 nucleotides of a guide sequence (e.g., gRNA) and that have a protospacer-adjacent motif (PAM) following the target sequence. 
     CRISPR/Cas system comprises Class 1 or Class 2 system components, including ribonucleic acid protein complexes. The Class 2 Cas nuclease families of proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, as described further herein. A Class 2 CRISPR/Cas system component may be from a Type II, Type IIA, Type IIB, Type IIC, Type V, or Type VI system. Class 2 Cas nucleases include, for example, Cas9 (also known as Csn1 or Csx12), Csn2, Cas4, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas13a (C2c2), Cas13b, Cas13c, and Cas13d proteins. In some embodiments, the Cas protein is from a Type II CRISPR/Cas system, i.e., a Cas9 protein from a CRISPR/Cas9 system, or a Type V CRISPR/Cas system, e.g., a Cas12a protein. In some embodiments, the Cas protein is from a Class 2 CRISPR/Cas system, i.e., a single-protein Cas nuclease such as a Cas9 protein or a Cas12a protein. 
     Other non-limiting examples of Cas proteins can include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, Cas9HiFi, homologues thereof, or modified versions thereof. 
     A base editor system provided herein may comprise a Cas9 or a Cas9 nuclease. A Cas9 protein or a Cas9 nuclease refers to an RNA-guided nuclease comprising a Cas9 protein, a fragment, or a variant thereof. A Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)—associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek et al., Science 337:816-821(2012), which is incorporated herein by reference in its entirety. 
     The term “Cas9” refers to an RNA guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to as a Casn1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat) associated nuclease. Cas9 can refer to a polypeptide with at least or at least about 50%, 60%, 70%, 80%, 90%, 100% sequence identity and/or sequence similarity to a wild type exemplary Cas9 polypeptide (e.g., Cas9 from  S. pyogenes ). Cas9 can refer to a polypeptide with at most or at most about 50%, 60%, 70%, 80%, 90%, 100% sequence identity and/or sequence similarity to a wild type exemplary Cas9 polypeptide (e.g., from  S. pyogenes ). Cas9 can refer to the wild type or a modified form of the Cas9 protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof. 
     Cas9 nuclease sequences and structures of variant Cas9 orthologs have been described in various species. Exemplary species that the Cas9 protein or other components can be from include, but are not limited to,  Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus  sp.,  Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gamma proteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina , Burkholderiales bacterium, Polar omonas  naphthalenivorans, Polar omonas  sp.,  Crocosphaera watsonii, Cyanothece  sp.,  Microcystis aeruginosa, Synechococcus  sp.,  Acetohalobium arabaticum, Ammonifex degensii , Caldicelulosiruptor becscii, Candidatus Desulforudis,  Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus , Pelotomaculum thermopropionium,  Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter  sp.,  Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc  sp.,  Arthrospira maxima, Arthrospira platensis, Arthrospira  sp.,  Lyngbya  sp.,  Microcoleus chthonoplastes, Oscillator  ia sp.,  Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Coryne  bacterium diphtheria, or  Acaryochloris marina . In some embodiments, the Cas9 protein is from  Streptococcus pyogenes . In some embodiments, the Cas9 protein may be from  Streptococcus thermophilus . In some embodiments, the Cas9 protein is from  Staphylococcus aureus.    
     Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski et al., (2013) RNA Biology 10:5, 726-737; which are incorporated herein by reference. 
     In some embodiments, wild-type Cas9 corresponds to Cas9 from  Streptococcus pyogenes  (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence as follows); and Uniprot Reference Sequence: 99ZW2 amino acid sequence as follows 
     
       
         
           
               
            
               
                 (SEQ ID NO: 37) 
               
               
                 ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGG 
               
               
                   
               
               
                 ATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGG 
               
               
                   
               
               
                 TTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCT 
               
               
                   
               
               
                 CTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGAC 
               
               
                   
               
               
                 AGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGG 
               
               
                   
               
               
                 AGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGA 
               
               
                   
               
               
                 CTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCC 
               
               
                   
               
               
                 TATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA 
               
               
                   
               
               
                 CTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGAT 
               
               
                   
               
               
                 TTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCA 
               
               
                   
               
               
                 TTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAAC 
               
               
                   
               
               
                 TATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCT 
               
               
                   
               
               
                 ATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAG 
               
               
                   
               
               
                 TAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGA 
               
               
                   
               
               
                 AAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCT 
               
               
                   
               
               
                 AATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC 
               
               
                   
               
               
                 AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAG 
               
               
                   
               
               
                 ATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATT 
               
               
                   
               
               
                 TTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCT 
               
               
                   
               
               
                 ATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTC 
               
               
                   
               
               
                 TTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATC 
               
               
                   
               
               
                 TTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGC 
               
               
                   
               
               
                 TAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGG 
               
               
                   
               
               
                 ATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC 
               
               
                   
               
               
                 AAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGG 
               
               
                   
               
               
                 TGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAA 
               
               
                   
               
               
                 AAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTAT 
               
               
                   
               
               
                 TATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCG 
               
               
                   
               
               
                 GAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATA 
               
               
                   
               
               
                 AAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAA 
               
               
                   
               
               
                 AATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTA 
               
               
                   
               
               
                 TTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAA 
               
               
                   
               
               
                 TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGAT 
               
               
                   
               
               
                 TTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGA 
               
               
                   
               
               
                 TTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTG 
               
               
                   
               
               
                 AAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATT 
               
               
                   
               
               
                 ATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGA 
               
               
                   
               
               
                 GGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGG 
               
               
                   
               
               
                 AAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAG 
               
               
                   
               
               
                 CTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT 
               
               
                   
               
               
                 TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGA 
               
               
                   
               
               
                 AATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGAT 
               
               
                   
               
               
                 AGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGG 
               
               
                   
               
               
                 CGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTA 
               
               
                   
               
               
                 AAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTA 
               
               
                   
               
               
                 ATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAA 
               
               
                   
               
               
                 TCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAA 
               
               
                   
               
               
                 TCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCT 
               
               
                   
               
               
                 GTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCA 
               
               
                   
               
               
                 AAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAA 
               
               
                   
               
               
                 GTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGAT 
               
               
                   
               
               
                 TCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATC 
               
               
                   
               
               
                 GGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGA 
               
               
                   
               
               
                 GACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTA 
               
               
                   
               
               
                 ACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTAT 
               
               
                   
               
               
                 CAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAA 
               
               
                   
               
               
                 TTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATT 
               
               
                   
               
               
                 CGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCG 
               
               
                   
               
               
                 AAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATG 
               
               
                   
               
               
                 CCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAA 
               
               
                   
               
               
                 TATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGA 
               
               
                   
               
               
                 TGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCG 
               
               
                   
               
               
                 CAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATT 
               
               
                   
               
               
                 ACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGG 
               
               
                   
               
               
                 GGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGC 
               
               
                   
               
               
                 GCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTA 
               
               
                   
               
               
                 CAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGA 
               
               
                   
               
               
                 CAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTT 
               
               
                   
               
               
                 TTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAA 
               
               
                   
               
               
                 AAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCAC 
               
               
                   
               
               
                 AATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAG 
               
               
                   
               
               
                 CTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAA 
               
               
                   
               
               
                 TATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGC 
               
               
                   
               
               
                 CGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGA 
               
               
                   
               
               
                 ATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAA 
               
               
                   
               
               
                 GATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGA 
               
               
                   
               
               
                 TGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAG 
               
               
                   
               
               
                 ATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAA 
               
               
                   
               
               
                 CCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAA 
               
               
                   
               
               
                 TCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTA 
               
               
                   
               
               
                 AACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAA 
               
               
                   
               
               
                 TCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGG 
               
               
                   
               
               
                 TGACTGA 
               
               
                   
               
               
                 (SEQ ID NO: 38) 
               
               
                 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE 
               
               
                   
               
               
                 DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK 
               
               
                   
               
               
                 PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ 
               
               
                   
               
               
                 SITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     In some embodiments, Cas9 is a Cas9 protein from species  Corynebacterium ulcerans  (NCBI Refs: NC_015683.1, NC_017317.1);  Corynebacterium diphtheria  (NCBI Refs. NC 016782.1, NC_016786.1);  Spiroplasma syrphidicola  (NCBI Ref: NC_021284.1);  Prevotella intermedia  (NCBI Ref: NC_017861.1);  Spiroplasma taiwanense  (NCBI Ref: NC_021846.1);  Streptococcus iniae  (NCBI Ref: NC_021314.1);  Belliella baltica  (NCBI Ref: NC_018010.1);  Psychroflexus torquisl  (NCBI Ref: NC_018721.1);  Listeria innocua  (NCBI Ref: NP_472073.1),  Campylobacter jejuni  (NCBI Ref: YP_002344900.1) or  Neisseria, meningitidis  (NCBI Ref: YP_002342100.1). 
     In some embodiments, a base editor provided herein comprises a programmable DNA binding protein, e.g. a Cas nuclease, with reduced or abolished nuclease activity. For example a Ca9 protein may be nuclease inactive or may be a Cas9 nickase. Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al, (2013) Cell. 28; 152(5): 1173-83, each of which are incorporated herein by reference in its entirety. For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of  S. pyogenes  Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al, Cell. 28;152(5): 1173-83 (2013)). The Cas9 nickase suitable for use in accordance with the present disclosure has an active HNH domain and an inactive RuvC domain and is able to cleave only the strand of the target DNA that is bound by the sgRNA (which is the opposite strand of the strand that is being edited via cytidine deamination). The Cas9 nickase of the present disclosure may comprise mutations that inactivate the RuvC domain, e.g., a D10A mutation. It is to be understood that any mutation that inactivates the RuvC domain may be included in a Cas9 nickase, e.g., insertion, deletion, or single or multiple amino acid substitution in the RuvC domain. In a Cas9 nickase described herein, while the RuvC domain is inactivated, the HNH domain remains activate. Thus, while the Cas9 nickase may comprise mutations other than those that inactivate the RuvC domain (e.g., D10A), those mutations do not affect the activity of the HNH domain. In a non-limiting Cas9 nickase example, the histidine at position 840 remains unchanged. 
     In some embodiments, a nuclease inactive Cas9 comprises the amino acid sequence of dCas9 (D10A and H840A) provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 39) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE 
               
               
                   
               
               
                 DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK 
               
               
                   
               
               
                 PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ 
               
               
                   
               
               
                 SITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     In some embodiments, a Cas9 nickase comprises the amino acid sequence of an exemplary catalytically Cas9 nickase (nCas9) is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 40) 
               
               
                 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL 
               
               
                   
               
               
                 LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL 
               
               
                   
               
               
                 EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL 
               
               
                   
               
               
                 RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI 
               
               
                   
               
               
                 NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN 
               
               
                   
               
               
                 FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL 
               
               
                   
               
               
                 LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF 
               
               
                   
               
               
                 FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK 
               
               
                   
               
               
                 QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY 
               
               
                   
               
               
                 VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN 
               
               
                   
               
               
                 LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL 
               
               
                   
               
               
                 LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII 
               
               
                   
               
               
                 KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL 
               
               
                   
               
               
                 KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS 
               
               
                   
               
               
                 LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM 
               
               
                   
               
               
                 GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV 
               
               
                   
               
               
                 ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS 
               
               
                   
               
               
                 IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 
               
               
                   
               
               
                 KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR 
               
               
                   
               
               
                 EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY 
               
               
                   
               
               
                 PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT 
               
               
                   
               
               
                 LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ 
               
               
                   
               
               
                 TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK 
               
               
                   
               
               
                 GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY 
               
               
                   
               
               
                 SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED 
               
               
                   
               
               
                 NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP 
               
               
                   
               
               
                 IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS 
               
               
                   
               
               
                 ITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     Additional suitable mutations that inactivate Cas9 will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D839A and/or N863A (See, e.g., Prashant et al, Nature Biotechnology. 2013; 31(9): 833-838, which are incorporated herein by reference), or), or K603R {See, e.g., Chavez et al., Nature Methods 12, 326-328, 2015, which is incorporated herein by reference). Cas9, dCas9, or Cas9 variant also encompasses Cas9, dCas9, or Cas9 variants from any organism. Also appreciated is that dCas9, Cas9 nickase, or other appropriate Cas9 variants from any organisms may be used in accordance with the present disclosure. 
     In some embodiments, Cas9 refers to a Cas9 from archaea (e.g. nanoarchaea), which constitute a domain and kingdom of single-celled prokaryotic microbes. 
     In some embodiments, the programmable DNA binding protein comprises a CasX or CasY, or a variant thereof, which have been described in, for example, Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb. 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. 
     Some aspects of the disclosure provide high fidelity Cas9 domains of the nucleobase editors provided herein. In some embodiments, high fidelity Cas9 domains are engineered Cas9 domains comprising one or more mutations that decrease electrostatic interactions between the Cas9 domain and the sugar-phosphate backbone of DNA, as compared to a corresponding wild-type Cas9 domain. Without wishing to be bound by any particular theory, high fidelity Cas9 domains that have decreased electrostatic interactions with the sugar-phosphate backbone of DNA may have less off-target effects. In some embodiments, the Cas9 domain comprises one or more mutations that decreases the association between the Cas9 domain and the sugar-phosphate backbone of DNA. In some embodiments, a Cas9 domain comprises one or more mutations that decreases the association between the Cas9 domain and the sugar-phosphate backbone of DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or more. In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of N497X, R661X, Q695X, and/or Q926X mutation as numbered in the wild type Cas9 amino acid sequence or a corresponding amino acid in another Cas9, wherein X is any amino acid. In some embodiments, any of the Cas9 or Cas9 fusion proteins provided herein comprise one or more of N497A, R661A, Q695A, and/or Q926A mutation of the amino acid sequence provided in the wild type Cas9 sequence, or a corresponding mutation as numbered in the wild type Cas9 amino acid sequence or a corresponding amino acid in another Cas9. Cas9 domains with high fidelity have been described in Kleinstiver, B. P., et al. “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495 (2016); and Slaymaker, I. M., et al. “Rationally engineered Cas9 nucleases with improved specificity.” Science 351, 84-88 (2015); the entire contents of each are incorporated herein by reference. 
     It should be appreciated that any of the base editors provided herein, for example, any of the adenosine deaminase base editors provided herein, may be converted into high fidelity base editors by modifying the Cas9 domain as described herein to generate high fidelity base editors, for example, a high fidelity adenosine base editor. In some embodiments, the high fidelity Cas9 domain is a nuclease inactive Cas9 domain. In some embodiments, the high fidelity Cas9 domain is a Cas9 nickase domain. 
     In some embodiments, a Cas protein comprises a CasX or CasY, or a variant thereof, which have been described in, for example, Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb. 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. 
     Alternatives to  S. pyogenes  Cas9 may include RNA-guided endonucleases from the Cpf1 family that exhibit cleavage activity in mammalian cells. Cpf1-mediated DNA cleavage is a double-strand break with a short 3′ overhang, which is different from Cas9-mediated DNA cleavage. The staggered cleavage pattern of Cpf1 may open up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Like the Cas9 variants and orthologues described above, Cpf1 may also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9. 
     In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the amino acid sequences listed in Table 23. In some embodiments, the Cas9 protein comprises any one of the amino acid sequences listed in Table 23. In some embodiments, the sequence of the Cas9 protein is any one of the amino acid sequences listed in Table 23. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the amino acid sequences of SEQ ID NOs: 2137, 2149, 2154, 2158, 2188, 2140, 40, 2146, 2152, 2156, and 2160. In some embodiments, the Cas9 protein comprises any one of the amino acid sequences of SEQ ID NOs: 2137, 2149, 2154, 2158, 2188, 2140, 40, 2146, 2152, 2156, and 2160. In some embodiments, the sequence of the Cas9 protein is any one of the amino acid sequences of SEQ ID NOs: 2137, 2149, 2154, 2158, and 2188. 
     In some embodiments, the Cas9 protein is encoded by the polynucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences listed in Table 23. In some embodiments, the Cas9 protein is encoded by any one of the polynucleotide sequences listed in Table 23. In some embodiments, the Cas9 protein is expressed by the polynucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences listed in Table 23. In some embodiments, the Cas9 protein is expressed by any one of the polynucleotide sequences listed in Table 23. In some embodiments, the Cas9 protein is encoded by the polynucleotide sequence that is at In some embodiments, the Cas9 protein is encoded by the polynucleotide sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. In some embodiments, the Cas9 protein is encoded by the polynucleotide sequence that comprises any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. In some embodiments, the Cas9 protein is encoded by any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, and 2189. In some embodiments, the Cas9 protein is expressed by the polynucleotide sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2190, 2138, 2147, 2158, or a combination thereof: In some embodiments, the Cas9 protein is encoded by the polynucleotide sequence that comprises any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2190, 2138, 2147, 2158, or a combination thereof. In some embodiments, the Cas9 protein is expressed by any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, and 2189. In some embodiments, the polynucleotide sequence further comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the polynucleotide sequences of SEQ ID NOs: 2138, 2147, 2158, or a combination thereof. In some embodiments, the polynucleotide sequence further comprises any one of the polynucleotide sequences of SEQ ID NOs: 2138, 2147, 2158, or a combination thereof. 
     Linkers 
     Base editors provided herein may comprises linkers that connect one or more components of the base editors. In certain embodiments, linkers may be used to link any of the protein or protein domains described herein. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In some embodiments, the linker is carbon bond, disulfide bond, carbon-heteroatom bond, etc. In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, alkyl halides, aryl halides, acyl halides, and isothiocyanates. 
     In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is a bond (e.g., a covalent bond), an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, a linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 17), which may also be referred to as the XTEN linker. In some embodiments, a linker comprises the amino acid sequence SGGS. In some embodiments, a linker comprises (SGGS) n  (SEQ ID NO: 41), (GGGS) n  (SEQ ID NO: 42), (GGGGS) n  (SEQ ID NO: 43), (G) n  (SEQ ID NO: 44), (EAAAK) n  (SEQ ID NO: 45), (GGS) n  (SEQ ID NO: 46), SGSETPGTSESATPES (SEQ ID NO: 17), or (XP) n  (SEQ ID NO: 47) motif, or a combination of any of these, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, a linker comprises SGSETPGTSESATPES (SEQ ID NO: 17), SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 19). In some embodiments, a linker comprises SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 20). In some embodiments, a linker comprises GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 21). In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 23). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 24). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS (SEQ ID NO: 25). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 26). It should be appreciated that any of the linkers provided herein may be used to link a first adenosine deaminase and a second adenosine deaminase; a deaminase (e.g., a first or a second adenosine deaminase) and a RNA guided programmable DNA binding protein; a RNA guided programmable DNA binding protein and an NLS; or a deaminase (e.g., a first or a second adenosine deaminase) and an NLS. 
     Various linker lengths and flexibilities between a deaminase (e.g., an engineered ecTadA) and a RNA guided programmable DNA binding protein (e.g., a Cas9 domain), and/or between a first adenosine deaminase and a second adenosine deaminase can be employed (e.g., ranging from very flexible linkers of the form (GGGGS) n  (SEQ ID NO: 22), (GGGGS) n  (SEQ ID NO: 22), and (G) n  to more rigid linkers of the form (EAAAK) n  (SEQ ID NO: 48), (SGGS) n  (SEQ ID NO: 49), and (XP) n ) in order to achieve the optimal length for deaminase activity for the specific application. In some embodiments, n is any integer between 3 and 30. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS) n  (SEQ ID NO: 51) motif, wherein n is 1, 3, or 7. 
     Protospacer Adjacent Motifs 
     CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). For example, in type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) (see, e.g., “Complete genome sequence of an Ml strain of  Streptococcus pyogenes .” Ferretti et ah, J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C, Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to,  S. pyogenes  and  S. thermophilus.    
     Some aspects of the disclosure provide programmed DNA binding protein domains, e.g. Cas9 domains that have altered PAM specificities. In some embodiments, a Cas9 derived from  S. pyogenes  (spCas9) recognizes a canonical NGG PAM sequence where the “N” in “NGG” is adenine (A), thymine (T), guanine (G), or cytosine (C), and the G is guanine. In some embodiments, the base editing fusion proteins provided herein function by deaminating a target nucleobase within a 4 base region (e.g., a “deamination window”), which is approximately 15 bases upstream of the PAM. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM. Accordingly, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et ah, “Broadening the targeting range of  Staphylococcus aureus  CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference. In some embodiments, the Cas9 domain is a Cas9 domain from  Staphylococcus aureus  (SaCas9). In some embodiments, the SaCas9 domain is a nuclease active SaCas9, a nuclease inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). A wild type SaCas9 amino acid sequence is provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 52) 
               
               
                 KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR 
               
               
                   
               
               
                 GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS 
               
               
                   
               
               
                 EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA 
               
               
                   
               
               
                 ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY 
               
               
                   
               
               
                 IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY 
               
               
                   
               
               
                 NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK 
               
               
                   
               
               
                 EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI 
               
               
                   
               
               
                 AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN 
               
               
                   
               
               
                 LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK 
               
               
                   
               
               
                 RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT 
               
               
                   
               
               
                 NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF 
               
               
                   
               
               
                 NYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISY 
               
               
                   
               
               
                 ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY 
               
               
                   
               
               
                 ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH 
               
               
                   
               
               
                 AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK 
               
               
                   
               
               
                 EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI 
               
               
                   
               
               
                 VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK 
               
               
                   
               
               
                 NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR 
               
               
                   
               
               
                 NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK 
               
               
                   
               
               
                 KLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY 
               
               
                   
               
               
                 REYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK 
               
               
                   
               
               
                 KG 
               
            
           
         
       
     
     In some embodiments, the SaCas9 comprises a N579X mutation, or a corresponding mutation in any of the amino acid sequences as numbered in the wild type SaCas9 sequence, wherein X is any amino acid except for N. In some embodiments, the SaCas9 comprises a N579A mutation as numbered in the wild type SaCas9 sequence or a corresponding mutation in another SaCas9 protein. 
     In some embodiments, the SaCas9 domain, the nuclease inactive SaCas9 domain, or the SaCas9 nickase domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a NNGRRT PAM sequence, where N=A, T, C, or G, and R=A or G. In some embodiments, the SaCas9 domain comprises one or more of E781X, N967X, and R1014X as numbered in the wild type SaCas9 sequence or a corresponding mutation in another SaCas9 protein wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of a E781K, a N967K, and a R1014H mutation as numbered in the wild type SaCas9 sequence or a corresponding mutation in another SaCas9 protein. In some embodiments, the SaCas9 domain comprises a E781K, a N967K, or a R1014H mutation as numbered in the wild type SaCas9 sequence or a corresponding mutation in another SaCas9 protein. 
     In some embodiments, the Cas9 domain is a Cas9 domain from  Streptococcus pyogenes  (SpCas9). In some embodiments, the SpCas9 domain is a nuclease active SpCas9, a nuclease inactive SpCas9 (SpCas9d), or a SpCas9 nickase (SpCas9n). In some embodiments, the SpCas9 comprises a D10X mutation as numbered in the wild type SpCas9 amino acid sequence or a corresponding mutation in another SapCas9 protein, wherein X is any amino acid except for D. In some embodiments, the SpCas9 comprises a D10A mutation as numbered in the wild type SpCas9 amino acid sequence or a corresponding mutation in another SpCas9 protein. In some embodiments, the SpCas9 domain, the nuclease in active SpCas9 domain, or the SpCas9 nickase domain can bind to a nucleic acid sequence having a non-NGG PAM. In some embodiments, the SpCas9 domain, the SpCas9 domain, the nuclease in active SpCas9 domain, or the SpCas9 nickase domain can bind to a nucleic acid sequence having a NGG, a NGA, or a NGCG PAM sequence. In some embodiments, the SpCas9 domain comprises one or more of a D1135X, a R1335X, and a T1337X mutation as numbered in the wild type SpCas9 amino acid sequence or a corresponding mutation in another SpCas9 protein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1135E, R1335Q, and T1337R mutation as numbered in the wild type SpCas9 amino acid sequence or a corresponding mutation in another SpCas9 protein. In some embodiments, the SpCas9 domain comprises one or more of a D1 134V, a R1334Q, and a T1336R mutation as numbered in the wild type Cas9 amino acid sequence, or a corresponding mutation thereof. In some embodiments, the SpCas9 domain comprises a D1135V, a R1335Q, and a T1337R mutation as numbered in the wild type SpCas9 amino acid sequence or a corresponding mutation in another SpCas9 protein. In some embodiments, the SpCas9 domain comprises one or more of a D1135X, a G1218X, a R1335X, and a T1337X mutation as numbered in the wild type SpCas9 amino acid sequence or a corresponding mutation in another SpCas9 protein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1135V, a G1218R, a R1335Q, and a T1337R mutation as numbered in the wild type SpCas9 amino acid sequence or a corresponding mutation in another SpCas9 protein. In some embodiments, the SpCas9 domain comprises a D1135V, a G1218R, a R1335Q, and a T1337R mutation as numbered in the wild type SpCas9 amino acid sequence or a corresponding mutation in another SpCas9 protein. 
     In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 sequences provided herein. 
     In some embodiments, a base editor provided herein comprises a RNA guide programmable DNA binding protein that may be used to target and modify a specific nucleic acid (e.g., DNA or RNA) sequence. Nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., nuclease inactive Cas9 and Cas9 nickase), CasX, CasY, Cas12a (Cpf1), Cas12b, C2c1, C2c2, C2C3, and Argonaute. One example of an nucleic acid programmable DNA-binding protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from  Prevotella  and  Francisella  1 (Cpf1). Similar to Cas9, Cpf1 is also a class 2 CRISPR effector. It has been shown that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). Cpf1 proteins described in Yamano et ah, “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference. 
     In some embodiments, a base editor provided herein comprises a nuclease inactive Cpf1 protein or a variant thereof. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alfa-helical recognition lobe of Cas9. In some embodiments, the Cpf1 nickase comprises one or more mutations corresponding to D917A, E1006A, or D1255A as numbered in the  Francisella novicida  Cpf1 protein. 
     Wild type  Francisella novicida  Cpf1 amino acid sequence is provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 53) 
               
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKA 
               
               
                   
               
               
                 KQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKS 
               
               
                   
               
               
                 AKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGI 
               
               
                   
               
               
                 ELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII 
               
               
                   
               
               
                 YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT 
               
               
                   
               
               
                 SEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI 
               
               
                   
               
               
                 NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT 
               
               
                   
               
               
                 TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT 
               
               
                   
               
               
                 DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY 
               
               
                   
               
               
                 LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA 
               
               
                   
               
               
                 QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSED 
               
               
                   
               
               
                 KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF 
               
               
                   
               
               
                 ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENK 
               
               
                   
               
               
                 GEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSI 
               
               
                   
               
               
                 DEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR 
               
               
                   
               
               
                 PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIA 
               
               
                   
               
               
                 NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI 
               
               
                   
               
               
                 NLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK 
               
               
                   
               
               
                 TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN 
               
               
                   
               
               
                 AIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG 
               
               
                   
               
               
                 VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYE 
               
               
                   
               
               
                 SVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR 
               
               
                   
               
               
                 LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD 
               
               
                   
               
               
                 KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM 
               
               
                   
               
               
                 PQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
            
           
         
       
     
     In some embodiments, the base editor comprises a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase. In some embodiments, the Cpf1 protein is a nuclease inactive Cpf1. In some embodiments, the Cpf1 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to the FnCpf1 sequence provided herein. In some embodiments, the Cpf1 protein comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A as numbered in the wild type FnCpf1 sequence provided herein. It should be appreciated that Cpf1 from other bacterial species may also be used in accordance with the present disclosure. 
     In some embodiments, the nucleic acid programmable DNA binding protein comprises a Cas12b (C2c1), C2c2, or C2c3 protein or a variant thereof. Additional features of Cas proteins in Class 2 CRISPR-Cas systems described in Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov. 5; 60(3): 385-397, the entire contents of which is hereby incorporated by reference. Effectors of two of the systems, C2c1 and C2c3, contain RuvC-like endonuclease domains related to Cpf1. A third system, C2c2 contains an effector with two predicated HEPN RNase domains. Production of mature CRISPR RNA is tracrRNA-independent, unlike production of CRISPR RNA by C2c1. C2c1 depends on both CRISPR RNA and tracrRNA for DNA cleavage. The crystal structure of Alicyclobaccillus acidoterrastris C2c1 (AacC2c1) in complex with a chimeric single-molecule guide RNA (sgRNA) is described in Liu et al., “C2c1-sgRNA Complex Structure Reveals RNA→Guided DNA Cleavage Mechanism”, Mol. Cell, 2017 Jan. 19;65(2):310-322; Yang et al., “PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas, the entire contents of which are hereby incorporated by reference. 
     An exemplary Cas12b amino acid sequence ( Bacillus  hisashii Cas12b) is provided below: 
     
       
         
           
               
               
            
               
                   
                 BhCasl2b (Bacillus hisashii) NCBI 
               
               
                   
                 Reference Sequence: WP_095142515 
               
               
                   
                 (SEQ ID NO: 54) 
               
               
                   
                 MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNH 
               
               
                   
               
               
                   
                 GIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVL 
               
               
                   
               
               
                   
                 KMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGEANQLSNK 
               
               
                   
               
               
                   
                 FLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEE 
               
               
                   
               
               
                   
                 DKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRN 
               
               
                   
               
               
                   
                 QSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEYKTLEER 
               
               
                   
               
               
                   
                 IKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREI 
               
               
                   
               
               
                   
                 IQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKEN 
               
               
                   
               
               
                   
                 HFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWV 
               
               
                   
               
               
                   
                 RFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGW 
               
               
                   
               
               
                   
                 EEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKG 
               
               
                   
               
               
                   
                 TLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVS 
               
               
                   
               
               
                   
                 KSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSGIESLEIGLR 
               
               
                   
               
               
                   
                 VMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRA 
               
               
                   
               
               
                   
                 SFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQF 
               
               
                   
               
               
                   
                 EDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWV 
               
               
                   
               
               
                   
                 AFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDR 
               
               
                   
               
               
                   
                 TRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKK 
               
               
                   
               
               
                   
                 MANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEER 
               
               
                   
               
               
                   
                 SRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAK 
               
               
                   
               
               
                   
                 TGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLY 
               
               
                   
               
               
                   
                 PDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVY 
               
               
                   
               
               
                   
                 CKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAG 
               
               
                   
               
               
                   
                 KLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSG 
               
               
                   
               
               
                   
                 NVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSMK 
               
               
                   
               
               
                   
                 RPAATKKAGQAKKKK 
               
            
           
         
       
     
     In some embodiments, the programmable DNA binding protein comprises an argonaute protein. One example of such a nucleic acid programmable DNA binding protein is an Argonaute protein from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5′ phosphorylated ssDNA of −24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at the gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described in Gao et al., Nat Biotechnol., 2016 July; 34(7):768-73. PubMed PMID: 27136078; Swarts et al., Nature. 507(7491) (2014):258-61; and Swarts et al., Nucleic Acids Res. 43(10) (2015):5120-9, each of which is incorporated herein by reference. 
     In some embodiments, the protospacer sequence comprises a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the protospacer sequences listed in Table 1 or Table 24. In some embodiments, the protospacer sequence comprises any one of the protospacer sequences listed in Table 1 or Table 24. In some embodiments, the protospacer sequence is any one of the protospacer sequences listed in Table 1 or Table 24. In some embodiments, the protospacer sequence comprises a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the sequences of SEQ ID NOs: 13-15, 50, 66-69, 81-252, and 1594-1617. In some embodiments, the protospacer sequence comprises any one of the sequences of SEQ ID NOs: 13-15, 50, 66-69, 81-252, and 1594-1617. In some embodiments, the protospacer sequence is any one of the sequences of SEQ ID NOs: 13-15, 50, 66-69, 81-252, and 1594-1617. 
     Guide Polynucleotides 
     Some aspects of this disclosure provide complexes comprising any of the fusion proteins provided herein, and a guide polynucleotide bound to a programmable DNA binding protein, e.g. a Cas9 domain of the base editor or fusion protein. 
     In some embodiments, a guide polynucleotide is a guide polynucleotide, a guide RNA (gRNA), or a nucleic acid encoding the same. 
     In some embodiments, the guide polynucleotide comprises a single nucleic acid sequence. In some embodiments, the guide polynucleotide comprises two nucleic acid sequences. In some embodiments, the guide nucleic acid (e.g., guide RNA) is from 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is a sequence in the genome of a mammal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence associated with a disease or disorder. In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence associated with a disease or disorder having a mutation in a PCSK9 gene, a ANGPTL3 gene, or a APOC3 gene. 
     Some aspects of this disclosure provide methods of using the fusion proteins, or complexes comprising a guide nucleic acid (e.g., gRNA) and a nucleobase editor provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA, or RNA molecule with any of the fusion proteins provided herein, and with at least one guide nucleic acid (e.g., guide RNA), wherein the guide nucleic acid, (e.g., guide RNA) is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is not immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence. 
     In some embodiments, a guide polynucleotide is a DNA. In some embodiments, a guide polynucleotide is an RNA. In some embodiments, a guide polynucleotide is a modified, artificial polynucleotides. In some embodiments, a guide polynucleotide is a single or single molecule polynucleotide. In some embodiments, a guide polynucleotide comprises dual polynucleotides. In some embodiments, a guide polynucleotide is a dual polynucleotides connected by a linker. In some embodiments, a guide polynucleotide is a dual polynucleotides connected by a non-nucleic acid linker. In some embodiments, a guide polynucleotide is a dual polynucleotides connected by a peptide linker or a chemical linker. In some embodiments, the guide polynucleotide is a single guide RNA. The guide RNA (gRNA) may guide the programmable DNA binding protein, e.g., a Class 2 Cas nuclease, e.g. a Cas9 to a target sequence on a target nucleic acid molecule, where the gRNA hybridizes with and the programmable DNA binding protein and modifies the target sequence. In some embodiments, the gRNA and the base editor fusion protein may form a ribonucleoprotein (RNP), e.g., a CRISPR/Cas complex. In some embodiments, the CRISPR complex may be a Type II CRISPR/Cas9 complex. In some embodiments, the CRISPR/Cas complex may be a Type V CRISPR/Cas complex, such as a Cpf1/guide RNA complex. 
     A gRNA can comprise at least three regions: a first region at the 5′ end that can bind to the complementary strand of a target site in a chromosomal sequence (spacer region), a second internal region that can form a stem loop structure, and a third 3′ region that can be single-stranded. A first region of each guide RNA can also be different such that each gRNA guides a protein, e.g., Cas9, to a specific target site. Further, second and third regions of each gRNA can be identical in all gRNAs. A second region of a gRNA may form a secondary structure. In some embodiments, a secondary structure formed by a gRNA can comprise a stem (or hairpin) and a loop. A length of a loop and a stem can vary. In some embodiments, a loop can range from about 3 to about 10 nucleotides in length. In some embodiments, a stem can range from about 6 to about 20 nucleotides in length. A stem can comprise one or more bulges of 1 to 10 nucleotides or about 10 nucleotides. In some embodiments, the overall length of a second region can range from about 16 to 60 nucleotides in length. In some embodiments, a loop can be about 4 nucleotides in length. In some embodiments, a stem can be about 12 in length. A third region of gRNA at the 3′ end can be essentially single-stranded. In some embodiments, a third region is sometimes not complementary to any chromosomal sequence in a cell of interest and is sometimes not complementary to the rest of a gRNA. In addition, the length of a third region can vary. In some embodiments, a third region can be more than 3 or more than 4 nucleotides in length. For example, the length of a third region can range from about 5 to 60 nucleotides in length. 
     A gRNA for a base editor system can comprise a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). In some embodiments, the crRNA may comprise a targeting sequence (or spacer sequence) that hybridizes with the complementary strand of the target sequence on the target nucleic acid molecule. The crRNA may also comprise a flagpole that is complementary to and hybridizes with a portion of the tracrRNA. In some embodiments, the crRNA may parallel the structure of a naturally occurring crRNA transcribed from a CRISPR locus of a bacteria, where the targeting sequence acts as the protospacer of the Cas9 in the base editor system. The gRNA may target any sequence of interest via the targeting sequence of the crRNA. In some embodiments, the degree of complementarity between the targeting sequence of the gRNA and the target sequence on the target nucleic acid molecule is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the targeting sequence of the gRNA and the target sequence on the target nucleic acid molecule may be 100% complementary. In other embodiments, the targeting sequence of the gRNA and the target sequence on the target nucleic acid molecule may contain at least one mismatch. For example, the targeting sequence of the gRNA and the target sequence on the target nucleic acid molecule may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. 
     In some embodiments, the length of the targeting sequence depends on the CRISPR/Cas component of the base editor system and components used. For example, different Cas proteins from different bacterial species have varying optimal targeting sequence lengths. Accordingly, the targeting sequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50 nucleotides in length. In some embodiments, the targeting sequence comprised 18-24 nucleotides in length. In some embodiments, the targeting sequence comprises 19-21 nucleotides in length. In some embodiments, the targeting sequence comprises 20 nucleotides in length. 
     In some embodiments, the guide RNA is a “dual guide RNA” or “dgRNA”. In some embodiments, the dgRNA comprises a first RNA molecule comprising a crRNA, and a second RNA molecule comprising a tracrRNA. The first and second RNA molecules may form a RNA duplex via the base pairing between the crRNA and the tracrRNA (e.g. the repeat and anti repeat). In some embodiments, the guide RNA is a “single guide RNA” or “sgRNA.” In some embodiments, the sgRNA may comprise a crRNA covalently linked to a tracrRNA. In some embodiments, the crRNA and the tracrRNA may be covalently linked via a linker. In some embodiments, the single-molecule guide RNA may comprise a stem-loop structure via the base pairing between the crRNA and the tracrRNA. In some embodiments, the sgRNA is a “Cas9 sgRNA” capable of directing a Cas9 protein. In certain embodiments, the guide RNA comprises a crRNA and tracrRNA sufficient for forming an active complex with a Cas9 protein and mediating RNA-guided DNA modification. The term “gRNA” and “sgRNA” are used interchangeably throughout this application. In some embodiments, more than one guide RNAs can be used; each guide RNA contains a different targeting sequence, such that the base editor system modifies more than one target sequence. In some embodiments, one or more guide RNAs may have the same or differing properties such as activity or stability within a base editor complex. Where more than one guide RNA is used, each guide RNA can be encoded on the same or on different expression cassettes. The promoters used to drive expression of the more than one guide RNA may be the same or different. 
     In some embodiments, the gRNA or mRNA encoding Cas protein is modified. The modifications can comprise chemical alterations, synthetic modifications, nucleotide additions, and/or nucleotide subtractions and modified nucleosides or nucleotides can be present in a gRNA. A gRNA or Cas protein-encoding mRNA comprising one or more modified nucleosides or nucleotides is called a “modified” RNA to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified RNA is synthesized with a non-canonical nucleoside or nucleotide. Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification); (vi) modification of the 3′ end or 5′ end of the oligonucleotide, e.g., removal, addition, modification, or replacement of a terminal phosphate group or conjugation of a moiety, cap, or linker (such 3′ or 5′ cap modifications may comprise a sugar and/or backbone modification); and (vii) modification or replacement of the sugar (an exemplary sugar modification). The modifications can enhance genome editing by CRISPR/Cas. A modification can alter chirality of a gRNA. In some cases, chirality may be uniform or stereopure after a modification. A guide RNA can also be truncated. Truncation can be used to reduce undesired off-target mutagenesis. The truncation can comprise any number of nucleotide deletions. For example, the truncation can comprise 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more nucleotides. In some embodiments, the term “cap,” as used herein, may be used to refer to one or more specially altered or modified nucleotides, e.g., specially altered or modified nucleotides on the 5′ end of mRNA. In some embodiments, the cap comprises to a modified guanine (G) nucleotide. In some embodiments, the cap refers to the 5′ end modification of mRNAs by addition of 7-Methylguanosine (N7-methyl guanosine or m7G). In some embodiments, “capping” of the mRNA structure plays a crucial role in a variety of cellular processes which include translation initiation, splicing, intracellular transport, and turnover. In some embodiment, the 5′ cap enhances mRNA stability as well as translation efficiency. Exemplary cap analogs include, but are not limited to, standard cap analog m7G(5′)ppp(5′)G, anti-reverse cap analog (ARCA) m7,3′-OGpppG (or 3′-O-Me-m7G(5′) ppp(5′)G), unmethylated cap analog G (5′)ppp(5′)G, methylated cap analog for A+1 sites m7G(5′)ppp(5′)A, and unmethylated cap analog for A+1 sites G(5′)ppp(5′)A. Additional methods of obtaining mRNA cap analogs are described in Kowalska et al., RNA 2008 14(6): 1119-1131, which is incorporated herein in its entirety. 
     In some embodiments, the guide RNA sequence comprises a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the guide sequences listed in Table 1 or Table 24. In some embodiments, the guide RNA sequence comprises any one of the guide RNA sequences listed in Table 1 or Table 24. In some embodiments, the guide RNA sequence is any one of the guide RNA sequences listed in Table 1 or Table 24. In some embodiments, the guide RNA sequence comprises a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the sequences of SEQ ID NOs: 9-11, 55, 59, 253-452, 1618-1635, 1637-1800, 1802-2135, and 2191. In some embodiments, the guide RNA sequence comprises any one of the sequences of SEQ ID NOs: 9-11, 55, 59, 253-452, 1618-1635, 1637-1800, 1802-2135, and 2191. In some embodiments, the guide RNA sequence is any one of the sequences of SEQ ID NOs: 9-11, 55, 59, 253-452, 1618-1635, 1637-1800, 1802-2135, and 2191. 
     Off-Target Effects 
     With standard CRISPR-Cas9 genome editing, off-target mutagenesis—i.e., edits at genomic sites other than the intended target site—can occur at sites that share a high degree of sequence similarity with the protospacer sequence encoded in the gRNA. 
     As used herein, off-target effect may be used refer to modifications, including indels, at genomic sites other than intended target site, for example, a protospacer sequence recognized by the guide RNA sequence. In the context of base editing, off-target editing may occur at genomic sites remote from an intended target site, or in close proximity to the intended target site. For example, one or more bases besides the target nucleobase in the base editing window may be edited (“bystander editing”). In certain embodiments, off-target base editing, e.g. bystander editing does not impact expression or function of the target gene. 
     Potential off-target sequence candidates in the genome may be predicted by bioinformatic analyses of the human genome using, e.g., the MIT Specificity Score (calculated by http://crispor.tefor.net/; minimum score of 50) or by in vitro biochemical assays, e.g., ONE-seq. 
     Off-target editing may be determined by sequencing analysis. In some embodiments, off-target editing is calculated using net nucleobase editing at potential off-target sites in the genome. For example, net nucleobase editing efficiency may be determined using no-base editor or no-guide RNA as a control, where net nucleobase editing efficiency is obtained by editing rate observed in cells contacted with a LNP enclosing a base editor mRNA and a guide RNA subjected by editing rate observed in control cells at predicted off-target sites. Additional methods for estimating and reducing off-target editing are described in Rees et al., Nat. Rev. Genet. 2018 19(12): 770-788, which is incorporated herein by reference in its entirety. 
     Methods for selecting, designing, and validating gRNAs and targeting sequences (or spacer sequences) are described herein and known to those skilled in the art. Software tools can be used to optimize the gRNAs corresponding to a target nucleic acid sequence, e.g., to minimize total off-target activity across the genome. For example, for each possible targeting domain choice using  S. pyogenes  Cas9, all off-target sequences (preceding selected PAMs, e.g., NAG or NGG) may be identified across the genome that contain up to certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. First regions of gRNAs complementary to a target site can be identified, and all first regions (e.g., crRNAs) can be ranked according to its total predicted off-target score; the top-ranked targeting domains represent those that are likely to have the greatest on-target and the least off-target activity. In some embodiments, a DNA sequence searching algorithm can be used to identify a target sequence in crRNAs of a gRNA for use with Cas9. A custom gRNA design software based on the public tool cas-offinder, which scores guides after calculating their genome-wide off-target propensity, can be also used to design a gRNA (Bae, et al., Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475 (2014)). In some embodiments, RepeatMasker program can be used to screen repeat elements and regions of low complexity in the input DNA sequences. In addition, the number of residues that could unintentionally be targeted (e.g., off-target residues that could potentially reside on ssDNA within the target nucleic acid locus) may be minimized to reduce the impact of potential substrate promiscuity of a deaminase domain in the nucleobase editor system. Candidate gRNAs can be functionally evaluated by using methods known in the art and/or as set forth herein. 
     In some embodiments, target sequences for a Cas9 protein in a base editor system can be selected based on the presence of target sequences of a gene of interest in one or more species. For example, a sequence may be selected as a target sequence if the sequence match target sequences in both human and cynomolgus monkey orthologs of a gene of interest. In some embodiments, target sequences for a Cas9 nuclease can be selected based on predicted off-target profiles, as judged by the MIT Specificity Score (calculated by http://crispor.tefor.net/). For example, a sequence may be selected as a target sequence if the sequence has a favorable predicted off-target profiles (e.g., minimum score of 50 when judged by the MIT Specificity Score). In some embodiments, target sequences for an adenine base editor (ABE) can be selected based on the ability of editing an adenine base within a splice donor site or a splice acceptor site in a target gene. In some embodiments, target sequences for an ABE can be selected based on the position of an adenine, e.g., if the adenine lies within the editing window of the ABE. 
     The gRNAs described herein can be synthesized chemically, enzymatically, or a combination thereof. For example, the gRNA can be synthesized using standard phosphoramidite-based solid-phase synthesis methods. Alternatively, the gRNA can be synthesized in vitro by operably linking DNA encoding the gRNA to a promoter control sequence that is recognized by a phage RNA polymerase. Examples of suitable phage promoter sequences include, but are not limited to, T7, T3, SP6 promoter sequences, or variations thereof. In some embodiments, gRNA comprises two separate molecules (e.g., crRNA and tracrRNA) and one molecule (e.g., crRNA) can be chemically synthesized and the other molecule (e.g., tracrRNA) can be enzymatically synthesized. 
     In some aspects, provided herein, is a composition for gene modification comprising a single guide RNA (sgRNA) described herein and the base editor fusion protein or a nucleic acid sequence encoding the base editor fusion protein. In some embodiments, the composition further comprises a vector that comprises the nucleic acid sequence encoding the base editor fusion protein. In some embodiments, base editor fusion protein comprises a Cas9 protein. In some embodiments, the Cas9 protein is a the Cas9 protein is a  Streptococcus pyogenes  Cas9. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers are listed in “Pharmaceutical composition and methods of treatment” section of this application. In some embodiments, the composition described herein can be provided in a lipid nanoparticle. 
     Chemically Modified Guide RNAs (2RNAs) 
     The present disclosure, provided herein, relates to a chemically modified CRISPR guide RNA (gRNA) that may be used in association with any general CRISPR/Cas system, for example, a base editor system as described herein. gRNAs (e.g., sgRNAs, short-sgRNAs, crRNAs, tracrRNAs, or dgRNAs) may comprise modifications at various nucleotide positions. In some aspects, provided herein, is a guide RNA (gRNA) or a single guide RNA (sgRNA) that comprises (i) a chemical modification at a nucleotide and (ii) an unmodified nucleotide. In some embodiments, the gRNA or the sgRNA may comprise a modification at a specific nucleotide position. In some embodiments, the gRNA or the sgRNA may comprise one or more modifications at one or more specific positions. 
     In an aspect, chemically modified CRISPR guide RNAs provided herein comprise modifications based on crystal structures. Not intended to be bound by a particular theory, X-ray crystal structure based guide design may be used to optimize gRNA modification in complex with the Cas protein, e.g. Cas9. In some embodiments, a nucleotide where a 2′-OH is in close proximity with a Cas9 protein in the Cas9-gRNA complex is unmodified. In some embodiments, a nucleotide where a 2′-OH is in contact with a Cas9 protein in the Cas9-gRNA complex is unmodified. In some embodiments, a nucleotide where a 2′-OH is in contact with a Cas9 protein with a hydrogen bond in the Cas9-gRNA complex is unmodified. In some embodiments, the Cas9-gRNA complex is a pre-catalytic ternary complex. In some embodiments, a nucleotide where a 2′-OH is in close proximity with another part of the gRNA in the Cas9-gRNA complex is unmodified. In some embodiments, a nucleotide where a 2′-OH is in contact with another part of the gRNA in the Cas9-gRNA complex is unmodified. In some embodiments, a nucleotide where a 2′-OH is in contact with another part of the gRNA with a hydrogen bond in the Cas9-gRNA complex is unmodified. In some embodiments, the Cas9-gRNA complex is a pre-catalytic ternary complex. As a person of skill in the art would understand, the term “clash” as used herein refers to physically-unlikely overlapping atomic volumes in a structure. Incorporation of a 2′O-Me modification in these situations would potentially result in structural rearrangement(s) that could be detrimental to RNP function. As a person of skill in the art would understand, the term “contact” as used herein means a stable, non-covalent interaction between two functional groups, e.g. a hydrogen bond. In some embodiments, a nucleotide where a clash with another part of the gRNA is predicted if a 2′-OH is replaced with a 2′-OMe in the Cas9-gRNA complex is unmodified. In some embodiments, a nucleotide where a clash with another 2′-OH in the gRNA is predicted if a 2′-OH is replaced with a 2′-OMe in the Cas9-gRNA complex is unmodified. In some embodiments, the Cas9-gRNA complex is a pre-catalytic ternary complex. In some embodiments, a nucleotide that is solvent-exposed or otherwise distant from Cas protein residues in the Cas9-gRNA complex comprises a chemical modification. In some embodiments, a nucleotide that is solvent-exposed or otherwise distant from another nucleotide in the gRNA in the Cas9-gRNA complex comprises a chemical modification. In some embodiments, the chemical modification is a 2′-OMe modification. Additional description of Cas9-gRNA crystal structure is included in Jiang F, Taylor D W, Chen J S, Kornfeld J E, Zhou K, Thompson A J, Nogales E, Doudna J A. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science. 2016 Feb. 19;351(6275):867-71, incorporated herein by reference in its entirety. 
     Modifications to guide RNA nucleotides can include, but are not limited to, 2′-O-methyl modifications, 2′-O-(2-methoxyethyl) modifications, 2′-fluoro modifications, phosphorothioate modifications, inverted abasic modifications, deoxyribonucleotides, bicylic ribose analog (e.g., locked nucleic acid (LNA), C-ethylene-bridged nucleic acid (ENA), bridged nucleic acid (BNA), unlocked nucleic acid (UNA)), base or nucleobase modifications, internucleoside linkage modifications, ribonebularine, 2′-O-methylnebularine, or 2′-deoxynebularine. Other examples of modifications include, but are not limited to, 5′adenylate, 5′ guanosine-triphosphate cap, 5′N7-Methylguanosine-triphosphate cap, 5′triphosphate cap, 3′phosphate, 3′thiophosphate, 5′phosphate, 5′thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9,3′-3′ modifications, 5′-5′ modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3′DABCYL, black hole quencher 1, black hole quencer 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linkers, 2′deoxyribonucleoside analog purine, 2′deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2′-O-methyl ribonucleoside analog, sugar modified analogs, wobble/universal bases, fluorescent dye label, 2′fluoro RNA, 2′O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate, 2-O-methyl 3phosphorothioate or any combinations thereof. 
     In some embodiments, the ribose group (or sugar) may be modified. In some embodiments, modified ribose group may control oligonucleotide binding affinity for complementary strands, duplex formation, or interaction with nucleases. Examples of chemical modifications to the ribose group include, but are not limited to, 2′-O-methyl (2′-OMe), 2′-fluoro (2′-F), 2′-deoxy, 2′-O-(2-methoxyethyl) (2′-MOE), 2′-NH2, 2′-O-Allyl, 2′-O-Ethylamine, 2′-O-Cyanoethyl, 2′-O-Acetalester, or a bicyclic nucleotide such as locked nucleic acid (LNA), 2′-(5-constrained ethyl (S-cEt)), constrained MOE, or 2′-0,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNANC). In some embodiments, 2′-O-methyl modification can increase binding affinity of oligonucleotides. In some embodiments, 2′-O-methyl modification can enhance nuclease stability of oligonucleotides. In some embodiments, 2′-fluoro modification can increase oligonucleotide binding affinity and nuclease stability. In this application, a ribonucleotide in lowercase in gRNA sequence depicts 2′-OMe modification (e.g., Table 1 or Table 24) (lowercase letter s indicates a phosphorothioate linkage). 
     In some embodiments, the phosphate group may be chemically modified. Examples of chemical modifications to the phosphate group includes, but are not limited to, a phosphorothioate (PS), phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, or phosphotriester modification. In some embodiments, PS linkage can refer to a bond where a sulfur is substituted for one nonbridging phosphate oxygen in a phosphodiester linkage, e.g., between nucleotides. An “s” may be used to depict a PS modification in gRNA sequences in this application (e.g., Table 1 or Table 24). In some embodiments, a gRNA or an sgRNA may comprise a phosphorothioate (PS) linkage at a 5′ end or at a 3′ end. In some embodiments, a gRNA or an sgRNA may comprise a phosphorothioate (PS) linkage at a 5′ end. In some embodiments, a gRNA or an sgRNA may comprise a phosphorothioate (PS) linkage at a 3′ end. In some embodiments, a gRNA or an sgRNA may comprise a phosphorothioate (PS) linkage at a 5′ end and at a 3′ end. In some embodiments, a gRNA or an sgRNA may comprise one, two, or three, or more than three phosphorothioate linkages at the 5′ end or at the 3′ end. In some embodiments, a gRNA or an sgRNA may comprise three phosphorothioate (PS) linkages at the 5′ end or at the 3′ end. In some embodiments, a gRNA or an sgRNA may comprise three phosphorothioate linkages at the 3′ end. In some embodiments, a gRNA or an sgRNA may comprise two and no more than two (i.e., only two) contiguous phosphorothioate (PS) linkages at the 5′ end or at the 3′ end. In some embodiments, a gRNA or an sgRNA may comprise three contiguous phosphorothioate (PS) linkages at the 5′ end or at the 3′ end. In some embodiments, a gRNA or an sgRNA may comprise the sequence 5′-UsUsU-3′ at the 3′end or at the 5′ end, wherein U indicates a uridine and wherein s indicates a phosphorothioate (PS) linkage. In some embodiments, a gRNA or an sgRNA may comprise the sequence 5′-ususu-3′ at the 3′end or at the 5′ end, wherein u indicates a 2′-O-methyluridine and wherein s indicates a phosphorothioate (PS) linkage. In some embodiments, a gRNA or an sgRNA may comprise the sequence 5′-ususuUUU-3′ at the 3′end or at the 5′ end, wherein U and u indicate uridine and 2′-O-methyluridine respectively, and wherein s indicates a phosphorothioate (PS) linkage. In some embodiments, a gRNA or an sgRNA may comprise the sequence 5′-ususuUuU-3′ at the 3′end or at the 5′ end, wherein U and u indicate uridine and 2′-O-methyluridine respectively, and wherein s indicates a phosphorothioate (PS) linkage. In some embodiments, a gRNA or an sgRNA may comprise the sequence 5′-usususuUuU-3′ at the 3′end, wherein U and u indicate uridine and 2′-O-methyluridine respectively, and wherein s indicates a phosphorothioate (PS) linkage. In some embodiments, a gRNA or an sgRNA may comprise the sequence 5′-ususuuuu-3′ at the 3′end, wherein u indicates 2′-O-methyluridine, and wherein s indicates a phosphorothioate (PS) linkage. 
     In some embodiments, the nucleobase may be chemically modified. Examples of chemical modifications to the nucleobase include, but are not limited to, 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, or halogenated aromatic groups. 
     In some embodiments the chemically modified gRNAs comprise nebularine. Nebularine is a purine ribonucleoside that is derived from a beta-D-ribose and is 9H-purine attached to a beta-D-ribofuranosyl residue at position 9 via a glycosidic (N-glycosyl) linkage. Nebularine is a purine ribonucleoside with no exocyclic functional moiety or substitution. In some embodiments, it is a purine ribonucleoside. In some embodiments, it is a purine D-ribonucleoside. In some embodiments, nebularine is further modified chemically. In this application, “X,” “x,” and “dX” may be used to depict a ribonebularine modification, a 2′-O-methylnebularine modification, and a 2′-deoxynebularine modification, respectively, in gRNA sequences (e.g., Table 1 or Table 24). In some embodiments, substitution of a nucleotide (e.g., A) of a gRNA sequence (e.g., spacer region or tracr region) with nebularine may reduce off-target effects without affecting the gRNA activity. In some embodiments, the nebularine, the deoxynebularine, or 2′-O-methylnebularine replaces an adenine in an unmodified gRNA or sgRNA. In some embodiments, the nebularine, the deoxynebularine, or 2′-O-methylnebularine is in the spacer sequence. In some embodiments, the nebularine, the deoxynebularine, or 2′-O-methylnebularine is in the tracrRNA sequence. In some embodiments, the nebularine, the deoxynebularine, or 2′-O-methylnebularine is in a tracrRNA sequence in the tracrRNA sequence. In some embodiments, the nebularine, the deoxynebularine, or 2′-O-methylnebularine is in a crRNA sequence in the tracrRNA sequence. In some embodiments, the nebularine, the deoxynebularine, or 2′-O-methylnebularine is in a stem loop structure in the tracrRNA sequence. 
     In some embodiments, the chemically modified gRNAs may comprise a total of 50-150 base pairs in length. In some embodiments, the chemically modified gRNAs may comprise a total of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 base pairs in length. In some embodiments, the chemically modified gRNAs may comprise a total of about 50 to about 140 base pairs in length. In some embodiments, the chemically modified gRNAs may comprise a total of about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 110, about 50 to about 120, about 50 to about 130, about 50 to about 140, about 60 to about 70, about 60 to about 80, about 60 to about 90, about 60 to about 100, about 60 to about 110, about 60 to about 120, about 60 to about 130, about 60 to about 140, about 70 to about 80, about 70 to about 90, about 70 to about 100, about 70 to about 110, about 70 to about 120, about 70 to about 130, about 70 to about 140, about 80 to about 90, about 80 to about 100, about 80 to about 110, about 80 to about 120, about 80 to about 130, about 80 to about 140, about 90 to about 100, about 90 to about 110, about 90 to about 120, about 90 to about 130, about 90 to about 140, about 100 to about 110, about 100 to about 120, about 100 to about 130, about 100 to about 140, about 110 to about 120, about 110 to about 130, about 110 to about 140, about 120 to about 130, about 120 to about 140, or about 130 to about 140 base pairs in length. In some embodiments, the chemically modified gRNAs may comprise a total of about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, or about 140 base pairs in length. In some embodiments, the chemically modified gRNAs may comprise a total of at least about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, or about 130 base pairs in length. In some embodiments, the chemically modified gRNAs may comprise a total of at most about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, or about 140 base pairs in length. In one embodiment, the chemically modified gRNAs may comprise a total of 100 base pairs in length. In another embodiment, the chemically modified gRNAs may comprise a total of 103 base pairs in length. 
     In some embodiments, the chemically modified gRNAs may comprise from 1 to 150 chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise a total of about 1 to about 45 chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise a total of about 1 to about 3, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 30, about 1 to about 35, about 1 to about 40, about 1 to about 45, about 3 to about 5, about 3 to about 10, about 3 to about 15, about 3 to about 20, about 3 to about 25, about 3 to about 30, about 3 to about 35, about 3 to about 40, about 3 to about 45, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 30, about 5 to about 35, about 5 to about 40, about 5 to about 45, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 10 to about 35, about 10 to about 40, about 10 to about 45, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 15 to about 35, about 15 to about 40, about 15 to about 45, about 20 to about 25, about 20 to about 30, about 20 to about 35, about 20 to about 40, about 20 to about 45, about 25 to about 30, about 25 to about 35, about 25 to about 40, about 25 to about 45, about 30 to about 35, about 30 to about 40, about 30 to about 45, about 35 to about 40, about 35 to about 45, about 40 to about 45, or about 45 to about 50 chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise a total of about 1, about 3, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise a total of at least about 1, about 3, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise a total of at most about 3, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise a total of about 50 to about 140 chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise a total of about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 110, about 50 to about 120, about 50 to about 130, about 50 to about 140, about 60 to about 70, about 60 to about 80, about 60 to about 90, about 60 to about 100, about 60 to about 110, about 60 to about 120, about 60 to about 130, about 60 to about 140, about 70 to about 80, about 70 to about 90, about 70 to about 100, about 70 to about 110, about 70 to about 120, about 70 to about 130, about 70 to about 140, about 80 to about 90, about 80 to about 100, about 80 to about 110, about 80 to about 120, about 80 to about 130, about 80 to about 140, about 90 to about 100, about 90 to about 110, about 90 to about 120, about 90 to about 130, about 90 to about 140, about 100 to about 110, about 100 to about 120, about 100 to about 130, about 100 to about 140, about 110 to about 120, about 110 to about 130, about 110 to about 140, about 120 to about 130, about 120 to about 140, or about 130 to about 140 chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise a total of about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, or about 140 chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise a total of at least about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, or about 130 chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise a total of at most about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, or about 140 chemically modified nucleotides. In one embodiment, the chemically modified gRNAs may comprise a total of 54 chemically modified nucleotides. In another embodiment, the chemically modified gRNAs may comprise a total of 62 chemically modified nucleotides. 
     In some embodiments, the chemically modified gRNAs may comprise about 1% to about 100% chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise about 1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 1% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 95%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 95%, about 40% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 90% to about 95%, about 90% to about 100%, or about 95% to about 100% chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise at least about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% chemically modified nucleotides. In some embodiments, the chemically modified gRNAs may comprise at most about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% chemically modified nucleotides. 
     In some embodiments, the chemically modified gRNAs may have chemically modified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 in the 5′-3′ direction in the gRNA sequence. 
     In some embodiments, the chemically modified gRNAs may have chemically modified nucleotide on one or more of the positions which are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 base pairs from the 5′ end of the gRNA sequence. 
     In some embodiments, the chemically modified gRNAs may have chemically modified nucleotide on one or more the positions where are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 base pairs from the 3′ end of the gRNA sequence. 
     In some embodiments, the chemically modified gRNA may be a sgRNA. The term “gRNA” and “sgRNA” are used interchangeably in this application. In some embodiments, the sgRNA may comprise one or more chemically modified nucleotides. In some embodiments, the chemically modified sgRNA may comprise a total of 50-150 base pairs in length. In some embodiments, the chemically modified sgRNA may comprise a total of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 base pairs in length. In some embodiments, the chemically modified sgRNA may comprise a total of about 50 to about 140 base pairs in length. In some embodiments, the chemically modified sgRNA may comprise a total of about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 110, about 50 to about 120, about 50 to about 130, about 50 to about 140, about 60 to about 70, about 60 to about 80, about 60 to about 90, about 60 to about 100, about 60 to about 110, about 60 to about 120, about 60 to about 130, about 60 to about 140, about 70 to about 80, about 70 to about 90, about 70 to about 100, about 70 to about 110, about 70 to about 120, about 70 to about 130, about 70 to about 140, about 80 to about 90, about 80 to about 100, about 80 to about 110, about 80 to about 120, about 80 to about 130, about 80 to about 140, about 90 to about 100, about 90 to about 110, about 90 to about 120, about 90 to about 130, about 90 to about 140, about 100 to about 110, about 100 to about 120, about 100 to about 130, about 100 to about 140, about 110 to about 120, about 110 to about 130, about 110 to about 140, about 120 to about 130, about 120 to about 140, or about 130 to about 140 base pairs in length. In some embodiments, the chemically modified sgRNAs may comprise a total of about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, or about 140 base pairs in length. In some embodiments, the chemically modified sgRNAs may comprise a total of at least about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, or about 130 base pairs in length. In some embodiments, the chemically modified sgRNA may comprise a total of at most about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, or about 140 base pairs in length. In one embodiment, the chemically modified sgRNA may comprise a total of 100 base pairs in length. In another embodiment, the chemically modified sgRNA may comprise a total of 103 base pairs in length. 
     In some embodiments, the chemically modified sgRNAs may comprise a total of from 1 to 150 chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise a total of about 1 to about 45 chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise a total of about 1 to about 3, about 1 to about 5, about 1 to about 10, about 1 to about 15, about 1 to about 20, about 1 to about 25, about 1 to about 30, about 1 to about 35, about 1 to about 40, about 1 to about 45, about 3 to about 5, about 3 to about 10, about 3 to about 15, about 3 to about 20, about 3 to about 25, about 3 to about 30, about 3 to about 35, about 3 to about 40, about 3 to about 45, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 30, about 5 to about 35, about 5 to about 40, about 5 to about 45, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 10 to about 35, about 10 to about 40, about 10 to about 45, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 15 to about 35, about 15 to about 40, about 15 to about 45, about 20 to about 25, about 20 to about 30, about 20 to about 35, about 20 to about 40, about 20 to about 45, about 25 to about 30, about 25 to about 35, about 25 to about 40, about 25 to about 45, about 30 to about 35, about 30 to about 40, about 30 to about 45, about 35 to about 40, about 35 to about 45, about 40 to about 45, or about 45 to about 50 chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise a total of about 1, about 3, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise a total of at least about 1, about 3, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise a total of at most about 3, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise a total of about 50 to about 140 chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise a total of about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 110, about 50 to about 120, about 50 to about 130, about 50 to about 140, about 60 to about 70, about 60 to about 80, about 60 to about 90, about 60 to about 100, about 60 to about 110, about 60 to about 120, about 60 to about 130, about 60 to about 140, about 70 to about 80, about 70 to about 90, about 70 to about 100, about 70 to about 110, about 70 to about 120, about 70 to about 130, about 70 to about 140, about 80 to about 90, about 80 to about 100, about 80 to about 110, about 80 to about 120, about 80 to about 130, about 80 to about 140, about 90 to about 100, about 90 to about 110, about 90 to about 120, about 90 to about 130, about 90 to about 140, about 100 to about 110, about 100 to about 120, about 100 to about 130, about 100 to about 140, about 110 to about 120, about 110 to about 130, about 110 to about 140, about 120 to about 130, about 120 to about 140, or about 130 to about 140 chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise a total of about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, or about 140 chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise a total of at least about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, or about 130 chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise a total of at most about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, or about 140 chemically modified nucleotides. In one embodiment, the chemically modified sgRNAs may comprise a total of 54 chemically modified nucleotides. In another embodiment, the chemically modified sgRNAs may comprise a total of 62 chemically modified nucleotides. 
     In some embodiments, the chemically modified sgRNAs may comprise about 1% to about 100% chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise about 1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 1% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 95%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 95%, about 40% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 90% to about 95%, about 90% to about 100%, or about 95% to about 100% chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise at least about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% chemically modified nucleotides. In some embodiments, the chemically modified sgRNAs may comprise at most about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% chemically modified nucleotides. 
     In some embodiments, the chemically modified sgRNAs may have chemically modified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 in the 5′-3′ direction in the sgRNA sequence. 
     In some embodiments, the chemically modified sgRNAs may have chemically modified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 base pairs from the 5′ end of the sgRNA sequence. 
     In some embodiments, the chemically modified sgRNAs may have chemically modified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 base pairs from the 3′ end of the sgRNA sequence. 
     In some embodiments, the chemically modified gRNAs or sgRNAs may have one or more chemical modifications in the spacer or protospacer region, i.e., the target sequence. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a spacer sequence of from 10 to 30 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a spacer sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a spacer sequence of about 10 to about 30 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a spacer sequence of about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 20 to about 25, about 20 to about 30, or about 25 to about 30 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a spacer sequence of about 10, about 15, about 20, about 25, or about 30 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a spacer sequence of at least about 10, about 15, about 20, or about 25 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a spacer sequence of at most about 15, about 20, about 25, or about 30 base pairs in length. In one embodiment, the chemically modified gRNAs or sgRNAs may comprise a spacer sequence of 18 base pairs in length. In another embodiment, the chemically modified gRNAs or sgRNAs may comprise a spacer sequence of 22 base pairs in length. In a preferred embodiment, the chemically modified gRNAs or sgRNAs may comprise a spacer sequence of 20 base pairs in length. 
     In some embodiments, the spacer sequence may comprise a total of from 1 to 30 chemically modified nucleotides. In some embodiments, the spacer region may comprise a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 chemically modified nucleotides. In some embodiments, the spacer sequence may comprise a total of about 1 to about 10 chemically modified nucleotides. In some embodiments, the spacer sequence may comprise a total of about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 6, about 1 to about 7, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 to about 7, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 to about 7, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 to about 10, or about 9 to about 10 chemically modified nucleotides. In some embodiments, the spacer sequence may comprise a total of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 chemically modified nucleotides. In some embodiments, the spacer sequence may comprise a total of at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9 chemically modified nucleotides. In some embodiments, the spacer sequence may comprise a total of at most about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 chemically modified nucleotides. In some embodiments, the spacer sequence may comprise a total of about 10 to about 30 chemically modified nucleotides. In some embodiments, the spacer sequence may comprise a total of about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 20 to about 25, about 20 to about 30, or about 25 to about 30 chemically modified nucleotides. In some embodiments, the spacer sequence may comprise a total of about 10, about 15, about 20, about 25, or about 30 chemically modified nucleotides. In some embodiments, the spacer sequence may comprise a total of at least about 10, about 15, about 20, or about 25 chemically modified nucleotides. In some embodiments, the spacer sequence may comprise a total of at most about 15, about 20, about 25, or about 30 chemically modified nucleotides. In one embodiment, the spacer sequence may comprise a total of 3 chemically modified nucleotides. In another embodiment, the spacer sequence may comprise a total of 5 chemically modified nucleotides. 
     In some embodiments, the spacer sequence may comprise from 1% to 100% chemically modified nucleotides. In some embodiments, the spacer sequence may comprise about 1% to about 100% chemically modified nucleotides. In some embodiments, the spacer sequence may comprise about 1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 1% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 95%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 95%, about 40% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 90% to about 95%, about 90% to about 100%, or about 95% to about 100% chemically modified nucleotides. In some embodiments, the spacer sequence may comprise about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% chemically modified nucleotides. In some embodiments, the spacer sequence may comprise at least about 1%, about 10%, about 200%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% chemically modified nucleotides. In some embodiments, the spacer sequence may comprise at most about 10%, about 20%, about 30%, about 40%, about 50%, about 600%, about 70%, about 80%, about 90%, about 95%, or about 100% chemically modified nucleotides. 
     In some embodiments, the chemically modified spacer sequence may have chemically modified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 in the 5′-3′ direction in the spacer sequence. 
     In some embodiments, the chemically modified spacer region may have chemically modified nucleotide on one or more of the positions which are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 base pairs from the 5′ end ofthe spacer sequence. 
     In some embodiments, the chemically modified spacer region may have chemically modified nucleotide on one or more the positions which are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 base pairs from the 3′ end of the spacer sequence. 
     In some embodiments, the chemically modified gRNAs or sgRNAs may have one or more chemical modifications in the tracrRNA sequence. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence of from 30 to 100 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence of from about 30 to about 130 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence of from about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 75, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 110, about 30 to about 120, about 30 to about 130, about 40 to about 50, about 40 to about 60, about 40 to about 70, about 40 to about 75, about 40 to about 80, about 40 to about 90, about 40 to about 100, about 40 to about 110, about 40 to about 120, about 40 to about 130, about 50 to about 60, about 50 to about 70, about 50 to about 75, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 110, about 50 to about 120, about 50 to about 130, about 60 to about 70, about 60 to about 75, about 60 to about 80, about 60 to about 90, about 60 to about 100, about 60 to about 110, about 60 to about 120, about 60 to about 130, about 70 to about 75, about 70 to about 80, about 70 to about 90, about 70 to about 100, about 70 to about 110, about 70 to about 120, about 70 to about 130, about 75 to about 80, about 75 to about 90, about 75 to about 100, about 75 to about 110, about 75 to about 120, about 75 to about 130, about 80 to about 90, about 80 to about 100, about 80 to about 110, about 80 to about 120, about 80 to about 130, about 90 to about 100, about 90 to about 110, about 90 to about 120, about 90 to about 130, about 100 to about 110, about 100 to about 120, about 100 to about 130, about 110 to about 120, about 110 to about 130, or about 120 to about 130 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence of from about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, about 120, or about 130 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence of from at least about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, or about 120 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence of from at most about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, about 120, or about 130 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence of 70 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence of 73 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence of 68 base pairs in length. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence of 71 base pairs in length. 
     In some embodiments, the tracrRNA sequence may comprise a total of from 1 to 130 chemically modified nucleotides. In some embodiments, the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence of from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from about 1 to about 10 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 6, about 1 to about 7, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 to about 7, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 to about 7, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 to about 10, or about 9 to about 10 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from at most about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from about 10 to about 30 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 20 to about 25, about 20 to about 30, or about 25 to about 30 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from about 10, about 15, about 20, about 25, or about 30 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from at least about 10, about 15, about 20, or about 25 chemically modified nucleotides. In some embodiments, the spacer region may comprise a total of at most about 15, about 20, about 25, or about 30 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from about 30 to about 130 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 75, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 110, about 30 to about 120, about 30 to about 130, about 40 to about 50, about 40 to about 60, about 40 to about 70, about 40 to about 75, about 40 to about 80, about 40 to about 90, about 40 to about 100, about 40 to about 110, about 40 to about 120, about 40 to about 130, about 50 to about 60, about 50 to about 70, about 50 to about 75, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 110, about 50 to about 120, about 50 to about 130, about 60 to about 70, about 60 to about 75, about 60 to about 80, about 60 to about 90, about 60 to about 100, about 60 to about 110, about 60 to about 120, about 60 to about 130, about 70 to about 75, about 70 to about 80, about 70 to about 90, about 70 to about 100, about 70 to about 110, about 70 to about 120, about 70 to about 130, about 75 to about 80, about 75 to about 90, about 75 to about 100, about 75 to about 110, about 75 to about 120, about 75 to about 130, about 80 to about 90, about 80 to about 100, about 80 to about 110, about 80 to about 120, about 80 to about 130, about 90 to about 100, about 90 to about 110, about 90 to about 120, about 90 to about 130, about 100 to about 110, about 100 to about 120, about 100 to about 130, about 110 to about 120, about 110 to about 130, or about 120 to about 130 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, about 120, or about 130 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from at least about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, or about 120 chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise a total of from at most about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, about 120, or about 130 chemically modified nucleotides. In one embodiment, the tracrRNA sequence may comprise a total of 51 chemically modified nucleotides. In another embodiment, the tracrRNA sequence may comprise a total of 59 chemically modified nucleotides. 
     In some embodiments, the tracrRNA sequence may comprise from 1% to 100% chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise about 1% to about 100% chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise about 1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 1% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 95%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 95%, about 40% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 90% to about 95%, about 90% to about 100%, or about 95% to about 100% chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise at least about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% chemically modified nucleotides. In some embodiments, the tracrRNA sequence may comprise at most about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% chemically modified nucleotides. 
     In some embodiments, the chemically modified tracrRNA sequence may have chemically modified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130 in the 5′-3′ direction in the tracrRNA sequence. 
     In some embodiments, the chemically modified tracr region may have chemically modified nucleotide on one or more of the positions which are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 129 base pairs from the 5′ end of the tracrRNA sequence. 
     In some embodiments, the chemically modified tracr region may have chemically modified nucleotide on one or more the positions which are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 129 base pairs from the 3′ end of the tracrRNA sequence. 
     In some embodiments, the chemically modified gRNAs may have one or more chemical modifications in the 5′ end of the gRNA sequence. In some embodiments, the chemically modified gRNAs may comprise one or more chemically modified nucleotides in the 5′ end of the gRNA sequence. In some embodiments, the chemically modified gRNAs may have one or more chemical modifications in the 3′ end of the gRNA sequence. In some embodiments, the chemically modified gRNAs may comprise one or more chemically modified nucleotides in the 3′ end of the gRNA sequence. 
     In some embodiments, the chemically modified gRNAs may comprise a crRNA. In some embodiments, the crRNA may comprise one or more chemically modified nucleotides. In some embodiments, the chemically modified crRNA may be of from 30 to 50 base pairs in length. In some embodiments, the chemically modified crRNA may be of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 base pairs in length. In some embodiments, the chemically modified crRNA may be of from about 30 to about 50 base pairs in length. In some embodiments, the chemically modified crRNA may be of from about 30 to about 32, about 30 to about 34, about 30 to about 36, about 30 to about 38, about 30 to about 40, about 30 to about 42, about 30 to about 44, about 30 to about 46, about 30 to about 48, about 30 to about 50, about 32 to about 34, about 32 to about 36, about 32 to about 38, about 32 to about 40, about 32 to about 42, about 32 to about 44, about 32 to about 46, about 32 to about 48, about 32 to about 50, about 34 to about 36, about 34 to about 38, about 34 to about 40, about 34 to about 42, about 34 to about 44, about 34 to about 46, about 34 to about 48, about 34 to about 50, about 36 to about 38, about 36 to about 40, about 36 to about 42, about 36 to about 44, about 36 to about 46, about 36 to about 48, about 36 to about 50, about 38 to about 40, about 38 to about 42, about 38 to about 44, about 38 to about 46, about 38 to about 48, about 38 to about 50, about 40 to about 42, about 40 to about 44, about 40 to about 46, about 40 to about 48, about 40 to about 50, about 42 to about 44, about 42 to about 46, about 42 to about 48, about 42 to about 50, about 44 to about 46, about 44 to about 48, about 44 to about 50, about 46 to about 48, about 46 to about 50, or about 48 to about 50 base pairs in length. In some embodiments, the chemically modified crRNA may be of from about 30, about 32, about 34, about 36, about 38, about 40, about 42, about 44, about 46, about 48, or about 50 base pairs in length. In some embodiments, the chemically modified crRNA may be of from at least about 30, about 32, about 34, about 36, about 38, about 40, about 42, about 44, about 46, or about 48 base pairs in length. In some embodiments, the chemically modified crRNA may be of from at most about 32, about 34, about 36, about 38, about 40, about 42, about 44, about 46, about 48, or about 50 base pairs in length. 
     In some embodiments, the crRNA may comprise a total of from 1 to 50 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of about 1 to about 10 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 6, about 1 to about 7, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 to about 7, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 to about 7, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 to about 10, or about 9 to about 10 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of at most about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of about 10 to about 30 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 20 to about 25, about 20 to about 30, or about 25 to about 30 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of about 10, about 15, about 20, about 25, or about 30 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of at least about 10, about 15, about 20, or about 25 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of at most about 15, about 20, about 25, or about 30 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of from about 30 to about 50 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of from about 30 to about 32, about 30 to about 34, about 30 to about 36, about 30 to about 38, about 30 to about 40, about 30 to about 42, about 30 to about 44, about 30 to about 46, about 30 to about 48, about 30 to about 50, about 32 to about 34, about 32 to about 36, about 32 to about 38, about 32 to about 40, about 32 to about 42, about 32 to about 44, about 32 to about 46, about 32 to about 48, about 32 to about 50, about 34 to about 36, about 34 to about 38, about 34 to about 40, about 34 to about 42, about 34 to about 44, about 34 to about 46, about 34 to about 48, about 34 to about 50, about 36 to about 38, about 36 to about 40, about 36 to about 42, about 36 to about 44, about 36 to about 46, about 36 to about 48, about 36 to about 50, about 38 to about 40, about 38 to about 42, about 38 to about 44, about 38 to about 46, about 38 to about 48, about 38 to about 50, about 40 to about 42, about 40 to about 44, about 40 to about 46, about 40 to about 48, about 40 to about 50, about 42 to about 44, about 42 to about 46, about 42 to about 48, about 42 to about 50, about 44 to about 46, about 44 to about 48, about 44 to about 50, about 46 to about 48, about 46 to about 50, or about 48 to about 50 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of from about 30, about 32, about 34, about 36, about 38, about 40, about 42, about 44, about 46, about 48, or about 50 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of from at least about 30, about 32, about 34, about 36, about 38, about 40, about 42, about 44, about 46, or about 48 chemically modified nucleotides. In some embodiments, the chemically modified crRNA may comprise a total of from at most about 32, about 34, about 36, about 38, about 40, about 42, about 44, about 46, about 48, or about 50 chemically modified nucleotides. 
     In some embodiments, the crRNA may comprise from 1% to 100% chemically modified nucleotides. In some embodiments, the crRNA may comprise about 1% to about 100% chemically modified nucleotides. In some embodiments, the crRNA may comprise about 1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 1% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 95%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 95%, about 40% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 90% to about 95%, about 90% to about 100%, or about 95% to about 100% chemically modified nucleotides. In some embodiments, the crRNA may comprise about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% chemically modified nucleotides. In some embodiments, the crRNA may comprise at least about 1%, about 10%, about 20%, about 30%, about 40%, about 500%, about 60%, about 70%, about 80%, about 90%, or about 95% chemically modified nucleotides. In some embodiments, the crRNA may comprise at most about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% about 95%, or about 100% chemically modified nucleotides. 
     In some embodiments, the chemically modified crRNA may have chemically modified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 in the 5′-3′ direction in the crRNA sequence. 
     In some embodiments, the chemically modified crRNA may have chemically modified nucleotide on one or more of the positions which are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 base pairs from the 5′ end of the crRNA sequence. 
     In some embodiments, the chemically modified crRNA may have chemically modified nucleotide on one or more of the positions which are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 base pairs from the 3′ end of the crRNA sequence. 
     In some embodiments, the chemically modified gRNAs may comprise a tracrRNA. In some embodiments, the tracrRNA may comprise one or more chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of from 50 to 130 base pairs in length. In some embodiments, the chemically modified tracrRNA may comprise a total of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130 base pairs in length. In some embodiments, the chemically modified tracrRNA may comprise a total of about 30 to about 130 base pairs in length. In some embodiments, the chemically modified tracrRNA may comprise a total of about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 75, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 110, about 30 to about 120, about 30 to about 130, about 40 to about 50, about 40 to about 60, about 40 to about 70, about 40 to about 75, about 40 to about 80, about 40 to about 90, about 40 to about 100, about 40 to about 110, about 40 to about 120, about 40 to about 130, about 50 to about 60, about 50 to about 70, about 50 to about 75, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 110, about 50 to about 120, about 50 to about 130, about 60 to about 70, about 60 to about 75, about 60 to about 80, about 60 to about 90, about 60 to about 100, about 60 to about 110, about 60 to about 120, about 60 to about 130, about 70 to about 75, about 70 to about 80, about 70 to about 90, about 70 to about 100, about 70 to about 110, about 70 to about 120, about 70 to about 130, about 75 to about 80, about 75 to about 90, about 75 to about 100, about 75 to about 110, about 75 to about 120, about 75 to about 130, about 80 to about 90, about 80 to about 100, about 80 to about 110, about 80 to about 120, about 80 to about 130, about 90 to about 100, about 90 to about 110, about 90 to about 120, about 90 to about 130, about 100 to about 110, about 100 to about 120, about 100 to about 130, about 110 to about 120, about 110 to about 130, or about 120 to about 130 base pairs in length. In some embodiments, the chemically modified tracrRNA may comprise a total of about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, about 120, or about 130 base pairs in length. In some embodiments, the chemically modified tracrRNA may comprise a total of at least about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, or about 120 base pairs in length. In some embodiments, the chemically modified tracrRNA may comprise a total of at most about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, about 120, or about 130 base pairs in length. 
     In some embodiments, the chemically modified tracrRNA may comprise a total of from 1 to 130 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of about 1 to about 10 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 6, about 1 to about 7, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 to about 7, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 to about 7, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 to about 10, or about 9 to about 10 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of at most about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of about 10 to about 30 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 20 to about 25, about 20 to about 30, or about 25 to about 30 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of about 10, about 15, about 20, about 25, or about 30 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of at least about 10, about 15, about 20, or about 25 chemically modified nucleotides. In some embodiments, the spacer region may comprise a total of at most about 15, about 20, about 25, or about 30 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of about 30 to about 130 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 75, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 110, about 30 to about 120, about 30 to about 130, about 40 to about 50, about 40 to about 60, about 40 to about 70, about 40 to about 75, about 40 to about 80, about 40 to about 90, about 40 to about 100, about 40 to about 110, about 40 to about 120, about 40 to about 130, about 50 to about 60, about 50 to about 70, about 50 to about 75, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 110, about 50 to about 120, about 50 to about 130, about 60 to about 70, about 60 to about 75, about 60 to about 80, about 60 to about 90, about 60 to about 100, about 60 to about 110, about 60 to about 120, about 60 to about 130, about 70 to about 75, about 70 to about 80, about 70 to about 90, about 70 to about 100, about 70 to about 110, about 70 to about 120, about 70 to about 130, about 75 to about 80, about 75 to about 90, about 75 to about 100, about 75 to about 110, about 75 to about 120, about 75 to about 130, about 80 to about 90, about 80 to about 100, about 80 to about 110, about 80 to about 120, about 80 to about 130, about 90 to about 100, about 90 to about 110, about 90 to about 120, about 90 to about 130, about 100 to about 110, about 100 to about 120, about 100 to about 130, about 110 to about 120, about 110 to about 130, or about 120 to about 130 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, about 120, or about 130 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of at least about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, or about 120 chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise a total of at most about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, about 120, or about 130 chemically modified nucleotides. 
     In some embodiments, the chemically modified tracrRNA may comprise about 1% to about 100% chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise about 1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 1% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 95%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 95%, about 40% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 90% to about 95%, about 90% to about 100%, or about 95% to about 100% chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise at least about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% chemically modified nucleotides. In some embodiments, the chemically modified tracrRNA may comprise at most about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% chemically modified nucleotides. 
     In some embodiments, the chemically modified tracrRNA may have chemically modified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130 in the 5′-3′ direction in the tracrRNA sequence. 
     In some embodiments, the chemically modified tracrRNA may have chemically modified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 129 base pairs from the 5′ end of the tracrRNA sequence. 
     In some embodiments, the chemically modified tracrRNA may have chemically modified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, or 129 base pairs from the 3′ end of the tracrRNA sequence. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises phosphorothioate (PS) linkages at the 5′ or 3′ end of the gRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 0, 1, 2, 3, 4, or 5 PS linkages at the 5′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 0, 1, 2, 3, 4, or 5 PS linkages at the 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 0, 1, 2, 3, 4, or 5 PS linkages, or any combinations thereof at each of 5′ and 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 0, 1, 2 or 3 PS linkages, or any combinations thereof at each of 5′ and 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 1 PS linkage at each of 5′ and 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at each of 5′ and 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at each of 5′ and 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 4 PS linkages at each of 5′ and 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 5 PS linkages at each of 5′ and 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end and 0 PS linkage (i.e., no modification) at the 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end and 1 PS linkage at the 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end and 2 PS linkages at the 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end and 4 PS linkages at the 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end and 5 PS linkages at the 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end and 0 PS linkage (i.e., no modification) at the 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end and 1 PS linkage at the 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end and 3 PS linkages at the 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end and 4 PS linkages at the 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end and 5 PS linkages at the 3′ end of the gRNA or sgRNA sequence. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises 2 and no more than 2 contiguous phosphorothioate (PS) linkages at the 5′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 and no more than 2 contiguous phosphorothioate (PS) linkages at the 3′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 contiguous phosphorothioate (PS) linkages at the 5′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 contiguous phosphorothioate (PS) linkages at the 3′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises the sequence 5′-UsUsU-3′ at the 3′end or at the 5′ end, wherein U indicates a uridine and wherein s indicates a phosphorothioate (PS) linkage. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 phosphorothioate (PS) linkages at the 5′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 phosphorothioate (PS) linkages at the 3′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises the two phosphorothioate linkages at the 5′ end, wherein the two phosphorothioate (PS) linkages are two contiguous phosphorothioate (PS) linkages at the first two nucleotide positions of the 5′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises the two phosphorothioate linkages at the 5′ end, wherein the two phosphorothioate (PS) linkages are within the first 3-10 nucleotides of the 5′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises the two phosphorothioate (PS) linkages at the 3′ end, wherein the two phosphorothioate (PS) linkages are two contiguous phosphorothioate (PS) linkages at the first two nucleotide positions of the 3′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises the two phosphorothioate (PS) linkages at the 3′ end, wherein the two phosphorothioate (PS) linkages are within the first 3-10 nucleotides of the 3′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises the sequence 5′-UsUsUs-3′ at the 3′ end, wherein U indicates a uridine and s indicates a phosphorothioate (PS) linkage. In some embodiments, the chemically modified gRNA or sgRNA comprises the sequence 5′-UsUsU-3′ at the 3′end, wherein U indicates a uridine and s indicates a phosphorothioate (PS) linkage. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 1 PS linkage at the internal position of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 4 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 5 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 6 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 7 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 8 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 9 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 10 PS linkages at the internal positions of the gRNA or sgRNA sequence. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises PS linkages at the 5′ end, the 3′ end, or at the internal positions, or any combination thereof, of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 PS linkages at the 5′end, the 3′ end, or at the internal positions, or any combination thereof, of the gRNA or sgRNA sequence. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end, 3 PS linkages at the 3′ end, and 0 PS linkage (i.e., no modification) at the internal position of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end, 3 PS linkages at the 3′ end, and 1 PS linkage at the internal position of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end, 3 PS linkages at the 3′ end, and 2 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end, 3 PS linkages at the 3′ end, and 3 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end, 3 PS linkages at the 3′ end, and 4 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end, 3 PS linkages at the 3′ end, and 5 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end, 3 PS linkages at the 3′ end, and 6 PS linkages at the internal positions of the gRNA or sgRNA sequence. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end, 2 PS linkages at the 3′ end, and 0 PS linkage (i.e., no modification) at the internal position of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end, 2 PS linkages at the 3′ end, and 1 PS linkage at the internal position of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end, 2 PS linkages at the 3′ end, and 2 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end, 2 PS linkages at the 3′ end, and 3 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end, 2 PS linkages at the 3′ end, and 4 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end, 2 PS linkages at the 3′ end, and 5 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end, 2 PS linkages at the 3′ end, and 6 PS linkages at the internal positions of the gRNA or sgRNA sequence. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end, 2 PS linkages at the 3′ end, and 0 PS linkage (i.e., no modification) at the internal position of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end and 2 PS linkages at the 3′ end and 1 PS linkage at the internal position of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end and 2 PS linkages at the 3′ end and 2 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end and 2 PS linkages at the 3′ end and 3 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end and 2 PS linkages at the 3′ end and 4 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end and 2 PS linkages at the 3′ end and 5 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 PS linkages at the 5′ end and 2 PS linkages at the 3′ end and 6 PS linkages at the internal positions of the gRNA or sgRNA sequence. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end, 3 PS linkages at the 3′ end, and 0 PS linkage (i.e., no modification) at the internal position of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end and 3 PS linkages at the 3′ end and 1 PS linkage at the internal position of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end and 3 PS linkages at the 3′ end and 2 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end and 3 PS linkages at the 3′ end and 3 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end and 3 PS linkages at the 3′ end and 4 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end and 3 PS linkages at the 3′ end and 5 PS linkages at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 PS linkages at the 5′ end and 3 PS linkages at the 3′ end and 6 PS linkages at the internal positions of the gRNA or sgRNA sequence. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises additional modified or unmodified nucleotide (N) with phosphodiester linkage, wherein N is an A, C, G, U, dA (deoxyA), dC (deoxyC), dG (deoxyG), or T, and any combinations thereof. In some embodiments, the chemically modified gRNA or sgRNA comprises 1, 2, 3, 4, or 5 additional N with phosphodiester linkage, wherein N is an A, G, U, dA, dG, dC, or T, and any combinations thereof. In some embodiments, the chemically modified gRNA or sgRNA comprises 1, 2, 3, 4, or 5 additional N with phosphodiester linkage at the 5′ or 3′ end. In one embodiment, the chemically modified gRNA or sgRNA comprises 1, 2, 3, 4, or 5 additional N with phosphodiester linkage at the 5′ end. In a preferred embodiment, the chemically modified gRNA or sgRNA comprises 1, 2, 3, 4, or 5 additional N with phosphodiester linkage at the 3′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 1, 2, 3, 4, or 5 additional N with phosphodiester linkage and each N is the same modified or unmodified nucleotide. For example, the chemically modified gRNA or sgRNA may comprise 4 additional N with phosphodiester linkage and each N of the 4 additional N is A, C, G, U, dA, dC, dG, or T. For example, the chemically modified gRNA or sgRNA may comprise 4 additional N with phosphodiester linkage and each N of the 4 additional N is A. For example, the chemically modified gRNA or sgRNA may comprise 3 additional N with phosphodiester linkage and each N of the 3 additional N is A, C, G, U, dA, dC, dG, or T. For example, the chemically modified gRNA or sgRNA may comprise 3 additional N with phosphodiester linkage and each N of the 3 additional N is A. For example, the chemically modified gRNA or sgRNA may comprise 3 additional N with phosphodiester linkage and each N of the 3 additional N is C. For example, the chemically modified gRNA or sgRNA may comprise 3 additional N with phosphodiester linkage and each N of the 3 additional N is G. For example, the chemically modified gRNA or sgRNA may comprise 3 additional N with phosphodiester linkage and each N of the 3 additional N is U. For example, the chemically modified gRNA or sgRNA may comprise 3 additional N with phosphodiester linkage and each N of the 3 additional N is dA. For example, the chemically modified gRNA or sgRNA may comprise 3 additional N with phosphodiester linkage and each N of the 3 additional N is dC. For example, the chemically modified gRNA or sgRNA may comprise 3 additional N with phosphodiester linkage and each N of the 3 additional N is dG. For example, the chemically modified gRNA or sgRNA may comprise 3 additional N with phosphodiester linkage and each N of the 3 additional N is T. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises additional Uracil (U) with phosphodiester linkage at the 5′ or 3′ end. In one embodiment, the chemically modified gRNA or sgRNA comprises additional U with phosphodiester linkage at the 5′ end. In a preferred embodiment, the chemically modified gRNA or sgRNA comprises additional U with phosphodiester linkage at the 3′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 1, 2, 3, 4, or 5 additional U with phosphodiester linkage at the 5′ or 3′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 1 additional U with phosphodiester linkage at the 5′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 1 additional U with phosphodiester linkage at the 3′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 additional U with phosphodiester linkage at the 5′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 additional U with phosphodiester linkage at the 3′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 additional U with phosphodiester linkage at the 5′ end. In a preferred embodiment, the chemically modified gRNA or sgRNA comprises 3 additional U with phosphodiester linkage at the 3′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 4 additional U with phosphodiester linkage at the 5′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 4 additional U with phosphodiester linkage at the 3′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 5 additional U with phosphodiester linkage at the 5′ end. In some embodiments, the chemically modified gRNA or sgRNA comprises 5 additional U with phosphodiester linkage at the 3′ end. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises a ribonebularine (depicted as “X” in this application, e.g., in Table 1 or Table 24). In some embodiments, the chemically modified gRNA or sgRNA comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonebularines. In some embodiments, the chemically modified gRNA or sgRNA does not comprise ribonebularine. In some embodiments, the chemically modified gRNA or sgRNA comprises 1 ribonebularine. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 ribonebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 ribonebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 4 ribonebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 5 ribonebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 6 ribonebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 7 ribonebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 8 ribonebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 9 ribonebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 10 ribonebularines. In some embodiments, the nebularine replaces an adenine in an unmodified gRNA or sgRNA or sgRNA or sgRNA. In some embodiments, the nebularine is in the spacer sequence. In some embodiments, the nebularine is in a tracrRNA sequence. In some embodiments, the nebularine is in a crRNA sequence in the tracrRNA sequence. In some embodiments, the nebularine is in a stem loop structure in the tracrRNA sequence. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises a 2′-O-methylnebularine (depicted as “x” in this application, e.g., in Table 1 or Table 24). In some embodiments, the chemically modified gRNA or sgRNA comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2′-O-methylnebularines. In some embodiments, the chemically modified gRNA or sgRNA does not comprise 2′-O-methylnebularine. In some embodiments, the chemically modified gRNA or sgRNA comprises 1 2′-O-methylnebularine. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 2′-O-methylnebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 2′-O-methylnebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 4 2′-O-methylnebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 5 2′-O-methylnebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 6 2′-O-methylnebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 7 2′-O-methylnebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 8 2′-O-methylnebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 9 2′-O-methylnebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 10 2′-O-methylnebularines. In some embodiments, the 2′-O-methylnebularine replaces an adenine in an unmodified gRNA or sgRNA or sgRNA or sgRNA. In some embodiments, the 2′-O-methylnebularine is in the spacer sequence. In some embodiments, the 2′-O-methylnebularine is in a tracrRNA sequence. In some embodiments, the 2′-O-methylnebularine is in a crRNA sequence in the tracrRNA sequence. In some embodiments, the 2′-O-methylnebularine is in a stem loop structure in the tracrRNA sequence. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises a 2′-deoxynebularine (depicted as “dX” in this application, e.g., in Table 1 or Table 24). In some embodiments, the chemically modified gRNA or sgRNA comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2′-deoxynebularine. In some embodiments, the chemically modified gRNA or sgRNA does not comprise 2′-deoxynebularine. In some embodiments, the chemically modified gRNA or sgRNA comprises 1 2′-deoxynebularine. In some embodiments, the chemically modified gRNA or sgRNA comprises 2 2′-deoxynebularine. In some embodiments, the chemically modified gRNA or sgRNA comprises 3 2′-deoxynebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 4 2′-deoxynebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 5 2′-deoxynebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 6 2′-deoxynebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 7 2′-deoxynebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 8 2′-deoxynebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 9 2′-deoxynebularines. In some embodiments, the chemically modified gRNA or sgRNA comprises 10 2′-deoxynebularines. In some embodiments, the 2′-deoxynebularine replaces an adenine in an unmodified gRNA or sgRNA or sgRNA or sgRNA. In some embodiments, the 2′-deoxynebularine is in the spacer sequence. In some embodiments, the 2′-deoxynebularine is in a tracrRNA sequence. In some embodiments, the 2′-deoxynebularine is in a crRNA sequence in the tracrRNA sequence. In some embodiments, the 2′-deoxynebularine is in a stem loop structure in the tracrRNA sequence. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises a 2′-O-methylribonucleotide (2′-OMe). In some embodiments, the chemically modified gRNA or sgRNA comprises from about 0 to about 70 2′-OMe. In some embodiments, the chemically modified gRNA or sgRNA comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 2′-OMe. In some embodiments, the chemically modified gRNA or sgRNAs may comprise from about 0 to about 70 2′-OMe. In some embodiments, the chemically modified gRNA or sgRNAs may comprise from about 0 to about 10, about 0 to about 20, about 0 to about 30, about 0 to about 35, about 0 to about 40, about 0 to about 45, about 0 to about 50, about 0 to about 55, about 0 to about 60, about 0 to about 65, about 0 to about 70, about 10 to about 20, about 10 to about 30, about 10 to about 35, about 10 to about 40, about 10 to about 45, about 10 to about 50, about 10 to about 55, about 10 to about 60, about 10 to about 65, about 10 to about 70, about 20 to about 30, about 20 to about 35, about 20 to about 40, about 20 to about 45, about 20 to about 50, about 20 to about 55, about 20 to about 60, about 20 to about 65, about 20 to about 70, about 30 to about 35, about 30 to about 40, about 30 to about 45, about 30 to about 50, about 30 to about 55, about 30 to about 60, about 30 to about 65, about 30 to about 70, about 35 to about 40, about 35 to about 45, about 35 to about 50, about 35 to about 55, about 35 to about 60, about 35 to about 65, about 35 to about 70, about 40 to about 45, about 40 to about 50, about 40 to about 55, about 40 to about 60, about 40 to about 65, about 40 to about 70, about 45 to about 50, about 45 to about 55, about 45 to about 60, about 45 to about 65, about 45 to about 70, about 50 to about 55, about 50 to about 60, about 50 to about 65, about 50 to about 70, about 55 to about 60, about 55 to about 65, about 55 to about 70, about 60 to about 65, about 60 to about 70, or about 65 to about 70 2′-OMe. In some embodiments, the chemically modified gRNA or sgRNAs may comprise from about 0, about 10, about 20, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, or about 70 2′-OMe. In some embodiments, the chemically modified gRNA or sgRNAs may comprise from at least about 0, about 10, about 20, about 30, about 35, about 40, about 45, about 50, about 55, about 60, or about 65 2′-OMe. In some embodiments, the chemically modified gRNA or sgRNAs may comprise from at most about 10, about 20, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, or about 70 2′-OMe. In some embodiments, the chemically modified gRNA or sgRNA does not comprise 2′-OMe. In some embodiments, the chemically modified gRNA or sgRNA comprises 62 2′-OMe. In some embodiments, the chemically modified gRNA or sgRNA comprises 54 2′-OMe. 
     In some embodiments, the chemically modified gRNA or sgRNA comprises 2′-OMe at the 5′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2′-OMe at the 3′ end of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2′-OMe at the internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2′-OMe at the 5′ end, 3′ end, or internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA comprises 2′-OMe at the 5′ end, 3′ end, and internal positions of the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA does not comprise 2′-OMe at the positions 2, 3, 4, 23, 24, 25, 27, 31, or 42, or any combinations thereof, in the 5′-3′ direction in the gRNA or sgRNA sequence. In some embodiments, the chemically modified gRNA or sgRNA does not comprise 2′-OMe at the positions 2, 3, 4, 23, 24, 25, 27, 31, and 42 in the 5′-3′ direction in the gRNA or sgRNA sequence. 
     An exemplary chemically modified gRNA or sgRNA sequence is shown below: 
                            (SEQ ID NO: 55)           5′-csasgsGUUCCAUGGGAUGCUCUgUUUUAGagcuaGaaauag                   caaGUUaAaAuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgca                   ccgAgUCggugcusususu-3′            
wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine, uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate 2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s” denotes phosphorothioate (PS) linkage.
 
     An exemplary chemically modified spacer sequence is shown below: 
                            (SEQ ID NO: 56)           5′-csasgsGUUCCAUGGGAUGCUCU-3′            
wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine, uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate 2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s” denotes phosphorothioate (PS) linkage.
 
     An exemplary chemically modified tracrRNA sequence is shown below: 
                            (SEQ ID NO: 57)           5′-gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcG                   UUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu-3′            
wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine, uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate 2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s” denotes phosphorothioate (PS) linkage.
 
     Another exemplary chemically modified gRNA or sgRNA sequence is shown below: 
                            (SEQ ID NO: 55)           5′-csasgsGUUCCAUGGGAUGCUCUgUUUUAGagcuagaaauag                   caaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGca                   ccgagucggugcusususu-3′            
wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine, uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate 2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s” denotes phosphorothioate (PS) linkage.
 
     Another exemplary chemically modified tracrRNA sequence is shown below: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 57) 
               
               
                   
                 5′-gUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccG 
               
               
                   
               
               
                   
                 UUAucAAcuugaaaaagugGcaccgagucggugcusususu-3′ 
               
            
           
         
       
     
     Another exemplary chemically modified guide RNA sequence is shown below: 
                            (SEQ ID NO: 58)           5′-cscscsGCACCTTGGCGCAGCGGGUUUUAGAGCUAGAAAUAG                   CAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA                   CCGAGUCGGUGCUsususu-3′,            
wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine, uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate 2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s” denotes phosphorothioate (PS) linkage.
 
     Another exemplary chemically modified guide RNA sequence is shown below: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 59) 
               
               
                   
                 5′-asasgsAUACCUGAAUAACCCUCGUUUUAGAGCUAGAAAUAG 
               
               
                   
               
               
                   
                 CAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA 
               
               
                   
               
               
                   
                 CCGAGUCGGUGCUsususu-3′ 
               
               
                   
                 (GA091) 
               
            
           
         
       
     
     Another exemplary chemically modified guide RNA sequence is shown below: 
                            (SEQ ID NO: 60)           5′-asasgsAUACCUGAAUAACCCUCGUUUUAGAgcuagaaauag                   cAAGUUAAAAUAAGGCUAGUCCGUUAUCAacuugaaaaaguggca                   ccgagucggugcusususu3′           (GA098)            
wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine, uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate 2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s” denotes phosphorothioate (PS) linkage.
 
     Another exemplary chemically modified guide RNA sequence is shown below: 
                            (SEQ ID NO: 59)           5′-asasgsAUACCUGAAUAACCCUCgUUUUAGagcuaGaaauag                   caaGUUaAaAuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgca                   ccgAgUCggugcusususu-3′           (GAO99)            
wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine, uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate 2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s” denotes phosphorothioate (PS) linkage.
 
     Another exemplary chemically modified guide RNA sequence is shown below: 
                            (SEQ ID NO: 59)           5′-           asasgsAUACCUGAAUAACCCUCgUUUUAGagcuagaaaua                   gcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGc                   accgagucggugcusususu-3′           (GA100)            
wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine, uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate 2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s” denotes phosphorothioate (PS) linkage.
 
     Another exemplary chemically modified guide RNA sequence is shown below: 
                            (SEQ ID NO: 11)           5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauag                   caaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGca                   ccgagucggugcusususuuuu-3′           (GA385)            
wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine, uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate 2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s” denotes phosphorothioate (PS) linkage.
 
     Another exemplary chemically modified guide RNA sequence is shown below: 
                            (SEQ ID NO: 11)           5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauag                   caaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGca                   ccgagucggugcusususuuUu-3′           (GA386),            
wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine, uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate 2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s” denotes phosphorothioate (PS) linkage.
 
     Another exemplary chemically modified guide RNA sequence is shown below: 
                            (SEQ ID NO: 12)           5′-cscscsGCACCUUGGCGCAGCGgUUUUAGagcuaGaaauagc                   aaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcac                   cgagucggugcuususuuuu-3′,           (GA387)            
wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine, uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate 2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s” denotes phosphorothioate (PS) linkage.
 
     Additional chemically modified guide RNA sequences and target gene information are provided in Table 1 or Table 24. 
     In some embodiments, the chemical modification comprises a phosphorothioate linkage (PS). In some embodiments, the sgRNA comprises a phosphorothioate linkage (PS) at a 5′ end or at a 3′ end. In some embodiments, the sgRNA comprises two and no more than two contiguous phosphorothioate linkages (PS) at the 5′ end or at the 3′ end. In some embodiments, the sgRNA comprises three contiguous phosphorothioate linkages (PS) at the 5′ end or at the 3′ end. In some embodiments, the sgRNA comprises the sequence 5′-UsUsU-3′ at the 3′end or at the 5′ end, wherein U indicates a uridine and wherein s indicates a phosphorothioate linkage (PS). 
     In some aspects, provided herein, is an sgRNA that comprises (i) a spacer sequence and (ii) a tracrRNA sequence, wherein the spacer sequence hybridizes with a target polynucleotide sequence in a PCSK9 gene or an ANGPTL3 gene when contacted with the target polynucleotide sequence, wherein the tracrRNA sequence binds a Cas protein in a base editor system when contacted with the base editor system, and wherein the sgRNA comprises a nebularine or a deoxynebularine. In some embodiments, the nebularine or the deoxynebularine replaces an adenine in an unmodified sgRNA. In some embodiments, the nebularine or the deoxynebularine is in the spacer sequence. In some embodiments, wherein the nebularine or the deoxynebularine is in the tracrRNA sequence. In some embodiments, the nebularine or the deoxynebularine is in a tracrRNA sequence. In some embodiments, the nebularine or the deoxynebularine is in a crRNA sequence in the tracrRNA sequence. In some embodiments, the nebularine or the deoxynebularine is in a stem loop structure in the tracrRNA sequence. 
     In some aspects, provided herein, is a single guide RNA that comprises a sequence selected from Table 1 or Table 24, wherein a, u, g, and c indicate 2′-OMe modified adenine, uridine, guanine, and cytidine, wherein s indicates a phosphorothioate (PS) linkage. 
     In some embodiments, the chemically modified guide RNA comprises an unmodified tracrRNA sequence GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 61). In some embodiments, the chemically modified guide RNA comprises a tracrRNA sequence GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU GAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 61), which comprises 1 or more modification. 
     In some embodiments, the chemically modified guide RNA comprises an oligonucleotide that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the chemically modified guide RNAs listed in Table 1 or Table 24. In some embodiments, the chemically modified guide RNA comprises any one of the chemically modified guide RNAs listed in Table 1 or Table 24. In some embodiments, the chemically modified guide RNA is any one of the chemically modified guide RNAs listed in Table 1 or Table 24. In some embodiments, the chemically modified guide RNA comprises an oligonucleotide that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to any one of the chemically modified guide RNAs of SEQ ID NOs: 9-11, 55, 59, 253-452, 1618-1635, 1637-1800, 1802-2135, and 2191. In some embodiments, the chemically modified guide RNA comprises any one of the chemically modified guide RNAs of SEQ ID NOs: 9-11, 55, 59, 253-452, 1618-1635, 1637-1800, 1802-2135, and 2191. In some embodiments, the chemically modified guide RNA is any one of the chemically modified guide RNAs of SEQ ID NOs: 9-11, 55, 59, 253-452, 1618-1635, 1637-1800, 1802-2135, and 2191. 
     Base Editing at Splice Sites 
     In an aspect, provided herein are base editor systems and methods of using same for modifying target genes by generating a genetic alteration in the DNA sequence that occurs at the boundary of an exon and an intron (splice site) at a splice site of a target gene. In some embodiments, the base editor system modifies a nucleobase at a splice acceptor site of a target gene. In some embodiments, the base editor system modifies a nucleobase at a splice donor site of a target gene. Modification at a splice donor or splice acceptor site may result in alternated transcripts. In some embodiments, modification at a splice donor or splice acceptor site results in aberrant transcripts. In some embodiments, modification at a splice donor or splice acceptor site results in unstable transcripts that are subject to degradation upon transcription. In some embodiments, modification at a splice donor or splice acceptor site results in a premature stop codon in the transcript. In certain embodiments, the methods comprise knocking out or knocking down genes by targeting splice acceptor-splice donor (SA-SD) sites or premature STOP (pmSTOP) sites. For such methods, guide polynucleotides are designed to disrupt one or more slice acceptor/donor sites within the target nucleotide sequence. In some embodiments, a guide polynucleotide, e.g. a single guide RNA comprises a sequence of at least 10 contiguous nucleotides or a sequence of 17-23 contiguous nucleotides, that is complementary to a target sequence in the genome of an organism and comprises a target base pair. 
     Disruptions at SA-SD sites are particularly advantageous because one may knock out coding sequence and non-coding RNAs (ncRNAs) without stop codon read through. Efficiency of base editing can be determined on the genomic level by EditR analysis of Sanger sequencing traces or by next generation sequencing (NGS), and also on the protein level by flow cytometry. Splice acceptor-splice donor base editing gRNAs that target the splice donor regions and the splice acceptor region exhibit base conversion efficiency of at least 5% and, in some cases, at least 80% or greater (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%). In some cases, SA-SD gRNAs are significantly more efficient at C-to-T conversions than gRNAs that introduce premature stop codons disrupting. 
     Guide RNAs for targeting SA-SD sites can be designed using an R based program that identifies gRNAs targeting all ncRNAs and protein coding gene SA-SD sites. In some cases, the user supplies the reference genome, Ensembl transcript ID of the reference sequence, protospacer adjacent motif (PAM) site, and distance to subset upstream and downstream of exon-intron boundary. The program extracts sequences of 20 base pairs+the PAM length upstream and 15 base pairs downstream of an exon-intron boundary, as well as the splice site motif. In some embodiments, a guide molecule can be from 20 to 120 bases in length, or more. In certain embodiments, a guide molecule can be from 20 to 60 bases in length, or 20 to 50 bases, or 30 to 50 bases, or 39 to 46 bases. 
     In some cases, it is advantageous to use chemically modified gRNAs having increased stability when transfected into mammalian cells. For example, gRNAs can be chemically modified to comprise 2′-O-methyl phosphorthioate modifications on at least one 5′ nucleotide and at least one 3′ nucleotide of each gRNA. In some cases, the three terminal 5′ nucleotides and three terminal 3′ nucleotides are chemically modified to comprise 2′-O-methyl phosphorthioate modifications. 
     In an aspect, provided herein are base editing systems and methods for targeting diseases for base editing disruption. The target sequence can be any disease-associated polynucleotide or gene, as have been established in the art. 
     Without being bound by a particular theory, where the goal is to disrupt a gene in vivo for a therapeutic purpose, cytosine base editors may be used to directly introduce stop codons into the coding sequence of the gene (nonsense mutations) by altering specific codons for glutamine (CAG-TAG, CAA-TAA), arginine (CGA-TGA), and tryptophan (TGG-TAG/TAA/TGA, with editing of cytosines on the antisense strand). In certain embodiments, adenine base editors may be used to disrupt gene function and/or expression by modifying nucleobases at splice sites of target genes. The more favorable off-target profile of adenine base editors, particularly with respect to gRNA-independent off-target DNA base editing, recommend the use of adenine base editors over cytosine base editors for therapeutic purposes. In some embodiments, a base editor, e.g. an adenosine nucleobase editor as describe herein may be used to disrupt a splice donors at the 5′ ends of introns or splice acceptors at the 3′ ends of introns. In some embodiments, splice site disruption results in the inclusion of intronic sequences in messenger RNA (mRNA)—potentially introducing nonsense, frameshift, or in-frame indel mutations that result in premature stop codons or in insertion/deletion of amino acids that disrupt protein activity—or in the exclusion of exonic sequences, which can also introduce nonsense, frameshift, or in-frame indel mutations. 
     Canonical splice donors comprise the DNA sequence GT on the sense strand, whereas canonical splice acceptors comprise the DNA sequence AG. In some embodiments, a base editor, e.g. an adenosine nucleobase editor as described herein can be used to generate alteration of the sequence disrupts normal splicing. In some embodiments, the adenosine base editor disrupts a complementary base in the second position in the antisense strand (GT-GC). In some embodiments, the adenosine base editor disrupts the first position in the sense strand (AG-GG). 
     Examples of useful applications of mutation or disruption of an endogenous gene sequence that reduces or abolishes expression of the target gene related to the disease, with significantly reduced off-target effect, indels frequency, and/or other unintended genetic interruptions related to double stranded breaks that result from traditional CRISPR-Cas9 nuclease modification. In some embodiments, the target gene is a PCSK9 gene. In some embodiments, the target gene is an ANGPTL3 gene. 
     The chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the binding affinity between a programmable DNA binding component of a base editor fusion protein, such as a Cas protein (e.g., a Cas9 protein, a nuclease inactive Cas9 protein, a Cas9 nickase) and guide RNAs (e.g., gRNAs or sgRNAs). In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the binding affinity between a Cas protein and gRNAs or sgRNAs by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, %14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the binding affinity between a Cas protein, e.g. a Cas9 protein, a nuclease inactive Cas9 protein, a Cas9 nickase, and gRNAs or sgRNAs by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the binding affinity between a base editor fusion or Cas protein and gRNAs or sgRNAs by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the binding affinity between a Cas protein or component of a base editor fusion protein and gRNAs or sgRNAs by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold compared to unmodified gRNAs or sgRNAs. 
     The chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the binding affinity between a programmable DNA binding component of a base editor fusion protein, such as a Cas protein (e.g., a Cas9 protein, a nuclease inactive Cas9 protein, a Cas9 nickase) and the tracrRNA sequence of the guide RNAs (e.g., gRNAs or sgRNAs). In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the binding affinity between a base editor fusion or Cas protein and the tracrRNA sequence of the gRNAs or sgRNAs by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the binding affinity between a base editor fusion or Cas protein and the tracrRNA sequence of the gRNAs or sgRNAs by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the binding affinity between a base editor fusion or Cas protein and the tracrRNA sequence of the gRNAs or sgRNAs by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the binding affinity between a base editor fusion or Cas protein and the tracrRNA sequence of the gRNAs or sgRNAs by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold compared to unmodified gRNAs or sgRNAs. 
     In some embodiments, the tracrRNA sequence of the gRNAs or sgRNAs may bind a programmable DNA binding component of a base editor fusion protein, such as a Cas protein (,) with increased binding affinity compared to a tracrRNA sequence in an unmodified sgRNA. The type II Cas protein may be used as a system or base editor as described herein. In some embodiments, the tracrRNA sequence of the gRNAs or sgRNAs may bind a Type II Cas protein with at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% increased binding affinity compared to a tracrRNA sequence in an unmodified sgRNA. In some embodiments, the tracrRNA sequence of the gRNAs or sgRNAs may bind a base editor comprising a Type II Cas protein with at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold increased binding affinity compared to a tracrRNA sequence in an unmodified sgRNA. In some embodiments, the tracrRNA sequence of the gRNAs or sgRNAs may bind a base editor fusion protein comprising a Cas9 protein with increased binding affinity compared to a tracrRNA sequence in an unmodified sgRNA. In some embodiments, the tracrRNA sequence of the gRNAs or sgRNAs may bind a Cas9 protein with at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, or 300% increased binding affinity compared to a tracrRNA sequence in an unmodified sgRNA. In some embodiments, the tracrRNA sequence of the gRNAs or sgRNAs may bind a base editor fusion protein comprising a Cas9 protein with at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold increased binding affinity compared to a tracrRNA sequence in an unmodified sgRNA. 
     The chemically modified gRNAs or sgRNAs of the present disclosure can reduce off-target effect in gene modification (e.g., gene or genome editing) of the target sequence. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can reduce off-target effect in gene modification of the target sequence by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can reduce off-target effect in gene modification of the target sequence by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can reduce off-target effect in gene modification of the target sequence by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can reduce off-target effect in gene modification of the target sequence by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold compared to unmodified gRNAs or sgRNAs. 
     In some embodiments, the target polynucleotide sequence is in a genome, wherein the gRNA or sgRNA is capable of directing a component (e.g. Cas9) of a base editor fusion protein to effect a modification in the target polynucleotide sequence, and wherein the modification results in less off-target effect in the genome as compared to an unmodified gRNA or sgRNA. In some embodiments, the gRNA or sgRNA is capable of directing a Type II Cas protein to effect a modification in the target polynucleotide sequence wherein the modification results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% less off-target effect in the genome as compared to an unmodified gRNA or sgRNA. In some embodiments, the gRNA or sgRNA is capable of directing a Type II Cas protein of a base editor fusion protein to effect a modification in the target polynucleotide sequence wherein the modification results in at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less off-target effect in the genome as compared to an unmodified gRNA or sgRNA. In some embodiments, the target polynucleotide sequence is in a genome, wherein the gRNA or sgRNA is capable of directing a Cas9 protein to effect a modification in the target polynucleotide sequence, and wherein the modification results in less off-target effect in the genome as compared to an unmodified gRNA or sgRNA. In some embodiments, the gRNA or sgRNA is capable of directing a Cas9 protein to effect a modification in the target polynucleotide sequence wherein the modification results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% less off-target effect in the genome as compared to an unmodified gRNA or sgRNA. In some embodiments, the gRNA or sgRNA is capable of directing a Cas9. In some embodiments, the target polynucleotide sequence is in a genome, wherein the gRNA or sgRNA is capable of directing a base editor fusion protein to effect a modification in the target polynucleotide sequence, and wherein the modification results in less off-target effect in the genome as compared to an unmodified gRNA or sgRNA. In some embodiments, the gRNA or sgRNA is capable of directing a base editor fusion protein to effect a modification in the target polynucleotide sequence wherein the modification results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, %14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% less off-target effect in the genome as compared to an unmodified gRNA or sgRNA. In some embodiments, the gRNA or sgRNA is capable of directing a base editor fusion protein to effect a modification in the target polynucleotide sequence wherein the modification results in at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less off-target effect in the genome as compared to an unmodified gRNA or sgRNA. In some embodiments, the modification using the chemically modified gRNAs or sgRNAs results in less off-target effect in a cell as compared to an unmodified gRNA or sgRNA. In some embodiments, the modification using the chemically modified gRNAs or sgRNAs results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% less off-target effect in a cell as compared to an unmodified gRNA or sgRNA. In some embodiments, the modification using the chemically modified gRNAs or sgRNAs results in at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less off-target effect in a cell as compared to an unmodified gRNA or sgRNA. 
     The chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the gene modification (e.g., gene or genome editing) efficiency. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the gene modification (e.g., gene or genome editing) efficiency by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the gene modification (e.g., gene or genome editing) efficiency by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the gene modification (e.g., gene or genome editing) efficiency by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the gene modification (e.g., gene or genome editing) efficiency by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold compared to unmodified gRNAs or sgRNAs. 
     The chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the complex of a base editor protein or component (e.g., Cas9 protein) and the guide RNAs (e.g., gRNAs or sgRNAs), for example, the stability of base editor complex. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the Cas protein-gRNA complex by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, %, 11%, 12%, 13%, %14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the base editor fusion or Cas protein-gRNA complex by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the base editor fusion or Cas protein-gRNA complex by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the base editor fusion or Cas protein-gRNA complex by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold compared to unmodified gRNAs or sgRNAs. 
     In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the base editor complex by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% compared to unmodified sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the base editor complex by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared to unmodified sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the base editor complex by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared unmodified sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the base editor-sgRNA or Cas9-sgRNA complex by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold compared to unmodified sgRNAs. 
     The chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the guide RNAs (e.g., gRNAs or sgRNAs) in vitro, in vivo, and/or ex vivo. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the gRNAs or sgRNAs in vitro, in vivo, and/or ex vivo by at least 1%, 2%, 3%, 4%, 5%, 6%, 7% 8%, 9% 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the gRNAs or sgRNAs in vitro, in vivo, and/or ex vivo by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the gRNAs or sgRNAs in vitro, in vivo, and/or ex vivo by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can enhance or increase the stability of the gRNAs or sgRNAs in vitro, in vivo, and/or ex vivo by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold compared to unmodified gRNAs or sgRNAs. 
     In some embodiments, the chemically modified gRNAs or sgRNAs exhibit increased stability in a cell as compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs exhibit at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% higher or increased stability in a cell as compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs exhibit be at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold higher or increased stability in a cell as compared to unmodified gRNAs or sgRNAs. 
     In some embodiments, the stability of the chemically modified gRNAs or sgRNAs is measured by half-life of the gRNAs or sgRNAs in the cell. In some embodiments, the chemically modified gRNAs or sgRNAs exhibit increased half-life in the cell compared to unmodified gRNAs or sgRNAs. In some embodiments, the gRNAs or sgRNAs exhibit increased half-life in the cell by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% as compared to unmodified gRNAs or sgRNAs. In some embodiments, the gRNAs or sgRNAs exhibit increased half-life in the cell by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% as compared to unmodified gRNAs or sgRNAs. In some embodiments, the gRNAs or sgRNAs exhibit increased half-life in the cell by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold as compared to unmodified gRNAs or sgRNAs. 
     The chemically modified gRNAs or sgRNAs of the present disclosure can be more resistant to degradation as compared to an unmodified gRNA or sgRNA. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% more resistant to degradation compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can be about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% more resistant to degradation compared to unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can be about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% more resistant to degradation compared unmodified gRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs of the present disclosure can be at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold more resistant to degradation compared to unmodified gRNAs or sgRNAs. 
     Target Gene Modification 
     In some aspects, provided herein, is a method for treating or preventing a condition in a subject in need thereof, the method comprising administering to the subject the composition or lipid nanoparticle comprising chemically modified gRNAs or sgRNAs described herein, wherein the gRNAs or sgRNAs direct a base editor protein to effect a modification in a target polynucleotide sequence in a cell of the subject, thereby treating or preventing the condition. In some embodiments, the target polynucleotide is in a PCSK9 gene. In some embodiments, the target polynucleotide is in an ANGPTL3 gene. In some embodiments, the modification is at a splice site of the target polynucleotide. In some embodiments, the modification is at a splice donor site of the target polynucleotide. In some embodiments, the modification is at a splice acceptor site of the target polynucleotide. In some embodiments, the modification reduces expression of functional PCSK9 protein encoded by the PCSK9 gene in the subject. In some embodiments, the modification reduces expression of functional ANGPTL3 protein encoded by the ANGPTL3 gene in the subject. In some embodiments, the condition is atherosclerotic vascular disease. In some embodiments, the condition is an atherosclerotic vascular disease, hypertriglyceridemia, or diabetes. In some embodiments, the subject exhibits a reduced blood LDL cholesterol level and/or a reduced blood triglycerides level as compared to before the administration. 
     In some embodiments, the disclosure provides base-editing systems, compositions and methods for editing a polynucleotide encoding an Apolipoprotein C3 (APOC3) protein and variants thereof. In some embodiments, provided herein are genome/base-editing systems, compositions and methods for editing a polynucleotide encoding Proprotein convertase subtilisin/kexin type 9 (PCSK9) and variants thereof. In some embodiments, provided herein are genome/base-editing systems, compositions and methods for editing a polynucleotide encoding Angiopoietin-like 3 (ANGPTL3) and variants thereof. To edit a target gene, a target gene polynucleotide may contact the compositions disclosed herein comprising a sgRNA and a adenosine base editor protein, wherein the sgRNA comprises a spacer sequence and a tracrRNA sequence, wherein the spacer sequence hybridizes with a target polynucleotide sequence in a PCSK9 or ANGPTL3 gene and the tracrRNA sequence binds the adenosine base editor protein (e.g. a Cas9 component of the adenosine base editor). In some embodiments, the sgRNA therefore directs the base editor protein to the target polynucleotide sequence to result in a to G modification in the target gene. In some embodiments, the target gene or target polynucleotide is selected from a gene encoding PCSK9, APOC3, LPA, and ANGPTL3. In some embodiments, the target polynucleotide sequence is in a PCSK9 gene. In some embodiments, the target polynucleotide sequence is in an ANGPTL3 gene. In some embodiments, the modification reduces or abolishes expression of functional PCSK9 protein encoded by the PCSK9 gene in the cell. In some embodiments, the modification reduces or abolishes expression of functional ANGPTL3 protein encoded by the ANGPTL3 gene in the cell. In some embodiments, the introduction is performed via a lipid nanoparticle that comprises the composition. 
     For example, the sgRNA and the Adenosine base editor protein may be expressed in a cell where a target gene editing is desired (e.g., a liver cell), to thereby allowing contact of the target gene with the composition disclosed herein (e.g., sgRNA and the Adenosine base editor protein). In some embodiments, the binding of the Adenosine base editor protein to its target polynucleotide sequence in the target gene is directed by a single guide RNA disclosed herein, e.g., a single guide RNA comprising (i) a spacer sequence and (ii) a tracrRNA sequence, wherein the spacer sequence hybridizes with a target polynucleotide sequence in a target gene. Thus, by designing the guide RNA sequence, the Adenosine base editor protein can be directed to edit any target polynucleotide sequence in the target gene (e.g., target gene encoding PCSK9, APOC3 and ANGPTL3). In some embodiments, the guide RNA sequence is co-expressed with the Adenosine base editor protein in a cell where editing is desired. 
     In some embodiments, a target gene comprising more than one mutations described herein are contemplated. For example, a target gene encoding a variant protein can be produced using the methods described herein that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. To make multiple mutations in the target gene, a plurality of guide RNA sequences can be used, each guide RNA sequence targeting one target polynucleotide sequence in the target gene. The Adenosine base editor protein is capable of editing each and every target polynucleotide sequence dictated by the guide RNA sequence. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more guide RNA sequences can be used in a gene editing reaction. In some embodiments, the guide RNA sequences as used (e.g., gRNA). In some embodiments, DNA molecule encoding the guide RNA sequences can also be used. 
     In some embodiments, simultaneous modifications into more than one target genes (e.g., more than one target gene in the LDL-mediated cholesterol clearance pathway) are also contemplated herein. For example, in some embodiments, a modification may be simultaneously introduced into PCSK9 and APOC3 gene. In some embodiments, a modification may be simultaneously introduced into PCSK9 and LDL-R gene. In some embodiments, a modification may be simultaneously introduced into PCSK9 and IODL gene. In some embodiments, a modification may be simultaneously introduced into PCSK9 and LPA gene. In some embodiments, a modification may be simultaneously introduced into APOC3 and IODL gene. In some embodiments, a modification may be simultaneously introduced into LDL-R and APOC3 gene. In some embodiments, a modification may be simultaneously introduced into LDL-R and IDOL gene. In some embodiments, a modification may be simultaneously introduced into PCSK9, APOC3, LDL-R and IDOL gene. In some embodiments, a modification can be simultaneously introduced into PCSK9 and ANGPTL3 gene. To simultaneously introduce of modifications into more than one target genes, multiple guide nucleotide sequences are used. 
     To edit a gene encoding the PCSK9 or ANGPTL3 protein, the gene is contacted with the composition described herein. In some embodiments, a target polynucleotide sequence in a target gene is contacted with the single guide RNA disclosed herein and a Adenosine base editor protein or a nucleic acid sequence encoding the Adenosine base editor protein, wherein the single guide RNA directs the Adenosine base editor protein to effect a modification in the target gene (e.g., target gene encoding PCSK9, APOC3 and ANGPTL3). In some embodiments, the target polynucleotide sequence is the gene locus in the genomic DNA of a cell. In some embodiments, the cell is a cultured cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is in vitro. In some embodiments, the cell is ex vivo. 
     In some embodiments, the cell is from a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a rodent. In some embodiments, the rodent is a mouse. In some embodiments, the rodent is a rat. As would be understood be those skilled in the art, a target polynucleotide sequence may be a DNA molecule comprising a coding strand and a complementary strand, e.g., the PCSK9 or ANGPTL3 gene locus in a genome. As such, the target polynucleotide sequence may also include coding regions (e.g., exons) and non-coding regions (e.g., introns or splicing sites). In some embodiments, the target polynucleotide sequence is located in the coding region (e.g., an exon) of the target gene (e.g., the PCSK9 or ANGPTL3 gene locus). As such, the modification in the coding region may result in an amino acid change in the protein encoded by the target gene, i.e., a mutation. In some embodiments, the mutation is a loss of function mutation. In some embodiments, the loss-of-function mutation is a naturally occurring loss-of-function mutation. In some embodiments, the target polynucleotide sequence is located in a non-coding region of the target gene, e.g., in an intron or a splicing site. In some embodiments, the base editor system provided herein results in a A•T to G•C alteration at a splice site in a PCSK9 gene or a ANGPTL3 gene. In some embodiments, the A•T to G•C alteration is at a splice acceptor site of the PCSK9 gene. In some embodiments, the A•T to G•C alteration results in an aberrant PCSK9 transcript encoded by the PCSK9 gene. In some embodiments, the A•T to G•C alteration results in a non-functional PCSK9 polypeptide encoded by the PCSK9 gene when expressed in a cell. In some embodiments, the A•T to G•C alteration is at a 5′ end of a splice donor site of an intron 1 of the PCSK9 gene. In some embodiments, the A•T to G•C alteration is at a 5′ end of a splice donor site of an intron 4 of the PCSK9 gene. In some embodiments, the base editor system provided herein results in a A•T to G•C alteration at a splice site ANGPTL3 gene, or the second A•T to G•C alteration is at a splice donor site of the ANGPTL3 gene. In some embodiments, the A•T to G•C alteration or the second A•T to G•C alteration is at a splice acceptor site of the ANTPTL3 gene. In some embodiments, the A•T to G•C alteration or the second A•T to G•C alteration results in an aberrant ANGPTL3 transcript encoded by the ANGPTL3 gene. In some embodiments, the A•T to G•C alteration or the second A•T to G•C alteration results in a non-functional ANGPTL3 polypeptide encoded by the ANGPTL3 gene. In some embodiments, the A•T to G•C alteration or the second A•T to G•C alteration is at a 5′ end of a splice donor site of an intron 6 of the ANGPLT3 gene. 
     In some embodiments, a target polynucleotide sequence is located in a splicing site and the editing of such sequence causes alternative splicing of the mRNA of a target gene. In some embodiments, the alternative splicing leads to leading to loss-of-function mutants. In some embodiments, the alternative splicing leads to the introduction of a premature stop codon in a mRNA encoded by the target gene, resulting in truncated and unstable proteins. In some embodiments, mutants that are defective in folding are produced. A loss-of-function variant generated by a gene that is modified using the compositions and methods disclosed herein, may have reduced activity compared to a wild type protein encoded by an unmodified target gene. Activity refers to any known biological activity of the wild-type protein in the art. 
     In some embodiments, the activity of a loss-of-function variant may be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or more. In some embodiments, the loss-of-function variant has no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, no more than 1% or less activity compared to a wild type protein. 
     In some embodiments, cellular activity of a protein encoded by a target gene may be reduced by reducing the level of properly folded and active protein. Introducing destabilizing mutations into the wild type protein may cause misfolding or deactivation of the protein. A variant generated by modifying a target gene using the compositions and methods disclosed herein comprises one or more destabilizing mutations may have reduced activity compared to the wild type protein encoded by an unmodified target gene. For example, the activity of a variant comprising one or more destabilizing mutations may be reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more. 
     In some embodiments, the methods and composition disclosed herein reduces or abolishes expression and/or function of protein encoded by a target gene. For example, the methods and composition disclosed herein reduces expression and/or function of protein encoded by the target gene by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to a control. 
     In some embodiments, the methods and composition disclosed herein reduces or abolishes expression and/or function of the protein encoded by a target gene by at least 2-fold relative to a control. For example, the methods and composition disclosed herein reduces or abolishes expression and/or function of the protein encoded by a target gene by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to a control. 
     Some aspects of the present disclosure provide strategies of editing target gene to reduce the amount of full-length, functional protein being produced. In some embodiments, stop codons may be introduced into the coding sequence of target gene upstream of the normal stop codon (referred to as a “premature stop codon”). Premature stop codons cause premature translation termination, in turn resulting in truncated and nonfunctional proteins and induces rapid degradation of the mRNA via the non-sense mediated mRNA decay pathway. See, e.g., Baker et al., Current Opinion in Cell Biology 16 (3): 293-299, 2004; Chang et al, Annual Review of Biochemistry 76: 51-74, 2007; and Behm-Ansmant et ah, Genes &amp; Development 20 (4): 391-398, 2006, each of which is incorporated herein by reference. 
     Proprotein Convertase Subtilisin-Kexin Type 9 (PCSK9) 
     In some embodiments, the target gene for modification using the compositions and methods disclosed herein is gene encoding PCSK9. Proprotein convertase subtilisin-kexin type 9 (PCSK9), also known as neural apoptosis-regulated convertase 1 (NARC-I), is a proteinase K-like subtilase identified as the 9th member of the secretory subtilase family. “Proprotein convertase subtilisin/kexin type 9 (PCSK9)” refers to an enzyme encoded by the PCSK9 gene. PCSK9 binds to the receptor for low-density lipoprotein (LDL) particles. In the liver, the LDL receptor removes LDL particles from the blood through the endocytosis pathway. When PCSK9 binds to the LDL receptor, the receptor is channeled towards the lysosomal pathway and broken down by proteolytic enzymes, limiting the number of times that a given LDL receptor is able to uptake LDL particles from the blood. Thus, blocking PCSK9 activity may lead to more LDL receptors being recycled and present on the surface of the liver cells, and will remove more LDL cholesterol from the blood. 
     Therefore, blocking PCSK9 can lower blood cholesterol levels. PCSK9 orthologs are found across many species. PCSK9 is inactive when first synthesized, a pre-pro enzyme, because a section of the peptide chain blocks its activity; proprotein convertases remove that section to activate the enzyme. Pro-PCSK9 is a secreted, globular, serine protease capable of proteolytic auto-processing of its N-terminal pro-domain into a potent endogenous inhibitor of PCSK9, which blocks its catalytic site. PCSK9&#39;s role in cholesterol homeostasis has been exploited medically. Drugs that block PCSK9 can lower the blood level of low-density lipoprotein cholesterol (LDL-C). The first two PCSK9 inhibitors, alirocumab and evolocumab, were approved by the U.S. Food and Drug Administration in 2015 for lowering cholesterol where statins and other drugs were insufficient. 
     The human gene for PCSK9 localizes to human chromosome Ip33-p34.3. PCSK9 is expressed in cells capable of proliferation and differentiation including, for example, hepatocytes, kidney mesenchymal cells, intestinal ileum, and colon epithelia as well as embryonic brain telencephalon neurons. See, e.g., Seidah et al., 2003 PNAS 100:928-933, which is incorporated herein by reference. 
     Original synthesis of PCSK9 is in the form of an inactive enzyme precursor, or zymogen, of 72-kDa, which undergoes autocatalytic, intramolecular processing in the endoplasmic reticulum (ER) to activate its functionality. This internal processing event has been reported to occur at the SSVFAQ jSIP motif, and has been reported as a requirement of exit from the ER. “j” indicates cleavage site. See, Benjannet et al., 2004 J. Biol. Chem. 279:48865-48875, and Seidah et al, 2003 PNAS 100:928-933, each of which are incorporated herein by reference. The cleaved protein is then secreted. The cleaved peptide remains associated with the activated and secreted enzyme. 
     The gene sequence for human PCSK9 is ˜22-kb long with 12 exons encoding a 692 amino acid protein. The protein sequence of human PCSK9 can be found, for example, at Deposit No. NP_777596.2, which sequence is incorporated herein in its entirety. Human, mouse and rat PCSK9 nucleic acid sequences have been deposited; see, e.g., GenBank Accession Nos.: AX127530 (also AX207686), AX207688, and AX207690, respectively, each of which sequence is incorporated herein in its entirety. The gene sequence of  Macaca fascicularis  can be found publically, for example, NCBI Gene ID: 102142788, which sequence is incorporated herein in their entirety.  Macaca fascicularis  proprotein convertase subtilisin/kexin type 9 isoform X2 sequence can be found publically, for example, at NCBI Reference Sequence: XP_005543317.1, which sequence is incorporated herein in its entirety. 
     The translated protein contains a signal peptide in the NH2-terminus, and in cells and tissues an about 74 kDa zymogen (precursor) form of the full-length protein is found in the endoplasmic reticulum. During initial processing in the cell, the about 14 kDa prodomain peptide is autocatalytically cleaved to yield a mature about 60 kDa protein containing the catalytic domain and a C-terminal domain often referred to as the cysteine-histidine rich domain (CHRD). This about 60 kDa form of PCSK9 is secreted from liver cells. The secreted form of PCSK9 appears to be the physiologically active species, although an intracellular functional role of the about 60 kDa form has not been ruled out. 
     Numerous PCSK9 variants are disclosed and/or claimed in several patent publications including, but not limited to the following: PCT Publication Nos. WO2001031007, WO2001057081, WO2002014358, WO2001098468, WO2002102993, WO2002102994, WO2002046383, WO2002090526, WO2001077137, and WO2001034768; US Publication Nos. US 2004/0009553 and US 2003/0119038, and European Publication Nos. EP 1 440 981, EP 1 067 182, and EP 1 471 152, each of which are incorporated herein by reference. 
     Several mutant forms of PCSK9 are well characterized, including S 127R, N157K, F216L, R218S, and D374Y, with S 127R, F216L, and D374Y being linked to autosomal dominant hypercholesterolemia (ADH). Benjannet et al. (J. Biol. Chem., 279(47):48865-48875 (2004)) demonstrated that the S 127R and D374Y mutations result in a significant decrease in the level of pro-PCSK9 processed in the ER to form the active secreted zymogen. As a consequence, it is believed that wild-type PCSK9 increases the turnover rate of the LDL receptor causing inhibition of LDL clearance (Maxwell et al, PNAS, 102(6):2069-2074 (2005); Benjannet et al, and Lalanne et al), while PCSK9 autosomal dominant mutations result in increased levels of LDLR, increased clearance of circulating LDL, and a corresponding decrease in plasma cholesterol levels. See, Rashid et al, PNAS, 102(15):5374-5379 (2005); Abifadel et al, 2003 Nature Genetics 34: 154-156; Timms et al, 2004 Hum. Genet. 114:349-353; and Leren, 2004 Clin. Genet. 65:419-422, each of which are incorporated herein by reference. 
     A later-published study on the S127R mutation of Abifadel et al, reported that patients carrying such a mutation exhibited higher total cholesterol and apoB 100 in the plasma attributed to (1) an overproduction of apoB 100-containing lipoproteins, such as low density lipoprotein (LDL), very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL), and (2) an associated reduction in clearance or conversion of said lipoproteins. Together, the studies referenced above evidence the fact that PCSK9 plays a role in the regulation of LDL production. Expression or upregulation of PCSK9 is associated with increased plasma levels of LDL cholesterol, and inhibition or the lack of expression of PCSK9 is associated with low LDL cholesterol plasma levels. Significantly, lower levels of LDL cholesterol associated with sequence variations in PCSK9 have conferred protection against coronary heart disease; Cohen et al, 2006 N. Engl. J. Med. 354: 1264-1272. 
     Lalanne et al. demonstrated that LDL catabolism was impaired and apolipoprotein B-containing lipoprotein synthesis was enhanced in two patients harboring S 127R mutations in PCSK9 (J. Lipid Research, 46: 1312-1319 (2005)). Sun et al. also provided evidence that mutant forms of PCSK9 are also the cause of unusually severe dominant hypercholesterolaemia as a consequence of its effect of increasing apolipoprotein B secretion (Sun et al, Hum. Mol. Genet, 14(9): 1161-1169 (2005)). These results were consistent with earlier results which demonstrated adenovirus-mediated overexpression of PCSK9 in mice results in severe hypercholesteromia due to drastic decreases in the amount of LDL receptor Dubuc et al., Thromb. Vase. Biol., 24: 1454-1459 (2004), in addition to results demonstrating mutant forms of PCSK9 also reduce the level of LDL receptor (Park et al., J. Biol. Chem., 279:50630-50638 (2004). The overexpression of PCSK9 in cell lines, including liver-derived cells, and in livers of mice in vivo, results in a pronounced reduction in LDLR protein levels and LDLR functional activity without changes in LDLR mRNA level (Maxwell et al., Proc. Nat. Amer. Set, 101:7100-7105 (2004); Benjannet S. et al, J. Bio. Chem. 279: 48865-48875 (2004)). 
     Various therapeutic approaches to the inhibition of PSCK9 have been proposed, including: inhibition of PSCK9 synthesis by gene silencing agents, e.g., RNAi; inhibition of PCSK9 binding to LDLR by monoclonal antibodies, small peptides or adnectins; and inhibition of PCSK9 autocatalytic processing by small molecule inhibitors. These strategies have been described in Hedrick et al., Curr Opin Investig Drugs 2009; 10:938-46; Hooper et al, Expert Opin Biol Ther, 2013; 13:429-35; Rhainds et al, Clin Lipid, 2012; 7:621-40; Seidah et al, Expert Opin Ther Targets 2009; 13:19-28; and Seidah et al, Nat Rev Drug Discov 2012; 11:367-83, each of which are incorporated herein by reference. 
     In some embodiments, the loss of function mutation induced in PCSK9 e.g., G106R, L253F, A443T, R93C, etc. In some embodiments, the loss-of-function mutation is engineered (i.e., not naturally occurring), e.g., G24D, S47F, R46H, S 153N, H193Y, etc. 
     PCSK9 variants that can be useful in the present disclosure are loss-of-function variants that may boost LDL receptor-mediated clearance of LDL cholesterol, alone or in combination with other genes involved in the pathway, e.g., APOC3, LDL-R, or Idol. In some embodiments, the PCSK9 loss-of-function variants produced using the methods of the present disclosure express efficiently in a cell. In some embodiments, the PCKS9 loss-of-function variants produced using the methods of the present disclosure is activated and exported to engage the clathrin-coated pits from unmodified cells in a paracrine mechanism, thus competing with the wild-type PCSK9 protein. In some embodiments, the PCSK9 loss-of-function variant comprises mutations in residues in the LDL-R bonding region that make direct contact with the LDL-R protein. In some embodiments, the residues in the LDL-R bonding region that make direct contact with the LDL-R protein are selected from the group consisting of R194, R237, F379, S372, D374, D375, D378, R46, R237, and A443. 
     As described herein, a loss-of-function PCSK9 variant, may have reduced activity compared to a wild type PCSK9 protein. PCSK9 activity refers to any known biological activity of the PCSK9 protein in the art. For example, in some embodiments, PCSK9 activity refers to its protease activity. In some embodiments, PCSK9 activity refers to its ability to be secreted through the cellular secretory pathway. In some embodiments, PCSK9 activity refers to its ability to act as a protein-binding adaptor in clathrin-coated vesicles. In some embodiments, PCSK9 activity refers to its ability to interact with LDL receptor. In some embodiments, PCSK9 activity refers to its ability to prevent LDL receptor recycling. These examples are not meant to be limiting. 
     In some embodiments, the activity of a loss-of-function PCSK9 variant may be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or more. In some embodiments, the loss-of-function PCSK9 variant has no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, no more than 1% or less activity compared to a wild type PCSK9 protein. Non-limiting, exemplary assays for determining PCSK9 activity have been described in the art, e.g., in US Patent Application Publication US20120082680, which are incorporated herein by reference. 
     In some embodiments, cellular PCSK9 activity may be reduced by reducing the level of properly folded and active PCSK9 protein. Introducing destabilizing mutations into the wild type PCSK9 protein may cause misfolding or deactivation of the protein. A PCSK9 variant comprising one or more destabilizing mutations described herein may have reduced activity compared to the wild type PCSK9 protein. For example, the activity of a PCSK9 variant comprising one or more destabilizing mutations described herein may be reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more. 
     In some embodiments, the methods and composition disclosed herein reduce or abolish expression of protein encoded by a target gene and/or function thereof. For example, the methods and composition disclosed herein reduces expression and/or function of PCSK9 protein encoded by the PCSK9 gene by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to a control. For example, the methods and composition disclosed herein reduces expression and/or function of APOC3 protein encoded by the APOC3 gene by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to a control. For example, the methods and composition disclosed herein reduces expression and/or function of ANGPTL3 protein encoded by the ANGPTL3 gene by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to a control. 
     In some embodiments, the gene modification methods and compositions disclosed herein reduces expression of functional PCSK9 protein encoded by the PCSK9 gene in the cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100%. In some embodiments, the modification reduces expression of functional PCSK9 protein encoded by the PCSK9 gene in the cell by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold. In some embodiments, the modification abolishes expression of functional PCSK9 protein encoded by the PCSK9 gene in the cell. 
     Some aspects of the present disclosure provide strategies of reducing cellular PCSK9 activity via preventing PCSK9 mRNA maturation and production. In some embodiments, such strategies involve alterations of splicing sites in the PCSK9 gene. Altered splicing site may lead to altered splicing and maturation of the PCSK9 mRNA. For example, in some embodiments, an altered splicing site may lead to the skipping of an exon, in turn leading to a truncated protein product or an altered reading frame. In some embodiments, an altered splicing site may lead to translation of an intron sequence and premature translation termination when an in frame stop codon is encountered by the translating ribosome in the intron. In some embodiments, a start codon is edited and protein translation initiates at the next ATG codon, which may not be in the correct coding frame. 
     The splicing sites typically comprises an intron donor site, a Lariat branch point, and an intron acceptor site. The mechanism of splicing is familiar to those skilled in the art. As a non-limiting example, the intron donor site has a consensus sequence of GGGTRAGT, and the C bases paired with the G bases in the intron donor site consensus sequence may be targeted by the methods and compositions described herein, thereby altering the intron donor site. The Lariat branch point also has consensus sequences, e.g., YTRAC, wherein Y is a pyrimidine and R is a purine. The C base in the Lariat branch point consensus sequence may be targeted by the nucleobase editors described herein, leading to the skipping of the following exon. The intron acceptor site has a consensus sequence of YNCAGG, wherein Y is a pyrimidine and N is any nucleotide. The C base of the consensus sequence of the intron acceptor site, and the C base paired with the G bases in the consensus sequence of the intron acceptor site may be targeted by the nucleobase editors described herein, thereby altering the intron acceptor site, in turn leading the skipping of an exon. As described herein, gene sequence for human PCSK9 is −22-kb long and contains 12 exons and 11 introns. Each of the exon-intron junction may be altered to disrupt the processing and maturation of the PCSK9 mRNA. 
     In some embodiments, a splice site disruption generated by a base editor system disclosed herein can result in the inclusion of intronic sequences in messenger RNA (mRNA) encoded by the PCSK9 gene. In some embodiments, the splice site disruption generates a nonsense, frameshift, or an in-frame indel mutation that result in premature stop codons or in insertion/deletion of amino acids that disrupt protein activity. In some embodiments, the splice site disruption generates exclusion of exonic sequences. In some embodiments, the splice site disruption generates exclusion of exonic sequences that results in nonsense, frameshift, or in-frame indel mutations in the PCSK9 transcript. Canonical splice donors comprise the DNA sequence GT on the sense strand, whereas canonical splice acceptors comprise the DNA sequence AG. Alteration of the sequence disrupts normal splicing. Splice donors can be disrupted by adenine base editing of the complementary base in the second position in the antisense strand (GT to GC), and splice acceptors can be disrupted by adenine base editing of the first position in the sense strand (AG to GG). 
     Further, the present disclosure also contemplates the use of destabilizing mutations to counteract the effect of gain-of-function PCSK9 variant. Gain-of-function PCSK9 variants (e.g., the gain-of-function variants have been described in the art and are found to be associated with hypercholesterolemia (e.g., in Peterson et al., J Lipid Res. 2008 June; 49(6): 1152-1156; Benjannet et al., J Biol Chem. 2012 Sep. 28;287(40):33745-55; Abifadel et al, Atherosclerosis. 2012 August; 223(2):394-400; and Cameron et al, Hum. Mol. Genet. (1 May 2006) 15(9): 1551-1558, each of which is incorporated herein by reference). Introducing destabilizing mutations into these gain-of-function PCSK9 variants may cause misfolding and deactivation of these gain-of-function variants, thereby counteracting the hyper-activity caused by the gain-of-function mutation. Further, gain-of-function mutations in several other key factors in the LDL-R mediated cholesterol clearance pathway, e.g., LDL-R, APOB, or APOC, have also been described in the art. Thus, making destabilizing mutations in these factors to counteract the deleterious effect of the gain-of-function mutation using the compositions and methods described herein, is also within the scope of the present disclosure. As such, the present disclosure further provides mutations that cause misfolding of PCSK9 protein or structurally destabilization of PCSK9 protein. 
     The polypeptide and coding nucleic acid sequences of PCSK9 and of other members of the family of human origin and those of a number of animals are publicly available, e.g., from the NCBI website or ENSEMBL website. Examples include, but are not limited to the following sequences, each of which sequences are incorporated herein in their entireties; 
     
       
         
           
               
            
               
                 Wild Type PCSK9 Gene (NG 009061.1), 
               
               
                   Homo   sapiens  proprotein convertase 
               
               
                 subtilisin/kexin type 9 (PCSK9), 
               
               
                 RefSeqGene (LRG 275) on chromosome 1 
               
               
                 (SEQ ID NO: 5) 
               
               
                 GTCCGATGGGGCTCTGGTGGCGTGATCTGCGCGCCCCAGGCGTCAAGCAC 
               
               
                   
               
               
                 CCACACCCTAGAAGGTTTCCGCAGCGACGTCGAGGCGCTCATGGTTGCAG 
               
               
                   
               
               
                 GCGGGCGCCGCCGTTCAGTTCAGGGTCTGAGCCTGGAGGAGTGAGCCAGG 
               
               
                   
               
               
                 CAGTGAGACTGGCTCGGGCGGGCCGGGACGCGTCGTTGCAGCAGCGGCTC 
               
               
                   
               
               
                 CCAGCTCCCAGCCAGGATTCCGCGCGCCCCTTCACGCGCCCTGCTCCTGA 
               
               
                   
               
               
                 ACTTCAGCTCCTGCACAGTCCTCCCCACCGCAAGGCTCAAGGCGCCGCCG 
               
               
                   
               
               
                 GCGTGGACCGCGCACGGCCTCTAGGTCTCCTCGCCAGGACAGCAACCTCT 
               
               
                   
               
               
                 CCCCTGGCCCTCATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCT 
               
               
                   
               
               
                 GCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTCCCGCGGGCGCCCGTG 
               
               
                   
               
               
                 CGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGT 
               
               
                   
               
               
                 TCCGAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCAC 
               
               
                   
               
               
                 CTTCCACCGCTGCGCCAAGGTGCGGGTGTAGGGATGGGAGGCCGGGGCGA 
               
               
                   
               
               
                 ACCCGCAGCCGGGACGGTGCGGTGCTGTTTCCTCTCGGGCCTCAGTTTCC 
               
               
                   
               
               
                 CCCCATGTAAGAGAGGAAGTGGAGTGCAGGTCGCCGAGGGCTCTTCGCTT 
               
               
                   
               
               
                 GGCACGATCTTGGGGACTGCAGGCAAGGCGGCGGGGGAGGACGGGTAGTG 
               
               
                   
               
               
                 GGGAGCACGGTGGAGAGCGGGGACGGCCGGCTCTTTGGGGACTTGCTGGG 
               
               
                   
               
               
                 GCGTGCGGCTGCGCTATTCAGTGGGAAGGTTCGCGGGGTTGGGAGACCCG 
               
               
                   
               
               
                 GAGGCCGAGGAAGGGCGAGCAGAGCACTGCCAGGATATCCTGCCCAGATT 
               
               
                   
               
               
                 TCCCAGTTTCTGCCTCGCCGCGGCACAGGTGGGTGAAGGAGTGAATGCCT 
               
               
                   
               
               
                 GGAACGTACTGGGAACTGCACCAGGCACAGAGAAAGCGGGCTTGCCATTA 
               
               
                   
               
               
                 TAGTGGGTTCCGATTTGGTTTGGAAAACATGGGCAGCGGAGGGTGGAGGG 
               
               
                   
               
               
                 CCTGGAGAGAAGGCCCTACCCGAGACAGGGGCGGGGTGGGAAGGACGGCA 
               
               
                   
               
               
                 GATGCTGGGAGCACGAGGCAATTTCTTTATGACACAGAACTCATGCTCTA 
               
               
                   
               
               
                 GTATTCCATCTGTTTCAGCCGAAGAAAAGAACCAGCTGAAGGGGCAGGGG 
               
               
                   
               
               
                 AGAAGGGGCGGAGGTATTCTCGAGGCCCATTGGCGTCCTTTAGGACTCAG 
               
               
                   
               
               
                 GCAGGGAAGGGCCCTTGGTGCTCTGGAGCCGGAGGTGGTGCGCCTGGTAC 
               
               
                   
               
               
                 TGGGACCCCGGAGCTGAGCCCGGCGCCTCAGCCCACCTGGCTGTCTGCCG 
               
               
                   
               
               
                 ACCGTGTGCGGGGCGAGTTTGCTCAACAACTCTGCCAGCTTCTGGCCCTC 
               
               
                   
               
               
                 AGGCTGTGGGAAGCTTCTTCCCGGGGCGAGACCACTAGCTTTTTCTAAGT 
               
               
                   
               
               
                 ATTACCAGCCCAGGACTTGGCTGAGGTTCTGTGTCCCCCAGCTTGGAGTC 
               
               
                   
               
               
                 AGATGTGGGGTTGAATCTTGGCTTCCTCTCACTAGCTGTGGTGCTTGACA 
               
               
                   
               
               
                 AGTCACTTATCCTTGAGCCTCCATTGCCTAATCTTTAAAAGGGAGGTGAC 
               
               
                   
               
               
                 AATCGTCCCTACGGCTCAGTGGCAGCAGATGGGGAGATGAAGGGAAAGTT 
               
               
                   
               
               
                 CTGTTGACCATGAGTGAACTTACAATGCAAGCCCCGGGGGGATCACTTGC 
               
               
                   
               
               
                 AGTTTTGTCCCTGTCTGCAGTGTGACCTGTTGGTGACATTGTCTTTGCTC 
               
               
                   
               
               
                 CAAACCACAGCTCCTGGGGCAGAGGGGAAAATTCTGCCACTCACAGCTGC 
               
               
                   
               
               
                 CTGCCCACGCTTCTGTCTGAGTGTGCTGGGTGGCAGGATGGCAAGTCCTT 
               
               
                   
               
               
                 ACTCAGCTCAGTATAGCCCTCTTCCTTGTTCCCTGAGCCTTTGACTTTCT 
               
               
                   
               
               
                 CGAGGGATGTTGTGGGGTTGTGGCCAGGATAAGAAAGGGCATTTCAAGTT 
               
               
                   
               
               
                 ACCACTGCTCCAAAACAACTGTTCTGGAAATAGTGAGTACCCCATCCTGA 
               
               
                   
               
               
                 GAGGTGAGTAAGCAGAGGCTGTATGACCACCTGAACCAAGCCCTTGAGGA 
               
               
                   
               
               
                 TGTTTCTTCTCTGGTGGAAGTTTGGAACAGGAGCCTCCTCAAGTTCATTT 
               
               
                   
               
               
                 ATTCATTCATTCAATGGTTATTTTGTGGGAATCGAATTTAGAATGAAAAT 
               
               
                   
               
               
                 ATTTTTTGGCAAGCAGAAAATAATTTTTAGACCAATCCTTTTCTTTTAGT 
               
               
                   
               
               
                 CATGAGAAACTGAGGCCCAGAGAGAGGAGGTCACCCCAGGTGCATTAGAA 
               
               
                   
               
               
                 CTGGGTTTCCAGAACTGACACTCCACTGCACAGAGTACTCTCCCAATTCA 
               
               
                   
               
               
                 TTCAATTTTTATTTAGCGGAAGGCATTTTCAGATGGGTCTTTGAAGCATT 
               
               
                   
               
               
                 AGTAGGAGTTCAGCGATGATGGTGTCATGAGAATTTTATTCTAGGATTAG 
               
               
                   
               
               
                 GAGGTACCATGAACAAAGATACAGAGCTGGGAAAACCAGAGGTGGAAGAT 
               
               
                   
               
               
                 AAGGAGCACATGTCCACAGTTCTTTTTCTTTTTTTTTTGAGATGGAGTTT 
               
               
                   
               
               
                 CGCTCTTGTTGCCCAGGCTGGAGTGCAATGGTGCAGTCTCAGCTCACTGC 
               
               
                   
               
               
                 AACATCTGTCTCCCGGGTTCAAGTGGTTCTCCTGCCTCAGCCTCCCAAGA 
               
               
                   
               
               
                 AGCTGGGATTACAGGTACCTGCCACCACGCCCGGCTAATTTTTGTATTTT 
               
               
                   
               
               
                 TAGTAGAGAAGGGGTTTCACCACGTTGGCCAGGCTAGTCGCAAACTCCTG 
               
               
                   
               
               
                 ACCTCCTCAGTGGATCCGAGGAGGTGATCCTCCCGCCTCAGCCTCCCAAA 
               
               
                   
               
               
                 GTGCTCGAATTACAGGTGTGAGCCACCACGCCTGGCCTCCACAGTTCTTT 
               
               
                   
               
               
                 ATCCACCGTCTGAAATGTAAAATGTTACGAAAACCAAAAGTTTTTTTTGT 
               
               
                   
               
               
                 GATTTATTTGATGGTAGCACCTGACGTGAACTGACATGAGATTATTTTTA 
               
               
                   
               
               
                 ATTTAGTTGTGTGAATATGCATATTCATATATTTTGCTGCATAGATTACA 
               
               
                   
               
               
                 GTATGCAGCTCCAGATTCTTCCAAGCAGACTCTGATTGCCCATTACTGCC 
               
               
                   
               
               
                 TTTCTAAAATCCAAACAAGTTCTGAGGTTCAAAACCGTTTTGGCCCTAAG 
               
               
                   
               
               
                 GCTTTGGGTAAAGGGGGTGGACTCTGTTCTACTCTGACTGGAGTCCAAGA 
               
               
                   
               
               
                 TGCATATATACAGAGATATGGGTGATGGGGCTGCAAGGTAGGTTGAGGTA 
               
               
                   
               
               
                 GGGGCCAAGGAGGAGCATGGAGTTTGGACTTGATTCATGAGGCTGTGGGG 
               
               
                   
               
               
                 AGCCAGTGAAGGTTCTTAAGCAGGTATGTCTGCCTGAGAGCAGTTGGAGC 
               
               
                   
               
               
                 AGACAAGAGCTAAAAACCAAACAAATCACCATAGATAGTGGCTGCTATAA 
               
               
                   
               
               
                 TTTGTTTGTCCCCTCCAAATCTCATGTGGAAATTTGGTCCTCAGTGTTGG 
               
               
                   
               
               
                 AAGTGGGGCCTAATGGGAGGTGTTTGGGTCATGGGGGAGGAACCCCTGTG 
               
               
                   
               
               
                 AAAGGCTTGGTGCCGTCCTTGTGATAATGAGTAAGTTCTCCCGCTATGAT 
               
               
                   
               
               
                 TTCCCTTGAAGGCTGATTATTAAAAAGAGCTTGGCACCTCCCTCTCTTCT 
               
               
                   
               
               
                 CTCTTGCTTCTTCTCTTGCCATGTGATTGATCTCTGCACATGTAGGCTCC 
               
               
                   
               
               
                 CCTTCACCTTCTGCCATCAGTGAAAGCAGCTTAAGGCCCTCACCAGAAGC 
               
               
                   
               
               
                 AGATGCTGGTGCCATGCTTCCTGGAGAGCTTGCAGAATCATGAGCTGAAT 
               
               
                   
               
               
                 AAATCCCTTTTCCTTGTAAATTACTCACCTTCAGGTATTCCTTTATATAG 
               
               
                   
               
               
                 CAACACAAAAGGACTAAGACAGTGGCCTTGACTTTTCTCTCTCTTTAAGA 
               
               
                   
               
               
                 AGTGTTGCCTTTGCTCACTTAGTCATCCCTTCTGCCTGCATTTGTAGAGC 
               
               
                   
               
               
                 ATCTGGATGGGAGATTTATATAACCGTCACTCTTGACTTTCCCAGCAGGC 
               
               
                   
               
               
                 CTATGTCATAGGTACTGTGGTCTCTACAATACAGCAGAGGTATCTGAGGC 
               
               
                   
               
               
                 TCCGAGAGGTTGAGTGACTTGCTCATGGCTGCACAACCAGTAAATATTGG 
               
               
                   
               
               
                 AGCTGGAATTCAGGTCCACGGTTTCCTGGCTCCAAAGCCCATGATTTTTT 
               
               
                   
               
               
                 CCCTCAATTTATTCTGACTGGGGCATGGGGGAGGGGGTGGCCTTTGGGCA 
               
               
                   
               
               
                 GGGCCACCAGGAGCGACCAGGCCCGTAGAGAGCTGGGTGCAGGTACAGAG 
               
               
                   
               
               
                 GAAAACCTGTTGTCGAGTGTGGCCCGTAGTTCCCATTTTTGCCTGAATGG 
               
               
                   
               
               
                 CACATTTGAAAGTGTTATATAACCATGTGAATAATAATAGTTGGCCTATA 
               
               
                   
               
               
                 TGAGTTCTTTAATTTGCTTTTTGGTCCGCATTTGGTAACTTCTTTATCAT 
               
               
                   
               
               
                 CTACTATACTCTGTTGTGTCTCTTTTGTTGTAATTTGTAAGTAGGGGTGA 
               
               
                   
               
               
                 GATAAAGTACACCTAGGGTTTGCTGGGTTTCTTCCATGTCATCATGTTCC 
               
               
                   
               
               
                 TCCTTGCATGGGGCCAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTG 
               
               
                   
               
               
                 GTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCG 
               
               
                   
               
               
                 CCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATG 
               
               
                   
               
               
                 TCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTG 
               
               
                   
               
               
                 CTGGAGCTGGTGAGCCACCCTTTTTGGGAATGGCACTTCCTGATAGGGCT 
               
               
                   
               
               
                 GGGCCACTGCATATACACTGGGGACTGTGCTTAGTAGGCCCATTGCTGAA 
               
               
                   
               
               
                 AATCAGAAGGGGACAGCAAGTATGTATTGAGCACTTATCGGGTACCAAGC 
               
               
                   
               
               
                 ACAGTAACTACTGGCTTTCTGTATAGAATTCCCTTTAAGCCTGGCCATGC 
               
               
                   
               
               
                 CCCAGTGGTACGTCTATCTTCATTTGAAAGACGAGGAGACTGAAGTTCAG 
               
               
                   
               
               
                 AGGGGACCACACAGACAGCTAGGGGTAGAGCCTGGATCAAACCCATTGGT 
               
               
                   
               
               
                 CTGCCTGCCAGCCATTCTTGTGCCAATGCATCTGCTGCCTACGGAAACCT 
               
               
                   
               
               
                 GTAGGGACAAGGCCCTGGGATGTTCAGTGGAGCCTGAGTCATTTTATAAA 
               
               
                   
               
               
                 AAAGCATGACTCTAGGGTCCAAAATTCCTTTGAAGCTGTTGCTATCCAGA 
               
               
                   
               
               
                 GTGAAGTCCCTTCTTTAGGACAGGGTGGCCCTCCTCCCTCCTGGATGTCA 
               
               
                   
               
               
                 CATCTTCGGTGGAGGGGCAGAAAGGGGACTGGGTATTCTCCTCACCCTGG 
               
               
                   
               
               
                 CCCTAGTGCTTCAAATCTTAAAAAAACGTTTTTATTTGTGCTTCTGCACC 
               
               
                   
               
               
                 ACCTTCTAGCCCACCTCGTTTCCTGGCCTCTAACTTGATGAGAGCGTGTG 
               
               
                   
               
               
                 TCATTTTCACACTGATTCTCCACATGGCAGGCGGTGCTTCTTAGCCTCCT 
               
               
                   
               
               
                 GCAGACAGTGAGGCCCCACGGTCTTGTCCAAGGTCACACAGCGTGTAATG 
               
               
                   
               
               
                 GGCAGGGTCAGAGTCTGGAGTCTGGACCTGGGTCTCCTAGCTGCACTGCA 
               
               
                   
               
               
                 CTGCTGCCCCATGGGTTAATCAGCTCAGCATACCGTGGCTGAACAGCTAC 
               
               
                   
               
               
                 CTCATACCAAGGCCTGTGGCGCCATGACAGGGATTGACAGGGTCCCTGCC 
               
               
                   
               
               
                 TTGGAAACCCGTAGTCTAAGTAGAGGAGACTGACAAGTCAATGCCTTCCA 
               
               
                   
               
               
                 TCAGTCTGCTCAACACACGTTTACCAAGTGCCTACTGTGTGCTGCAGAGG 
               
               
                   
               
               
                 CGAAGATGACACAGCTCAGGCCTTTCCCTTGAGCTTACAGTTCAGGAGGA 
               
               
                   
               
               
                 GAGACTGACCAGTGACTGCCAGTACAGTTGACTATGGGACAATGTGCTCA 
               
               
                   
               
               
                 GCCTTGGGGAGAGACGAAGAAGGTACCCGTATAGCACCAGATGACAGGCA 
               
               
                   
               
               
                 CGAGCCCCACAGGCCAGGGCAGCTGCTCAGAGGAGAGTAGGCCAAGCAGA 
               
               
                   
               
               
                 AGGCAAACAGAAGGCTGCAGGCATTTGCCATCGAGAGCTGGACTTCAAAC 
               
               
                   
               
               
                 TGGGCATCATACCAGCCTGGGTTCGAGTCCTGCCCAGCCCCTTATTGGCT 
               
               
                   
               
               
                 GTCTAACCCTGAGCAAATCCCTTCACCTCTCTGAGCCTCATTCCTCTATC 
               
               
                   
               
               
                 TGTAAACCAGTTATAATAATTGGAACATTCATTTAAGGACTAAATGAGGT 
               
               
                   
               
               
                 CGTGAAGCATTCAGCAGATGCTAGGTACGGAAACTCGCTGAAGTGGGGGC 
               
               
                   
               
               
                 AGGTTAAGAAGCCTCTGGGGATACGAAGGCATCCAGGGACTAGTTGTGGC 
               
               
                   
               
               
                 AGGAGGCTGTTACCACTTAGGTCTGAAGGGTAAGGAGAGGGAATAGCTTT 
               
               
                   
               
               
                 CCCTCTGCCCAGTTGGAGCCGGTGGCATGGAGGAGAGGCTGCCTGTGGGG 
               
               
                   
               
               
                 AATCACCCGAGGGTTCACCGCTGCCATGCGCAGGGAGTCAGGAGGTAGGG 
               
               
                   
               
               
                 AGGGAGTGGGGCAGATGCACACCATTTTTTTTTTTTTTTGAGACTCTGTT 
               
               
                   
               
               
                 GCCCAGACTGGAGTGCAGTGGTGCCATATCTGCACCTCTGCCTCCCGGGT 
               
               
                   
               
               
                 TCAAGCTCACTGCAACCTCTGCCTCCCGGGTTCAAGCGATTCTCCTGCCT 
               
               
                   
               
               
                 CAGCCTCCCGAGTAGCTGGGACTACAGGTGTGTGCCACCATGCCTGGCTA 
               
               
                   
               
               
                 ATTTTTGTATTTTTAATAGAGATGGGGTTTCACCATGTTGGCCAGGCTGG 
               
               
                   
               
               
                 TCTCGAACTCTCGACCTCAGGTGATCCCCCACCTCGGCCTCCCAAAGTGC 
               
               
                   
               
               
                 TGGGATTACAGGCGTGAGTCACCGCTCCCAGCTGCTGATGCACTCTTGTC 
               
               
                   
               
               
                 CTTCTAACTCCTGCTAGTGCCTCCCATTGGCTGAGCCCAACTGGAAGCTT 
               
               
                   
               
               
                 TGCAAGGGAGCTGGTGCTGCAGTTTGCACTGAGCAGGCTGGAGAAGGCTG 
               
               
                   
               
               
                 GAGAATAGACTAGGGGACAAACCGAATTGCCAGTGCTGTTATGTCATGAT 
               
               
                   
               
               
                 TTAGGCATGGAGTCCAGGGCCTGAGCTTCACTCCATGTCCATCCTGCCCA 
               
               
                   
               
               
                 GAGCCTTGGCACAGCCTGGCTCCCAGACAAGATGTCAAGTTCAGAATCCT 
               
               
                   
               
               
                 TCCTAAAAGGAATCCTCTATGCCAGACCGTGTTGCAGGGATATGGGAGTG 
               
               
                   
               
               
                 CTGGGCTCCCAGCCTGATCAAGGAGCGAGAAAACTCAGGCTCCTAGTCTG 
               
               
                   
               
               
                 TCCTCCGGGGCACTAGCAGGGACAAGGTGGGAGGCTGCTGGGCTGGGATG 
               
               
                   
               
               
                 TGGGGACAGGTTTGATCAGGTAAGGCCAGGCTGTGGCTGTGTTTGCTGCT 
               
               
                   
               
               
                 GTCCAAATGGCTTAAGCAGAGTCCCCCGGCCTCTCTGGCTTCTGCAGGCC 
               
               
                   
               
               
                 TTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGC 
               
               
                   
               
               
                 CCAGAGCATCCCGTGGAACCTGGAGCGGATTACCCCTCCACGGTACCGGG 
               
               
                   
               
               
                 CGGATGAATACCAGCCCCCCGGTAAGACCCCCATCTGTGCCCTGCCCCAC 
               
               
                   
               
               
                 CCCATCTGAGCTGAATCCATTTGCTCTGCCCTGGCCTGGCCTCCCTGCTG 
               
               
                   
               
               
                 GTGGTTTCCACTTCTCGGGGGGCTTTGGGACTCAGCACCTCCACTGACCC 
               
               
                   
               
               
                 CTTTTTTTCTGTCCCATCCCCATCCCCTGCAGCCCCCACTGCCTGCCTTC 
               
               
                   
               
               
                 CTGTTGCCCCACAAATGCAAAAGTCTTGCCTTAAATGATCCTCTTTTCCT 
               
               
                   
               
               
                 TCTTTTCTCTTGTTTTCCTTTTCTCACCATTTGGAATGGCCCAGCAGGCT 
               
               
                   
               
               
                 GCACTTACCTTGGAAGGAGGGTTCATCTGATGGTGACTCTACCTAGGGCC 
               
               
                   
               
               
                 CCCAGGCCTCTATAACTCCCAGTGCCCTGCAGACTGGACCAGATCCTTTA 
               
               
                   
               
               
                 ATGGGATAGACACAACCCTGTCTGGGATGCCTCTGCCTACCTTCCTGTTT 
               
               
                   
               
               
                 TGCTGCTCCACCTGCCTCCAGCTCCGTTTGGCTTCCTGGGGCTCCCTGCC 
               
               
                   
               
               
                 TGGGCCACTTTGTGTCTTCCCTCTAGGCCTTTCTTTCCACTGTTCCCTCT 
               
               
                   
               
               
                 GCCTGGTGTGGCCTGGCTATGGAAGGGAGGGAGGAGGAGCGGCCATGGAA 
               
               
                   
               
               
                 AACGGTCTGCATTCTAGCAGGGACTTGCAGGTGGCAATTCAGTCGGGGAA 
               
               
                   
               
               
                 GACTCTAGATGCACCTGGCCTGAGGAGAGAATGAAGGGTTCTAGTTGGAC 
               
               
                   
               
               
                 TGTGTTAAGTTTGAGGTGCCCATGGTGTGAGGTCTGGAGCTCAGCGCAGA 
               
               
                   
               
               
                 GATGATGCAATGTGGTGGGTCCATGCAACATGGTGCCAGGACGCAGAGCT 
               
               
                   
               
               
                 TGGGGTGAACTCAGCTTTCACCCCTTACCGGTTCTCGTGGGATCTTGGGA 
               
               
                   
               
               
                 AGCCACTTTCTTCTATGAGCTTTGTCGTTCTTGTCTGTAAAATGGGCACA 
               
               
                   
               
               
                 TAACCCTGTCCCTGTCCTTCTCACAGGTTGCTGTGAGACTCCAATGAGTT 
               
               
                   
               
               
                 GAAGGATGTGCAGATGCTTTTGGAAGTGAAAAGTTGGGGGGCTACTGTGT 
               
               
                   
               
               
                 GACTTTGCATACACCCAAACTGTGTGACCTTGCATATGTCTGAGTTGCTG 
               
               
                   
               
               
                 CCATTGCAACAGATCAGAGCTGGTGGGCTGGGTGTGGAGAAAGGGTTTGT 
               
               
                   
               
               
                 GTGGGGGACATCCTCTGGCAAGGGTGGCAGCAGCAGAAGTGAGGGGCCTG 
               
               
                   
               
               
                 GTCGGTCATGTGTGCTGACCCGGCCTGGGCAGCCTGTGGCCAGGGAGAGG 
               
               
                   
               
               
                 ACAGCTCCTCTGTAGGAAGAGCCTGTTCCTTTCCAACCAGGTGAGACCTC 
               
               
                   
               
               
                 TTCAGTGGAGCCCTGGAGCCCCCTGTACTCCACATCAGTGCCTCAGGGAC 
               
               
                   
               
               
                 CTCCCGGAGCAGGCTAATATCAGAGACCAAGAGGGACACTGGCAGAGGAT 
               
               
                   
               
               
                 CACAGAGACCCCAGTCCAGGCAGGGACTGAGAAGATCTTGCCCCCTAAGT 
               
               
                   
               
               
                 TAGTTTCCTAGCACTGCTGTGACAAATTACCACCCCCTCGGTTGGAACAA 
               
               
                   
               
               
                 GTTGATTCTCTGCAGTCCTGGAGGCCAGAAGCCTGAATCAGTGTCGGCAG 
               
               
                   
               
               
                 GACCACTTTCTCCCGGGGGGCTCCAGGGAGAAGCTTCTCTTGCCTCTTCC 
               
               
                   
               
               
                 GTGTCCCAACAGCGGCAGCACACCAATCCCAGCCTCTGTCTTCACACAGC 
               
               
                   
               
               
                 CTTCTCTGTGTCTCTCTCCTCTTCATTGTCTCATAAGGACACTTGTCATT 
               
               
                   
               
               
                 GGATTTAGGGCCCACTGGATCCTCCAGGATGATCTCATGTGGGGAACCTT 
               
               
                   
               
               
                 AACCACATCTGCAAGGACCCTTTTTCCAAATAAGGTCACAGCCACAGGTT 
               
               
                   
               
               
                 GTGGGGGTTAGGATGTGAGTGTATCTCTTTGGCAGCCACTGTTCCCTCCT 
               
               
                   
               
               
                 CTCCCTTGGGCCAGAAGCAGACGTGGGGCCCTTTCTTCCCCATAGGATGC 
               
               
                   
               
               
                 CCATGGATTGCCCCCCTTCCCGCTTCCCCCGAGTGTCTGTGGGAGGTGGC 
               
               
                   
               
               
                 AGGAATGGCAGGCAGGGGTGTGGAACCCCTTCTGGAGTCATATCAAGGGC 
               
               
                   
               
               
                 TTGGCTGGAGGAAGTCCTCCTGGAGCTGTTGGGCTGGCATGGGGCAGGCT 
               
               
                   
               
               
                 GGCTGGGCCCAGCAGCAGCTTCTTCATTCATGGGGAGGCCACAAGCATGG 
               
               
                   
               
               
                 GCCCTAGAGCTGGCTGCCGCCCTCAAACCCAGACCCTGCACTCTTAACTG 
               
               
                   
               
               
                 TGTGACCTTGCATACGTCACTCACCCTCTCTGATCTTCAGGTTCCTCTGC 
               
               
                   
               
               
                 AAAAGGGAGGTAATGATAACCCTCACTCTGGGGGGCTGTTTGGAGGGTTA 
               
               
                   
               
               
                 AATCAGTTATTGCTGTAGCATGCATTTCTCTGTCAGGTATTGAGTGAGGT 
               
               
                   
               
               
                 GCTGTGATTTTAGCCCTGCATTTTTCTTTTCTTACCATTCAATAATAACG 
               
               
                   
               
               
                 TTTTGAGCACCCACTGTGCGCCAGGCACCATATTAGGTGCTGGGGATACA 
               
               
                   
               
               
                 AATGTGAATGAAATGAATGTGGTCTCTTCCCCCAACAGTGTATCCAGAAG 
               
               
                   
               
               
                 ATTAATCCATTCCTTAAACAAATGCTACTTGACACAGATTAGTTCTGGAT 
               
               
                   
               
               
                 AGGCTGAGAGCTCTGAAGGAGTGCAGGCAGCTGCGAGCCTGTGTATCCAG 
               
               
                   
               
               
                 CAGAAGGATCAGGAAAGGATTCCTGGAGGAAGCGCTGTTCTAGCCAAGAC 
               
               
                   
               
               
                 CTACGGGGGCATTATTAACCAGGCAAAGGGGACGGTGTCCAAGCAGTGGA 
               
               
                   
               
               
                 ATGAACGTGGATTGAAGCTGTGAGGCAGGAGGGAGTGTGGCCTGTGCAGA 
               
               
                   
               
               
                 AGGGACCGAGGCTGGTGAGACCAGGAGGGCCTGGGTGGCCTCCAGGTCAG 
               
               
                   
               
               
                 ATGTGAAAGGAAGAACTTGGCCACAGTCTGAGCTTCTCAGGCGTATGGCA 
               
               
                   
               
               
                 GGGCTGCCTGGTGAGAGGGAATGAGCTCCCTGCTCTGGAGGTATGCAAGC 
               
               
                   
               
               
                 AGGACTGGGCTCTCACCTGCCAGAGGCCACAGAGCTTTCCAGAGGCTGGA 
               
               
                   
               
               
                 AGAGGCCACTCCAAGGCCTCTTTGCCCCTGAGAGTGGTGGCTCTTCTTGA 
               
               
                   
               
               
                 GGCCACCTTGCCACGCTGTCACAGGGAACTAGCAGCCCCTGCCTCACCCG 
               
               
                   
               
               
                 GGGGTTTGGAAGATAGAGGGAGGCCTAGGAAGGGCCCTGTGTCTCATCCG 
               
               
                   
               
               
                 AGCTGGGCCCCTTTCCAGCCTCTCACTGGAAGGAAGCCCAAGGATGTTCC 
               
               
                   
               
               
                 TGTGGGGGCTTTTACCAGGCCCACCTGCCCTCTGCTGGCCATGCTTGCAG 
               
               
                   
               
               
                 CCTCCTGACCCTGTCCCAGCAGGACAGTGGGCTGGTGTGAGCGGGCAGGA 
               
               
                   
               
               
                 ACCGCCTGCACTTAGAAGGTGTGGGGCTGCCTCCCCGAGCTTCCATCTGC 
               
               
                   
               
               
                 CGCTGGGGCCACACCCCAGGCCCAGGGATGGGACCCCACAGTGGTCACAT 
               
               
                   
               
               
                 CATCTTGCAGCAGAACCCAGGTACAGCTCCTGGAGCAGATGGTGGTCCCA 
               
               
                   
               
               
                 AGCACGGGTGGGACCAGAAAGGACTCTCACCTGGGCTAACTCAGCTGCAG 
               
               
                   
               
               
                 CCTCAGTTCCCTCCTCACACACGACGAGGAACATGGACTGGAAGCCTGCC 
               
               
                   
               
               
                 CAGCAGGCCTTCTGCTCGATGTGCGTTGTGTGGCTTACGTCCAGGGAGGG 
               
               
                   
               
               
                 AAGCAGCCTCTGTGCTGTCTTCTAGATAAGCCTGTATTCCCCGGGCTGTC 
               
               
                   
               
               
                 TGCCAATGTATCCAGTTGTCCCGTCAGCCTGGAAGCTCTGAGGGAAAACC 
               
               
                   
               
               
                 TTGGGCTGCTTCCTGAGCACCTGTATCCCCTGCAGCCAGCCCGGGGCCTC 
               
               
                   
               
               
                 TGCTAGGAGCAGACTGAGCATGGCTTATGGGCCTGGCACCATCTGGCCTC 
               
               
                   
               
               
                 TGCCCACCTTGCTGGCCTTGTCTTGTGTCTGCCCCTTCGACATTCCATAG 
               
               
                   
               
               
                 CCCAGCTCAATATCTAGTGGTTCCTCTAGGGTGGCGAGCACTGTTTGGTC 
               
               
                   
               
               
                 TCCAGATGTCTTCAGGTCGGAGCTCACAGCGCTCTCAGCCACCCCTTCCC 
               
               
                   
               
               
                 AGTGTAGCACCGGGCACATGGTAGATGCCTATTGATGAGTGAAAGCTCCT 
               
               
                   
               
               
                 AACACACTCAGAGAGCAAGGACTCCGCCTCATCCCACAGCCTGGGAGGAG 
               
               
                   
               
               
                 AGGCAGACTGCCAAGGACCTGCTCAGCATGCTACAGAAGAAACCAAAGTG 
               
               
                   
               
               
                 CCCACGGGACTGATCAGTGGAGCTTCCTGCCGAGACTGGAGGCCTTAGGG 
               
               
                   
               
               
                 CAGGGTAGACAGTGTGTGTGCAGGCTGGGGACTCACAGTTCGGACTGTGC 
               
               
                   
               
               
                 CCAGACCTACTAGCATAGTGGGTGGGTGGGAGGATGCGGGACTGGGGGCC 
               
               
                   
               
               
                 GACCTTGCCTGAAATTCATGTGGGATCTCAGAGCAGCCACTGAATTGCTC 
               
               
                   
               
               
                 TGTAGGGGGCTAAATAGTGGCCCCCACAGATACACACACCCAGACAGAGC 
               
               
                   
               
               
                 CTGTGAGCCAGACCTTATTTGGAGAAAAGGTCTTTGTAGATGTAATTAAG 
               
               
                   
               
               
                 CATCTCAAGATGGCATCATCTGGATTATGCGGTGGGCTGTAAGTCCTGTG 
               
               
                   
               
               
                 ATGTGTCTTTATGAGAGAAAGGCAGAGGGAGATTTGACACACACAGGAGG 
               
               
                   
               
               
                 GGCCACGTGGAGACAGAGGTGGAGATTGGAGAAATGTGGCCACAAGCCAG 
               
               
                   
               
               
                 GGAACACCAGCAGCCACCAGAAGCCGGAAGACGTGAGGCAGGGTTCTTCC 
               
               
                   
               
               
                 CAGAGCCTTCGCTGCTGAGTCTGGGAATTTGTGACCGAAGCCATAAGAAG 
               
               
                   
               
               
                 TGGGTACACGCCCTGAGCCTCCCACACTTGCTCACCTGTCCTGAGATGAG 
               
               
                   
               
               
                 AATCTCTACTCTGCAGCATATTTGGAGGATCACTGCGGGGGCCACAGAGG 
               
               
                   
               
               
                 TGCTGTTCAGATGGCACTTCAGAAGACTCAGGAGACCCTGGGGCAGGAGC 
               
               
                   
               
               
                 AGTTTGACTGACAGCCCAGAGGGCTGCCCTCTGATTCCACCTGAGGCCCT 
               
               
                   
               
               
                 GCTTTTCCTGGCTGCAGGGGTTCCAGGGCCAGGCCATTTCCGCTGGCGCA 
               
               
                   
               
               
                 GGACTCTGCTAGCAGCAACCTGCCTGAAGTCTTCCTTTGGCCTGGCTGAG 
               
               
                   
               
               
                 AGTTTCTGAGACCTGCGCTGGAGCGGAGGTGCTTCCTTCCTTGCTTCCTT 
               
               
                   
               
               
                 TCTTCCTCTCTCCCTTCTCCATCCAGCAGGCTGGACCTGCCTGGCATCTG 
               
               
                   
               
               
                 TGAGCTCTCCCTACTTTCTCCTATACCCTAACCTTTGTCCTGCATGGGCG 
               
               
                   
               
               
                 ACTCCCCCAGTGAGTCTCTTGCAGCTTTTACCCCAGTGCCTGCTTCTTGG 
               
               
                   
               
               
                 AGAATCCAAACTGATCCAGTTAGGGATGATAAAGTGTAGGGTAGGCGCTC 
               
               
                   
               
               
                 GGTGACTGTTTTCTCTGAGGTTGTGACTCGTGTGAGGCAGAAGCAGTCCC 
               
               
                   
               
               
                 CGTGAGCCCTCCTGGTATCTTGTGGAGTGGAGAACGCTTGGACCTGGAGC 
               
               
                   
               
               
                 CAGGAGGCCCAGACATACATCCTGTCCGAGCTGCAGCTTCCTGTCTCTAA 
               
               
                   
               
               
                 AATGAGCCGGCCAGCGCAGGTGGCCAGACATCACTGTTATTCTCCTTTGA 
               
               
                   
               
               
                 GTCTTTAAATCTTGTTGTCTTTCTTGCAGACTCGGTGAGCTGTGAAAGGC 
               
               
                   
               
               
                 TATAATAGGGGCTTTATTTTACACTTTGATACTATTTTTTGAACATTCAT 
               
               
                   
               
               
                 ATTATTGTTAGATATTGATATTCATATGAAGGAGCAGGATGACTTGGGTC 
               
               
                   
               
               
                 CTTCTTGGCAGTAGCATTGCCAGCTGATGGCCTTGGACAGTTACCTGCCC 
               
               
                   
               
               
                 TCTCTAGGCCTCCCTTTCCTTGTCTATGAAATACATTATAGAATAGGATG 
               
               
                   
               
               
                 TAGTGTGTGAGGATTTTTTGGAGGTTAAACGAGTGAATATATTTAAGGCG 
               
               
                   
               
               
                 CTTTCACCAGTGCCTGGGATGTGCTCTGTAGTTTCTGTGTGTTAACTATA 
               
               
                   
               
               
                 AGGTTGACTTTATGCTCATTCCCTCCTCTCCCACAAATGTCGCCTTGGAA 
               
               
                   
               
               
                 AGACGGAGGCAGCCTGGTGGAGGTGTATCTCCTAGACACCAGCATACAGA 
               
               
                   
               
               
                 GTGACCACCGGGAAATCGAGGGCAGGGTCATGGTCACCGACTTCGAGAAT 
               
               
                   
               
               
                 GTGCCCGAGGAGGACGGGACCCGCTTCCACAGACAGGTAAGCACGGCCGT 
               
               
                   
               
               
                 CTGATGGGAGGGCTGCCTCTGCCCATATCCCCATCCTGGAGGTGGGTGGG 
               
               
                   
               
               
                 GACTGCCACCCCAGAGCGTTGCAGCTGTACTCCTGGGTTGCACCCCCCCC 
               
               
                   
               
               
                 AGCTGTCACTGTCCCCTCCCTGCCATCAGTTGTGGGAAGGGCGTTCATCC 
               
               
                   
               
               
                 ATCCAGCCACCTGCTGATTTGTTATAGGGTGGAGGGGGGGTCTTTCTCAT 
               
               
                   
               
               
                 GTGGTCCTTGTGTTCGTCGAGCAGGCCAGCAAGTGTGACAGTCATGGCAC 
               
               
                   
               
               
                 CCACCTGGCAGGGGTGGTCAGCGGCCGGGATGCCGGCGTGGCCAAGGGTG 
               
               
                   
               
               
                 CCAGCATGCGCAGCCTGCGCGTGCTCAACTGCCAAGGGAAGGGCACGGTT 
               
               
                   
               
               
                 AGCGGCACCCTCATAGGTAAGTGATGGCCCCAGACGCTGGTCTCTCTCCA 
               
               
                   
               
               
                 TCTGGACCTGGCCTGGGAGGTGGCTTGGGCTGGGCCCAGGGAGAGCTAAT 
               
               
                   
               
               
                 GTCTCCTAACCAAGAATGCTGTGGCAGCCTCTGCCGCAGAGCCAGAGAAC 
               
               
                   
               
               
                 CAGAGTGCCAAGGCTGGCAGGGTTCCCAGTGGCCACGAGTGCAGATGAAG 
               
               
                   
               
               
                 AAACCCAGGCCCCAAGAGGGTCATGCAGGTAGCCCAGGGAGTTCAGCCTT 
               
               
                   
               
               
                 GACCCTGGGTCAATGACCTTTCCACAGTTCCACACTGCTCCCCTTTTAAA 
               
               
                   
               
               
                 ATCCGGTGATGTCTTTATGTCTTTTGTTATGTTATCTTCAATGTGGAGGG 
               
               
                   
               
               
                 ACTCGAGGTGATCTAAGCAAACTTTTTCTATCTTCTGCTTGCATACCTCT 
               
               
                   
               
               
                 GAGACCAGGGGACTCACTCACTTGCATGACTGGGCCCTGCAGGTCACACT 
               
               
                   
               
               
                 GGCCAGGCAGATGTGGTGGAGGAACTGGCAGAGGACTTTTTCTAGACTGT 
               
               
                   
               
               
                 GACTACATTTAGTCCACCCAGCGGCCCCCCTATGAAGTCCAGTTGAGAAC 
               
               
                   
               
               
                 TAGGACTCTGGGGGCCGGTGGACAGAGAAGAGGGAGGGTTCTCTCCCTTA 
               
               
                   
               
               
                 CTGACTTCCTTCTGTGGCCAGACATTGAGCAAGGCCTCTGTACAGCATGT 
               
               
                   
               
               
                 CCTGGGGCTGGCCTTGCCGTAGCTGCTAAATAGTTGACGAAACCAGTCCA 
               
               
                   
               
               
                 GAGAGGGGAGGTGACTGCCAGGGTCGCACAGCTCAAGCTGGGGAACTCGC 
               
               
                   
               
               
                 TGGGAAAACTGTCAGCTCTGGGCAGCAGCTTGACTTCCACTGTAAGCCCC 
               
               
                   
               
               
                 AGCCCCCAGGGTCAAACACTGGCTCTGGTGCTGGCAGAGGCAGCCCACTA 
               
               
                   
               
               
                 GCCTGTTTCAAAGGCTGAGAAGGCCCAGGAGTCTGCCCTGTGCTCCACCA 
               
               
                   
               
               
                 GTTCTGCCCTGAGACTTTCCTACAGAGTACAGGTTTTGATGTTCAGTTTT 
               
               
                   
               
               
                 AAAGGCAAGAATCAATAACCTTCTGCCCCATCAGGTGACCCCTTGTGCCT 
               
               
                   
               
               
                 GTCCCACCCCTTTATTGACTGACCTCGGCTCAGTCAGGTCAGTTCCTGAA 
               
               
                   
               
               
                 GGTCAGTGTGTGGAGGGGAGGCTGTTCTTTCCCAGAAAGGCCTTCCCCAG 
               
               
                   
               
               
                 GCCTGGTGCTCTGGCCTCTGGAGGACTTCCTGGAGAAGTCCCTTCTTTGG 
               
               
                   
               
               
                 GGTCCCAGTCAGTGTATGGGAAGCCCTTATTGCATGACCTGGCACGGGGC 
               
               
                   
               
               
                 AGGGGCTCAACAGTCACTATTGCCTTCCTTGCCACTGCCATTTCCTCCTC 
               
               
                   
               
               
                 TGTAAGCAGGTGATTGTGTGTCCAGTCTGAGCACAGAGATAAGCACACAG 
               
               
                   
               
               
                 CAGGTGCTTAATAACTAGCAGCTGTAGGCTGGGCGCGGTGGCTCATGCCT 
               
               
                   
               
               
                 GTAATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCACCTGAGGTCAG 
               
               
                   
               
               
                 GAGTTCGAGACCAGCCTGTTCAACATGGTGAAACCCCGTCTCTACTAAAA 
               
               
                   
               
               
                 ATACAAAAATTAGCCAGGCATGGTGGTGGGTGTCTGTATCCCAGCTACTT 
               
               
                   
               
               
                 GGGAGGCTAAGGCAGGAGAATCGCTTGAACCCAGGAGGTGGAGGTTGCAG 
               
               
                   
               
               
                 TGAGCTGAGATCGTGCCACTGCAATCCAGCCTGAGTGATAGAGCGAGATT 
               
               
                   
               
               
                 CCATCTCAAAAATAAATAAGTAAATAACTAGCAGCTGTAAATGTGGCTGT 
               
               
                   
               
               
                 TGTTCTTCACCTCCACACTCAGTGCCACTCCACTCCCTCCCTCCGTGGTG 
               
               
                   
               
               
                 TGAGGGGCCTCACTAGCTGTCTCCTAGGAGGAGCATGGCTGTGAGATTCC 
               
               
                   
               
               
                 AGCTCCATCCTTGGCCACGGCTCCTGGAGACATCTTAGAGGCCAGGATCC 
               
               
                   
               
               
                 AGAAGGCTCCCACACCTCATTTGACAGGGGAGAAGCTGTCAGTTCCAGGT 
               
               
                   
               
               
                 CCCCTTGCACATCAGGGCCAGAGCTGCGTTAGGCCTCCAGTCTCCAGGCC 
               
               
                   
               
               
                 ACTGGGCCAGAGCTCACAGGCTGGCAGAGGGTTAGAACTGTTACTGGTGG 
               
               
                   
               
               
                 CTGGGTGCAGTGGCTCACGCCTGTAATCTTAGCACTTTGGGAGGGCAAGG 
               
               
                   
               
               
                 CGGGAGGATCATGAGGTCAGGACATCGAGACCATCCTTGCTAACACGGTG 
               
               
                   
               
               
                 AAGCCCCGTCTCTACTAAAACTACAAAAAATTAGCCGGGCGTGGTGGCAG 
               
               
                   
               
               
                 GCGCCTGTAGTCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATGGCGTGA 
               
               
                   
               
               
                 ACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATTGCGCCACTGCACTCCA 
               
               
                   
               
               
                 GCCTGGGCAATAGAGCGAGACTCCGTCTGGAAAGAAAAAAAAAAAAAAGA 
               
               
                   
               
               
                 GCTGTTACTGTTGACAGTAGCATGAGGTAGACCATGGCCTGCACCAAAAT 
               
               
                   
               
               
                 GGGGGAGTGGAGTGCCACTGAGGCCAGAAGGAACCACACCCTCAAGGGTG 
               
               
                   
               
               
                 GGGAGTTATGGTATGGGGGGTCCTAGGCATGGAGTCTTTTAATTCTTTAG 
               
               
                   
               
               
                 ACAATCCTGGGAGCAACTGTCCCTGTTTCACAGAGGGCGGGGCCACACAG 
               
               
                   
               
               
                 CTGGTGAGTGGGCAGCCAAGACTCTGTTCAAGTTTGTGTGGGTCCAACAC 
               
               
                   
               
               
                 TTGCGGCCACGGTGGAGGGGCATCTGAGCCAGGCCTCAGAGAGTGGCGGG 
               
               
                   
               
               
                 GGAAGTTGGGTGGGGAAGTGTGCCCTTCTCATTCCTCTGAGGCTCATCCT 
               
               
                   
               
               
                 CTTGGTGCCTCTCTTTCATGGAAAGGGATAATAAGGTTATTGTGAGGATC 
               
               
                   
               
               
                 CCCTGAGTTCGTATATTCAGACGCTTAGACAGAGCCAGGCACAGAGAAGG 
               
               
                   
               
               
                 GCCCGGGGTTGGCTAGTTTGATTGCTGGTGTAATTGCTAATATCTTCCAG 
               
               
                   
               
               
                 TTTGTATTGGTCAAGGTTCTGCAGAGAAGCAGAACCAGTAGGATGTATAT 
               
               
                   
               
               
                 ATTAAGAGTTTCAAGCTCATGTGACCGTGCGGGCTGGCAAGTCTGAAATC 
               
               
                   
               
               
                 CGCAGGGCAGGCCAGGCAGGCTGGCAATTCCTGCAGAATTTGATGTTGCA 
               
               
                   
               
               
                 ATACTGAGTCCTAAGGCAGTCCTGGGGCAGAATTCCTTCTTCCCTGGGAG 
               
               
                   
               
               
                 GCCTCAGTCTGTTCTCTTAAGGCCTTCAACTGATTAAATGAGGCCTGCCC 
               
               
                   
               
               
                 AAGTTATAGAGAGTAACCTGCCTTACTCCGTCTTCTGATTTAAATGTTAG 
               
               
                   
               
               
                 TCACATCTAAAAAATATTTTCGCAGCAGCATTTCCACTGGCTTTTGACCA 
               
               
                   
               
               
                 AACATCAGGCCACAAAGTTGATCCCCAAAATTAACCATCACTCTGTGCCT 
               
               
                   
               
               
                 GTAAGGGAGGGGCTGGGAAAGGGGAGCAGGTCTCCCCAAGGGGTGACCTT 
               
               
                   
               
               
                 GGCTTTGTTCCTCCCAGGCCTGGAGTTTATTCGGAAAAGCCAGCTGGTCC 
               
               
                   
               
               
                 AGCCTGTGGGGCCACTGGTGGTGCTGCTGCCCCTGGCGGGTGGGTACAGC 
               
               
                   
               
               
                 CGCGTCCTCAACGCCGCCTGCCAGCGCCTGGCGAGGGCTGGGGTCGTGCT 
               
               
                   
               
               
                 GGTCACCGCTGCCGGCAACTTCCGGGACGATGCCTGCCTCTACTCCCCAG 
               
               
                   
               
               
                 CCTCAGCTCCCGAGGTAGGTGCTGGGGCTGCTGCCCCAAGGCGCGGGTAG 
               
               
                   
               
               
                 GGGGCGGAGGGCGGAGGGCGGAGGGAGGGCGGGCGGGCAGGCGGGCTTCT 
               
               
                   
               
               
                 TGTGGCACGTGGGCTTCTTGTGGCACGTTCCTGGAGGCCGAACCCTTCTG 
               
               
                   
               
               
                 GCTTTGGAAGGAGTCGTCAGAGACCCCCGCCATGCGGGAGGCTGGGGAGG 
               
               
                   
               
               
                 AAGGGGCTCGAAACCTCCATCATCGCAGAGTCTGAATAGCAGTGGCCCCG 
               
               
                   
               
               
                 CCATGCGCCCACGTAGCGGCGCCTACGTAGCCACGCCCCCACACCCCGTC 
               
               
                   
               
               
                 CTGGCCACTCTCCCTCCTGAAGGTCTTCTGGTACCCGCCCCCTCCCCATC 
               
               
                   
               
               
                 TCCATCCCCAGGCCCTGCGTCCTCTGCCCAATACTCTTTGGGCCTCCCTG 
               
               
                   
               
               
                 TTGTCCAGCTCTCTCCGCGGCTCCATGACTGACAACTTGAGCAAGGCTAA 
               
               
                   
               
               
                 TGTGAATGGGAGCGGTTGAGGGCTCAGACCTCTCACCCGAGGAACATCCA 
               
               
                   
               
               
                 CAGAGTGTGCCGCATGCCCGGTGCAGTGTGGCTGCGGGGACACAGACACG 
               
               
                   
               
               
                 GAGCCTCGGCCCTGAGGAGCTGGGGGGCAGTGACCGTCCCTCCTCTGACC 
               
               
                   
               
               
                 CACCACTCCTCCAGTGTCAGGACACTGCGGGTATCTAGGGGAAGGAATCT 
               
               
                   
               
               
                 TGTTCCACTTCAAGTCTGGAACTTCAAGTCTGTGTGTGTGCGTGCGCGCG 
               
               
                   
               
               
                 CGCGCGTTGGGGGTGGGGGTTGCAGAGCAGATGCGTACCTGACAGCGGTA 
               
               
                   
               
               
                 ACCTAGGTCCCCCCTGGCCTATCAAGGCTTCCCTGGCGGCCGAATTTAAA 
               
               
                   
               
               
                 GGCATCAAGCAAACAAAGCCCAACACATCTCTGCCTTGTCCTCTCAGTTT 
               
               
                   
               
               
                 CCCCCCGTGGCACTTAGAACCACTTGATACACCGAATAGTTTCCTATCTC 
               
               
                   
               
               
                 CCCCACTAGGATGTAAACTCCACAGGGGCATTGGGAATGCTGCCTGGCTA 
               
               
                   
               
               
                 TGGTAGGGACAGAGGGGAGCACCAGGGCGGGGCAGGGGTGCCAGAGTTCT 
               
               
                   
               
               
                 GCCTGGGCAGTCAGATTTTCCTTAGGAGGGGACATTTGAGTGGGACCCAA 
               
               
                   
               
               
                 ACAGGTGTATAGCAGTTGTCCAGCCCAGCTGGCAAGGCCTGAGTCTGCCT 
               
               
                   
               
               
                 CTGCAACCCCTCTCTTGGGCTCCTTTCTCTGCCACCCACCTCCTCACCTT 
               
               
                   
               
               
                 TCCAGGTCATCACAGTTGGGGCCACCAATGCCCAAGACCAGCCGGTGACC 
               
               
                   
               
               
                 CTGGGGACTTTGGGGACCAACTTTGGCCGCTGTGTGGACCTCTTTGCCCC 
               
               
                   
               
               
                 AGGGGAGGACATCATTGGTGCCTCCAGCGACTGCAGCACCTGCTTTGTGT 
               
               
                   
               
               
                 CACAGAGTGGGACATCACAGGCTGCTGCCCACGTGGCTGGTAAGTCACCA 
               
               
                   
               
               
                 CCCCACTGCCTCGGCCACCGTGATGCTAACAGCCCCTTTGGCAGTCAGGG 
               
               
                   
               
               
                 TCTGTGCCGGGACCTCCAGTGCCAGGCTCTGTGCAGGGGGACCAGAGATG 
               
               
                   
               
               
                 AAGTAGGCCTGATGGTGCCTTCAAGGACACTCAGTCTGATGAGGGAGGCG 
               
               
                   
               
               
                 AGTGCACAGAGGAAACACGAGGTCAGGGCTGTATTAGAGGGAGCCCAGAG 
               
               
                   
               
               
                 GAGGCACCTGCCCAGCCCGAGGGTCAGAGAAGGCATCTTGGAGGAGGGAC 
               
               
                   
               
               
                 ATTTGATCGGGAGCTTGATGGATGAATAGGAGTTCACCTGGCCGATAAGA 
               
               
                   
               
               
                 CAGCAACTACCAAGGCTTAGAGGTGTGAGAGGAGGCTGTCTTACCTCACT 
               
               
                   
               
               
                 GAGTAAGGACTGCAGGCGGCTTACCTTCGAGAAGAGAGCTTAGTGTCTGT 
               
               
                   
               
               
                 GTGCACGTGTGTTTGTGTGTATGTGTGTGCGTGTGTGCACTGGCAGGAGT 
               
               
                   
               
               
                 CCCCTGCTGGGGCAGGAGGGCCGGGCCATCACCATCTTTCACCATTCACC 
               
               
                   
               
               
                 CCTGCACCAGGCATTGCAGCCATGATGCTGTCTGCCGAGCCGGAGCTCAC 
               
               
                   
               
               
                 CCTGGCCGAGTTGAGGCAGAGACTGATCCACTTCTCTGCCAAAGATGTCA 
               
               
                   
               
               
                 TCAATGAGGCCTGGTTCCCTGAGGACCAGCGGGTACTGACCCCCAACCTG 
               
               
                   
               
               
                 GTGGCCGCCCTGCCCCCCAGCACCCATGGGGCAGGTAAGCAGGATGGCAG 
               
               
                   
               
               
                 GGTGGGCAAGTCCAGGCTGGGGCTTGGGAGGTCTGTGTGACCTTGACAGT 
               
               
                   
               
               
                 CTCTCCCTTCTCCCTTGTCTGTGTAAGGAGGATGACGCCACCTTAAATAG 
               
               
                   
               
               
                 GATTAAATGAGAATGGGGCTCTGAAAGGGCTGTGCAATATTTTCATAACG 
               
               
                   
               
               
                 TGTTTTTATAGAGACAGTTGAGTATGTTCTTTAAGCCCTCCTCTCTCCTA 
               
               
                   
               
               
                 CCATGAACTAAAGATTTCTGTGGAGGTCCCCTCACTCCCAGCACCCCCTC 
               
               
                   
               
               
                 CTCATCCCAGGCCCTTTTTGCAGGTTGGCAGCTGTTTTGCAGGACTGTAT 
               
               
                   
               
               
                 GGTCAGCACACTCGGGGCCTACACGGATGGCCACAGCCGTCGCCCGCTGC 
               
               
                   
               
               
                 GCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAA 
               
               
                   
               
               
                 GCGGCGGGGCGAGCGCATGGAGGTGACTGTACCCCTCCTTCGTGTGTGTG 
               
               
                   
               
               
                 TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCAGTGCT 
               
               
                   
               
               
                 GGGCCCTCAGGGACCCCCAGCAAGCCCCTCCATCCTCCAGACTCCAGCTC 
               
               
                   
               
               
                 TTCTGTAAGCTTACAGGGCTGGCCAGACCAGGAGTGGGGCACTCCTCACT 
               
               
                   
               
               
                 TCACGCGGCTGGGGGCTGCTGGAGAGAGCCACAGCGGGAAGGGTTTCCTA 
               
               
                   
               
               
                 GAGGCTGCAGGACAGTGCTGGATGGATTTTCAATGCTCACCTGGGTGTGA 
               
               
                   
               
               
                 GCGTGCGGCAGGGCCGCGTGAGGGTCAGCGATCTGCTACTCTGGACTCAG 
               
               
                   
               
               
                 CCATCTCTAGGCCCCTCTCACTCAGGTGCTCCATGGTTCTGGGAGCTGAG 
               
               
                   
               
               
                 AAATCTCAAACCAGCAAAAAAGTGGAATTGATGTTGATGCTACAGGATAG 
               
               
                   
               
               
                 TGCACAGATGCCATCTGGTTGCAGCATTTTGGTGGAAGGGCAGTGCCCAG 
               
               
                   
               
               
                 CTAGGAGAGTGAGGAGGGGCAGGCATTTCTGGCTTGAGGAGATGGGGTCT 
               
               
                   
               
               
                 TAATGCTCGTGTGAGAGGCAGAGTGGGTGGAGTGGAGCTGGCTGGATCCT 
               
               
                   
               
               
                 TGCTTTGGCCTCCTGGATTTCTCTCTATCTCCATTTTGAAACCACTCTGT 
               
               
                   
               
               
                 GTTTGGAAGAACTTTTGAGTATTCAGAGCTGCCCACTGGCAGAACAGTCT 
               
               
                   
               
               
                 TCCTTGGGCAGGAGTGAGCTCCTTGTCCCCAGAAGGCTGGGTCTGGCTGG 
               
               
                   
               
               
                 CCCCTGGCAGGGACACTGATGAGGGTGCTTGAGTTGATCCTGTCTAGTCC 
               
               
                   
               
               
                 CTTTCTGTGTTTTCAAAGCCCATTCTAAAGCAGATTCCCATTTCCGTCTT 
               
               
                   
               
               
                 TGACTCTAAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTT 
               
               
                   
               
               
                 TTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAG 
               
               
                   
               
               
                 GCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGAC 
               
               
                   
               
               
                 CCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGTAGGAGGCTGG 
               
               
                   
               
               
                 GCTTGCCCTGGGGTGAGGAGGGGTCTCTTTCTCCTTATGCACCCACTGCC 
               
               
                   
               
               
                 CGCGAGGCTTGGTCCTCACAAGTGTGATCCATGAGACTCAAGCCTGACTT 
               
               
                   
               
               
                 GCAGTTCCATACTCTGGTTCTGCCACTTCCATGCCCTTTGAGCCTGGGCA 
               
               
                   
               
               
                 GGTGACCTTACTTCTCCTCATCTCAGCTTCCTCCTCCATAAGAGGGAAAA 
               
               
                   
               
               
                 AGGTATTACCTGCCTCATTGTGTTGCAAGGAGATGGGCAGCATCTAGGGC 
               
               
                   
               
               
                 ACTGGCCTGGAGTATCGCAGGTGCTTTGCCTAAGGTGGTGCAGTCCAGGA 
               
               
                   
               
               
                 GAGGCAGCTCCAGAGAGAGGCCCCCGGCTGGGGCTGAAAGGAGGGCAGAC 
               
               
                   
               
               
                 CTCGGTTTGAATTTCACCCTGCCGCTCTATAGCTGTGTGACTTGGGCAAA 
               
               
                   
               
               
                 TTACTTAACATCTCTGTATGAGGAAATGATGAGTGCTAAGCACTTAGCTT 
               
               
                   
               
               
                 AGTGCCGGGACAATATAAATTCTAGCTATCGTTACTATTGTTTTCATCAC 
               
               
                   
               
               
                 CCGTTGCTTTAAAATCCAGCCTCTGGTATAGGCAACTATTGACGGGCTAC 
               
               
                   
               
               
                 CCTGTGTCGAAAACATGCCCAGGCAGGTAGCAGGAAGTCACAGATGGGGA 
               
               
                   
               
               
                 CCTCTTGGGGCATCAAGGGATGGTGCCCTGAGGCTGAGCTGTTCTGGTTG 
               
               
                   
               
               
                 GGTGGAGCATGAGAGGTCTGGGAAGACAGTGGGACTCCAGCCTGGAATAA 
               
               
                   
               
               
                 GAGGCTCAGAGTTGATTCTCGTCTGAGCACGTCCAGGGGAACCACTGAGG 
               
               
                   
               
               
                 GTTTGGGAACAGGAGAGTGAGGGTGAGAACCTGGTTCTGGGCACAGCAGG 
               
               
                   
               
               
                 CTGGCATGTAGGATGGATGTTCAGGAAAGATGAGCATAGTCAGGTGGCTG 
               
               
                   
               
               
                 GTGCCCTTGTCCAGGGGAGAGGCTCCGTCAGGTTCAGGGGTCCTGGCTTG 
               
               
                   
               
               
                 GAGGGAAGTCCGCCATGCTCTAATCACGCTCCCCTTTGGAAGTGCTCAGC 
               
               
                   
               
               
                 CGATGAGCTCACAGGCACATGTCAGTTTGAAGTCATGGAATCTGACTCCA 
               
               
                   
               
               
                 TGAAGCGCACCTCAAAGAGCACCATTTTGCAGCTAAGGGAACTGCAGGCT 
               
               
                   
               
               
                 GGACATGCTGAGTGGCTGCCCCGAGCCCTTGCAGCTAGGACATAGAGAAT 
               
               
                   
               
               
                 GCTAGTAACCACAACCCTACCATGTTCAGAGCACATGCCAGGCTCCATGC 
               
               
                   
               
               
                 TGGGGCTTCGCACGTGTCATCTTCACAGTGTCCCTGTGAGTAGGTGTGGT 
               
               
                   
               
               
                 TTCTCTTTCCATCTTACAAATGAGTAAACAGAGCCTCAGTGTAGCTAAGT 
               
               
                   
               
               
                 AACCACTATTTTAGGTTTCTTAGCCAATGGGTGTGTCTGACTCCTAAGCC 
               
               
                   
               
               
                 CATGGAGGGCATTCTGAGGTGGTTCAGACAGACCCCGGCTTACCCTTGAA 
               
               
                   
               
               
                 CTTCTGCCTGCTGGCTGCATAGGGAGGGGCTGGGGGGAGTTTGAGCATCT 
               
               
                   
               
               
                 CAGGCCATAGAGCCCCTGCCTCACTGTCTCCATCTCTGGGTGGAAAGATG 
               
               
                   
               
               
                 GTGTTTTCCCTGAGAAACTAAGGCTCAGAGAGGTTGAATGGCTCTCCCAA 
               
               
                   
               
               
                 GGTCACACAGCTGGTCAGCTGCAGAGTTGAGAACACAGGAGTCCTGGTGC 
               
               
                   
               
               
                 TCAGGCCAGCATCTCTTTTTTTCTTTGAGTTGTTTCTAGGTTTCCTAGCT 
               
               
                   
               
               
                 CTTGCCTCAGACCTTAAAGAGAGAGGGTCTGATGGGGATGGGCACTGGAG 
               
               
                   
               
               
                 ACGGAGCATCCCAGCATTTCACATCTGAGCTGGCTTTCCTCTGCCCCAGG 
               
               
                   
               
               
                 CTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTG 
               
               
                   
               
               
                 TGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCC 
               
               
                   
               
               
                 AGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAA 
               
               
                   
               
               
                 GGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGAAGAGGCCCGTGAGGC 
               
               
                   
               
               
                 CGGGTGGGTGGGGTGCTGCGTGTCTCTCCTGCACAGCTTTTCTGTGTCAG 
               
               
                   
               
               
                 TTTGTGCCACCACCATACCGCCATGCATCAGGGTGGCGGTTTGCCAGGTA 
               
               
                   
               
               
                 GATGCTGTGGGCAGCTTCCGCCATTGTGTGGACAGCATGTATATGTGTCT 
               
               
                   
               
               
                 CTGTGTGGCTGGGTCTGTTTTTGCTTTTGTCCAGATCAGTAAGGTTTGCT 
               
               
                   
               
               
                 ACCTGGGTACCCCACTCCACTTGGAGTAGAATGTGCATAAATATGGCATA 
               
               
                   
               
               
                 AAGAAATGCAATATGCATGCATTTATTGATTGATCTATTTTTTTCTGAGA 
               
               
                   
               
               
                 TGGGGTCTTGCTGTGTTGCCCAGGCTGGTCTCAAATTCCTGGGCTCAAGC 
               
               
                   
               
               
                 AATCCTCTGGTCTCAGCCTCCCCAAGTGTTGGGATTATAGGCATGAGCCG 
               
               
                   
               
               
                 CTGCACCTGGCCTCTCTGATCTATTTAACAAACCTGCTGGGAGGGTCTCA 
               
               
                   
               
               
                 GGGTCAGGAGCAGCACTGGGCTCTGAGGACACAGAGCTCACTCAGCCGTG 
               
               
                   
               
               
                 ACCCAGAGGGGGTGCCTGAGCTGCATGCTGAAGGTTGTTAGCATGACCAG 
               
               
                   
               
               
                 CAAGGCAAGAAAAGGCCCTGCCGAGATTAGCAAGGCATGTGCCAAGCCCT 
               
               
                   
               
               
                 GGAATGTGACAGCCGGGCCTTCTAGAAACCTGAGTGTATAACTCTCCTTA 
               
               
                   
               
               
                 AAAGCCAGTAGGAGCTCCTCAAAAGGCAGCCCTAAGGAGTCCACTCTTAA 
               
               
                   
               
               
                 ATGAACTCAGAGTCAGTTTTAAAATGCAAGTCTGTGTTGATTCTGGTCTG 
               
               
                   
               
               
                 GATGGTGCATTCCTCGAGAGCAAAAGACAGTCTTGGTCTTGGATCCACTT 
               
               
                   
               
               
                 GCCCTGGGTACACTGAGGGCTGCTAGGTTCCAGGTGCTCTTCCTGGCACT 
               
               
                   
               
               
                 GGGGAGGGATACAGGCCCAAGAGACATGCTGTTCTCCCTCCTGGAGCATC 
               
               
                   
               
               
                 TATTTTAGTGGAGGAAGACAGAAAACAAACCATTAATATAGAGTACTGAA 
               
               
                   
               
               
                 AAGATGCGATGGAGAAAACTATAGCAAGGAAGGGAATGGGGTGGGAGAGA 
               
               
                   
               
               
                 GGTCAGGAGAGGTCTCGCTGACAAGGTGGACGAAACAGGCCATGAGGCAG 
               
               
                   
               
               
                 AGAACATGTTCCAGGCAAAGCAAAGGCCCCCAGGTGGGGATGTGCAGGGA 
               
               
                   
               
               
                 GTACCAGGAAACCAGAGAGGTGGGAATAGTTATGAGATGGGGGGTGCCTC 
               
               
                   
               
               
                 AGAGGGGACAGGGCCAAGTCAGGTGAGACCTGAGGGTCACAGTCAGCAGT 
               
               
                   
               
               
                 GAGCTGGGGCCATGCAGGGGTCTGGCCTCAGAGGAGTGTGGTCTGGCCTG 
               
               
                   
               
               
                 GATCTGAACCTCTCACTGTGGCCTAGCTGCTGAGCTGAGAAGAGATGACA 
               
               
                   
               
               
                 AGGACCTTGGGCAGAAGCAGGGAGACTGGAGGGAGGCGGTGGAGGGTCCA 
               
               
                   
               
               
                 GGCGTTGGGGCGGGGCTCAGGCTGGAGTCTGAAGGGAGCCTGCAGGCCTG 
               
               
                   
               
               
                 GTGGGTGGATGTGGGTGGGAGAGGGGGAGGATGGCACCAAGGCTCGGGCC 
               
               
                   
               
               
                 CCTGGACAGATGGAGTTGCCATTAAGTGGGATGGGGCAGGCTATGGGGCC 
               
               
                   
               
               
                 ATCAGTTTCAGAGGGATGAGTTTGGCACTGGCATGGTAGGCATCTGTCTA 
               
               
                   
               
               
                 TCTCCACGGCCCTCAAACCAGGCATGAAGCAGGAGCTCACGTGTTTGGTC 
               
               
                   
               
               
                 AGCCATGGTGCAGAACCGCCTGGGTGGGAGGTGCGGGGTGGGAGATACAC 
               
               
                   
               
               
                 GGTTGTGTCCCAAATGGGCTCTGAGCCAGCGAGGGCCGTCTGCACTTTGG 
               
               
                   
               
               
                 CCTCACAGAAGGATGTCGGAGGGAGAAATGAAGTGTGGGTGGGGGTCCCG 
               
               
                   
               
               
                 GGCCACGCTAGACATGTGCTTTCTTTTCCTCGGGCTCTGGCAGGTGACCG 
               
               
                   
               
               
                 TGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGG 
               
               
                   
               
               
                 ACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAG 
               
               
                   
               
               
                 GAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGGGGCCGTGACAG 
               
               
                   
               
               
                 CCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAG 
               
               
                   
               
               
                 CTCCAGTGACAGCCCCATCCCAGGATGGGTGTCTGGGGAGGGTCAAGGGC 
               
               
                   
               
               
                 TGGGGCTGAGCTTTAAAATGGTTCCGACTTGTCCCTCTCTCAGCCCTCCA 
               
               
                   
               
               
                 TGGCCTGGCACGAGGGGATGGGGATGCTTCCGCCTTTCCGGGGCTGCTGG 
               
               
                   
               
               
                 CCTGGCCCTTGAGTGGGGCAGCCTCCTTGCCTGGAACTCACTCACTCTGG 
               
               
                   
               
               
                 GTGCCTCCTCCCCAGGTGGAGGTGCCAGGAAGCTCCCTCCCTCACTGTGG 
               
               
                   
               
               
                 GGCATTTCACCATTCAAACAGGTCGAGCTGTGCTCGGGTGCTGCCAGCTG 
               
               
                   
               
               
                 CTCCCAATGTGCCGATGTCCGTGGGCAGAATGACTTTTATTGAGCTCTTG 
               
               
                   
               
               
                 TTCCGTGCCAGGCATTCAATCCTCAGGTCTCCACCAAGGAGGCAGGATTC 
               
               
                   
               
               
                 TTCCCATGGATAGGGGAGGGGGCGGTAGGGGCTGCAGGGACAAACATCGT 
               
               
                   
               
               
                 TGGGGGGTGAGTGTGAAAGGTGCTGATGGCCCTCATCTCCAGCTAACTGT 
               
               
                   
               
               
                 GGAGAAGCCCCTGGGGGCTCCCTGATTAATGGAGGCTTAGCTTTCTGGAT 
               
               
                   
               
               
                 GGCATCTAGCCAGAGGCTGGAGACAGGTGCGCCCCTGGTGGTCACAGGCT 
               
               
                   
               
               
                 GTGCCTTGGTTTCCTGAGCCACCTTTACTCTGCTCTATGCCAGGCTGTGC 
               
               
                   
               
               
                 TAGCAACACCCAAAGGTGGCCTGCGGGGAGCCATCACCTAGGACTGACTC 
               
               
                   
               
               
                 GGCAGTGTGCAGTGGTGCATGCACTGTCTCAGCCAACCCGCTCCACTACC 
               
               
                   
               
               
                 CGGCAGGGTACACATTCGCACCCCTACTTCACAGAGGAAGAAACCTGGAA 
               
               
                   
               
               
                 CCAGAGGGGGCGTGCCTGCCAAGCTCACACAGCAGGAACTGAGCCAGAAA 
               
               
                   
               
               
                 CGCAGATTGGGCTGGCTCTGAAGCCAAGCCTCTTCTTACTTCACCCGGCT 
               
               
                   
               
               
                 GGGCTCCTCATTTTTACGGGTAACAGTGAGGCTGGGAAGGGGAACACAGA 
               
               
                   
               
               
                 CCAGGAAGCTCGGTGAGTGATGGCAGAACGATGCCTGCAGGCATGGAACT 
               
               
                   
               
               
                 TTTTCCGTTATCACCCAGGCCTGATTCACTGGCCTGGCGGAGATGCTTCT 
               
               
                   
               
               
                 AAGGCATGGTCGGGGGAGAGGGCCAACAACTGTCCCTCCTTGAGCACCAG 
               
               
                   
               
               
                 CCCCACCCAAGCAAGCAGACATTTATCTTTTGGGTCTGTCCTCTCTGTTG 
               
               
                   
               
               
                 CCTTTTTACAGCCAACTTTTCTAGACCTGTTTTGCTTTTGTAACTTGAAG 
               
               
                   
               
               
                 ATATTTATTCTGGGTTTTGTAGCATTTTTATTAATATGGTGACTTTTTAA 
               
               
                   
               
               
                 AATAAAAACAAACAAACGTTGTCCTAAC 
               
               
                   
               
               
                 Human PCSK9 Amino Acid Sequence (NP_777596.2) 
               
               
                 (SEQ ID NO: 6) 
               
               
                 MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEED 
               
               
                   
               
               
                 GLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQA 
               
               
                   
               
               
                 QAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVF 
               
               
                   
               
               
                 AQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRV 
               
               
                   
               
               
                 MVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSL 
               
               
                   
               
               
                 RVLNCQGKGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAA 
               
               
                   
               
               
                 CQRLARAGVVLVTAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGT 
               
               
                   
               
               
                 LGTNFGRCVDLFAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMML 
               
               
                   
               
               
                 SAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHG 
               
               
                   
               
               
                 AGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERM 
               
               
                   
               
               
                 EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRV 
               
               
                   
               
               
                 HCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASC 
               
               
                   
               
               
                 CHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAV 
               
               
                   
               
               
                 DNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ 
               
            
           
         
       
     
     Apolipoprotein C3 (APOC3) 
     In some embodiments, the target gene for modification using the compositions and methods disclosed herein is gene encoding APOC3. The LDL-R mediated cholesterol clearance pathway involves multiple players. Non-limiting examples of protein factors involved in this pathway include: Apolipoprotein C3 (APOC3), LDL receptor (LDL-R), and Increased Degradation of LDL Receptor Protein (IDOL). These protein factors and their respective function are described in the art. Further, loss-of-function variants of these factors have been identified and characterized, and are determined to have cardio protective functions. See, e.g., Jorgensen et al., N Engl J Med 2014; 371:32-41 Jul. 3, 2014; Scholtzl et al, Hum. Mol. Genet. (1999) 8 (11): 2025-2030; De Castro-Oros et al., BMC Medical Genomics, 20147: 17; and Gu et al., J Lipid Res. 2013, 54(12):3345-57, each of which are incorporated herein by reference. Thus, some aspects of the present disclosure provide the generation of loss-of-function variants of APOC3 (e.g., A43T and R19X), LDL-R, and IDOL (e.g., R266X) using the methods and compositions disclosed herein. 
     Apolipoprotein C-III (APOC3) is a protein that in humans is encoded by the APOC3 gene. APOC3 is a component of very low density lipoproteins (VLDL). APOC3 inhibits lipoprotein lipase and hepatic lipase. It is also thought to inhibit hepatic uptake of triglyceride-rich particles. An increase in APOC3 levels induces the development of hypertriglyceridemia. Recent evidence suggests an intracellular role for APOC3 in promoting the assembly and secretion of triglyceride-rich VLDL particles from hepatic cells under lipid-rich conditions. However, two naturally occurring point mutations in human apoC3 coding sequence, A23T and K58E have been shown to abolish the intracellular assembly and secretion of triglyceride-rich VLDL particles from hepatic cells. 
     Loss-of-function mutations that may be made in APOC3 gene using the methods and compositions described herein are also provided. The strategies to generate loss-of-function mutation are similar to that used for PCSK9 or ANGPTL3 (e.g., premature stop codons, destabilizing mutations, altering splicing, etc.). 
     In some embodiments, the gene modification methods and compositions described herein reduces expression of functional APOC3 protein encoded by the APOC3 gene in the cell. In some embodiments, the modification reduces expression of functional APOC3 protein encoded by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100%. In some embodiments, the modification reduces expression of functional ANGPTL3 protein encoded by the APOC3 or ANGPTL3 gene in the cell by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold. In some embodiments, the modification abolishes expression of functional APOC3 protein encoded by the APOC3 gene in the cell. 
     The protein sequence of human APOC3 can be found, for example, at Deposit No. NP_000031.1, which reference is incorporated herein in its entirety. Human nucleic acid sequences can be found at e.g., GenBank Accession Nos.: NG 008949.1, which sequence is incorporated herein in its entirety. Mouse, rat and monkey APOC3 nucleic acid sequences have been deposited; see, e.g., Ensembl accession number ENSMUSG00000032081, ENSRNOG00000047503, and ENSMFAG00000001837 respectively, each of which sequences is incorporated herein in its entirety. 
     The polypeptide and coding nucleic acid sequences of APOC3 and of other members of the family of human origin and those of a number of animals are publicly available, e.g., from the NCBI website or ENSEMBL website. Examples include, but are not limited to the following sequences, each of which sequences are incorporated herein in their entireties; 
     
       
         
           
               
               
            
               
                 NG_008949.1:5000-8165  Homo sapiens  apolipoprotein C3 (APOC3), 
                   
               
               
                 RefSeqGene on chromosome 11 
               
               
                 (SEQ ID NO: 62) 
                   
               
               
                 CTGCTCAGTTCATCCCTAGAGGCAGCTGCTCCAGGTAATGCCCTCTGGGGAGGGG 
                   
               
               
                   
               
               
                 AAAGAGGAGGGGAGGAGGATGAAGAGGGGCAAGAGGAGCTCCCTGCCCAGCCC 
               
               
                   
               
               
                 AGCCAGCAAGCCTGGAGAAGCACTTGCTAGAGCTAAGGAAGCCTCGGAGCTGGA 
               
               
                   
               
               
                 CGGGTGCCCCCCACCCCTCATCATAACCTGAAGAACATGGAGGCCCGGGAGGGG 
               
               
                   
               
               
                 TGTCACTTGCCCAAAGCTACACAGGGGGTGGGGCTGGAAGTGGCTCCAAGTGCA 
               
               
                   
               
               
                 GGTTCCCCCCTCATTCTTCAGGCTTAGGGCTGGAGGAAGCCTTAGACAGCCCAGT 
               
               
                   
               
               
                 CCTACCCCAGACAGGGAAACTGAGGCCTGGAGAGGGCCAGAAATCACCCAAAG 
               
               
                   
               
               
                 ACACACAGCATGTTGGCTGGACTGGACGGAGATCAGTCCAGACCGCAGGTGCCT 
               
               
                   
               
               
                 TGATGTTCAGTCTGGTGGGTTTTCTGCTCCATCCCACCCACCTCCCTTTGGGCCTC 
               
               
                   
               
               
                 GATCCCTCGCCCCTCACCAGTCCCCCTTCTGAGAGCCCGTATTAGCAGGGAGCCG 
               
               
                   
               
               
                 GCCCCTACTCCTTCTGGCAGACCCAGCTAAGGTTCTACCTTAGGGGCCACGCCAC 
               
               
                   
               
               
                 CTCCCCAGGGAGGGGTCCAGAGGCATGGGGACCTGGGGTGCCCCTCACAGGACA 
               
               
                   
               
               
                 CTTCCTTGCAGGAACAGAGGTGCCATGCAGCCCCGGGTACTCCTTGTTGTTGCCC 
               
               
                   
               
               
                 TCCTGGCGCTCCTGGCCTCTGCCCGTAAGCACTTGGTGGGACTGGGCTGGGGGCA 
               
               
                   
               
               
                 GGGTGGAGGCAACTTGGGGATCCCAGTCCCAATGGGTGGTCAAGCAGGAGCCCA 
               
               
                   
               
               
                 GGGCTCGTCCAGAGGCCGATCCACCCCACTCAGCCCTGCTCTTTCCTCAGGAGCT 
               
               
                   
               
               
                 TCAGAGGCCGAGGATGCCTCCCTTCTCAGCTTCATGCAGGGTTACATGAAGCACG 
               
               
                   
               
               
                 CCACCAAGACCGCCAAGGATGCACTGAGCAGCGTGCAGGAGTCCCAGGTGGCCC 
               
               
                   
               
               
                 AGCAGGCCAGGTACACCCGCTGGCCTCCCTCCCCATCCCCCCTGCCAGCTGCCTC 
               
               
                   
               
               
                 CATTCCCACCCGCCCCTGCCCTGGTGAGATCCCAACAATGGAATGGAGGTGCTCC 
               
               
                   
               
               
                 AGCCTCCCCTGGGCCTGTGCCTCTTCAGCCTCCTCTTTCCTCACAGGGCCTTTGTC 
               
               
                   
               
               
                 AGGCTGCTGCGGGAGAGATGACAGAGTTGAGACTGCATTCCTCCCAGGTCCCTC 
               
               
                   
               
               
                 CTTTCTCCCCGGAGCAGTCCTAGGGCGTGCCGTTTTAGCCCTCATTTCCATTTTCC 
               
               
                   
               
               
                 TTTCCTTTCCCTTTCTTTCTCTTTCTATTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTC 
               
               
                   
               
               
                 TTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCCTTTCTTTCTTTCCTTTCTTTCTT 
               
               
                   
               
               
                 TCCTTTCTTTCTTTCTTTCCTTTCTTTCTCTTTCTTTCTTTCTTTCCTTTTTCTTTCTTT 
               
               
                   
               
               
                 CCCTCTCTTCCTTTCTCTCTTTCTTTCTTCTTCTTTTTTTTTTAATGGAGTCTCCCTC 
               
               
                   
               
               
                 TGTCACCTAGGCTGGAGTGCAGTGGTGCCATCTCGGCTCACTGCAACCTCCGTCT 
               
               
                   
               
               
                 CCCGGGTTCAACCCATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGC 
               
               
                   
               
               
                 ACGCGCCACCACACCCAGCTAATTTTTGTATTTTTAGCAGAGATGGGGTTTCACC 
               
               
                   
               
               
                 ATGTTGGCCAGGTTGGTCTTGAATTCCTGACCTCAGGGGATCCTCCTGCCTCGGC 
               
               
                   
               
               
                 CTCCCAAAGTGCTGGGATTACAGGCATGAGCCACTGCGCCTGGCCCCATTTTCCT 
               
               
                   
               
               
                 TTTCTGAAGGTCTGGCTAGAGCAGTGGTCCTCAGCCTTTTTGGCACCAGGGACCA 
               
               
                   
               
               
                 GTTTTGTGGTGGACAATTTTTCCATGGGCCAGCGGGGATGGTTTTGGGATGAAGC 
               
               
                   
               
               
                 TGTTCCACCTCAGATCATCAGGCATTAGATTCTCATAAGGAGCCCTCCACCTAGA 
               
               
                   
               
               
                 TCCCTGGCATGTGCAGTTCACAATAGGGTTCACACTCCTATGAGAATGTAAGGCC 
               
               
                   
               
               
                 ACTTGATCTGACAGGAGGCGGAGCTCAGGCGGTATTGCTCACTCACCCACCACTC 
               
               
                   
               
               
                 ACTTCGTGCTGTGCAGCCCGGCTCCTAACAGTCCATGGACCAGTACCTATCTATG 
               
               
                   
               
               
                 ACTTGGGGGTTGGGGACCCCTGGGCTAGGGGTTTGCCTTGGGAGGCCCCACCTG 
               
               
                   
               
               
                 ACCCAATTCAAGCCCGTGAGTGCTTCTGCTTTGTTCTAAGACCTGGGGCCAGTGT 
               
               
                   
               
               
                 GAGCAGAAGTGTGTCCTTCCTCTCCCATCCTGCCCCTGCCCATCAGTACTCTCCTC 
               
               
                   
               
               
                 TCCCCTACTCCCTTCTCCACCTCACCCTGACTGGCATTAGCTGGCATAGCAGAGG 
               
               
                   
               
               
                 TGTTCATAAACATTCTTAGTCCCCAGAACCGGCTTTGGGGTAGGTGTTATTTTCTC 
               
               
                   
               
               
                 ACTTTGCAGATGAGAAAATTGAGGCTCAGAGCGATTAGGTGACCTGCCCCAGAT 
               
               
                   
               
               
                 CACACAACTAATCAATCCTCCAATGACTTTCCAAATGAGAGGCTGCCTCCCTCTG 
               
               
                   
               
               
                 TCCTACCCTGCTCAGAGCCACCAGGTTGTGCAACTCCAGGCGGTGCTGTTTGCAC 
               
               
                   
               
               
                 AGAAAACAATGACAGCCTTGACCTTTCACATCTCCCCACCCTGTCACTTTGTGCC 
               
               
                   
               
               
                 TCAGGCCCAGGGGCATAAACATCTGAGGTGACCTGGAGATGGCAGGGTTTGACT 
               
               
                   
               
               
                 TGTGCTGGGGTTCCTGCAAGGATATCTCTTCTCCCAGGGTGGCAGCTGTGGGGGA 
               
               
                   
               
               
                 TTCCTGCCTGAGGTCTCAGGGCTGTCGTCCAGTGAAGTTGAGAGGGTGGTGTGGT 
               
               
                   
               
               
                 CCTGACTGGTGTCGTCCAGTGGGGACATGGGTGTGGGTCCCATGGTTGCCTACAG 
               
               
                   
               
               
                 AGGAGTTCTCATGCCCTGCTCTGTTGCTTCCCCTGACTGATTTAGGGGCTGGGTG 
               
               
                   
               
               
                 ACCGATGGCTTCAGTTCCCTGAAAGACTACTGGAGCACCGTTAAGGACAAGTTCT 
               
               
                   
               
               
                 CTGAGTTCTGGGATTTGGACCCTGAGGTCAGACCAACTTCAGCCGTGGCTGCCTG 
               
               
                   
               
               
                 AGACCTCAATACCCCAAGTCCACCTGCCTATCCATCCTGCGAGCTCCTTGGGTCC 
               
               
                   
               
               
                 TGCAATCTCCAGGGCTGCCCCTGTAGGTTGCTTAAAAGGGACAGTATTCTCAGTG 
               
               
                   
               
               
                 CTCTCCTACCCCACCTCATGCCTGGCCCCCCTCCAGGCATGCTGGCCTCCCAATA 
               
               
                   
               
               
                 AAGCTGGACAAGAAGCTGCTATGA  
               
               
                   
               
               
                 NP_000031.1 Human apolipoprotein C-III precursor 
               
               
                 (SEQ ID NO: 63) 
                   
               
               
                 MQPRVLLVVALLALLASARASEAEDASLLSFMQGYMKHATKTAKDALSSVQESQV 
                   
               
               
                   
               
               
                 AQQARGWVTDGFSSLKDYWSTVKDKFSEFWDLDPEVRPTSAVAA  
               
            
           
         
       
     
     Angiopoietin-Like 3 (ANGPTL3) 
     In some embodiments, the target gene for modification using the compositions and methods disclosed herein is gene encoding ANGPTL3. ANGPTL3 has been associated with diseases and disorders such as, but not limited to, Arteriosclerosis, Atherosclerosis, Cardiovascular Diseases, Coronary heart disease, Diabetes, Diabetes Mellitus, Non-Insulin-Dependent Diabetes Mellitus, Fatty Liver, Hyperinsulinism, Hyperlipidemia, Hypertriglyceridemia, Hypobetalipoproteinemias, Inflammation, Insulin Resistance, Metabolic Diseases, Obesity, Malignant neoplasm of mouth, Lipid Metabolism Disorders, Lip and Oral Cavity Carcinoma, Dyslipidemias, Metabolic Syndrome X, Hypotriglyceridemia, Opitz trigonocephaly syndrome, Ischemic stroke, Hypertriglyceridemia result, Hypobetalipoproteinemia Familial 2, Familial hypobetalipoproteinemia, and Ischemic Cerebrovascular Accident. Editing the ANGPTL3 gene using any of the methods described herein may be used to treat, prevent and/or mitigate the symptoms of the diseases and disorders described herein. 
     The ANGPTL3 gene encodes the Angiopoietin-Like 3 protein, which is a determinant factor of high density lipoprotein (HDL) level in human. It positively correlates with plasma triglyceride and HDL cholesterol. The activity of ANGPTL3 is expressed predominantly in the liver. ANGPTL3 is associated with Dyslipidemias. Dyslipidemias is a genetic disease characterized by elevated level of lipids in the blood that contributes to the development of clogged arteries (atherosclerosis). These lipids include plasma cholesterol, triglycerides, or high-density lipoprotein. Dyslipidemia increases the risk of heart attacks, stroke, or other circulatory concerns. Current management includes lifestyle changes such as exercise and dietary modifications as well as use of lipid-lowering drugs such as statins. Non-statin lipid-lowering drugs include bile acid sequestrants, cholesterol absorption inhibitors, drugs for homozygous familial hypercholesteremia, fibrates, nicotinic acid, omega-3 fatty acids and/or combination products. Treatment options usually depend on the specific lipid abnormality, although different lipid abnormalities often coexist. Treatment of children is more challenging as dietary changes may be difficult to implement and lipid-lowering therapies have not been proven effective. 
     ANGPTL3 is also known to cause hypobetalipoproteinemia. Hypobetalipoproteinemia is an inherited disease (autosomal recessive) that affects between 1 in 1000 and 1 in 3000 people worldwide. Common symptoms of hypobetalipoproteinemia include plasma levels of LDL cholesterol or apolipoprotein B below the 5th percentile which impairs the body&#39;s ability to absorb and transport fats and can lead to retinal degeneration, neuropathy, coagulopathy, or abnormal buildup of fats in the liver called hepatic steatosis. In severely affected patients, hepatic steatosis may progress to chronic liver disease (cirrhosis). Current treatment of hypobetalipoproteinemia includes severe restriction of long-chain fatty acids to 15 grams per day to improve fat absorption. In infants with hypobetalipoproteinemia, brief supplementation with medium-chain triglycerides may be effective but amount must be closely monitored to avoid liver toxicity. Another option for treating hypobetalipoproteinemia is administration high doses of vitamin E to prevent neurologic complications. Alternatively, vitamin A (10,000-25,000 IU/d) supplementation may be effective if an elevated prothrombin time suggests vitamin K depletion. 
     In one example, the target tissue for the compositions and methods described herein is liver tissue. In one example, the gene is ANGPTL3 which may also be referred to as Angiopoietin 5, ANGPT5, ANG-5, Angiopoietin-Like Protein 3, Angiopoietin-5, FHBL2, and ANL3. ANGPTL3 has a cytogenetic location of 1p31.3 and the genomic coordinate are on Chromosome 1 on the forward strand at position 62,597,487-62,606,159. 
     Loss-of-function mutations that may be made in ANGPTL3 gene using the methods and compositions described herein are also provided. The strategies to generate loss-of-function mutation are similar to that used for PCSK9 (e.g., include, but are not limited to premature stop codons, destabilizing mutations, altering splicing, etc.). 
     In some embodiments, the modification reduces expression of functional ANGPTL3 protein encoded by the ANGPTL3 gene in the cell. In some embodiments, the modification reduces expression of functional ANGPTL3 protein encoded by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100%. In some embodiments, the modification reduces expression of functional ANGPTL3 protein encoded by the ANGPTL3 gene in the cell by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold. In some embodiments, the modification abolishes expression of functional ANGPTL3 protein encoded by the ANGPTL3 gene in the cell. 
     In some embodiments, a splice site disruption generated by a base editor system disclosed herein can result in the inclusion of intronic sequences in messenger RNA (mRNA) encoded by the ANGPTL3 gene. In some embodiments, the splice site disruption generates a nonsense, frameshift, or an in-frame indel mutation that result in premature stop codons or in insertion/deletion of amino acids that disrupt protein activity. In some embodiments, the splice site disruption generates exclusion of exonic sequences. In some embodiments, the splice site disruption generates exclusion of exonic sequences that results in nonsense, frameshift, or in-frame indel mutations in the ANGPTL3 transcript. Canonical splice donors comprise the DNA sequence GT on the sense strand, whereas canonical splice acceptors comprise the DNA sequence AG. Alteration of the sequence disrupts normal splicing. Splice donors can be disrupted by adenine base editing of the complementary base in the second position in the antisense strand (GT to GC), and splice acceptors can be disrupted by adenine base editing of the first position in the sense strand (AG to GG). 
     In some embodiments, a base editor system provided herein effects an A•T to G•C alteration in a ANGPTL3 gene when contacted with the ANGPTL3 gene. In some embodiments, the A•T to G•C alteration is at a splice donor site of the ANGPTL3 gene. In some embodiments, the A•T to G•C alteration is at a splice acceptor site of the ANTPTL3 gene. In some embodiments, the A•T to G•C alteration results in an aberrant ANGPTL3 transcript encoded by the ANGPTL3 gene. In some embodiments, the A•T to G•C alteration results in a non-functional ANGPTL3 polypeptide encoded by the ANGPTL3 gene. In some embodiments, the A•T to G•C alteration is at a 5′ end of a splice donor site of an intron 6 of the ANGPLT3 gene. 
     The nucleotide sequence of human ANGPTL3 is provided, for example, in NG_028169.1, which is incorporated herein in its entirety. The protein sequence of human ANGPTL3 is provided, for example, AAD34156.1, which is incorporated herein in its entirety. 
     Mouse, rat, and monkey ANGPTL3 nucleic acid sequences have been deposited; see, e.g., Ensembl accession number ENSMUSG00000028553, ENSRNOG00000008638, and ENSMFAG00000007083 respectively., each of which sequences are incorporated herein its entirety. 
     The polypeptide and coding nucleic acid sequences of ANGPTL3 and of other members of the family of human origin and those of a number of animals are publicly available, e.g., from the NCBI website or ENSEMBL website. Examples include, but are not limited to the following sequences, each of which sequences are incorporated herein in their entireties; 
     
       
         
           
               
               
            
               
                 NG_028169.1 Human angiopoietin like 3 (ANGPTL3),  
                   
               
               
                 RefSeqGene on chromosome 1 
               
               
                 (SEQ ID NO: 7) 
                   
               
               
                 AATGACAAACTGAAAAAATCTATTGTTTGTTATATATATAACAAAGAATTAGTAT 
                   
               
               
                   
               
               
                 CCACAATATGTAAATAATTCCTAAAATTAGTCAGAAAGAGACAAACTTAAAAAG 
               
               
                   
               
               
                 AGGGTAACAAGGAGGGGAGCAAATTATGTACATAACCAGATGATTCGCAAAGAC 
               
               
                   
               
               
                 GGCAACAGAGATGGCCAGCAAAACAAACTAGATATATACTTGTCTATTAGATTT 
               
               
                   
               
               
                 ATCAACATTTTTTGCCTTTTTCATTAAAAGCATTTGTAAAAGGATATAGGAAAAG 
               
               
                   
               
               
                 AGGAACTCTCATATACTCCTGGCAGGGATGTAAATTGGTACAACCCTTTTGAAGG 
               
               
                   
               
               
                 ACAATCTGACAAAAGCAATCGTAAGTTACAAGTCAACATCTATGAATGTATATG 
               
               
                   
               
               
                 AAAATATTTATATACATACATCACCACCATAAAAGCATTTTCTATACATACTGTTT 
               
               
                   
               
               
                 ATAATTAGAAAATTGGAAACAAATGATTAAAAGGGGGCTGATTAAATTAAGGTT 
               
               
                   
               
               
                 CATCTATATAACAGGATTATGCAGCTATTAAAAAGGACGTGGTAACTCTATAGAC 
               
               
                   
               
               
                 ATTCATAGGAAAATAAATTTTAAAATACTAAGATCCTGAATGATATATATATCAT 
               
               
                   
               
               
                 GAGCTATTATACATAACAAGATCCCACTTGTGTTATAAAAAATTATGTTTAGTCA 
               
               
                   
               
               
                 TTCAAAGGGTCTGGTATGATAGACCCAAAATGTTAATAGAGTCGAGATTTTTATT 
               
               
                   
               
               
                 TTTTATAGGTTTTTGAAATACCTGAATTTTCACAATAAGTACTTTGCACATTAAAA 
               
               
                   
               
               
                 ATCTTAGCTGGGCATGGTGGCTCACGCTTGTAATCCCAGCACTCTGGGAGGCCAA 
               
               
                   
               
               
                 GGTAGGCAGATCACCTGAGGTCAGGAGTTCGAGACCAGTCTGGCCAACATGGTG 
               
               
                   
               
               
                 AAACCCCGTCTCTACTAAAAATACAAAAATTAGCCAGGCTTGGTTGGGGGTGCCT 
               
               
                   
               
               
                 GTAATTCCAGATACTCGGGAAGCTGAGACAGGAGAATCGCTTGAACCCAGGAGG 
               
               
                   
               
               
                 GGGAAGTTGCAGTGAGCTGAGATCACGACGCTGCACTCCAGCCTGGGCAACAAA 
               
               
                   
               
               
                 GAGCAAAACTCCGTCTCAAAAATAAATAAAGAAAAAATCTTTACATGTCCAAAG 
               
               
                   
               
               
                 ATACGGCTGTTCAACTAAAAAATATATATGTATAAAACTTAACATGTTAATAGTG 
               
               
                   
               
               
                 AACACACAAAACAGTAAGATAGATAAAATTATTCCTTCAAAGCTCACTTAACCTC 
               
               
                   
               
               
                 TGGATCTACACTGTCCAAAAAGACGGTCTAATGAGACAATTGAGCACTTGATAG 
               
               
                   
               
               
                 GTGAGTGGTTCTAACTGAGATATGTTCTCAGTATAAAACATACAATAGGATCTTC 
               
               
                   
               
               
                 CTATACAACATTAATTAAAAAACAAACTATTGTAGTTAAAAAGGAAAAAATTAG 
               
               
                   
               
               
                 AGATACTATGTAAAAAAGAGCCAAAATACCTTGTATTTTATTTGAAAGACATATC 
               
               
                   
               
               
                 TCCATAAGATTACACAACCTCGTGTAGGATAAAGGACTTTGCTTTGCTTGGAATT 
               
               
                   
               
               
                 TAAACAATTTAGGCTCTTAAATGTCCTAAAATTCTCTGTAGCTAAGAAATTTTTAT 
               
               
                   
               
               
                 ATTGGTTCCTAGGAACTAGGAATCCTTAAATTAGGCCCTACATTTGCTTACAAGT 
               
               
                   
               
               
                 TTATTTTCCTTGGCATAAAATTTTTTAGTTTTTACATTACTGGTTATATTTGATCAG 
               
               
                   
               
               
                 GGTTCTATTTAAATAGGCACAAGTTCAAGCAAAGATCAGATTCTGCTTTTAGCAG 
               
               
                   
               
               
                 TGTGTACTCAGACAGGAAGTATTAAAAGGCAGGCAGAAAATCCTTTATAAAATT 
               
               
                   
               
               
                 ACTACTTTCAATGCATTTTCCCACGTTGAAATGCTTCTGCAGTTTATAATTAGGCA 
               
               
                   
               
               
                 AATTACTTTAATTATAATCAATAATGCTGTTCAAATTACTATAAGAATTATACAG 
               
               
                   
               
               
                 ATATTTATACCAAGAGACAATATACTAGAAACCAAGACTACGTGACCATTACCTC 
               
               
                   
               
               
                 TACTCTGTCAGTGTTATTTGTGAGAAATTGCACAAATTTTGCAAAAAGTGTTAGT 
               
               
                   
               
               
                 ATCCTACTACAGTAGGATATAATATAGAAGGAAATAATTTCATAAAGCCTGTCTT 
               
               
                   
               
               
                 TGGTACTAGTGCTCAGTTACTTTCATTAACTAAAAAAGGGGCTACTCTTCAAATT 
               
               
                   
               
               
                 CCCTTCTCTAAAAAGAATGTACTATATCAAAAGGGGGTAAACACTACTACGTATA 
               
               
                   
               
               
                 CATTCTGCACTTAGAAATCCCTATATGTTGATTTTATCATTCTCTTATTCAATAAA 
               
               
                   
               
               
                 TATTGTTTCTACAATGTGTAAGGCATTACTGTACTAAAGCATTATAAGGAATATA 
               
               
                   
               
               
                 AGTTAAAAACACATACAAATCTTGCCAATCAACAGCTTATAGTGTAATAGGGGA 
               
               
                   
               
               
                 GAGAAGCTGGCCCATCTATATTCTCCCTCAACTAGCAAGTGGATGAAATATCAGG 
               
               
                   
               
               
                 GTCAATAGTTATAAGCCACAAAAGCTGACAGCTTAATTAAGAGAAGTTTTGAAA 
               
               
                   
               
               
                 TATGTATTTCATGACCAATAATTACAACTGTAACTTTTCTATTTAAAGAAGGAGA 
               
               
                   
               
               
                 AAATTTGAATTTCTTCTCTAGCTCAACATACACTTCTATAATTCCATTACATGAAC 
               
               
                   
               
               
                 CAGAGTAAAGGGTAAGATGGAAATGAAGAATATTTTCTTACCCTTTTGTGGTTCT 
               
               
                   
               
               
                 ATATTGGACACTTAAAAATCATACACAACCTAATCAAAAGATGTAATTCTTTAAA 
               
               
                   
               
               
                 AAGGTACGAGACCAAAATTCAGAAAATCTAGACTATAACAAAATTTCTCAATTT 
               
               
                   
               
               
                 ACATTATCTTAATATGCAATTAATTTTCACCAGTAAAATACTATAGTATGGGTAC 
               
               
                   
               
               
                 AAATGCATTGATTAGTTCTAATTACAAAAATGGCTAATATATAATACTGTGTAGT 
               
               
                   
               
               
                 GTTTATGATACATCAGATAATGTTCTAAGTGCTCTGAAAATATAAACTTTTAATCT 
               
               
                   
               
               
                 TTTATACGACCCTATAAAATAGGTGTTATTCTCACTGGAGAGATGAGAAAACAGG 
               
               
                   
               
               
                 GGTTCAGAGATGTGAAGTAATTTGACCAAAGGTCACAAAGCTGAAGAATATGAA 
               
               
                   
               
               
                 ATCCGGGATTCTGATTCAGGCAGTCTTATTCCAGAATCATGCTCTTAACCACTAT 
               
               
                   
               
               
                 GGAATACTGCCTCTACTGTAACTATTATACCCAAAACCCTTAATCCTAAGTCATC 
               
               
                   
               
               
                 AAAAGGAAGAGCCTCTATTTTACACAATGAAGAGGCATTTCTAAGAATAGAAAT 
               
               
                   
               
               
                 TTAGGGACGAGCACAGTGGCTTACTCCTTTAATATCAGCACTTTGAGAGGCTGAT 
               
               
                   
               
               
                 ATGGGAGGTTCACTTGAAGTCAGGAGTTCAAGGTCAGCTTGGGCAACATAGTGA 
               
               
                   
               
               
                 AACACAGTCTCTACAAAATATTTAAAAATTAGCTGGGTGTGGTGGCATGCATCTA 
               
               
                   
               
               
                 TAGTCTCAGCTACTTGGGAGACAGAGGGAGGAGGCTTGCTCGAGCCCAGGAGTT 
               
               
                   
               
               
                 CGTGGCTATAGTGAGCTATGATCATGCCACTGCACTCCAGCCTGGACAACAGAG 
               
               
                   
               
               
                 CAAGACCCTGTCTCTAAAAAAGAAAAGAAATTTGGAAATGGTTTATTTTGTATTA 
               
               
                   
               
               
                 ACAATTTATAATTTACACTGAAATTTATTATGATAAAACTTTTCCCTGTGTTAAAA 
               
               
                   
               
               
                 AGCTATTAACTTTATGAAAAATTTCTTTTAGGTAAGGTTGATTATATATACCCACA 
               
               
                   
               
               
                 CACATACACAGGTTAAAAGTTAGTTTCATGTGACATAATAACTAGCATTTTGAGC 
               
               
                   
               
               
                 ACTACCTGTTTGCCCAGCACTGTTCTAAGTGCTCTACATGTATTATTGTTAAATTA 
               
               
                   
               
               
                 TCATAACACTATGAATTATGTACTATAATTACCCCAGCTTTACAGATGAGGAGAC 
               
               
                   
               
               
                 TAATCCATGGGGAGGTTAAGTAACTTGTCCAAGGCCAGACAGCTAGAGCCGGCT 
               
               
                   
               
               
                 TTTGGACCCACACCACAGTCTGACTCCAGCACCCATATTCTTAACAATTTCACCA 
               
               
                   
               
               
                 TATTAATATGTCAAGATTAAGCAGTTTTAAAGGATGCTATTTTCTCACAAATTTCT 
               
               
                   
               
               
                 TAATATGAACACTCAATAAGAATAATCACTAATATAAGCATTTAGTATTTTTTTA 
               
               
                   
               
               
                 ACACTAAGTTGGAAGCATAGTGGAACATTTATTTTTAGAAATATTATTAATTGGC 
               
               
                   
               
               
                 TGGGCTCACGCTTGTAATCGGCTGGGCTCATGCCTGTAAATTTTGGGAGGCCAAG 
               
               
                   
               
               
                 GTAAAAGAATTGCTTGAGCCCAGTATTTCCAGACCAGCATGGGCAATACATTAA 
               
               
                   
               
               
                 GACATCATCTTTAAAAAAAAAATGTTATTAATCTCCTCTTTTTGTTAAATGTATAT 
               
               
                   
               
               
                 TATCAAAATTGTTACTAAGCTAACAAACTTCAGAAAAACTTATGATGGGCAAGCT 
               
               
                   
               
               
                 GCTTGTGACATTGAAGGTATTTAAGATTCAATTCTAGTTTGGTCCTAGATGACCA 
               
               
                   
               
               
                 CATATCCATTGTTCCTTCAACGAGCACATGGTAAAGAGCCTAGAACACAGAGAC 
               
               
                   
               
               
                 ACAGAACACAGTGGAGAAAAGGGAGTGAAATGTCTTTAATGACACTTACTATAT 
               
               
                   
               
               
                 ATGGGATTTTGTGACAATATACAAGGATGGTTAAGACATATAAGGTGATGCAAA 
               
               
                   
               
               
                 AAAACATATTAACAATTATAGTGACAAAAAATGAGGAGCATATAATTATACATT 
               
               
                   
               
               
                 GATTTATACAGAGTACCAGAGGAACACAGCATTGAGAGCCGTAACACCACCTGA 
               
               
                   
               
               
                 GGGAGTGGAGAAAGGCTTCAGAGAGAAAGTGTTTTTTGGAATGGATCACTGTTT 
               
               
                   
               
               
                 CCAAAAGAACTAAAGTACAGTTTGAGAAATGCATACTTAATTCATTACTTTTTTC 
               
               
                   
               
               
                 CCCTCAACTTTAATAATAAATTTACCCAACAAAAAAGTTTATTTTTGACTTGTAA 
               
               
                   
               
               
                 ATCTCTTAAAATCATAAAAAAGTAAAATTAGCTTTTAAAAACAGGTAGTCACCAT 
               
               
                   
               
               
                 AGCATTGAATGTGTAGTTTATAATACAGCAAAGTTAAATACAATTTCAAATTACC 
               
               
                   
               
               
                 TATTAAGTTAGTTGCTCATTTCTTTGATTTCATTTAGCATTGATCTAACTCAATGT 
               
               
                   
               
               
                 GGAAGAAGGTTACATTCGTGCAAGTTAACACGGCTTAATGATTAACTATGTTCAC 
               
               
                   
               
               
                 CTACCAACCTTACCTTTTCTGGGCAAATATTGGTATATATAGAGTTAAGAAGTCT 
               
               
                   
               
               
                 AGGTCTGCTTCCAGAAGAAAACAGTTCCACGTTGCTTGAAATTGAAAATCAAGA 
               
               
                   
               
               
                 TAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCA 
               
               
                   
               
               
                 GAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAG 
               
               
                   
               
               
                 ATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGA 
               
               
                   
               
               
                 CATGGTCTTAAAGACTTTGTCCATAAGACGAAGGGCCAAATTAATGACATATTTC 
               
               
                   
               
               
                 AAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCGCTGCAAACCAGTGA 
               
               
                   
               
               
                 AATCAAAGAAGAAGAAAAGGAACTGAGAAGAACTACATATAAACTACAAGTCA 
               
               
                   
               
               
                 AAAATGAAGAGGTAAAGAATATGTCACTTGAACTCAACTCAAAACTTGAAAGCC 
               
               
                   
               
               
                 TCCTAGAAGAAAAAATTCTACTTCAACAAAAAGTGAAATATTTAGAAGAGCAAC 
               
               
                   
               
               
                 TAACTAACTTAATTCAAAATCAACCTGAAACTCCAGAACACCCAGAAGTAACTTC 
               
               
                   
               
               
                 ACTTAAAGTAAGTAGAAAATAAAGAGGGTTCATGTTTATGTTTTCAATGTGGATC 
               
               
                   
               
               
                 TTTTAAAAAAAATATTTCTAAGGCATGCCATTTGAAATACTTTGTTGCATTGTTGA 
               
               
                   
               
               
                 AATACTTTTTTTTCCAAGAAAAATAATCTCCAGAAAATAAAATTTCCTATTATAA 
               
               
                   
               
               
                 TTTCAAGTTAGTTTTTTGTTTCCCTAATGTTATATATGAAAACACTGAAAATTTGC 
               
               
                   
               
               
                 ATTTTATATGAAAATTACAAATCGGTTAAATTATACAATCTAGAACACTATGTCA 
               
               
                   
               
               
                 TTACACTATTGTAAATTACTGAAGGTAAGTAAAAAGTTAAAAAAAATTTAAAACT 
               
               
                   
               
               
                 ATTCTCCAGTGTTTAAAACAGATTAAATAATACAGTAAATGGAAAAGATTTATTC 
               
               
                   
               
               
                 ATATGAAAATATGCTGGGCTTTTTCTTTTAATTGAAGTTCAGAAAATCAAATTTTA 
               
               
                   
               
               
                 GAGATAGTACAATTTAAATAAAATGTTAAGGACAAAAATATGTGCTATTTGAAA 
               
               
                   
               
               
                 GAAGCATACAAGGGGAAGGAATTGCCAATATTCATTTTTCAAATCCATTATTAGT 
               
               
                   
               
               
                 TTAAAAATTTAGATTATGATAGTGTTACAGGAAATTAATAGAAAAGAAAGAGGA 
               
               
                   
               
               
                 AAGCAACTTATAACCAACCTACTCTCTATATCCAGACTTTTGTAGAAAAACAAGA 
               
               
                   
               
               
                 TAATAGCATCAAAGACCTTCTCCAGACCGTGGAAGACCAATATAAACAATTAAA 
               
               
                   
               
               
                 CCAACAGCATAGTCAAATAAAAGAAATAGAAAATCAGGTAAGTCAGTATTTTAA 
               
               
                   
               
               
                 TGGTATGTCCCATCTTTCACACAGGTCTGTAAAAACACTGAATCCTAAAATTATT 
               
               
                   
               
               
                 TACAAGCTTTAACTGGATCATGAGTAAAATTATCACATCAGCATAACTGTTAAAA 
               
               
                   
               
               
                 TTGCAGGCTCTGAAGCTAATAAACTACCTGCATTTAAACCATGGCTCTAAAACTT 
               
               
                   
               
               
                 TGTGTGACCTTGAATAAATTACTTCACCCCTTTATCTCTCAGTTTCCTCACATATA 
               
               
                   
               
               
                 CTACAAAGATAATAACAGAACTTATAGGATTATTGTAAGAAAAAAAATTAATTC 
               
               
                   
               
               
                 ATAGCAGCCAATGTCATCTTACTAAAATTCAAATTAGATCATGTTTCTCTTTGCTC 
               
               
                   
               
               
                 AAAACCACACAATAGCTTTCCATTTCACTCATATTGGCTCTTTAGACCAAGATTA 
               
               
                   
               
               
                 CCCAACCCTTCGTCATCTCACTGACTTCACCTCCTCTACTCTAGTTATTCTGACCG 
               
               
                   
               
               
                 CTTTACCAGTATTCAAACACATCAAACATACTGCCACCTCAAAGCCTTTGCCCTT 
               
               
                   
               
               
                 GTTGTTTCCTCTAACTGGAACGCTCTTCTGCCCTGGTATCTACGTGGCCCACTCTC 
               
               
                   
               
               
                 TGATTTCCCTTAGGGTCGTTATCAAACAAAAAATTCCCAATGAAGACTTACAAGG 
               
               
                   
               
               
                 TCACTTAACCAAAAATCACAACCGCCTGGTCCCATCCCTGAAAACTTCTACTTCC 
               
               
                   
               
               
                 TTAGCTACTTTTCTCCTGCACACTCACCTTTATTTAACATAACATAAATTTTAGTT 
               
               
                   
               
               
                 ATTTATCTCTTCTATTCCTGCACTAAAATGTAAGCTCTGTGAATACAGGGATTTTT 
               
               
                   
               
               
                 TCCATTATCTTCATATTTTCCATTATTTGTATATACTCCAGAATATAGAATACTGT 
               
               
                   
               
               
                 ATGGCACACAGTAGGCATTTCTGTTGAATTAATAAATGTAATGTCATATTCACAC 
               
               
                   
               
               
                 AGAAGCGTGTGCTATGATTATTATTACTTGGATTACTAGAAATAGTGTGCCTCAT 
               
               
                   
               
               
                 AATTAAAGGTCAACATTCAACAATGTAATTAATCTACAATGTAAACATCTGGTGA 
               
               
                   
               
               
                 AGTGACAGAGGGAAGCACTTGTTTAGAAAAAAGCTATGTCAGAATCCATGTATT 
               
               
                   
               
               
                 CTAATATGCAGTACAATAGTTTAAAAATATTAATAATACTCTCAAACAGCTATTC 
               
               
                   
               
               
                 AAGAGGATTCAAAAAACATAATATAAACTCAGAGAAACTGGTAAACAAAATCAT 
               
               
                   
               
               
                 TTTCAAGAGATATAAAACAAATATTATTACCAATTTCCACTAAACAAACATAATG 
               
               
                   
               
               
                 TTAGTAGTGCTGCTAAAAGGTTTTTTATCAACTACTTTTGGTTTCCATACTTTCCTT 
               
               
                   
               
               
                 CTTATGATGTTATTATTCTAAATTCTTTTCAATTATATCTTTTACTATGATTAAATG 
               
               
                   
               
               
                 AACCTGCTCCCCAAAGCAAAATGTTACTATAGTAATATACATTGTGTCTAAAAAT 
               
               
                   
               
               
                 AAAAATGTGTGAAGAAACCAAAACAATGAATTTCTGAGTTGGAAGAAGAGTTAG 
               
               
                   
               
               
                 ATCATTTAACTTTCTCATATTTAAATTAAAAAAACAAAACTCTAAAAATTTAAGT 
               
               
                   
               
               
                 AACTTTAAGATCACATAGTTACTTAGTAGAAAAGAGTAATACCCAGCAAGCAAA 
               
               
                   
               
               
                 CTTTACAATAGATCCTTTTAAATAAGGTCCTAGGAAATATCATTCATGCCAGCAT 
               
               
                   
               
               
                 CAAAAAACTAACACTAATAATGCAAGATATTATATATTCTGCTTTTCTTACTGTC 
               
               
                   
               
               
                 AATGAGAAAAACTATCATTCAATAAATTGCAAACCCAACACACTTAAATAAAAA 
               
               
                   
               
               
                 TAAAATGTTACTGCTAAACTAACGATAAACTACTGAATATATAGAAAGTAAGCA 
               
               
                   
               
               
                 AACAAACTTGCCAACCTGCCAACATCTACAGATATGTTTACAGGTCAAAAATTAT 
               
               
                   
               
               
                 CAAATTATCAAGAAAGCCTGGTTCAAATTATGTATTATGTCTTTATCACAGGTCT 
               
               
                   
               
               
                 GAAGATCAGTAAGACCTAAAACTGAAAATTATTAAACTTAAAATCTGAACAGAA 
               
               
                   
               
               
                 TATCAAATATATTTTATTCATATAAATAAAAGAATACATTACAATATTCTAAGCA 
               
               
                   
               
               
                 AAGCAGTCTCTACTTTTGGCCTTGCTCTGTTTTCCGACCAATGTCTGCTTTTTTGC 
               
               
                   
               
               
                 CTTGCTTTATTTTTTTATCTTATTAAATAATGTCCCTGATTAAATATTTTGAGAAC 
               
               
                   
               
               
                 AGGTAATCTGTACAATCTGAATAACACTGTTTATCTAAATATCAAACACCGTTAT 
               
               
                   
               
               
                 AACATTATGAACTGAAAGACAAACTGTACTTCTGACATCCTTACTCAGATTTCCC 
               
               
                   
               
               
                 CTAATTGTATATTCAGTATCATTTTAAAAAACAGATTTATATTCTTTTATCAGCTC 
               
               
                   
               
               
                 AGAAGGACTAGTATTCAAGAACCCACAGAAATTTCTCTATCTTCCAAGCCAAGA 
               
               
                   
               
               
                 GCACCAAGAACTACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATG 
               
               
                   
               
               
                 ATGGTAAGACACTTTGGTGGGTTTCCTTCTTGAAGCTATTATTATCAAATTCCCTA 
               
               
                   
               
               
                 TTCTTAGGACTTGTTCTAGACTAAAAGATAGTTAAGAGATATCCATCAAATACAA 
               
               
                   
               
               
                 TGTATCAACCTAAACTGGATGCTGGGGTTCTTTTTACACCCTATAAAAGACATAC 
               
               
                   
               
               
                 CTAAGACAATCAGAGAAATACAAATATGGACTTGATTATTAGATAATATAGAAG 
               
               
                   
               
               
                 GTTTATTAATTTTCTTAGATGTGATCATGGTATTGCAGTTTTAAAGGAGAACAATC 
               
               
                   
               
               
                 TCCTGTTTAAGAGATACATGCTGAAATATTTACGGAGTTAAAGGTCACTGGACTC 
               
               
                   
               
               
                 CAGACTGGTGATAGAACAAGACTCTGTCTCTAAAAAATAATTAATTTTTTAAAAG 
               
               
                   
               
               
                 AAAATAGTTTGGTAAGATGATTCTTACATTCTTAAATAACACGCCATCTAAGAAA 
               
               
                   
               
               
                 AATGCTTTAACATAAACATTACTGAAAAAATGCTACATTTGCCACAACTTCATAA 
               
               
                   
               
               
                 AATGTCAAGTGAAATCTCAAGCTCCAAAGATATTATTCCTATTACTAAATCTGAT 
               
               
                   
               
               
                 GTAATAACATTTTATTGATTCTAGGCATTCCTGCTGAATGTACCACCATTTATAAC 
               
               
                   
               
               
                 AGAGGTGAACATACAAGTGGCATGTATGCCATCAGACCCAGCAACTCTCAAGTT 
               
               
                   
               
               
                 TTTCATGTCTACTGTGATGTTATATCAGGTAAAACCTGTCTAAGGAGAATAGACA 
               
               
                   
               
               
                 GTAGTTAGTTCAACTTACTCATTACGTATTAGGAAGATTAACCTGGTTATCATTGT 
               
               
                   
               
               
                 TTTATACATATATATATGAAATATATATGAGTATTCGTATAAATATAATACTTTTA 
               
               
                   
               
               
                 CCTTGTTTATGTATTTACTCAATATTCTCCTTTTCCTCTAAAATAATCTGAAGTGA 
               
               
                   
               
               
                 CTATTATCAATAAGTTTACTATGCCAAAATTCATTAATTGCCTTTCACTTAACTTT 
               
               
                   
               
               
                 TGGGACCATAATAAATAATAAAATGTATTGCCATAACATTAATAAACTACCTTAC 
               
               
                   
               
               
                 AAAACCACCAATTAAAATCAAACAAACAAAAAAGTGTTATTTACATCTGTCAAC 
               
               
                   
               
               
                 ATAAATCTACTAAAAATACATGATTTCATTCATTATATTCAGGTAGTCCATGGAC 
               
               
                   
               
               
                 ATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAA 
               
               
                   
               
               
                 CTACAAATATGGTTTTGGGAGGCTTGATGGTAAGGGGACTACATTCAATCATTCA 
               
               
                   
               
               
                 TTCACTTGCTAATCTACAAATATTTACTGAGAACCTCTTATGGACCAGGTATTAG 
               
               
                   
               
               
                 GAAAAGTAGTAACGAACGAGAAGCAGTCTCAGCCTTCATATAATTTATTATCAA 
               
               
                   
               
               
                 ACAATTACACATTTGTTAGTAAATTACACTTATTACAACTGTTATTATTTGAATTA 
               
               
                   
               
               
                 TATTTATCACAATTACATGTCTGTTCTTAAATATACTTATCACAATTTAATTCCAC 
               
               
                   
               
               
                 GGCTTACAATGATCATAACTATAATTATTAAAGACAATTTTGATTAAATGTTATG 
               
               
                   
               
               
                 TCATAAGTAGTAACTGTTACAAATAAGCTGTGAAAAGAACCACTCCTAGCATTA 
               
               
                   
               
               
                 GTCACTCTATTCTCTCATTAACGTTTTACATATCAATTAATTGGAAGTTAAAAGGA 
               
               
                   
               
               
                 CCAGGAAACTCAGACATACAGTATACATTTTAAAATTTCAATTATTTAAATATAA 
               
               
                   
               
               
                 TATATAGAATGTATGGCTTATAATGAATTAGTTAACTCAATGCAAATTATTCTATT 
               
               
                   
               
               
                 TTGATTACAAATAGTAAAATAAGCAAGATAAAATAACAGATGTTTAAAATCCAA 
               
               
                   
               
               
                 AAAGCACATACAAAAATCCATGAATGATGTCTAAGTACTCACTTATAAAGTAGA 
               
               
                   
               
               
                 AGACATTCATTATTATATCAAATTTTTAAATGCTCAGTACTATTTGACCATTTAAA 
               
               
                   
               
               
                 AATTTTGTATTCAAACTACCAGTGAAAGCCCTACCTAGAAGGTATACTCAGTGAT 
               
               
                   
               
               
                 AAGTTTTGTAGCTCCAAATCTTCTAATAGTGAGTGTAACCCCAAAATAAAAGGCT 
               
               
                   
               
               
                 GACAGGTAAGTCGAGAATACTCACTTAATTCTGGTAAGAAAGCAACCCATTTGT 
               
               
                   
               
               
                 ACTTGTATTTACCAGCAATCCTTAAAATGAAGCTTCCTACTAACTCAATAGCAAT 
               
               
                   
               
               
                 AAGACAATAGTGAATGTTTAATGAAAACAGTATTTTATAAATACTTTAATAAAAA 
               
               
                   
               
               
                 GGATTGTGATGAAGAACAATCTATTTATATTTGTTATTTGTTTTTAATTCCAATAA 
               
               
                   
               
               
                 AAATAATTTTTAAAATTACAGAAAAAAGTTATTAAGAACCATGCTTTTAAATTTA 
               
               
                   
               
               
                 AAATGATTTTTTAAATTTATTCCTGTCTTTTTCTACAAAGAAAGCATACATTAAGC 
               
               
                   
               
               
                 AAATACCAAAGGCCAGGTTTACATTTGAAGAAAGTGACATTATTATTACTCAAGT 
               
               
                   
               
               
                 CTCTAGGAATACTTAACACATCTCTTGACTGTATATGGATGTTAATAAATAGCTG 
               
               
                   
               
               
                 ACAGTAAAGTTTATCCATATAAAGACTTGCAAATATTCCTCTACCAATGACGAGA 
               
               
                   
               
               
                 CTTTAAAATATCTATAATAATGTAACACATTTCACTGGTGAAACATGTCTTGTCAT 
               
               
                   
               
               
                 ATGCATTATAGAAAGGATAATCAGACTTTCAGTTATATTAATATTTTTAACATTTT 
               
               
                   
               
               
                 TGTGCACATAGCTATCTTCAATAAAATTGTTTTAAAAGGTATTATTTTAAGATAC 
               
               
                   
               
               
                 ACTAAAATGATCAAGGGATTCAAGACTAAACAACTCAATTAGTTGCACCAATAA 
               
               
                   
               
               
                 AAAACACTTAAAAAAACTGTCAGTGTCCAACCTGTACTTAATAACTCACAGATTT 
               
               
                   
               
               
                 TTAAAACTTTTCTTTTCAGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCA 
               
               
                   
               
               
                 TAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACA 
               
               
                   
               
               
                 ACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATAC 
               
               
                   
               
               
                 GCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAA 
               
               
                   
               
               
                 GATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAG 
               
               
                   
               
               
                 AGGGTTATTCAGGTATCTTTTTCTGATACCAATACTTTATTTTCATATCTTCAAAG 
               
               
                   
               
               
                 TATCTTCCCACATTATTAGCTATTATCTGCAATGACAACTTTTAAAAATCCGAATC 
               
               
                   
               
               
                 CCAAATAAGCGTTTTCTCTCTAGACGAAAACCTCTTAACTATAATGAAAGTGTTC 
               
               
                   
               
               
                 ATTCTAGTTCAATCAGGTATTTTACCTCTAATCTTCCTCAGATTTTCTATTTTTTGG 
               
               
                   
               
               
                 TAGTGTATAGATTATTTATACAGATTATTTAAAATTGGGACTTATACAGATTATTT 
               
               
                   
               
               
                 AAAACTGGGATACATGCATCTAAAACACTGTAATATTTATAAGAAAGGAAGATA 
               
               
                   
               
               
                 AACTTACGGGGAAATACAGTAACAGTAACTACATACGAGTCTGTACCCATTAAA 
               
               
                   
               
               
                 TTGCATATCTATCTCCTTTAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAA 
               
               
                   
               
               
                 CAACCTAAATGGTAAATATAACAAACCAAGAGCAAAATCTAAGCCAGAGAGGA 
               
               
                   
               
               
                 GAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAA 
               
               
                   
               
               
                 CCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAATGAACTGAGGCAA 
               
               
                   
               
               
                 ATTTAAAAGGCAATAATTTAAACATTAACCTCATTCCAAGTTAATGTGGTCTAAT 
               
               
                   
               
               
                 AATCTGGTATTAAATCCTTAAGAGAAAGCTTGAGAAATAGATTTTTTTTATCTTA 
               
               
                   
               
               
                 AAGTCACTGTCTATTTAAGATTAAACATACAATCACATAACCTTAAAGAATACCG 
               
               
                   
               
               
                 TTTACATTTCTCAATCAAAATTCTTATAATACTATTTGTTTTAAATTTTGTGATGTG 
               
               
                   
               
               
                 GGAATCAATTTTAGATGGTCACAATCTAGATTATAATCAATAGGTGAACTTATTA 
               
               
                   
               
               
                 AATAACTTTTCTAAATAAAAAATTTAGAGACTTTTATTTTAAAAGGCATCATATG 
               
               
                   
               
               
                 AGCTAATATCACAACTTTCCCAGTTTAAAAAACTAGTACTCTTGTTAAAACTCTA 
               
               
                   
               
               
                 AACTTGACTAAATACAGAGGACTGGTAATTGTACAGTTCTTAAATGTTGTAGTAT 
               
               
                   
               
               
                 TAATTTCAAAACTAAAAATCGTCAGCACAGAGTATGTGTAAAAATCTGTAATACA 
               
               
                   
               
               
                 AATTTTTAAACTGATGCTTCATTTTGCTACAAAATAATTTGGAGTAAATGTTTGAT 
               
               
                   
               
               
                 ATGATTTATTTATGAAACCTAATGAAGCAGAATTAAATACTGTATTAAAATAAGT 
               
               
                   
               
               
                 TCGCTGTCTTTAAACAAATGGAGATGACTACTAAGTCACATTGACTTTAACATGA 
               
               
                   
               
               
                 GGTATCACTATACCTTATTTGTTAAAATATATACTGTATACATTTTATATATTTTA 
               
               
                   
               
               
                 ACACTTAATACTATGAAAACAAATAATTGTAAAGGAATCTTGTCAGATTACAGTA 
               
               
                   
               
               
                 AGAATGAACATATTTGTGGCATCGAGTTAAAGTTTATATTTCCCCTAAATATGCT 
               
               
                   
               
               
                 GTGATTCTAATACATTCGTGTAGGTTTTCAAGTAGAAATAAACCTCGTAACAAGT 
               
               
                   
               
               
                 TACTGAACGTTTAAACAGCCTGACAAGCATGTATATATGTTTAAAATTCAATAAA 
               
               
                   
               
               
                 CAAAGACCCAGTCCCTAAATTATAGAAATTTAAATTATTCTTGCATGTTTATCGA 
               
               
                   
               
               
                 CATCACAACAGATCCCTAAATCCCTAAATCCCTAAAGATTAGATACAAATTTTTT 
               
               
                   
               
               
                 ACCACAGTATCACTTGTCAGAATTTATTTTTAAATATGATTTTTTAAAACTGCCAG 
               
               
                   
               
               
                 TAAGAAATTTTAAATTAAACCCATTTGTTAAAGGATATAGTGCCCAAGTTATATG 
               
               
                   
               
               
                 GTGACCTACCTTTGTCAATACTTAGCATTATGTATTTCAAATTATCCAATATACAT 
               
               
                   
               
               
                 GTCATATATATTTTTATATGTCACATATATAAAAGATATGTATGATCTATGTGAAT 
               
               
                   
               
               
                 CCTAAGTAAATATTTTGTTCCAGAAAAGTACAAAATAATAAAGGTAAAAATAAT 
               
               
                   
               
               
                 CTATAATTTTCAGGACCACAGACTAAGCTGTCGAAATTAACGCTGATTTTTTTAG 
               
               
                   
               
               
                 GGCCAGAATACCAAAATGGCTCCTCTCTTCCCCCAAAATTGGACAATTTCAAATG 
               
               
                   
               
               
                 CAAAATAATTCATTATTTAATATATGAGTTGCTTCCTCTATTTGGTTTCCTTAAAA 
               
               
                   
               
               
                 AAAAAAAAAACTCTCATAGGACATGTTTCATTTTGTTCCTTTCAGGAGTAGTAAA 
               
               
                   
               
               
                 TTAGACGTTTTCCCCATATAAAGCTTTTTTCTACCAGAAAGATACTTCTGGTAGAA 
               
               
                   
               
               
                 GAAGAGAAAGGAGCTCTTTATGGTTCACACGACTGTCTCCTGTCCTAACTACTTT 
               
               
                   
               
               
                 GCTTAAAGTGCTCAAATTCCATCACTACTCACAGTTGTCTAATCTAAGTCTAATC 
               
               
                   
               
               
                 CCCTTTGATCTCTCAGACTACCTTCCCTTTTATCTCTCTACTACTTAATAATAAGA 
               
               
                   
               
               
                 ATATCTTTTTTTCAAACTTGACCTTCATTTTGCTTTCACAATACTATACTCTCCATG 
               
               
                   
               
               
                 GATTATCCCTTATCTGAATCCATCTTTATAACCCTATTCCTTTCTCATATTTAGTAC 
               
               
                   
               
               
                 TGTGGGCCAATGGACAACCTTCAATCATCTTTTCTACACTGACCCTCAGACATTC 
               
               
                   
               
               
                 TATCTGCTCTCACGGACTCCTTTATTTACCATGAATAAAGTTCCAAAATCTACATA 
               
               
                   
               
               
                 TTCATCCCAAGTCTCTTTCCAGTTCCCCTTCTTACATTGCCTATTTGCCATTTCTCC 
               
               
                   
               
               
                 CTTCAATACCCTATACTTCACTCAAATTCAACATACCAAAAATAAAAGGCCAGGC 
               
               
                   
               
               
                 ACGGTGGCTCACACCTGTAATCCCAGGACTTTGGGAGGCTGAGGCAGGTGGATC 
               
               
                   
               
               
                 ACCTGAGGTCAGGAGTCTGACCAGCCTGACCAATATGGTGAAACCCCGTCTCTAC 
               
               
                   
               
               
                 CTAAAATACAAAAATTAGCCAGGCGTGGTGGCATGTGCCTACAGTCCCAGCTAC 
               
               
                   
               
               
                 TCAAGAGGCTGAGACAGGAGAATCGCTTGAACCCAGGAGGCGGAGGTTGCAGTG 
               
               
                   
               
               
                 AGCTGAGATCACACCAATGCACTGGGTGACAGAACAAGACTGACTCAAAAAAAA 
               
               
                   
               
               
                 ATAAATAACAAATTCCCCAGCCCCTTACTGCTACTGCTATCCCTTTCTACCCACCT 
               
               
                   
               
               
                 TTCCCTCCTTTATACTCTTTCACACCATCTTCCTCACTTCTTTATATCCATTAATAT 
               
               
                   
               
               
                 GACCAGCATGTTCCCAGTCACAGAAGCCTGGAACCCGGAAGACATCTCTGGCTTT 
               
               
                   
               
               
                 TCACTCAACTTTGTAAACTACCTCTTTTGTATCATAAGCCACCAAGTTCAATACA 
               
               
                   
               
               
                 ATCTTCTCTTGAAACGTCTCTTAATCTTATAAGCTTTCTTCCCCAAAGACTGTCTT 
               
               
                   
               
               
                 TAACTTCAGTGCTAGATTATATAAGT  
               
               
                   
               
               
                 AAD34156.1 human angiopoietin-related protein 3 
               
               
                 (SEQ ID NO: 8) 
                   
               
               
                 MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKD 
                   
               
               
                   
               
               
                 FVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMS 
               
               
                   
               
               
                 LELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIK 
               
               
                   
               
               
                 DLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRN 
               
               
                   
               
               
                 VKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQN 
               
               
                   
               
               
                 FNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYL 
               
               
                   
               
               
                 GNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHD 
               
               
                   
               
               
                 ECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 
               
            
           
         
       
     
     Lipoprotein (a) (LPA) 
     In some embodiments, the target gene for modification using the compositions and methods disclosed herein is gene encoding lipoprotein a (LPA). LPA is a low-density lipoprotein variant. Genetic and epidemiologic studies have identified LPA as a risk factor for atherosclerosis and related diseases, such as coronary heart disease and stroke. LPA concentrations vary more than one thousand times between individuals: from &lt;0.2 to &gt;200 mg/dL. This range of concentrations is observed in all populations studied by scientists so far. The mean and median concentrations between different world populations show distinct particularities, the main being the two to threefold higher LPA plasma concentration of populations of African descent compared to Asian, Oceanic, or European populations. High LPA in blood correlates with coronary heart disease (CHD), cardiovascular disease (CVD), atherosclerosis, thrombosis, and stroke. Individuals without LPA or with very low LPA levels seem to be healthy. Thus, plasma LPA is not vital, at least under normal environmental conditions. Since apo(a)/LPA appeared rather recently in mammalian evolution—only old world monkeys and humans have been shown to harbor LPA—its function might not be vital, but just evolutionarily advantageous under certain environmental conditions, e.g. in case of exposure to certain infectious diseases. 
     An exemplary LPA amino acid sequence encoded by Human reference sequence NG_016147.1 is provided below: 
     
       
         
           
               
               
            
               
                 &gt;sp|P08519|APOA_HUMAN Apolipoprotein(a) OS =  Homo sapiens   
                   
               
               
                 OX = 9606 GN = LPA PE = 1 SV = 1 
               
               
                 (SEQ ID NO: 64) 
                   
               
               
                 MEHKEVVLLLLLFLKSAAPEQSHVVQDCYHGDGQSYRGTYSTTVTGRTCQAWSSM 
                   
               
               
                   
               
               
                 TPHQHNRTTENYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEG 
               
               
                   
               
               
                 TAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWS 
               
               
                   
               
               
                 SMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAE 
               
               
                   
               
               
                 GTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQA 
               
               
                   
               
               
                 WSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCS 
               
               
                   
               
               
                 DAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTC 
               
               
                   
               
               
                 QAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQ 
               
               
                   
               
               
                 CSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGR 
               
               
                   
               
               
                 TCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNL 
               
               
                   
               
               
                 TQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVT 
               
               
                   
               
               
                 GRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYC 
               
               
                   
               
               
                 NLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTT 
               
               
                   
               
               
                 VTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWE 
               
               
                   
               
               
                 YCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYS 
               
               
                   
               
               
                 TTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVR 
               
               
                   
               
               
                 WEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRG 
               
               
                   
               
               
                 TYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPG 
               
               
                   
               
               
                 VRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSY 
               
               
                   
               
               
                 RGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRD 
               
               
                   
               
               
                 PGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQ 
               
               
                   
               
               
                 SYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYT 
               
               
                   
               
               
                 RDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGN 
               
               
                   
               
               
                 GQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYC 
               
               
                   
               
               
                 YTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYH 
               
               
                   
               
               
                 GNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAP 
               
               
                   
               
               
                 YCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQEC 
               
               
                   
               
               
                 YHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAV 
               
               
                   
               
               
                 AAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGV 
               
               
                   
               
               
                 QECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPD 
               
               
                   
               
               
                 AVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRP 
               
               
                   
               
               
                 GVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCR 
               
               
                   
               
               
                 NPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTE 
               
               
                   
               
               
                 QRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNY 
               
               
                   
               
               
                 CRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAP 
               
               
                   
               
               
                 TEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIM 
               
               
                   
               
               
                 NYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQ 
               
               
                   
               
               
                 APTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGL 
               
               
                   
               
               
                 IMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPS 
               
               
                   
               
               
                 EQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNA 
               
               
                   
               
               
                 GLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEA 
               
               
                   
               
               
                 PSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYP 
               
               
                   
               
               
                 NAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSL 
               
               
                   
               
               
                 EAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEY 
               
               
                   
               
               
                 YPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVP 
               
               
                   
               
               
                 SLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPE 
               
               
                   
               
               
                 YYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTP 
               
               
                   
               
               
                 VPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSR 
               
               
                   
               
               
                 TPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPT 
               
               
                   
               
               
                 VTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHS 
               
               
                   
               
               
                 HSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVA 
               
               
                   
               
               
                 PPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTP 
               
               
                   
               
               
                 HSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTA 
               
               
                   
               
               
                 VAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSS 
               
               
                   
               
               
                 MTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAE 
               
               
                   
               
               
                 GTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQA 
               
               
                   
               
               
                 WSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCS 
               
               
                   
               
               
                 DAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTC 
               
               
                   
               
               
                 QAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQ 
               
               
                   
               
               
                 CSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGR 
               
               
                   
               
               
                 TCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDPVAAPYCYTRDPSVRWEYCNLT 
               
               
                   
               
               
                 QCSDAEGTAVAPPTITPIPSLEAPSEQAPTEQRPGVQECYHGNGQSYQGTYFITVTGR 
               
               
                   
               
               
                 TCQAWSSMTPHSHSRTPAYYPNAGLIKNYCRNPDPVAAPWCYTTDPSVRWEYCNLT 
               
               
                   
               
               
                 RCSDAEWTAFVPPNVILAPSLEAFFEQALTEETPGVQDCYYHYGQSYRGTYSTTVTG 
               
               
                   
               
               
                 RTCQAWSSMTPHQHSRTPENYPNAGLTRNYCRNPDAEIRPWCYTMDPSVRWEYCN 
               
               
                   
               
               
                 LTQCLVTESSVLATLTVVPDPSTEASSEEAPTEQSPGVQDCYHGDGQSYRGSFSTTVT 
               
               
                   
               
               
                 GRTCQSWSSMTPHWHQRTTEYYPNGGLTRNYCRNPDAEISPWCYTMDPNVRWEYC 
               
               
                   
               
               
                 NLTQCPVTESSVLATSTAVSEQAPTEQSPTVQDCYHGDGQSYRGSFSTTVTGRTCQS 
               
               
                   
               
               
                 WSSMTPHWHQRTTEYYPNGGLTRNYCRNPDAEIRPWCYTMDPSVRWEYCNLTQCP 
               
               
                   
               
               
                 VMESTLLTTPTVVPVPSTELPSEEAPTENSTGVQDCYRGDGQSYRGTLSTTITGRTCQ 
               
               
                   
               
               
                 SWSSMTPHWHRRIPLYYPNAGLTRNYCRNPDAEIRPWCYTMDPSVRWEYCNLTRCP 
               
               
                   
               
               
                 VTESSVLTTPTVAPVPSTEAPSEQAPPEKSPVVQDCYHGDGRSYRGISSTTVTGRTCQ 
               
               
                   
               
               
                 SWSSMIPHWHQRTPENYPNAGLTENYCRNPDSGKQPWCYTTDPCVRWEYCNLTQC 
               
               
                   
               
               
                 SETESGVLETPTVVPVPSMEAHSEAAPTEQTPVVRQCYHGNGQSYRGTFSTTVTGRT 
               
               
                   
               
               
                 CQSWSSMTPHRHQRTPENYPNDGLTMNYCRNPDADTGPWCFTMDPSIRWEYCNLT 
               
               
                   
               
               
                 RCSDTEGTVVAPPTVIQVPSLGPPSEQDCMFGNGKGYRGKKATTVTGTPCQEWAAQ 
               
               
                   
               
               
                 EPHRHSTFIPGTNKWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCASSSF 
               
               
                   
               
               
                 DCGKPQVEPKKCPGSIVGGCVAHPHSWPWQVSLRTRFGKHFCGGTLISPEWVLTAA 
               
               
                   
               
               
                 HCLKKSSRPSSYKVILGAHQEVNLESHVQEIEVSRLFLEPTQADIALLKLSRPAVITDK 
               
               
                   
               
               
                 VMPACLPSPDYMVTARTECYITGWGETQGTFGTGLLKEAQLLVIENEVCNHYKYIC 
               
               
                   
               
               
                 AEHLARGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYARVSRFVT 
               
               
                   
               
               
                 WIEGMMRNN  
               
            
           
         
       
     
     Base Editor Protein-2RNA Complexes 
     In another aspect, provided herein is a complex comprising the single guide RNA as provided herein in complex with the base editor fusion protein, e.g. the adenosine base editor fusion protein, wherein the complex comprises increased stability as compared to a complex with an unmodified single guide RNA and a base editor protein, wherein the stability is measured by half life of the complex ex vivo or in vitro. 
     In some embodiments, the complex comprises increased stability as compared to a complex with an unmodified single guide RNA and a Cas9 protein. In some embodiments, the complex comprises increased stability by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least 10000%, at least 20000%, at least 30000%, at least 40000%, at least 50000%, at least 60000%, at least 70000%, at least 80000%, at least 90000%, or at least 100000% as compared to a complex with an unmodified single guide RNA and a Cas9 protein. In some embodiments, the complex comprises increased stability by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared to a complex with an unmodified single guide RNA and a Cas9 protein. 
     In some embodiments, wherein the stability of the complex is measured by half life of the complex. In some embodiments, wherein the stability of the complex is measured by half life of the complex ex vivo. In some embodiments, wherein the stability of the complex is measured by half life of the complex in vitro. 
     In some embodiments, the complex comprises increased half life by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least 10000%, at least 20000%, at least 30000%, at least 40000%, at least 50000%, at least 60000%, at least 70000%, at least 80000%, at least 90000%, or at least 100000% as compared to a complex with an unmodified single guide RNA and a Cas9 protein wherein half life of the complex is measured ex vivo. In some embodiments, the single guide RNA exhibits increased half life of the complex by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared to a complex with an unmodified single guide RNA and a Cas9 protein, wherein half life of the complex is measured ex vivo. 
     In some embodiments, the complex comprises increased half life when measured in vitro by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least 10000%, at least 20000%, at least 30000%, at least 40000%, at least 50000%, at least 60000%, at least 70000%, at least 80000%, at least 90000%, or at least 100000% as compared to a complex with an unmodified single guide RNA and a Cas9 protein, wherein half life of the complex is measured in vitro. In some embodiments, the single guide RNA exhibits increased half life of the complex by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared to a complex with an unmodified single guide RNA and a Cas9 protein, wherein half life of the complex is measured in vitro. 
     In another aspect, provided herein is a cell comprising the complex as provided herein. In some embodiments, the cell may be an in vitro cell. In some embodiments, the cell may be an ex vivo cell. In some embodiments, the cell may be an in vivo cell. In some embodiments, the cell may be an isolated cell. 
     Gene Modification Compositions 
     In another aspect, provided herein is a composition for gene modification comprising the single guide RNA as provided herein and a base editor protein or a nucleic acid sequence encoding the base editor protein. In some embodiments, the composition further comprises a vector that comprises the nucleic acid sequence encoding the base editor protein. 
     In some embodiments, the nucleic acid sequence may be a DNA, an RNA or mRNA, or a modified nucleic acid sequence. In some embodiments, the vector may be an expression vector. In some embodiments, the nucleic acid is operatively linked to a promoter of the vector. In some embodiments, the vector is a plasmid or a viral vector. 
     Therapeutics and Methods of Treatment 
     In some aspects, provided herein is a method for treating or preventing a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (i) a guide polynucleotide or a nucleic acid encoding same, and (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same. In some embodiments, the base editor system comprises a guide polynucleotide. In some embodiments, the base editor system comprises a nucleic acid encoding a guide polynucleotide. In some embodiments, the base editor system comprises a base editor fusion protein comprising a programmable DNA binding domain and a deaminase. In some embodiments, the base editor system comprises a nucleic acid encoding a base editor fusion protein comprising a programmable DNA binding domain and a deaminase. 
     In some embodiments, the guide polynucleotide directs the base editor system to effect a nucleobase alteration in a ANGPTL3 gene in the subject. In some embodiments, the guide polynucleotide directs the base editor system to effect a nucleobase alteration in a PCSK9gene in the subject. 
     In some embodiments, the base alteration occurs in at least 35% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing, thereby treating or preventing the condition in the subject. 
     In some aspects, provided herein is a method for treating or preventing a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a first composition, comprising (i) a guide polynucleotide or a nucleic acid encoding same, and (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same, wherein the guide polynucleotide directs the base editor system to effect a nucleobase alteration in a PCSK9 gene in the subject, wherein the base alteration occurs in at least 35% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing; and a second composition, comprising (i) a guide polynucleotide or a nucleic acid encoding same, and (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same, wherein the guide polynucleotide directs the base editor system to effect a nucleobase alteration in a ANGPTL3 gene in the subject, wherein the base alteration occurs in at least 35% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. 
     In some embodiments, the first composition and the second composition are administered sequentially. In some embodiment, the first composition is administered for one or more doses, then the second composition is administered for one or more doses. In some embodiments, the first composition and the second composition interspersed. In some embodiments, the first composition and the second composition are administered concurrently. In some embodiments, the first composition and the second composition are administered for one or more doses. In some embodiments, the first composition and the second composition are administered over and interval of over an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours. In some embodiments, the first composition and the second composition are administered over and interval of over an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, the first composition and the second composition are administered over and interval of over an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks. 
     In some embodiments, the base alteration occurs in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 10%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 1%-90%, 5%-85%, 10%-80%, 15%-75%, 20%-70%, 25%-65%, 30%-60%, 35%-55%, or 40%-50% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 100% of whole liver cells in the subject as measured by next generation sequencing or Sanger sequencing. 
     In some embodiments, the base alteration occurs in hepatocytes in the subject. In some embodiments, the base alteration occurs in at least 30% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in hepatocytes in the subject. In some embodiments, the base alteration occurs in at least 35% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7% 99.8%, or 99.9% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in at most 1%, 2%, 3%, 4%, 5%0, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 100%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 1%-90%, 5%-85%, 10%-80%, 15%-75%, 20%-70%, 25%-65%, 30%-60%, 35%-55%, or 40%-50% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. In some embodiments, the base alteration occurs in 100% of hepatocytes in the subject as measured by next generation sequencing or Sanger sequencing. 
     In some embodiments, the base alteration occurred in whole liver cells in the subject is measured by next generation sequencing. In some embodiments, the base alteration occurred in whole liver cells in the subject is measured by Sanger sequencing. In some embodiments, the base alteration occurred in hepatocytes in the subject is measured by next generation sequencing. In some embodiments, the base alteration occurred in hepatocytes in the subject is measured by Sanger sequencing. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering (i) the guide polynucleotide and (ii) the nucleic acid encoding the base editor fusion protein to the subject. In some embodiments, the base editor system comprises a guide polynucleotide. In some embodiments, the base editor system comprises a nucleic acid encoding a guide polynucleotide. 
     In some embodiments, the nucleic acid encoding the base editor fusion protein is a mRNA. In some embodiments, the mRNA generates the base editor fusion protein upon translation in the subject after the administration. In some embodiments, the base editor fusion protein forms a RNP complex in the subject. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein further comprises administering a lipid nanoparticle (LNP) enclosing a guide polynucleotide or a nucleic acid encoding the guide polynucleotide (i). In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein further comprises administering a second LNP enclosing a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same (ii). In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein further comprises administering a LNP enclosing a guide polynucleotide or a nucleic acid encoding the guide polynucleotide (i) and a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same (ii). In some embodiments, the RNP complex is formed upon uptake of the LNP or the second LNP by a liver cell in the subject. 
     In some embodiments, the ratio of a guide polynucleotide or a nucleic acid encoding the guide polynucleotide (i) and a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same (ii) is about 1:10 to about 10:1 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1:1, 1.5:1, 2:1, 3:1, 4:1, 1:1.5, 1:2, 1:3, or 1:4 by weight. In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 500:1 to about 1:500. 
     In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1000:1 to about 1:1000 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. 
     In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 10000:1 to about 1:10000. In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. 
     In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 10:1 to about 1:10 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 4:1, 3;1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, or 1:4 by weight. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 500:1 to about 1:500. 
     In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1000:1 to about 1:1000 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3: 1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. 
     In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 10000:1 to about 1:10000. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at least about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of a nucleic acid encoding the guide polynucleotide and the nucleic acid encoding the base editor fusion protein is at most about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1: 14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. 
     In some embodiments, the nucleobase alteration results in a reduction of at least 35% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. 
     In some embodiments, the nucleobase alteration results in a reduction of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. 
     In some embodiments, the nucleobase alteration results in a reduction of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 10%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 31%-99.9%, 32%-99.9%, 33%-99.9%, 34%-99.9%, 35%-99.9%, 36%-99.9%, 37%-99.9%, 38%-99.9%, 39%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-79%, 1%-78%, 1%-77%, 1%-76%, 1%-75%, 1%-74%, 1%-73%, 1%-72%, 1%-71%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-39%, 1%-38%, 1%-37%, 1%-36%, 1%-35%, 1%-34%, 1%-33%, 1%-32%, 1%-31%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%, 25%-90%, 30%-85%, 31%-80%, 32%-79%, 33%-78%, 34%-77%, 35%-76%, 36%-76%, 37%-75%, 38%-74%, 39%-73%, 40%-72%, 45%-71%, 50%-70%, or 55%-65% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 100% in blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. 
     In some embodiments, the nucleobase alteration results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, 1000% less blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less blood PCSK9 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. 
     In some embodiments, the reduction of blood PCSK9 protein level or the blood PCSK9 protein level in the subject as compared to prior to the administration is measured by ELISA (enzyme-linked immunosorbent assay). In some embodiments, the reduction of blood PCSK9 protein level or the blood PCSK9 protein level in the subject as compared to prior to the administration is measured by Western blot analysis. In some embodiments, the reduction of blood PCSK9 protein level or the blood PCSK9 protein level in the subject as compared to prior to the administration is measured by LC-MS/MS (liquid chromatography-tandem mass spectrometry). 
     In some embodiments, the nucleobase alteration results in a reduction of at least 35% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. 
     In some embodiments, the nucleobase alteration results in a reduction of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. 
     In some embodiments, the nucleobase alteration results in a reduction of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 10%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 31%-99.9%, 32%-99.9%, 33%-99.9%, 34%-99.9%, 35%-99.9%, 36%-99.9%, 37%-99.9%, 38%-99.9%, 39%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-79%, 1%-78%, 1%-77%, 1%-76%, 1%-75%, 1%-74%, 1%-73%, 1%-72%, 1%-71%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-39%, 1%-38%, 1%-37%, 1%-36%, 1%-35%, 1%-34%, 1%-33%, 1%-32%, 1%-31%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%, 25%-90%, 30%-85%, 31%-80%, 32%-79%, 33%-78%, 34%-77%, 35%-76%, 36%-76%, 37%-75%, 38%-74%, 39%-73%, 40%-72%, 45%-71%, 50%-70%, or 55%-65% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in a reduction of 100% in blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. 
     In some embodiments, the nucleobase alteration results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, 1000% less blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobase alteration results in at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less blood ANGPTL3 protein level in the subject as compared to prior to the administration as measured by ELISA, Western blots, or LC-MS/MS. 
     In some embodiments, the reduction of blood ANGPTL3 protein level or the blood ANGPTL3 protein level in the subject as compared to prior to the administration is measured by ELISA (enzyme-linked immunosorbent assay). In some embodiments, the reduction of blood ANGPTL3 protein level or the blood ANGPTL3 protein level in the subject as compared to prior to the administration is measured by Western blot analysis. In some embodiments, the reduction of blood ANGPTL3 protein level or the blood ANGPTL3 protein level in the subject as compared to prior to the administration is measured by LC-MS/MS (liquid chromatography-tandem mass spectrometry). 
     In some embodiments, the nucleobase alteration results in a reduction of at least 35% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. 
     In some embodiments, the nucleobase alteration results in a reduction of at least 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 10%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%, 25%-90%, 30%-85%, 35%-80%, 40%-75%, 45%-70%, 50%-65%, or 55%-60% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 100% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1%, 2%, %13%, %14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, 1000% less blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration. 
     In some embodiments, the nucleobase alteration results in a reduction of at least 35% in blood triglyceride level in the subject as compared to prior to the administration. 
     In some embodiments, the nucleobase alteration results in a reduction of at least 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 10%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%, 25%-90%, 30%-85%, 35%-80%, 40%-75%, 45%-70%, 50%-65%, or 55%-60% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in a reduction of 100% in blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, 1000% less blood triglyceride level in the subject as compared to prior to the administration. In some embodiments, the nucleobase alteration results in at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less blood triglyceride level in the subject as compared to prior to the administration. 
     In some embodiments, the blood triglyceride level or the reduction of blood triglyceride level in the subject as compared to prior to the administration is measured by any standard technique. In some embodiments, the blood low-density lipoprotein cholesterol (LDL-C) level or the reduction of blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration is measured by any standard technique. For example, a clinical analyzer instrument may be used to measure a ‘lipid panel’ in serum samples which entails the direct measurement of cholesterol (total C), triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C) enzymatically. Reagent kits specific for each analyte contain buffers, calibrators, blanks and controls. As used in the present disclosure, cholesterol, triglycerides and HDL-C may be quantified using absorbance measurements of specific enzymatic reaction products. LDL-C may be determined indirectly. In some instances, most of circulating cholesterol can be found in three major lipoprotein fractions: very low-density lipoproteins (VLDL), LDL and HDL. In some embodiments, total circulating cholesterol may be estimated with the formula [Total C]=[VLDL-C]+[LDL-C]+[HDL-C]. Thus the LDL-C can be calculated from measured values of total cholesterol, triglycerides and HDL-C according to the relationship: [LDL-C]=[total C]− [HDL-C]-[TG]/5, where [TG]/5 is an estimate of VLDL-cholesterol. A reagent kit specific for triglycerides containing buffers, calibrators, blanks and controls. As used herein, serum samples from the study may be analyzed and triglycerides may be measured using a series of coupled enzymatic reactions. In some embodiments, H 2 O 2  may be used to quantify the analyte.as the end product of the last one and its absorbance at 500 nm, and the color intensity is proportional to triglyceride concentrations. 
     In some embodiments, the guide polynucleotide is a guide RNA. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with 0, 1, or 2 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with no mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with 1 mismatch. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with 2 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with 3 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with 4 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the ANGPTL3 gene with 5 mismatches. 
     In some embodiments, the guide polynucleotide is a guide RNA. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with 0, 1, or 2 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with no mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with 1 mismatch. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with 2 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with 3 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with 4 mismatches. In some embodiments, the guide RNA comprises a spacer sequence that binds to the complementary strand of a protospacer sequence of the PCSK9 gene with 5 mismatches. 
     In some embodiments, the nucleobase alteration is outside of the protospacer sequence in less than 1% of whole liver cells in the subject as measured by net nucleobase editing. In some embodiments, the nucleobase alteration is outside of the protospacer sequence in less than 1% of hepatocytes in the subject as measured by net nucleobase editing. In some embodiments, the nucleobase alteration is only within the protospacer sequence as measured by net nucleobase editing. 
     In some embodiments, the nucleobase alteration is outside of the protospacer sequence in less than 0.01%. 0.02%, 0.03% 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%0, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%0, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 65%, 80%, 85%, 90% of whole liver cells in the subject as measured by net nucleobase editing. In some embodiments, the nucleobase alteration is outside of the protospacer sequence in less than 0.01%. 0.02%, 0.03% 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%0, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 65%, 80%, 85%, 90% of hepatocytes in the subject as measured by net nucleobase editing. In some embodiments, the nucleobase alteration is outside of the protospacer sequence in less than 0.01%. 0.02%, 0.03% 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%0, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 65%, 80%, 85%, 90% of cells in the subject as measured by net nucleobase editing. 
     In some embodiments, the administration is via intravenous infusion. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises sequential administration of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding the guide polynucleotide and the second LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises concurrent administration of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding the guide polynucleotide and the second LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same followed by staggered doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding the guide polynucleotide over an interval of 1, 2, 3, 4, 5, 6, or 7 days. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same followed by staggered doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding the guide polynucleotide over an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same followed by staggered doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding the guide polynucleotide over an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same followed by staggered doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding the guide polynucleotide over an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months. 
     The method as described herein may be used for treating or preventing a condition in a subject in need thereof. In some embodiments, the method comprises administering a single dose of a LNP enclosing (i) a guide polynucleotide, e.g., a guide RNA, or a nucleic acid encoding the guide polynucleotide and (ii) a base editor fusion protein or a nucleic acid encoding the base editor fusion protein, e.g., an mRNA encoding the base editor fusion protein. For example, the method may comprise administering an LNP enclosing (i) a guide RNA and (i) an mRNA encoding the base editor fusion protein. The LNP may be administered at a single dose or multiple doses. In other embodiments, the method comprises administering a LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding the guide polynucleotide, and a second LNP enclosing (ii) the base editor fusion protein or a nucleic acid encoding the base editor fusion protein. The LNP may be administered at a single dose or multiple doses. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 1 day, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 1 day, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 1 day, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 1 day, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 1 day, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 1 day, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 1 day, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 2 days, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 2 days, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 2 days, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 2 days, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 2 days, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 2 days, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 2 days, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 3 days, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 3 days, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 3 days, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 3 days, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 3 days, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 3 days, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 3 days, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 4 days, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 4 days, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 4 days, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 4 days, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 4 days, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 4 days, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 4 days, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 5 days, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 5 days, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 5 days, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 5 days, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 5 days, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 5 days, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 5 days, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 6 days, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 6 days, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 6 days, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 6 days, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 6 days, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 6 days, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 6 days, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 7 days, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 7 days, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 7 days, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 7 days, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 7 days, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 7 days, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same after 7 days, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 1 day, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 1 day, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 1 day, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 1 day, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 1 day, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 1 day, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 1 day, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 2 days, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 2 days, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 2 days, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 2 days, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 2 days, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 2 days, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 2 days, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 3 days, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 3 days, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 3 days, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 3 days, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 3 days, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 3 days, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 3 days, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 4 days, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 4 days, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 4 days, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 4 days, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 4 days, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 4 days, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 4 days, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 5 days, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 5 days, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 5 days, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 5 days, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 5 days, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 5 days, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 5 days, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 6 days, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 6 days, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 6 days, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 6 days, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 6 days, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 6 days, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 6 days, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 7 days, and the multiple doses are given at 1 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 7 days, and the multiple doses are given at 2 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 7 days, and the multiple doses are given at 3 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 7 days, and the multiple doses are given at 4 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 7 days, and the multiple doses are given at 5 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 7 days, and the multiple doses are given at 6 day intervals. In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same and administering multiple doses of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding same after 7 days, and the multiple doses are given at 7 day intervals. 
     In some embodiments, the method for treating or preventing a condition in a subject in need thereof as described herein comprises administering a single dose of the LNP enclosing (i) a guide polynucleotide or a nucleic acid encoding the guide polynucleotide and (ii) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a nucleic acid encoding same. In some embodiments, the single dose of the LNP is at 0.3 mg/kg. In some embodiments, the single dose of the LNP is at 0.5 mg/kg. In some embodiments, the single dose of the LNP is at 1 mg/kg. In some embodiments, the single dose of the LNP is at 1 mg/kg. In some embodiments, the single dose of the LNP is at about 0.3 to about 3 mg/kg. 
     Methods for treatment of a condition as provided herein may comprise a treatment course of one or more treatments, with each treatment comprising a single dose or multiple doses of a base editor system, or a LNP enclosing one or more components of the base editor system. For example, a subject in need thereof may be administered a single dose of a LNP enclosing a mRNA encoding a base editor fusion protein and a guide RNA for a treatment. In some embodiments, a subject may be administered a treatment course of one or more treatments, wherein each treatment comprises one or more of the single doses of the LNP, for example, a subject may also be administered 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more doses of a LNP enclosing a mRNA encoding a base editor fusion protein and a guide RNA for a treatment. The subject may receive a treatment course of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more treatments, where each dose may be administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 24 months, 48 months or apart. A single dose for the treatment as described herein may comprise a LNP enclosing a guide RNA or a mRNA encoding the base editor fusion protein, or both. 
     In some embodiments, the single dose of the LNP comprises 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg, 3.4 mg/kg, 3.5 mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, 4.0 mg/kg, 4.1 mg/kg, 4.2 mg/kg, 4.3 mg/kg, 4.4 mg/kg, 4.5 mg/kg, 4.6 mg/kg, 4.7 mg/kg, 4.8 mg/kg, 4.9 mg/kg, 5.0 mg/kg, 5.1 mg/kg, 5.2 mg/kg, 5.3 mg/kg, 5.4 mg/kg, 5.5 mg/kg, 5.6 mg/kg, 5.7 mg/kg, 5.8 mg/kg, 5.9 mg/kg, 6.0 mg/kg, 6.1 mg/kg, 6.2 mg/kg, 6.3 mg/kg, 6.4 mg/kg, 6.5 mg/kg, 6.6 mg/kg, 6.7 mg/kg, 6.8 mg/kg, 6.9 mg/kg, 7.0 mg/kg, 7.1 mg/kg, 7.2 mg/kg, 7.3 mg/kg, 7.4 mg/kg, 7.5 mg/kg, 7.6 mg/kg, 7.7 mg/kg, 7.8 mg/kg, 7.9 mg/kg, 8.0 mg/kg, 8.1 mg/kg, 8.2 mg/kg, 8.3 mg/kg, 8.4 mg/kg, 8.5 mg/kg, 8.6 mg/kg, 8.7 mg/kg, 8.8 mg/kg, 8.9 mg/kg, 9.0 mg/kg, 9.1 mg/kg, 9.2 mg/kg, 9.3 mg/kg, 9.4 mg/kg, 9.5 mg/kg, 9.6 mg/kg, 9.7 mg/kg, 9.8 mg/kg, 9.9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 105 mg/kg, 110 mg/kg, 115 mg/kg, 120 mg/kg, 125 mg/kg, 130 mg/kg, 135 mg/kg, 140 mg/kg, 145 mg/kg, 150 mg/kg, 155 mg/kg, 160 mg/kg, 165 mg/kg, 170 mg/kg, 175 mg/kg, 180 mg/kg, 185 mg/kg, 190 mg/kg, 195 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, or 500 mg/kg. 
     In some embodiments, the condition is a atherosclerotic cardiovascular disease. In some embodiments, the condition is a cardiovascular disease, or diabetes. In some embodiments, the condition is a atherosclerotic vascular disease. 
     In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a monkey. In some embodiments, the subject is a human. In some embodiments, the deaminase is an adenine deaminase. In some embodiments, the nucleobase alteration is a A•T to G•C alteration. In some embodiments, the deaminase is an adenine deaminase and the nucleobase alteration is a A•T to G•C alteration. In some embodiments, the programmable DNA binding domain comprises a nuclease inactive Cas9. In some embodiments, the programmable DNA binding domain comprises a Cas9 nickase. In some embodiments, the programmable DNA binding domain comprises a Cas9. 
     In some embodiments, the nucleobase alteration is at a splice site of the PCSK9 gene. In some embodiments, the nucleobase alteration is at a splice donor site of the PCSK9 gene. In some embodiments, the splice donor site is at 5′ end of PCSK9 intron 1 as referenced in SEQ ID NO: 5. In some embodiments, the nucleobase alteration is at a splice acceptor site of the PCSK9 gene. In some embodiments, the nucleobase alteration results in a frame shift, a premature stop codon, a insertion or deletion in a transcript encoded by the PCSK9 gene. In some embodiments, the nucleobase alteration results in an aberrant transcript encoded by the PCSK9 gene. In some embodiments, the guide polynucleotide is a guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the guide RNA comprises a tracrRNA sequence. In some embodiments, the guide RNA comprises a chemical modification as set forth in Table 1 or Table 24. 
     In some embodiments, the nucleobase alteration is at a splice site of the ANGPTL3 gene. In some embodiments, the nucleobase alteration is at a splice donor site of the ANGPTL3 gene. In some embodiments, the splice donor site is at 5′ end of ANGPTL3 intron 6 as referenced in SEQ ID NO: 7. In some embodiments, the nucleobase alteration is at a splice acceptor site of the ANGPTL3 gene. In some embodiments, the nucleobase alteration results in a frame shift, a premature stop codon, a insertion or deletion in a transcript encoded by the ANGPTL3 gene. In some embodiments, the nucleobase alteration results in an aberrant transcript encoded by the ANGPTL3 gene. In some embodiments, the guide polynucleotide is a guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the guide RNA comprises a tracrRNA sequence. In some embodiments, the guide RNA comprises a chemical modification as set forth in Table 1 or Table 24. 
     In some embodiments, the guide RNA comprises a guide RNA sequence set forth in Table 1 or Table 24. In some embodiments, the guide RNA comprises the sequence 5′-5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUC cG UUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu-3′ (SEQ ID NO: 9), 5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUcc GU UAucAAcuugaaaaagugGcaccgagucggugcusususu-3′ (SEQ ID NO: 9), 5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUcc GUUAucAAcuuGaaaaagugGcaccgagucggugcusususu-3′ (SEQ ID NO: 9) (GA346), 5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUcc GUUAacAAcuugaaaaagugGcaccgagucggugcusususu-3 (SEQ ID NO: 65) (GA374), 5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUcc GUUAucAAcuugaaaaagugGcaccgagucggugcusususuuuu-3′ (SEQ ID NO: 11) (GA385), 5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUcc GUUAucAAcuugaaaaagugGcaccgagucggugcusususuuUu-3′ (SEQ ID NO: 11) (GA386) or 5′-cscscsGCACCUUGGCGCAGCGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccG UUAucAAcuuGaaaaagugGcaccgagucggugcuususuuuu-3′ (SEQ ID NO: 12) (GA387). 
     In some embodiments, the protospacer sequence comprises a protospacer sequence set forth in Table 1 or Table 24. In some embodiments, the protospacer comprises the sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 13) 
               
               
                   
                 5′-CCCGCACCTTGGCGCAGCGG-3′, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 14) 
               
               
                   
                 AAGATACCTGAATAACTCTC-3′ 
               
               
                   
                 or 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 15) 
               
               
                   
                 5′-AAGATACCTGAATAACCCTC-3′. 
               
            
           
         
       
     
     In some embodiments, the base editor fusion protein comprises the sequence of SEQ ID NO: 3. In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 3 or to any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identity plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to the amino acid sequence set forth in SEQ ID NO: 3 or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NO: 3 or any of the adenosine deaminases provided herein. 
     In some embodiments, the mRNA comprises a cap analog. 
     In some embodiments, the mRNA comprises at least 1, 2, or 3 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 1, 2, or 3 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 1 nucleotide at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 2 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 3 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 4 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 5 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 6 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 7 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 8 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 9 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. In some embodiments, the mRNA comprises at least 10 nucleotides at the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemical modification or a combination thereof. 
     In some embodiments, the mRNA comprises a poly A tail. 
     The compositions described herein, may be administered to a subject in need thereof, in a therapeutically effective amount, to treat conditions related to high circulating cholesterol levels. Conditions related to high circulating cholesterol level that may be treated using the compositions and methods described herein include, without limitation: hypercholesterolemia, elevated total cholesterol levels, elevated low-density lipoprotein (LDL) levels, elevated blood LDL-cholesterol levels, reduced blood high-density lipoprotein cholesterol level, liver steatosis, coronary heart disease, vascular disease, ischemia, stroke, peripheral vascular disease, thrombosis, type 2 diabetes, hypertriglyceridemia, high elevated blood pressure, atherosclerosis, obesity, Alzheimer&#39;s disease, neurodegeneration, and combinations thereof. The compositions and methods disclosed herein are effective in reducing the circulating cholesterol level in the subject, thus treating the conditions. The compositions and methods disclosed herein are effective in reducing blood LDL cholesterol level and/or a reducing blood triglycerides level as compared to before the administration. 
     In another aspect, provided herein is a method for treating or preventing a condition in a subject in need thereof, the method comprising administering to the subject the complex as provided herein, the composition as provided herein, or the lipid nanoparticle as provided herein, wherein the sgRNA directs the adenosine base editor protein to effect a modification in a target polynucleotide sequence in a cell of the subject, thereby treating or preventing the condition. 
     In some embodiments, the target polynucleotide sequence is in a PCSK9 gene. In some embodiments, the modification reduces expression of functional PCSK9 protein encoded by the PCSK9 gene in the subject. In some embodiments, the condition is atherosclerotic vascular disease. In some embodiments, the target polynucleotide sequence is in an ANGPTL3 gene. In some embodiments, the modification reduces expression of functional ANGPTL3 protein encoded by the ANGPTL3 gene in the subject. In some embodiments, the condition is an atherosclerotic vascular disease, hypertriglyceridemia, or diabetes. 
     A patient who is being treated for a condition, a disease or a disorder is one who a medical practitioner has diagnosed as having such a condition. Diagnosis may be by any suitable means. Diagnosis and monitoring may involve, for example, detecting the presence of diseased, dying or dead cells in a biological sample (e.g., tissue biopsy, blood test, or urine test), detecting the presence of plaques, detecting the level of a surrogate marker in a biological sample, or detecting symptoms associated with a condition. A patient in whom the development of a condition is being prevented may or may not have received such a diagnosis. One in the art will understand that these patients may have been subjected to the same standard tests as described above or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors (e.g., family history or genetic predisposition). 
     The therapeutic methods of the disclosure may be carried out on subjects displaying pathology resulting from a disease or a condition, subjects suspected of displaying pathology resulting from a disease or a condition, and subjects at risk of displaying pathology resulting from a disease or a condition. For example, subjects that have a genetic predisposition to a disease or a condition can be treated prophylactically. Subjects exhibiting symptoms associated with a condition, a disease or a disorder may be treated to decrease the symptoms or to slow down or prevent further progression of the symptoms. The physical changes associated with the increasing severity of a disease or a condition are shown herein to be progressive. Thus, in embodiments of the disclosure, subjects exhibiting mild signs of the pathology associated with a condition or a disease may be treated to improve the symptoms and/or prevent further progression of the symptoms. 
     In some embodiments, the subject exhibits a reduced blood LDL cholesterol level and/or a reduced blood triglycerides level as compared to before the administration. 
     In some embodiments, after the administration, the subject exhibits a reduced blood low-density lipoprotein (LDL) cholesterol level by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% as compared to before the administration. In some embodiments, after the administration, the subject exhibits a reduced blood low-density lipoprotein (LDL) cholesterol level by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared to before the administration. 
     In some embodiments, after the administration, the subject exhibits a reduced blood triglycerides level by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% as compared to before the administration. 
     In some embodiments, after the administration, the subject exhibits a reduced blood triglycerides level by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% as compared to before the administration. In some embodiments, after the administration, the subject exhibits a reduced blood triglycerides level by at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold as compared to before the administration. 
     The dosage and frequency (single or multiple doses) administered to a mammal can vary depending upon a variety of factors, for example, whether the mammal suffers from another disease, and its route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated, kind of concurrent treatment, complications from the disease being treated or other health-related problems. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the ability of those skilled in the art. The treatment, such as those disclosed herein, can be administered to the subject on a daily, twice daily, biweekly, monthly or any applicable basis that is therapeutically effective. In embodiments, the treatment is only on an as-needed basis, e.g., upon appearance of signs or symptoms of a condition or a disease, e.g., an atherosclerotic vascular disease, hypertriglyceridemia, or diabetes. 
     The compositions described herein, may be administered to a subject in need thereof, in a therapeutically effective amount, to treat conditions related to high circulating cholesterol levels. Conditions related to high circulating cholesterol level that may be treated using the compositions and methods described herein include, without limitation: hypercholesterolemia, elevated total cholesterol levels, elevated low-density lipoprotein (LDL) levels, elevated blood LDL-cholesterol levels, reduced blood high-density lipoprotein cholesterol level, liver steatosis, coronary heart disease, vascular disease, ischemia, stroke, peripheral vascular disease, thrombosis, type 2 diabetes, hypertriglyceridemia, high elevated blood pressure, atherosclerosis, obesity, Alzheimer&#39;s disease, neurodegeneration, and combinations thereof. The compositions and methods disclosed herein are effective in reducing the circulating cholesterol level in the subject, thus treating the conditions. The compositions and methods disclosed herein are effective in reducing blood LDL cholesterol level and/or a reducing blood triglycerides level as compared to before the administration. 
     Toxicity and therapeutic efficacy of the compositions of the disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects (the ratio LD50/ED50) is the therapeutic index. Agents that exhibit high therapeutic indices are preferred. The dosage of agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. While agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. 
     The skilled artisan will appreciate that certain factors may influence the dosage and frequency of administration required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general characteristics of the subject including health, sex, weight and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compositions can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of the composition of the disclosure used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein. The therapeutically-effective dosage will generally be dependent on the patient&#39;s status at the time of administration. The precise amount can be determined by routine experimentation but may ultimately lie with the judgment of the clinician, for example, by monitoring the patient for signs of disease and adjusting the treatment accordingly. 
     Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a disease. Alternatively, sustained continuous release formulations of a polypeptide or a polynucleotide may be appropriate. Various formulations and devices for achieving sustained release are known in the art. In some embodiments, dosage is daily, every other day, every three days, every four days, every five days, or every six days. In some embodiments, dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. 
     The dosing regimen (including a composition disclosed herein) can vary over time. In some embodiments, for an adult subject of normal weight, doses ranging from about 0.01 to 1000 mg/kg may be administered. In some embodiments, the dose is between 1 to 200 mg. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular subject and that subject&#39;s medical history, as well as the properties of the polypeptide or the polynucleotide (such as the half-life of the polypeptide or the polynucleotide, and other considerations well known in the art). 
     For the purpose of the present disclosure, the appropriate therapeutic dosage of a composition as described herein will depend on the specific agent (or compositions thereof) employed, the formulation and route of administration, the type and severity of the disease, whether the polypeptide or the polynucleotide is administered for preventive or therapeutic purposes, previous therapy, the subject&#39;s clinical history and response to the antagonist, and the discretion of the attending physician. Typically, the clinician will administer a polypeptide until a dosage is reached that achieves the desired result. 
     Administration of one or more compositions can be continuous or intermittent, depending, for example, upon the recipient&#39;s physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of a composition may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a disease. 
     The methods and compositions of the disclosure described herein including embodiments thereof can be administered with one or more additional therapeutic regimens or agents or treatments, which can be co-administered to the mammal. 
     Metabolic Syndrome and Cardiovascular Disease 
     The ability of gene modification, especially to edit bases directly allows for the precise edit, modification, and/or disruption of genes in vivo without the need to create DSBs and improved methods of treating human disease. For example, Chadwick et. al used cytosine base editing to introduce nonsense mutations into the Pcsk9 gene in adult mice. Delivery of BE3 and a gRNA via adenoviral vector into the liver caused a reduction in plasma PCSK9 levels by 56%. Furthermore, cholesterol levels in the base-edited mice were reduced by roughly 30%. In subsequent work, cytosine base editing to disrupt the Angptl3 gene in adult mice resulted in substantially reduced blood cholesterol and triglyceride levels, and cytosine base editing to disrupt the Pcsk9 gene or the Hpd gene in fetal mice resulted in postnatal reduction of cholesterol levels or cure of the disease hereditary tyrosinemia type 1. 
     Cardiovascular disease, obesity, type 2 diabetes, metabolic syndrome may share one or more similar underlying etiologies. The human PCSK9 gene encodes a protein that helps regulate the amount of cholesterol in the bloodstream. The liver protein Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in human is a secreted, globular, auto-activating serine protease that acts as a protein-binding adaptor within endosomal vesicles to bridge a pH-dependent interaction with the low-density lipoprotein receptor (LDL-R) during endocytosis of LDL particles, preventing recycling of the LDL-R to the cell surface and leading to reduction of LDL-cholesterol clearance. PCSK9 orthologs are found across many species. The PCSK9 protein breaks down low-density lipoprotein receptors before they reach the cell surface, so more cholesterol can remain in the bloodstream. As a result, the PCSK9 gene and the encoded PCSK9 protein are attractive targets for regulating cholesterol metabolism, especially in lowering blood cholesterol levels. 
     Other targets have also been shown that are associated with decreased plasma levels of triglycerides (TGs), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), which leads to a significant reduction in cardiovascular risk. For example, human ANGPTL3 is considered an important new pharmacological target for the treatment of cardiovascular diseases. Experimental evidence demonstrates that anti-ANGPTL3 therapies have an important anti-atherosclerotic effect. Results from phase I clinical trials with a monoclonal anti-ANGPTL3 antibody (evinacumab) and anti-sense oligonucleotide (ASO) clearly show a significant lipid lowering effect. 
     Human ANGPTL3 gene is located on chromosome 1p31. Genetic variants of human ANGPTL3 gene have been linked to different plasma lipid profiles. For example, homozygous loss of function (LOF) variants of ANGPTL3 gene cause the levels of all plasma lipoproteins to be greatly reduced. ANGPTL3 is exclusively expressed in the liver and is secreted into circulation where it goes through cleavage by hepatic proprotein convertases. The ANGPTL3 protein is a 460-amino-acid (aa) polypeptide with a distinctive signal peptide sequence, a N-terminal helical domain and a C-terminal globular fibrinogen homology domain. The N-terminal coiled-coil region (17-207aa) affects plasma TG levels via reversibly inhibiting catalytic activity of LPL while the fibrinogen-like domain (207-460aa) binds to integrin αvβ3 receptor and affects angiogenesis, which is similar to the function of angiopoietins. A short linker region (at 221-222 and 224-225) between N- and C-terminal domains is a special zone, which has been verified to be split by furin. Existed results have shown that the truncated form of cleavage ANGPTL3 could reinforce the inhibitory activity of LPL and endothelial lipase (EL), suggesting that the cleavage type of ANGPTL3 may function more effectively. 
     Moreover, human apolipoprotein C-III (APOC3) could potentially be another therapeutic target. APOC3 is a protein that in humans is encoded by the APOC3 gene. APOC3 is a component of VLDL. APOC3 inhibits lipoprotein lipase and hepatic lipase. It is also thought to inhibit hepatic uptake of triglyceride-rich particles. An increase in APOC3 levels induces the development of hypertriglyceridemia. Recent evidence suggests an intracellular role for APOC3 in promoting the assembly and secretion of triglyceride-rich VLDL particles from hepatic cells under lipid-rich conditions. However, two naturally occurring point mutations in human apoC3 coding sequence, A23T and K58E have been shown to abolish the intracellular assembly and secretion of triglyceride-rich VLDL particles from hepatic cells. 
     Targeted base editing with target genes involved in vascular disease and diabetes as described in Chadwick A C, Wang X, Musunuru K. In vivo base editing of PCSK9 (proprotein convertase subtilisin/kexin type 9) as a therapeutic alternative to genome editing. Arterioscler Thromb Vasc Biol, 2017, 37: 1741-7; Chadwick A C, Evitt N H, Lv W, et al. Reduced blood lipid levels with in vivo CRISPR-Cas9 base editing of ANGPTL3. Circulation, 2018, 137: 975-7; Rossidis A C, Stratigis J D, Chadwick A C, et al. In utero CRISPR-mediated therapeutic editing of metabolic genes. Nat Med, 2018, 24: 1513-8 is incorporated herein by reference in its entirety. While genes involved in lipid metabolism, including PCSK9, have been edited by targeted by current gene editing technologies, there remains a need for precise base editing targeting these genes for improved editing outcomes. 
     Pharmaceutical Composition 
     In some aspects, provided herein is a pharmaceutical composition comprising the base editor system as provided herein and a pharmaceutically acceptable carrier or excipient. 
     In some aspects, provided herein, is a pharmaceutical composition for gene modification comprising a gRNA or sgRNA described herein and a Base editor fusion protein or a nucleic acid sequence encoding the Base editor fusion protein and a pharmaceutically acceptable carrier. The composition for gene modification comprising a gRNA or sgRNA described herein and a Base editor fusion protein or a nucleic acid sequence encoding the Base editor fusion protein can be formulated into pharmaceutical compositions. Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Suitable formulations for use in the present disclosure and methods of delivery are generally well known in the art. Proper formulation is dependent upon the route of administration chosen. A summary of pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington&#39;s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &amp; Wilkins 1999), herein incorporated by reference for such disclosure. 
     A pharmaceutical composition can be a mixture of a gRNA or sgRNA described herein and a Base editor fusion protein or a nucleic acid sequence encoding the Base editor fusion protein with one or more of other chemical components (i.e., pharmaceutically acceptable ingredients), such as carriers, excipients, binders, filling agents, suspending agents, flavoring agents, sweetening agents, disintegrating agents, dispersing agents, surfactants, lubricants, colorants, diluents, solubilizers, moistening agents, plasticizers, stabilizers, penetration enhancers, wetting agents, anti-foaming agents, antioxidants, preservatives, or one or more combination thereof. The pharmaceutical composition facilitates administration of the gRNA or sgRNA described herein and the Base editor fusion protein or a nucleic acid sequence encoding the Base editor fusion protein to an organism or a subject in need thereof. 
     The pharmaceutical compositions of the present disclosure can be administered to a subject using any suitable methods known in the art. The pharmaceutical compositions described herein can be administered to the subject in a variety of ways, including parenterally, intravenously, intradermally, intramuscularly, colonically, rectally, or intraperitoneally. In some embodiments, the pharmaceutical compositions can be administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject. In some embodiments, the pharmaceutical compositions can be administered parenterally, intravenously, intramuscularly, or orally. 
     For administration by inhalation, the adenovirus described herein can be formulated for use as an aerosol, a mist, or a powder. For buccal or sublingual administration, the pharmaceutical compositions may be formulated in the form of tablets, lozenges, or gels formulated in a conventional manner. In some embodiments, the adenovirus described herein can be prepared as transdermal dosage forms. In some embodiments, the adenovirus described herein can be formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In some embodiments, the adenovirus described herein can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, or ointments. In some embodiments, the adenovirus described herein can be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas. In some embodiments, the adenovirus described herein can be formulated for oral administration such as a tablet, a capsule, or liquid in the form of aqueous suspensions or solutions selected from the group including, but not limited to, aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. 
     In some embodiments, the pharmaceutical composition for gene modification comprising a gRNA or sgRNA described herein and a Type II Cas protein or a nucleic acid sequence encoding the Type II Cas protein further comprises a therapeutic agent. The additional therapeutic agent may modulate different aspects of the disease, disorder, or condition being treated and provide a greater overall benefit than administration of either the replication competent recombinant adenovirus or the therapeutic agent alone. Therapeutic agents include, but are not limited to, a chemotherapeutic agent, a radiotherapeutic agent, a hormonal therapeutic agent, and/or an immunotherapeutic agent. In some embodiments, the therapeutic agent may be a radiotherapeutic agent. In some embodiments, the therapeutic agent may be a hormonal therapeutic agent. In some embodiments, the therapeutic agent may be an immunotherapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent. Preparation and dosing schedules for additional therapeutic agents can be used according to manufacturers&#39; instructions or as determined empirically by a skilled practitioner. For example, preparation and dosing schedules for chemotherapy are also described in The Chemotherapy Source Book, 4th Edition, 2008, M. C. Perry, Editor, Lippincott, Williams &amp; Wilkins, Philadelphia, Pa. 
     The subjects that can be treated with gene modification compositions of chemically modified gRNAs or sgRNAs described herein and a Type II Cas protein or a nucleic acid sequence encoding the Type II Cas protein and methods described herein can be any subject with a disease or a condition. For example, the subject may be a eukaryotic subject, such as an animal. In some embodiments, the subject is a mammal, e.g., human. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a fetus, an embryo, or a child. In some embodiments, the subject is a non-human primate such as chimpanzee, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs, and the like. 
     In some embodiments, the subject is prenatal (e.g., a fetus), a child (e.g., a neonate, an infant, a toddler, a preadolescent), an adolescent, a pubescent, or an adult (e.g., an early adult, a middle-aged adult, a senior citizen). The human subject can be between about 0 month and about 120 years old, or older. The human subject can be between about 0 and about 12 months old; for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months old. The human subject can be between about 0 and 12 years old; for example, between about 0 and 30 days old; between about 1 month and 12 months old; between about 1 year and 3 years old; between about 4 years and 5 years old; between about 4 years and 12 years old; about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years old. The human subject can be between about 13 years and 19 years old; for example, about 13, 14, 15, 16, 17, 18, or 19 years old. The human subject can be between about 20 and about 39 years old; for example, about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 years old. The human subject can be between about 40 to about 59 years old; for example, about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 years old. The human subject can be greater than 59 years old; for example, about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 years old. The human subjects can include male subjects and/or female subjects. 
     Lipid Nanoparticle (LNP) Composition 
     In some embodiments, LNPs are prepared in accordance with the methods described in Conway, A. et al. 2019 Mol. Ther. 27, 866-877, and Villiger, L. et al 2021  Nat. Biomed. Eng.  5, 179-18, which are incorporated by reference. In some embodiments, LNPs are composed of an amino lipid, a monomethoxypolyethylene glycol (or methoxypoluyethyle glycol) of average molecular weight 2000 Da conjugated to a lipid called PEG-Lipid, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, LNPs are composed of proprietary ionizable cationic lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and a PEG-lipid. 
     In some embodiments, LNPs have an average hydrodynamic diameter of about 30 about 160, about 35-about 160, about 40-about 160, about 45-about 160, about 50-about 160, about 55-about 160, about 60-about 160, about 65-about 160, about 70-about 160, about 75-about 160, about 80-about 160, about 85-about 160, about 90-about 160, about 95-about 160, about 100-about 160, about 105-about 160, about 110-about 160, about 115-about 160, about 120-about 160, about 125-about 160, about 130-about 160, about 135-about 160, about 140-about 160, about 145-about 160, about 150-about 160, about 30-about 155, about 30-about 150, about 30-about 145, about 30-about 140, about 30 about 135, about 30-about 130, about 30-about 125, about 30-about 120, about 30-about 115, about 30-about 110, about 30-about 105, about 30-about 100, about 30-about 95, about 30-about 90, about 30-about 85, about 30-about 80, about 30-about 75, about 30 about 70, about 30-about 65, about 30-about 60, about 30-about 55, about 30-about 50, about 30-about 45, about 30-about 40, about 30-about 45, about 35-about 45, about 35 about 50, about 40-about 50, about 40-about 55, about 45-about 55, about 45-about 60, about 50-about 60, about 50-about 65, about 55-about 60, about 55-about 65, about 55 about 70, about 60-about 70, about 60-about 75, about 65-about 75, about 65-about 80, about 70-about 80, about 70-about 85, about 75-about 85, about 75-about 90, about 80 about 90, about 80-about 95, about 85-about 95, about 85-about 100, about 90-about 100, about 90-about 105, about 95-about 105, about 95-about 110, about 100-about 110, about 100-about 115, about 105-about 115, about 105-about 120, about 110-about 120, about 110-about 125, about 115-about 125, about 115-about 130, about 120-about 130, about 120-about 135, about 125-about 135, about 125-about 140, about 130-about 140, about 130-about 145, about 35-about 140, about 45-about 130, about 55-about 120, about 65-about 110, about 75-about 100, or about 85-about 90 nm. 
     In some embodiments, LNPs comprise about 1-about 97, about 5-about 97, about 10-about 97, about 15-about 97, about 20-about 97, about 25-about 97, about 30-about 97, about 35-about 97, about 40-about 97, about 45-about 97, about 50-about 97, about 55-about 97, about 60-about 97, about 65-about 97, about 70-about 97, about 75- about 97, about 80-about 97, about 1-about 95, about 1-about 90, about 1-about 85, about 1 about 80, about 1-about 75, about 1-about 70, about 1-about 65, about 1-about 60, about 1-about 55, about 1-about 50, about 1-about 45, about 1-about 40, about 1-about 35, about 1-about 30, about 1-about 25, about 1-about 20, about 1-about 15, about 1-about 10, about 10-about 30, about 10-about 35, about 15-about 35, about 15-about 40, about 20-about 40, about 20-about 45, about 25-about 45, about 25-about 50, about 30-about 50, about 30-about 55, about 35-about 55, about 35-about 60, about 40-about 60, about 40-about 65, about 45-about 65, about 45-about 70, about 50-about 70, about 50-about 75, about 55-about 75, about 55-about 80, or about 60-about 80% of amino lipids (in mol %). 
     In some embodiments, LNPs comprise about 1-about 40, about 1-about 38, about 1-about 36, about 1-about 34, about 1-about 32, about 1-about 30, about 1-about 28, about 1-about 26, about 1-about 24, about 1-about 22, about 1-about 20, about 1-about 18, about 1-about 16, about 1-about 14, about 1-about 12, about 1-about 10, about 1 about 8, about 1-about 6, about 1-about 4, about 1-about 2, about 2-about 40, about 4 about 40, about 6-about 40, about 8-about 40, about 10-about 40, about 12-about 40, about 14-about 40, about 16-about 40, about 18-about 40, about 20-about 40, about 22 about 40, about 24-about 40, about 26-about 40, about 28-about 40, about 30-about 40, about 32-about 40, about 34-about 40, about 36-about 40, about 38-about 40, about 2 about 35, about 2-about 30, about 2-about 25, about 2-about 20, about 2-about 15, about 2-about 10, about 2-about 8, about 2-about 6, or about 2-about 4% DSPC (in mol %). 
     In some embodiments, LNPs comprise about 1-about 20, about 1-about 19, about 1-about 18, about 1-about 17, about 1-about 16, about 1-about 15, about 1-about 14, about 1-about 13, about 1-about 12, about 1-about 11, about 1-about 10, about 1-about 9, about 1-about 8, about 1-about 7, about 1-about 6, about 1-about 5, about 1-about 4, about 1-about 3, about 2-about 6, about 3-about 7, about 4-about 8, about 5-about 9, about 6-about 10, about 7-about 11, about 8-about 12, about 9-about 13, about 10 about 14, about 11-about 15, about 12-about 16, about 13-about 17, about 14-about 18, about 15-about 19, or about 16-about 20% PEG-Lipid (in mol %), 
     In some embodiments, LNPs comprise about 1-about 97, about 5-about 97, about 10-about 97, about 15-about 97, about 20-about 97, about 25-about 97, about 30-about 97, about 35-about 97, about 40-about 97, about 45-about 97, about 50-about 97, about 55-about 97, about 60-about 97, about 65-about 97, about 70-about 97, about 75-about 97, about 80-about 97, about 1-about 95, about 1-about 90, about 1-about 85, about 1 about 80, about 1-about 75, about 1-about 70, about 1-about 65, about 1-about 60, about 1-about 55, about 1-about 50, about 1-about 45, about 1-about 40, about 1-about 35, about 1-about 30, about 1-about 25, about 1-about 20, about 1-about 15, about 1-about 10, about 10-about 30, about 10-about 35, about 15-about 35, about 15-about 40, about 20-about 40, about 20-about 45, about 25-about 45, about 25-about 50, about 30-about 50, about 30-about 55, about 35-about 55, about 35-about 60, about 40-about 60, about 40-about 65, about 45-about 65, about 45-about 70, about 50-about 70, about 50-about 75, about 55-about 75, about 55-about 80, or about 60-about 80% of cholesterol (in mol %). 
     In some embodiments, LNPs comprise about 1-about 97, 5-about 97, 10-about 97, 15-about 97, 20-about 97, 25-about 97, 30-about 97, 35-about 97, 40-about 97, 45 about 97, 50-about 97, 55-about 97, 60-about 97, 65-about 97, 70-about 97, 75-about 97, 80-about 97, 1-about 95, 1-about 90, 1-about 85, 1-about 80, 1-about 75, 1-about 70, 1-about 65, 1-about 60, 1-about 55, 1-about 50, 1-about 45, 1-about 40, 1-about 35, 1-about 30, 1-about 25, 1-about 20, 1-about 15, 1-about 10, 10-about 30, 10 about 35, 15-about 35, 15-about 40, 20-about 40, 20-about 45, 25-about 45, 25-about 50, 30-about 50, 30-about 55, 35-about 55, 35-about 60, 40-about 60, 40-about 65, 45-about 65, 45-about 70, 50-about 70, 50-about 75, 55-about 75, 55-about 80, or 60 about 80% of amino lipids; 1-about 40, 1-about 38, 1-about 36, 1-about 34, 1-about 32, 1-about 30, 1-about 28, 1-about 26, 1-about 24, 1-about 22, 1-about 20, 1-about 18, 1-about 16, 1-about 14, 1-about 12, 1-about 10, 1-about 8, 1-about 6, 1-about 4, 1 about 2, 2-about 40, 4-about 40, 6-about 40, 8-about 40, 10-about 40, 12-about 40, 14-about 40, 16-about 40, 18-about 40, 20-about 40, 22-about 40, 24-about 40, 26 about 40, 28-about 40, 30-about 40, 32-about 40, 34-about 40, 36-about 40, 38-about 40, 2-about 35, 2-about 30, 2-about 25, 2-about 20, 2-about 15, 2-about 10, 2-about 8, 2-about 6, or 2-about 4% DSPC; 1-about 20, 1-about 19, 1-about 18, 1-about 17, 1 about 16, 1-about 15, 1-about 14, 1-about 13, 1-about 12, 1-about 11, 1-about 10, 1 about 9, 1-about 8, 1-about 7, 1-about 6, 1-about 5, 1-about 4, 1-about 3, 2-about 6, 3-about 7, 4-about 8, 5-about 9, 6-about 10, 7-about 11, 8-about 12, 9-about 13, 10 about 14, 11-about 15, 12-about 16, 13-about 17, 14-about 18, 15-about 19, and about 16-about 20% PEG-Lipid, with the balance being cholesterol (all in mol %). 
     Amino Lipid 
     Described herein are LNP compositions comprising an amino lipid, a phospholipid, a PEGlipid, a cholesterol or a derivative thereof, a payload, or any combination thereof. In some embodiments, the LNP composition comprises an amino lipid. In one aspect, disclosed herein is an amino lipid having the structure of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, 
     
       
         
         
             
             
         
       
     
     wherein
 
each of R 1  and R 2  is independently C 7 -C 22  alkyl, C 7 -C 22  alkenyl, C 3 -C 8  cycloalkyl, —C 2 -C 10  allkylene-L-R 6 , or
 
     
       
         
         
             
             
         
       
     
     wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted;
 
each of X, Y, and Z is independently —C(═O)NR 4 —, —NR 4 C(═O)—, —C(═O)O—, —OC(═O)—, —OC(═O)O—, —NR 4 C(═O)O—, —OC(═O)NR 4 —, —NR 4 C(═O)NR 4 —, —NR 4 C(═NR 4 )NR 4 —, —C(═S)NR 4 —, —NR 4 C(═S)—, —C(═O)O—, —OC(═S)—, OC(═S)O—, —NR 4 C(═S)O—, —OC(═S)NR 4 —, —NR 4 C(═S)NR 4 —, —C(═O)S—, —SC(═O)—, —OC(═O)S—, —NR 4 C(═O)S—. —SC(═O)NR 4 —, —C(═S)S—, —SC(═S)—, —SC(═S)O—, —NR 4 C(═S)S—, —SC(═S)NR 4 —, —C(═S)S—, —SC(═S)—, —SC(═O)S—, —SC(═S)S—, —NR 4 C(═S)S—, —SC(═S)NR 4 —O, S, or a bond;
 
each of L is independently —C(═O)NR 4 —, —NR 4 C(═O)—, —C(═O)O—. —OC(═O)O—, —NR 4 C(═O)O—, —OC(═O)NR 4 —, —NR 4 C(═O)NR 4 —, —NR 4 C(═NR 4 )NR 4 —, —C(═S)NR 4 —, —NR 4 C(═S)—, —C(═O)O—, —OC(═S)—, OC(═S)O—, —NR 4 C(═S)O—, —OC(═S)NR 4 —, —NR 4 C(═S)NR 4 —, —C(═O)S—, SC(═O)—, —OC(═O)S—, —NR 4 C(═O)S—, —SC(═O)NR 4 —, —C(═S)S—, —SC(═S)—, —SC(═S)O—, —NR 4 C(═S)S—, —SC(═S)NR 4 —, —C(═S)S—, —SC(═S)—, —SC(═O)S—, —SC(═S)S—, —NR 4 C(═S)S—, —SC(═S)NR 4 —, O, S, —C 1 -C 10  alkylene-O—, —C 1 -C 10  alkylene-C(═O)O—, —C 1 -C 10  alkylene-OC(═O)—, or a bond, wherein the alkylene is substituted or unsubstituted;
 
R 3  is —C 0 -C 10  alkylene-NR 7 R 8 , —C 0 -C 10  alkylene-heterocycloalkyl, or —C 0 -C 10  alkylene-heterocycloaryl,
 
wherein the alkylene, heterocycloalkyl and heterocycloaiyl is independently substituted or unsubstituted; each of R 4  is independently hydrogen or substituted or unsubstituted C 1 -C 6  alkyl;
 
R 5  is hydrogen or substituted or unsubstituted C 1 -C 6  alkyl;
 
each of R 6  is independently substituted or unsubstituted C 3 -C 22  alkyl or substituted or unsubstituted C 3 -C 22  alkenyl;
 
each of R 7  and R 8  is independently hydrogen or substituted or unsubstituted C 1 -C 6  alkyl, or
 
R 7  and R 8  taken together with the nitrogen to which they are attached form a substituted or unsubstituted C 2 -C 6  heterocyclyl;
 
p is an integer selected from 1 to 10; and
 
each of n, m, and q is independently 0, 1, 2, 3, 4, or 5.
 
     In some embodiments of Formula (I), if the structure carries more than one asymmetric C-atom, each asymmetric C-atom independently represents racemic, chirally pure R and/or chirally pure S isomer, or a combination thereof. 
     In some embodiments, each of n, in, and q in Formula (I) is independently 0, 1, 2, or 3. In some embodiments, each of n, m, and q in Formula (I) is 1. 
     In some embodiments, the compound of Formula (1) has a structure of Formula (Ia), or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof: 
     
       
         
         
             
             
         
       
     
     wherein
 
each of R 1  and R 2  is independently C 7 -C 22  alkyl, C 7 -C 22  alkenyl, C 3 -C 8  cycloalkyl, —C 2 -C 10  alkylene-L-R 6 , or
 
     
       
         
         
             
             
         
       
     
     wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted;
 
each of X, Y, and Z is independently C(═O)NR 4 —, —NR 4 C(D)—, —C(═O)O—, —OC(═O)—, —OC(═O)O—, —NR 4 C(═O)O—, —OC(═O)NR 4 —, —NR 4 C═O)NR 4 —, —NR 4 C(═NR 4 )NR 4 —. —C(═S)NR 4 —, —NR 4 C(═S)—, —C(E)O—, —OC(═S)—, OC(═S)O—, —NR 4 C(═S)O—, —OC(═S)NR 4 —, —NR 4 C(═S)NR 4 —, —C(═O)S—, —SC(═O)—, —OC(═O)S—, —NR 4 C(═O)S—, —SC(═O)NR 4 — —C(═S)S—, —SC(═S)—, —SC(═S)O—, —NR 4 C(═S)S—, —SC(═S)NR 4 —, —C(═S)S—. —SC(═S)—, —SC(═O)S—, —SC(═S)S—. —NR 4 C(═S)S—, —SC(═S)NR 4 —, O, S, —C 1 -C 10  alkylene-O—, or a bond, wherein the alkylene is substituted or unsubstituted;
 
each of L is independently —C(═O)NR 4 —, —NR 4 C(═O)—, —C(═O)O—, —OC(═O)—, —OC(═O)O—, —NR 4 C(═O)O—, —OC(═O)NR 4 —, —NR 4 C(═O)NR 4 —, —NR 4 C(═NR 4 )NR 4 —. —C(═S)NR 4 —, —NR 4 C(═S)—, —C(═O)O—, —OC(═S)—, OC(═S)O—, —NR 4 C(═S)O—, —OC(═S)NR 4 —, —NR 4 C(═S)NR 4 —, —C(═O)S—, —SC(═O)—, —OC(═O)S—, —NR 4 C(═O)S—, —SC(═O)NR 4 — —C(═S)S—, —SC(═S)—, —SC(═S)O—, —NR 4 C(═S)S—, —SC(═S)NR 4 —, —C(═S)S—, —SC(═S)—, —SC(═O)S—, —SC(═S)S—, —NR 4 C(═S)S—, —SC(═S)NR 4 —, O, S. —C 1 -C 10  alkylene-O—, —C 1 -C 10  alkylene-C(═O)O—, —C 1 -C 10  alkylene- OC(═O)—, or a bond, wherein the alkylene is substituted or unsubstituted;
 
R 3  is —C 0 -C 10  alkylene-NR 7 R 8 , —C 0 -C 10  alkylene-heterocycloalkyl, or —C 0 -C 10  alkylene-heterocyclowyl, wherein the alkylene, heterocycloalkyl and heterocycloaryl is independently substituted or unsubstituted;
 
each of R 4  is independently hydrogen or substituted or unsubstituted CI—C6 alkyl;
 
R 5  is hydrogen or substituted or unsubstituted C 1 -C 6  alkyl;
 
each of R 6  is independently substituted or unsubstituted C 3 -C 22  alkyl or substituted or unsubstituted C 3 -C 22  alkenyl;
 
each of R 7  and R 8  is independently hydrogen or substituted or unsubstituted C 1 -C 6  alkyl, or
 
R 7  and R 8  taken together with the nitrogen to which they are attached form a substituted or unsubstituted C 2 -C 6  heterocyclyl; and
 
p is an integer selected from 1 to 10.
 
     In some embodiments of Formula (1a), if the structure carries more than one asymmetric Catom, each asymmetric C-atom independently represents racemic, chirally pure R and/or chirally pure S isomer, or a combination thereof. 
     In some embodiments, R 1  and R 2  in Formula (I) and Formula (Ia) is independently C 7 -C 22  alkyl, C 7 -C 22  alkenyl, —C 2 -C 10  alkylene-L-R 6 , or 
     
       
         
         
             
             
         
       
     
     wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, R 1  and R 2  in Formula (I) and Formula (1a) is independently C 10 -C 20  alkyl, C 10 -C 20  alkenyl. —C 8 -C 7  alkylene-L-R 6 , or 
     
       
         
         
             
             
         
       
     
     wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, R 1  in Formula (I) and Formula (1a) is 
     
       
         
         
             
             
         
       
     
     In some embodiments, each of L in Formula (I) and Formula (Ia) is independently O, S, —C 1 -C 10  alkylene-O—, —C 1 -C 10  alkylene-C(═O)O—, —C 1 -C 10  alkylene-OC(═O)—, or a bond, wherein the alkylene is substituted or unsubstituted. In some embodiments, each of L in Formula (I) and Formula (Ia) is independently O, S, —C 1 -C 3  alkylene-O—, —C 1 -C 3  alkylene-C(═O)O—, —C 1 -C 3  alkylene-OC(═O)—, or a bond, wherein the alkylene is substituted or unsubstituted. In some embodiments, each of L in Formula (I) and Formula (1a) is independently O, S, —C 1 -C 3  alkylene-O—, —C 1 -C 3  alkylene-C(═O)O—, —C 1 -C 3  alkylene-OC(═O)—, or a bond, wherein the alkylene is linear or branched unsubstituted alkylene. 
     In some embodiments, each of R 6  in Formula (I) and Formula (1a) is independently substituted or unsubstituted linear C 3 -C 22  alkyl or substituted or unsubstituted linear C 3 -C 22  alkenyl. In some embodiments, each of R 6  in Formula (I) and Formula (Ia) is independently substituted or unsubstituted C 3 -C 20  alkyl or substituted or unsubstituted C 3 -C 20  alkenyl. In some embodiments, each of R 6  in Formula (I) and Formula (Ia) is independently substituted or unsubstituted C 3 -C 10  alkyl or substituted or unsubstituted C 3 -C 10  alkenyl. In some embodiments, each of R 6  in Formula (I) and Formula (Ia) is independently substituted or unsubstituted C 3 -C 10  alkyl. In some embodiments, each of R 6  in Formula (I) and Formula (1a) is independently substituted or unsubstituted linear C 3 -C 10  alkyl. In some embodiments, each of R 6  in Formula (I) and Formula (Ia) is independently substituted or unsubstituted n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, or n-dodecyl. In some embodiments, each of R 6  in Formula (I) and Formula (Ia) is independently substituted or unsubstituted n-octyl. In some embodiments, each of R 6  in Formula (I) and Formula (Ia) is n-octyl. 
     In some embodiments, each of L in Formula (I) and Formula (Ia) is independently —C(═O)O—, —OC(═O)—, —C 1 -C 10  alkylene-O—, or O. In some embodiments, each of L in Formula (I) and Formula (Ia) is O. In some embodiments, each of L in Formula (I) and Formula (Ia) is —C 1 -C 3  alkylene-O—. In some embodiments, p in Formula (I) and Formula (1a) is 1, 2, 3, 4, or 5. In some embodiments, p in Formula (I) and Formula (Ia) is 2. 
     In some embodiments, R 1  in Formula (I) and Formula (Ia) is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In some embodiments, each of R 4  in Formula (I) and Formula (Ia) is independently H or substituted or unsubstituted C 1 -C 4  alkyl. In some embodiments, each of. R 4  in Formula (I) and Formula (1a) is independently substituted or unsubstituted linear C 1 -C 4  alkyl. In some embodiments, each of R 4  in Formula (1) and Formula (1a) is H. In some embodiments, each of R 4  in Formula (I) and Formula (Ia) is independently H, —CH 3 , —CH 2 CH 3 , —CH 2 CH 2 CH 3 , or —CH(CH 3 ) 2 . In some embodiments, each of R 4  in Formula (I) and Formula (Ia) is independently H or —CH 3 . In some embodiments, each of R 4  in Formula (1) and Formula (Ia) is —CH 3 . 
     In some embodiments, X in Formula (I) and Formula (Ia) is —C(═O)O— or —OC(═O))—. In some embodiments, X in Formula (I) and Formula (Ia) is —C(═O)NR 4 — or —NR 4 C(═O)—. In some embodiments, X in Formula (I) and Formula (Ia) is —C(═O)N(CH 3 )—, —N(CH 3 )C(═O)—, —C(═O)NH—, or —NHC(═O)—. In some embodiments, X in Formula (I) and Formula (Ia) is —C(═O))NH—, —C(═O)N(CH 3 )—. —OC(═O))—, —NHC(═O)—, —N(CH 3 )C(═O))—, —C(═O)O—, —OC(═O)O—, —NHC(═O)O—, —N(CH 3 )C(═O)O—, —OC(═O))NH—, —OC(═O)N(CH 3 )—, —NHC(═O)NH—, —N(CH 3 )C(═O))NH—, —NHC(═O)N(CH 3 )—, —N(CH 3 )C(═O)N(CH 3 )—, NHC(═NH)NH—, —N(CH 3 )C(═NH)NH—, —NHC(═NH)N(CH 3 )—, —N(CH 3 )C(═NH)N(CH 3 )—, NHC(═NMe)NH—, —N(CH 3 )C(═NMe)NH—, —NHC(═NMe)N(CH 3 )—, or —N(CH 3 )C(═NMe)N(CH 3 )—. 
     In some embodiments. R 2  in Formula (I) and Formula (Ia) is C 7 -C 22  alkyl, C 7 -C 22  alkenyl, —C 2 -C 10  alkylene-L-R 6 , or 
     
       
         
         
             
             
         
       
     
     Wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl is independently substituted or unsubstituted. In some embodiments, R 2  in Formula (I) and Formula (Ia) is substituted or unsubstituted C 7 -C 22  alkyl or substituted or unsubstituted C 7 -C 22  alkenyl. In some embodiments, R 2  in Formula (I) and Formula (1a) is substituted or unsubstituted linear C 7 -C 22  alkyl or substituted or unsubstituted linear C 7 -C 22  alkenyl. In some embodiments, R 2  in Formula (I) and Formula (1a) is substituted or unsubstituted C 10 -C 20  alkyl or substituted or unsubstituted C 10 -C 20  alkenyl. In some embodiments, R 2  in Formula (I) and Formula (Ia) is unsubstituted C 10 -C 20  alkyl. In some embodiments, R 2  in Formula (I) and Formula (Ia) is unsubstituted C 10 -C 20  alkenyl. In some embodiments, R 2  in Formula (I) and Formula (1a) is —C 2 -C 10  alkylene-L-R 6 . In some embodiments, R 2  in Formula (I) and Formula (Ia) is —C 2 -C 10  alkylene- C(═O)O— R 6  or —C 2 -C 10  alkylene-OC(═O)— R 6 . 
     In some embodiments, R 2  in Formula (I) and Formula (Ia) is 
     
       
         
         
             
             
         
       
     
     In some embodiments, Y in Formula (I) and Formula (Ia) is —C(═O)O— or —OC(═O)—. In some embodiments, Y in Formula (I) and Formula (Ia) is —C(═O)NR 4 — or —NR 4 C(═O)—. In some embodiments, Y in Formula (I) and Formula (Ia) is —C(═O)N(CH 3 )—, —N(CH 3 )C(═O)—, —C(═O)NH—, or —NHC(═O)—. In some embodiments, Y in Formula (I) and Formula (Ia) is —OC(═O)O—, —NR 4 C(═O)O—, —OC(═O)NR 4 —, or —NR 4 C(═O)NR 4 —. In some embodiments. Y in Formula (I) and Formula (Ia) is —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, —NHC(═O)NH—, —N(CH 3 )C(═O)O—. —OC(═O)N(CH 3 )—, —N(CH 3 )C(═O)N(CH 3 )— or —N(CH 3 )C(═O)NH—. In some embodiments, Y in Formula (I) and Formula (Ia) is —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, or —NHC(═O)NH—. 
     In some embodiments, R 3  in Formula (I) and Formula (Ia) is —C 0 -C 10  alkylene-NR 7 R 8  or —C 0 -C 10  alkylene-heterocycloalkyl, wherein the alkylene and heterocycloalkyl is independently substituted or unsubstituted. In some embodiments, R 3  in Formula (I) and Formula (Ta) is —C 0 -C 10  alkylene-NR 7 R 8 . Tn some embodiments, R 3  in Formula (I) and Formula (Ia) is —C 1 -C 6  alkylene-NR 7 R 8 . In some embodiments, R 3  in Formula (I) and Formula (Ia) is —C 1 -C 4  alkylene-NR 7 R 8 . In some embodiments, R 3  in Formula (I) and Formula (Ia) is —C 2 — alkylene-NR 7 R 8 . In some embodiments, R 3  in Formula (I) and Formula (Ia) is —C 2 — alkylene-NR 7 R 8 . Tn some embodiments, R 3  in Formula (I) and Formula (Ia) is —C 3 — alkylene-NR 7 R 8 . In some embodiments, R 3  in Formula (I) and Formula (Ia) is —C 4 — alkylene- NR 7 R 8 . In some embodiments, R 3  in Formula (I) and Formula (Ia) is —C 5 — alkylene-NR 7 R 8 . In some embodiments, R 3  in Formula (I) and Formula (Ia) is —C 0 -C 10  alkylene-heterocycloalkyl. In some embodiments, R 3  in Formula (I) and Formula (Ia) is —C 1 -C 6  alkylene-heterocycloalkyl, wherein the heterocycloalkyl comprises 1 to 3 nitrogen and 0-2 oxygen. In some embodiments, R 3  in Formula (I) and Formula (1a) is —C 1 -C 6  alkylene-heterocycloaryl. 
     In some embodiments, each of R 7  and R 8  in Formula (I) and Formula (Ia) is independently hydrogen or substituted or unsubstituted C 1 -C 6  alkyl. Tn some embodiments, each of R 7  and R 8  is independently hydrogen or substituted or unsubstituted C 1 -C 3  alkyl. In some embodiments, each of R 7  and R 8  is independently substituted or unsubstituted C 1 -C 3  alkyl. In some embodiments, each of R 7  and R 8  is independently —CH 3 , —CH 2 CH 3 , —CH 2 CH 2 CH 3 , or —CH(CH 3 ) 2 . In some embodiments, each of R and R 8  is CH 3 . In some embodiments, each of R 7  and R 8  is —CH 2 CH 3 . 
     In some embodiments, R 7  and R 8  in Formula (I) and Formula (Ia) taken together with the nitrogen to which they are attached form a substituted or unsubstituted C 2 -C 6  heterocyclyl. In some embodiments, R 7  and R 8  taken together with the nitrogen to which they are attached form a substituted or unsubstituted C 2 -C 6  heterocycloalkyl. In some embodiments, R 7  and R 8  taken together with the nitrogen to which they are attached form a substituted or unsubstituted 3-7 membered heterocycloalkyl. 
     In some embodiments, R 3  in Formula (I) and Formula (1a) is 
     
       
         
         
             
             
         
       
     
     In some embodiments. R 3  in Formula (I) and Formula (Ia) is 
     
       
         
         
             
             
         
       
     
     In some embodiments. R 3  in Formula (1) and Formula (1a) is 
     
       
         
         
             
             
         
       
     
     In some embodiments, Z in Formula (I) and Formula (Ia) is —C(═O)O— or —OC(═O)—. In some embodiments, Z in Formula (1) and Formula (Ia) is —C(═O)NR 4 — or —NR 4 C(═O)—. In some embodiments, Z in Formula (I) and Formula (Ia) is —C(═O)N(CH 3 )—, —N(CH 3 )C(═O)—, —C(═O)NH—, or —NHC(═O)—. In some embodiments, Z in Formula (I) and Formula (Ia) is —OC(═O)O—, —NR 4 C(═O)O—, —OC(O)NR 4 —, or —NR 4 C(═O)NR 4 —. In some embodiments. Z in Formula (I) and Formula (Ia) is —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, —NHC(═O)NH—, —N(CH 3 )C(═O)O—, —OC(═O)N(CH 3 )—, —N(CH 3 )C(═O)N(CH 3 )—, —NHC(═O)N(CH 3 )— or —N(CH 3 )C(═O)NH—. In some embodiments, Y in Formula (I) and Formula (Ia) is —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, or —NHC(═O)NH—. 
     In some embodiments, R 5  in Formula (I) and Formula (Ia) is hydrogen or substituted or unsubstituted C 1 -C 3  alkyl. In some embodiments, R 5  in Formula (I) and Formula (Ia) is H, —CH 3 , —CH—)CH 3 , —CH 2 CH 2 CH 3 , or —CH(CH 3 ) 2 . In some embodiments, R 5  in Formula (I) and Formula (Ia) is H. 
     In some embodiments, the LNP comprises a plurality of amino lipids. For example, the LNP composition can comprise 2, 3, 4, 5, 6.7, 8, 9, 10, or more amino lipids. For another example, the LNP composition can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9, at least 10, or at least 20 amino lipids. For yet another example, the LNP composition can comprise at most 23, at most 4, at most 5, at most 6, at most 7, at most 9, at most 10, at most 20, or at most 30 amino lipids. 
     In some embodiments, the LNP composition comprises a first amino lipid. In some embodiments, the LNP composition comprises a first amino lipid and a second amino lipid. In some embodiments, the LNP composition comprises a first amino lipid, a second amino lipid, and a third amino lipid. In some embodiments, the LNP composition comprises a first amino lipid, a second amino lipid, a third amino lipid, and a fourth amino lipid. In some embodiments, the LNP composition does not comprise a fourth amino lipid. In some embodiments, the LNP composition does not comprise a third amino lipid. In some embodiments, a molar ratio of the first amino lipid to the second amino lipid is from about 0.1 to about 10. In some embodiments, a molar ratio of the first amino lipid to the second amino lipid is from about 0.20 to about 5. In some embodiments, a molar ratio of the first amino lipid to the second amino lipid is from about 0.25 to about 4. In some embodiments, a molar ratio of the first amino lipid to the second amino lipid is about 0.25, about 0.33, about 0.5, about 1, about 2, about 3, or about 4. 
     In some embodiments, a molar ratio of the first amino lipid:the second amino lipid:the third amino lipid is about 4:1:1. In some embodiments, a molar ratio of the first amino lipid:the second amino lipid:the third amino lipid is about 1:1:1. In some embodiments, a molar ratio of the first amino lipid:the second amino lipid: the third amino lipid is about 2:1:1. In some embodiments, a molar ratio of the first amino lipid:the second amino lipid: the third amino lipid is about 2:2:1. In some embodiments, a molar ratio of the first amino lipid:the second amino lipid:the third amino lipid is about 3:2:1. In some embodiments, a molar ratio of the first amino lipid:the second amino lipid: the third amino lipid is about 3:1:1. In some embodiments, a molar ratio of the first amino lipid:the second amino lipid:the third amino lipid is about 5:1:1. In some embodiments, a molar ratio of the first amino lipid:the second amino lipid:the third amino lipid is about 3:3:1. In some embodiments, a molar ratio of the first amino lipid:the second amino lipid: the third amino lipid is about 4:4:1. 
     In some embodiments, the LNP composition comprises one or more amino lipids. In some embodiments, the one or more amino lipids comprise from about 40 mol % to about 65 mol % of the total lipid present in the particle. In some embodiments, the one or more amino lipids comprise about 40 mol %, about 41 mol %, about 42 mol %, about 43 mol %, about 44 mol %, about 45 mol %, about 46 mol %, about 47 mol %, about 48 mol %, about 49 mol %, about 50 mol %, about 51 mol %, about 52 mol %, about 53 mol %, about 54 mol %, about 55 mol %, about 56 mol %, about 57 mol %, about 58 mol %, about 59 mol %, about 60 mol %, about 61 mol %, about 62 mol %, about 63 mol %, about 64 mol %, or about 65 mol % of the total lipid present in the particle. In some embodiments, the first amino lipid comprises from about 1 mol % to about 99 mol % of the total amino lipids present in the particle. In some embodiments, the first amino lipid comprises from about 16.7 mol % to about 66.7 mol % of the total amino lipids present in the particle. In some embodiments, the first amino lipid comprises from about 20 mol % to about 60 mol % of the total amino lipids present in the particle. 
     In some embodiments, the amino lipid is an ionizable lipid. An ionizable lipid can comprise one or more ionizable nitrogen atoms. In some embodiments, at least one of the one or more ionizable nitrogen atoms is positively charged. Tn some embodiments, at least 10 mol %, 20 mol %, 30 mol %, 40 mol %, 50 mol %, 60 mol %, 70 mol %, 80 mol %. 90 mol %, 95 mol %, or 99 mol % of the ionizable nitrogen atoms in the LNP composition are positively charged. In some embodiments, the amino lipid comprises a primary amine, a secondary amine, a tertiary amine, an imine, an amide, a guanidine moiety, a histidine residue, a lysine residue, an arginine residue, or any combination thereof. In some embodiments, the amino lipid comprises a primary amine, a secondary amine, a tertiary amine, a guanidine moiety, or any combination thereof. In some embodiments, the amino lipid comprises a tertiary amine. 
     In some embodiments, the amino lipid is a cationic lipid. Tn some embodiments, the amino lipid is an ionizable lipid. In some embodiments, the amino lipid comprises one or more nitrogen atoms. In some embodiments, the amino lipid comprises one or more ionizable nitrogen atoms. Exemplary cationic and/or ionizable lipids include, but are not limited to, 3-(didodecylamino)- N1,N1,4-tri dodecyl-1-piperazineethan amine (KL10), N142-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4—dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC 3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2R)), and (2S)—2-({8-[(33)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2S)). 
     In some embodiments, an amino lipid described herein can take the form of a salt, such as a pharmaceutically acceptable salt. All pharmaceutically acceptable salts of the amino lipid are encompassed by this disclosure. As used herein, amino lipid also includes its pharmaceutically acceptable salts, and its diastereomeric, enantiomeric, and epimeric forms. 
     In some embodiments, an amino lipid described herein, possesses one or more stereocenters and each stereocenter exists independently in either the R or S configuration. The lipids presented herein include all diastereomeric, enantiomeric, and epimeric forms as well as the appropriate mixtures thereof. The lipids provided herein include all cis. trans, syn, anti, entgegen (E), and zusammen (Z) isomers as well as the appropriate mixtures thereof. Tn certain embodiments, lipids described herein are prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds/salts, separating the diastereomers and recovering the optically pure enantiomers. In some embodiments, resolution of enantiomers is carried out using covalent diastereomeric derivatives of the compounds described herein. In another embodiment, diastereomers are separated by separation/resolution techniques based upon differences in solubility. In other embodiments, separation of stereoisomers is performed by chromatography or by the forming diastereomeric salts and separation by recrystallization, or chromatography, or any combination thereof. Jean Jacques, Andre Collet, Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John Wiley and Sons, Inc., 1981. In one aspect, stereoisomers are obtained by stereoselective synthesis. 
     In some embodiments, the lipids such as the amino lipids are substituted based on the structures disclosed herein. In some embodiments, the lipids such as the amino lipids are unsubstituted. In another embodiment, the lipids described herein are labeled isotopically (e.g. with a radioisotope) or by another other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels. 
     Lipids described herein include isotopically-labeled compounds, which are identical to those recited in the various formulae and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the present lipids include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine and chlorine, such as, for example,  2 H,  3 H,  13 C,  14 C,  15 N,  18 O,  17 O,  35 S  18 F,  36 Cl. In one aspect, isotopically-labeled lipids described herein, for example those into which radioactive isotopes such as  3 H and  14 C are incorporated, are useful in drug and/or substrate tissue distribution assays. In one aspect, substitution with isotopes such as deuterium affords certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements. 
     In some embodiments, the asymmetric carbon atom of the amino lipid is present in enantiomerically enriched form. In certain embodiments, the asymmetric carbon atom of the amino lipid has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (S)— or (R)-configuration. 
     In some embodiments, the disclosed amino lipids can be converted to N-oxides. In some embodiments, N-oxides are formed by a treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid and/or hydrogen peroxides). Accordingly, disclosed herein are N-oxide compounds of the described amino lipids, when allowed by valency and structure, which can be denated as N O or N + —O − . In some embodiments, the nitrogen in the compounds of the disclosure can be converted to N-hydroxy or N-alkoxy. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine by an oxidizing agent such as ra-CPBA. All shown and claimed nitrogencontaining compounds are also considered. Accordingly, also disclosed herein are N-hydroxy and Nal koxy (e.g., N—OR, wherein R is substituted or unsubstituted C 1 -C 6  alkyl, C 1 -C 6  alkenyl, C 1 -C 6  alkynyl, 3-14-membered carbocycle or 3-14-membered heterocycle) derivatives of the described amino lipids. 
     PEG-Lipid 
     As used herein, a “PEG lipid” or “PEG-lipid” refers to a lipid comprising a polyethylene glycol component. 
     In some embodiments, the described LNP composition comprises a PEG-lipid. In some embodiments, the described LNP composition comprises two or more PEG-lipids. Exemplary PEG—lipids include, but are not limited to, the lipids. Exemplary PEG-lipids also include, but are not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG—modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, the one or more PEG-lipids can comprise PEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, a PEG-DSPE lipid, or a combination thereof. In some embodiments, PEG moiety is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some embodiments, the PEG moiety is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, the PEG moiety includes PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, e.g., j. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)). In some embodiments, the PEG moiety does not include PEG copolymers, e.g., it may be a PEG monopolymer. Exemplary PEG-lipids include, but are not limited to, PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylgiycerol (PEG-DSPE), PEG-dipalmitoylglycerol, PEG-disteiylglycerol, PEG-dilawylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol, and PEG-DMB (3,4- Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). 
     In some embodiments, a PEG-lipid is a PEG-lipid conjugate, for example, PEG coupled to dialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides (see, e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g. POZ-DAA conjugates; see, e.g., WO 2010/006282), polyamide oligomers (e.g.,  ATTA -lipid conjugates), and mixtures thereof. 
     A PEG-lipid can comprise one or more ethylene glycol units, for example, at least 1, at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, or at least 150 ethylene glycol units. In some embodiments, a number average molecular weight of the PEG-lipids is from about 200 Da to about 5000 Da. In some embodiments, a number average molecular weight of the PEG-lipids is from about 500 Da to about 3000 Da. In some embodiments, a number average molecular weight of the PEG-lipids is from about 750 Da to about 2500 Da. In some embodiments, a number average molecular weight of the PEG- lipids is from about 750 Da to about 2500 Da. In some embodiments, a number average molecular weight of the PEG-lipids is about 500 Da, about 750 Da, about 1000 Da, about 1250 Da, about 1500 Da, about 1750 Da, or about 2000 Da. In some embodiments, a polydispersity index (PD1) of the one or more PEG-lipids is smaller than 2. In some embodiments, a PDI of the one or more PEG-lipids is at most 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In some embodiments, a PDI of the one or more PEG-lipids is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. 
     In some embodiments, the PEG-lipid comprises from about 0.1 mol % to about 10 mol % of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises from about 0.1 mol % to about 6 mol % of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises from about 0.5 mol % to about 5 mol % of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises from about 1 mol % to about 3 mol % of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises about 2.0 mol % to about 2.5 mol % of the total lipid present in the particle. In some embodiments, the PEG-lipid comprises about 1 mol %, about 1.1 mol %, about 1.2 mol %, about 1.3 mol %, about 1.4 mol %, about 1.5 mol %, about 1.6 mol %, about 1.7 mol %, about 1.8 mol %, about 1.9 mol %, about 2.0 mol %, about 2.1 mol %, about 2.2 mol %, about 2.3 mol %, about 2.4 mol %, about 2.5 mol %, about 2.6 mol %, about 2.7 mol %, about 2.8 mol %, about 2.9 mol %, or about 3.0 mol % of the total lipid present in the particle. 
     In some embodiments, the LNP composition comprises a plurality of PEG-lipids, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more distinct PEG-lipids. 
     Phospholipid 
     As used herein, a “phospholipid” refers to a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds. In some embodiments, a phospholipid may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of an LNP to pass through the membrane, i.e., delivery of the one or more elements to a cell. 
     In some embodiments, the described LNP composition comprises a phospholipid. In some embodiments, the phospholipid comprises a lipid selected from the group consisting of: phosphatidylcholine (PC), phosphatidylethanolamine amine, glycerophospholipid, sphingophospholipids, Guriserohosuhono, sphingolipids phosphono lipids, natural lecithins, and hydrogenated phospholipid. In some embodiments, the phospholipid comprises a phosphatidylcholine. Exemplary phosphatidylcholines include, but are not limited to, soybean phosphatidylcholine, egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dipalmitoyl phosphatidylcholine, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), dimyristoyl phosphatidylcholine (DMPC), and dioleoyl phosphatidylcholine (DOPC). In certain specific embodiments, the phospholipid is DSPC. 
     In some embodiments, the phospholipid comprises a phosphatidylethanolamine amine. In some embodiments, the phosphatidylethanolamine amine is distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dimyristoyl phosphoethanolamine (DMPE), 16-0-Monome Le PE, 16-0-dimethyl PE, 18-1-trans PE, palmitoyl oleoyl-phosphatidylethanolamine (POPE), or 1-stearoyl-2- oleoyl-phosphatidyl ethanolamine (SOPE). In some embodiments, the phospholipid comprises a glycerophospholipid. In some embodiments, the glycerophospholipid is plasmalogen, phosphatidate, or phosphatidylcholine. In some embodiments, the glycerophospholipid is phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyl oleoyl phosphatidylglycerol (POPG), or lysophosphatidylcholine. In some embodiments, the phospholipid comprises a sphingophospholipid. In some embodiments, the sphingophospholipid is sphingomyelin, ceramide phosphoethanolamine, ceramide phosphoglycerol, or ceramide phosphoglycerophosphoric acid. In some embodiments, the phospholipid comprises a natural lecithin. In some embodiments, the natural lecithin is egg yolk lecithin or soybean lecithin. Tn some embodiments, the phospholipid comprises a hydrogenated phospholipid. In some embodiments, the hydrogenated phospholipid is hydrogenated soybean phosphatidylcholine. In some embodiments, the phospholipid is selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. 
     In some embodiments, the phospholipid comprises a lipid selected from: 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC). 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 2-Oleoyl-1- pahnitoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-di arachidonoyl-sn-glycero-3-phosphocholine, 1,2-di docosahexaenoyl-sn- glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diaracliidonoyl-sn-gly cero-3- phosphoethanol amine, 1,2-di docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diol eoy I -sn- glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. 
     A phospholipid can comprise a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety can comprise phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, or a sphingomyelin. A fatty acid moiety can comprise lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, or docosahexaenoic acid. In some specific embodiments, a phospholipid can be functionalized with or cross-linked to one or more alkynes, which may undergo a copper-catalyzed cycloaddition upon exposure to an azide. 
     In some embodiments, the LNP composition comprises a plurality of phospholipids, for example, at least 2, 3, 4, 5, or more distinct phospholipids. In some embodiments, the phospholipid comprises from 1 mol % to 20 mol % of the total lipid present in the particle. In some embodiments, the phospholipid comprises from about 5 mol % to about 15 mol % of the total lipid present in the particle. In some embodiments, the phospholipid comprises from about 8 mol % to about 12 mol % of the total lipid present in the particle. In some embodiments, the phospholipid comprises from about 9 mol %, 10 mol %, or 11 mol % of the total lipid present in the particle. 
     Cholesterol 
     In some embodiments, the LNP composition comprises a cholesterol or a derivative thereof. In some embodiments, the LNP composition comprises a structural lipid. The structural lipid can be selected from steroid, sterol, alkyl resoreinol, cholesterol or derivative thereof, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alphatocopherol, and a combination thereof. In some embodiments, the structural lipid is a corticosteroid such as prednisolone, dexamethasone, prednisone, and hydrocortisone. In some embodiments, the cholesterol or derivative thereof is cholesterol, 5- heptadecylresorcinol, or cholesterol hemisuccinate. Tn some embodiments, the cholesterol or derivative thereof is cholesterol. 
     In some embodiments, the cholesterol or derivative thereof is a cholesterol derivative. In some embodiments, the cholesterol derivative is a polar cholesterol analogue. In some embodiments, the polar cholesterol analogue is 5a-cholestanol, 513-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether, cholestely1-(4′-hydroxy)-butyl ether, or 6-ketocholestanol. In some embodiments, the polar cholesterol analogue is cholesteryl-(4′-hydroxy)-butyl ether. In some embodiments, the cholesterol derivative is a non-polar cholesterol analogue. In some embodiments, the non-polar cholesterol analogue is 5acholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, or cholestetyl decanoate. 
     In some embodiments, the cholesterol or the derivative thereof comprises from 20 mol % to 50 mol % of the total lipid present in the particle. In some embodiments, the cholesterol or the derivative thereof comprises about 20 mol %, about 21 mol %, about 22 mol %, about 23 mol %, about 24 mol %, about 25 mol %, about 26 mol %, about 27 mol %, about 28 mol %, about 29 mol %, about 30 mol %, about 31 mol %, about 32 mol %, about 33 mol %, about 34 mol %, about 35 mol %, about 36 mol %, about 37 mol %, about 38 mol %, about 39 mol %, about 40 mol %, about 41 mol %, about 42 mol %, about 43 mol %, about 44 mol %. about 45 mol %. about 46 mol %, about 47 mol %, about 48 mol %, or about 50 mol % of the total lipid present in the particle. 
     Phosphate Charge Neutralizer 
     In some embodiments, the LNP described herein comprises a phosphate charge neutralizer. In some embodiments, the phosphate charge neutralizer comprises arginine, asparagine, glutamine, lysine, histidine, cationic dendrimers, polyamines, or a combination thereof. In some embodiments, the phosphate charge neutralizer comprises one or more nitrogen atoms. In some embodiments, the phosphate charge neutralizer comprises a polyamine. In some embodiments, the polyamine is 1,3- propanediamine, spermine, spermidine, Norspermidine, Tris(2-aminoethy)amine, Cyclen, 1,4,7- Triazacyclononane, 1,1,1-Tris(aminomethyl)ethane, Diethylenetriamine, Triethylenetetramine, or a combination thereof. In some embodiments, the polyamine is 1,3-propanediamine, 1,4-butanediamine, spermine, spermidine, or a combination thereof. In some embodiments, the N/P ratio for the phosphate charge neutralizer is from 0.01 to 10. In some embodiments, the N/P ratio for the phosphate charge neutralizer is from about 0.05 to about 2. In some embodiments, the N/P ratio for the phosphate charge neutralizer is from about 0.1 to about 1. In some embodiments, the N/P ratio for the phosphate charge neutralizer is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1. In some embodiments, the NIP ratio for the phosphate charge neutralizer is about 0.25, 0.5, or 0.75. 
     Antioxidants 
     In some embodiments, the LNP described herein comprises one or more antioxidants. In some embodiments, the one or more antioxidants function to reduce a degradation of the cationic lipids, the payload, or both. In some embodiments, the one or more antioxidants comprise a hydrophilic antioxidant. In some embodiments, the one or more antioxidants is a chelating agent such as ethylenediaminetetraacefic acid (EDTA) and citrate. In some embodiments, the one or more antioxidants is EDTA. In some embodiments, the one or more antioxidants comprise a lipophilic antioxidant. In some embodiments, the lipophilic antioxidant comprises a vitamin E isomer or a polyphenol. In some embodiments, the one or more antioxidants are present in the LNP composition at a concentration of at least 1 mM, at least 10 mM, at least 20 mM, at least 50 mM, or at least 100 mM. In some embodiments, the one or more antioxidants are present in the particle at a concentration of about 20 mM. 
     Payload 
     The LNPs described herein can be designed to deliver a payload, such as a therapeutic agent, or a target of interest. In some embodiments, an LNP described herein encloses one or more components of a base editor system as described herein. For example, a LNP may enclose one or more of a guide RNA, a nucleic acid encoding the guide RNA, a vector encoding the guide RNA, a base editor fusion protein, a nucleic acid encoding the base editor fusion protein, a programmable DNA binding domain, a nucleic acid encoding the programmable DNA binding domain, a deaminase, a nucleic acid encoding the deaminase, or all or any combination thereof. In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is a RNA, for example, a mRNA. 
     Additional exemplary therapeutic agents include, but are not limited to, antibodies (e.g., monoclonal, chimeric, humanized, nanobodies, and fragments thereof etc.), cholesterol, hormones, peptides, proteins, chemotherapeutics and other types of antineoplastic agents, low molecular weight drugs, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allozymes, aptamers, ribozyme, decoys and analogs thereof, plasmids and other types of expression vectors, and small nucleic acid molecules, RNAi agents, short interfering nucleic acid (siNA), messenger ribonucleic acid (messenger RNA, mRNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules, peptide nucleic acid (PNA), a locked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmented interfering RNA), aiRNA (asymetrical interfering RNA), and siRNA with 1, 2 or more mismatches between the sense and anti-sense strand to relevant cells and/or tissues, such as in a cell culture, subject or organism. Therapeutic agents can be purified or partially purified, and can be naturally occurring or synthetic, or chemically modified. In some embodiments, the therapeutic agent is an RNAi agent, short interfering nucleic acid (siNA), short interfering RNA (siRNA), double- stranded RNA (dsRNA), micro-RNA (miRNA), or a short hairpin RNA (shRNA) molecule. In some embodiments, the therapeutic agent is an mRNA. 
     In some embodiments, the payload comprises one or more nucleic acid(s) (i.e., one or more nucleic acid molecular entities). In some embodiments, the nucleic acid is a single-stranded nucleic acid. In some embodiments, single-stranded nucleic acid is a DNA. In some embodiments, single- stranded nucleic acid is an RNA. In some embodiments, the nucleic acid is a double-stranded nucleic acid. In some embodiments, the double-stranded nucleic acid is a DNA. In some embodiments, the double-stranded nucleic acid is an RNA. In some embodiments, the double-stranded nucleic acid is a DNA-RNA hybrid. In some embodiments, the nucleic acid is a messenger RNA (mRNA), a microRNA, an asymmetrical interfering RNA (aiRNA), a small hairpin RNA (shRNA), or a Dicer- Substrate dsRNA. 
     Other Lipids 
     In some embodiments, the disclosed LNP compositions comprise a helper lipid. In some embodiments, the disclosed LNP compositions comprise a neutral lipid. In some embodiments, the disclosed LNP compositions comprise a stealth lipid. In some embodiments, the disclosed LNP compositions comprises additional lipids. 
     As used herein, neutral lipids suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn- glycero-3- phosphocholine (DSPC), phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl- sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoylsn- glycero-3-phosphocholine (DBPC), 1- stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3- phosphocholine (DEPC), 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), lysophosphatidyl choline, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In some embodiments, the neutral phospholipid is selected from the group consisting of DSPC and dimyristoyl phosphatidyl ethanolamine (DMPE). In some embodiments, the neutral phospholipid is DSPC. Neutral lipids can function to stabilize and improve processing of the LNPs. 
     Helper lipids can refer to lipids that enhance transfection (e.g. transfection of the nanoparticle (LNP) comprising the composition as provided herein, including the biologically active agent). The mechanism by which the helper lipid enhances transfection includes enhancing particle stability. In some embodiments, the helper lipid enhances membrane fusogenicity. Helper lipids can include steroids, sterols, and alkyl resorcinols. Helper lipids suitable for use in the present disclosure can include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In some embodiments, the helper lipid is cholesterol. In some embodiments, the helper lipid is be cholesterol hemisuccinate. 
     Stealth lipids can refer to lipids that alter the length of time the nanoparticles can exist in vivo (e.g., in the blood). Stealth lipids can assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids used herein may modulate pharmacokinetic properties of the LNP. Stealth lipids suitable for use in a lipid composition of the disclosure can include, but are not limited to, stealth lipids having a hydrophilic head group linked to a lipid moiety. Stealth lipids suitable for use in a lipid composition of the present disclosure and information about the biochemistry of such lipids can be found in Romberg et al, Pharmaceutical Research, Vol. 25, No. 1, 2008, pg. 55-71 and I-Toekstra et al, Biochimica et Biophysica Acta 1660 (2004) 41-52. Additional suitable PEG lipids are disclosed, e.g., in WO 2006/007712. 
     In some embodiments, the stealth lipid is a PEG-lipid. In one embodiment, the hydrophilic head group of stealth lipid comprises a polymer moiety selected from polymers based on PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N- vinylpyrrolidone), polyaminoacids and poly N-(2-hydroxypropyl)methacrylamide]. Stealth lipids can comprise a lipid moiety. In some embodiments, the lipid moiety of the stealth lipid may be derived from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. The dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups. 
     The structures and properties of helper lipids, neutral lipids, stealth lipids, and/or other lipids are further described in W02017173054A1, W02019067999A1, US20180290965A1, US20180147298A1, US20160375134A1, U.S. Pat. Nos. 8,236,770, 8,021,686, 8,236,770B2, U.S. Pat. No. 7,371,404B2, U.S. Pat. No. 7,780,983B2, U.S. Pat. No. 7,858,117B2, US20180200186A1, US20070087045A1, W02018119514A1, and W02019067992A1, all of which are hereby incorporated by reference in their entirety. 
     LNP Formulations 
     The LNPs described herein can be designed for one or more specific applications or targets. The elements of a nanoparticle (LNP) composition or a composition can be selected based on a particular application or target, and/or based on the efficacy, toxicity, expense, ease of use, and availability. Similarly, the particular formulation of a nanoparticle composition is a composition comprising one or more described lipids. may be selected for the particular application or target. Suitable phosphate charge neutralizers to be used in formulations include, but are not limited to, Spermidine and 1,3-propanediamine. 
     The described LNP formulations can be designed for one or more specific applications or targets. For example, a nanoparticle composition may be designed to deliver a therapeutic agent such as an RNA to a particular cell, tissue, organ, or system or group thereof in a mammal&#39;s body. Physiochemical properties of nanoparticle compositions may be altered in order to increase selectivity for particular bodily targets. For instance, particle sizes may be adjusted based on the fenestration sizes of different organs. The therapeutic agent included in a nanoparticle composition may also be selected based on the desired delivery target or targets. For example, a therapeutic agent may be selected for a particular indication, condition, disease, or disorder and/or for delivery to a particular cell, tissue, organ, or system or group thereof (e.g., localized or specific delivery). In certain embodiments, a nanoparticle composition may include an mRNA encoding a polypeptide of interest capable of being translated within a cell to produce the polypeptide of interest. Such a composition may be designed to be specifically delivered to a particular organ. 
     The amount of a therapeutic agent in an LNP composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition. For example, the amount of an RNA comprised in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of a therapeutic agent and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some embodiments, the wt/wt ratio of the lipid component to a therapeutic agent in a nanoparticle composition may be from about 5:1 to about 60:1, such as about 5:1. 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt/wt ratio of the lipid component to a therapeutic agent may be from about 10:1 to about 40:1. In certain embodiments, the wt/wt ratio is about 20:1. The amount of a therapeutic agent in a nanoparticle composition can be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy). 
     In some embodiments, an LNP composition comprises one or more nucleic acids such as RNAs. In some embodiments, the one or more RNAs, lipids, and amounts thereof may be selected to provide a specific NIP ratio. The NIP ratio can be selected from about 1 to about 30. The N/P ratio can be selected from about 2 to about 10. In some embodiments, the NIP ratio is from about 0.1 to about 50. In some embodiments, the N/P ratio is from about 2 to about 8. hi some embodiments, the NIP ratio is from about 2 to about 15, from about 2 to about 10, from about 2 to about 8, from about 2 to about 6, from about 3 to about 15, from about 3 to about 10, from about 3 to about 8, from about 3 to about 6, from about 4 to about 15, from about 4 to about 10, from about 4 to about 8, or from about 4 to about 6. hi some embodiments, the N/P ratio is about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, or about 6.5. In some embodiments, the NIP ratio is from about 4 to about 6. In some embodiments, the NIP ratio is about 4, about 4.5, about 5, about 5.5, or about 6. 
     In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 70%, from about 70% to about 90%, or from about 90% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 75% to about 95%. 
     In another aspect, provided herein is a lipid nanoparticle (LNP) comprising the composition as provided herein. As used herein, a lipid nanoparticle (LNP) composition or a nanoparticle composition is a composition comprising one or more described lipids. LNP compositions are typically sized on the order of micrometers or smaller and may include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. In some embodiments, a LNP refers to any particle that has a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. In some embodiments, a nanoparticle may range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm. In some embodiments, a liposome having a lipid bilayer with a diameter of 500 rim or less. In some embodiments, the LNPs described herein can have a mean diameter of from about 1 nm to about 2500 nm, from about 10 nm to about 1500 nm, from about 20 nm to about 1000 nm, from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 rim to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm- or from about 70 nm to about 80 nm. The LNPs described herein can have a mean diameter of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, or greater. The LNPs described herein can be substantially non-toxic. 
     In some embodiments, an LNP may be made from cationic, anionic, or neutral lipids. In some embodiments, an LNP may comprise neutral lipids, such as the fusogenic phospholipid 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membrane component cholesterol, as helper lipids to enhance transfection activity and nanoparticle stability. In some embodiments, an LNP may comprise hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or combination of lipids that are known in the art can be used to produce an LNP. Examples of lipids used to produce LNPs include, but are not limited to DOTMA (N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOSPA (N,N-dimethyl-N-([2-sperminecarboxamido]ethyl)-2,3-bis(dioleyloxy)-1-propaniminium pentahydrochloride), DOTAP (1,2-Dioleoyl-3-trimethylammonium propane), DMRIE (N-(2-hydroxyethyl)- N,N-dimethyl-2,3-bis(tetradecyloxy-1-propanaminiumbromide), DC-cholesterol (3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol), DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE (2-Bis(dimethylphosphino)ethane)-polyethylene glycol (PEG). Examples of cationic lipids include, but are not limited to, 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3 -DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids include, but are not limited to, DPSC, DPPC (Dipalmitoylphosphatidylcholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE, and SM (sphingomyelin). Examples of PEG-modified lipids include, but are not limited to, PEG-DMG (Dimyristoyl glycerol), PEG-CerC14, and PEG-CerC20. In some embodiments, the lipids may be combined in any number of molar ratios to produce a LNP. In some embodiments, the polynucleotide may be combined with lipid(s) in a wide range of molar ratios to produce an LNP. 
     The term “substituted”, unless otherwise indicated, refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, oxo, thioxy, arylthio, alkylthioalkyl, arylthioallcyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, aiylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, aiylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and an aliphatic group. It is understood that the substituent may be further substituted. Exemplary• substituents include amino, alkylamino, and the like. 
     As used herein, the term “substituent” means positional variables on the atoms of a core molecule that are substituted at a designated atom position, replacing one or more hydrogens on the designated atom, provided that the designated atom&#39;s normal valency is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. A person of ordinal), skill in the art should note that any carbon as well as heteroatom with valences that appear to be unsatisfied as described or shown herein is assumed to have a sufficient number of hydrogen atom(s) to satisfy the valences described or shown. In certain instances one or more substituents having a double bond (e.g., “oxo” or “═O”) as the point of attachment may be described, shown or listed herein within a substituent group, wherein the structure may only show a single bond as the point of attachment to the core structure of Formula (I). A person of ordinary skill in the art would understand that, while only a single bond is shown, a double bond is intended for those substituents. 
     The term “alkyl” refers to a straight or branched hydrocarbon chain radical, having from one to twenty carbon atoms, and which is attached to the rest of the molecule by a single bond. An alkyl comprising up to 10 carbon atoms is referred to as a C 1 -C 10  alkyl, likewise, for example, an alkyl comprising up to 6 carbon atoms is a C 1 -C 6  alkyl. Alkyls (and other moieties defined herein) comprising other numbers of carbon atoms are represented similarly. Alkyl groups include, but are not limited to, C 1 -C 10  alkyl, C 1 -C 9  alkyl, C 1 -C 8  alkyl. C 1 -C 7  alkyl, C 1 -C 6  alkyl, C 1 -C 5  alkyl, C 1 -C 4  alkyl, C 1 -C 3  alkyl, C 1 -C 2  alkyl, C 2 -C 8  alkyl, C 3 -C 8  alkyl and C 4 -C 8  alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (i-propyl), n-butyl, i-butyl, s-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, 1-ethyl-propyl, and the like. In some embodiments, the alkyl is methyl or ethyl. In some embodiments, the alkyl is —CH(CH 3 ) 2  or —C(CH 3 ) 3 . Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted as described below. “Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group. In some embodiments, the alkylene is —CI-12—, —CH 2 CH 2 —, or —CH 2 CH 2 CH 2 —. In some embodiments, the alkylene is —CH 2 —. In some embodiments, the alkylene is —CH 2 CH 2 —. In some embodiments, the alkylene is —CH 2 CH 2 CH 2 —. 
     The term “alkenyl” refers to a type of alkyl group in which at least one carbon-carbon double bond is present. In one embodiment, an alkenyl group has the formula —C(R)═CR 2 , wherein R refers to the remaining portions of the alkenyl group, which may be the same or different. In some embodiments, R is H or an alkyl. In some embodiments, an alkenyl is selected from ethenyl (i.e., vinyl), propenyl (i.e., allyl), butenyl, pentenyl, pentadienyl, and the like. Non-limiting examples of an alkenyl group include —CH═CH 2 , —C(CH 3 )═CH 2 , —CH═CHCH 3 , —C(CH 3 )═CHCH 3 , and —CH 2 CH═CH 2 . 
     The term “cycloalkyl” refers to a monocyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. In some embodiments, cycloalkyls are saturated or partially unsaturated. In some embodiments, cycloalkyls are spirocyclic or bridged compounds. In some embodiments, cycloalkyls are fused with an aromatic ring (in which case the cycloalkyl is bonded through a non-aromatic ring carbon atom). Cycloalkyl groups include groups having from 3 to 10 ring atoms. Representative cycloalkyls include, but are not limited to, cycloalkyls having from three to ten carbon atoms, from three to eight carbon atoms, from three to six carbon atoms, or from three to five carbon atoms. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the monocyclic cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl or cyclohexenyl. In some embodiments, the monocyclic cycloalkyl is cyclopenteny 1. Polycyclic radicals include, for example, adamantyl, 1,2- dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetrainyl, decalinyl, 3,4-dihydronaphthalenyl-.1 (2H)— one. spiro[2.2]pentyl, norbomyl and bicycle[1.1.1]pentyl. Unless otherwise stated specifically in the specification, a cycloalkyl group may be optionally substituted. Depending on the structure, a cycloalkyl group can be monovalent or divalent (i.e., a cycloalkylene group). 
     The term “heterocycle” or “heterocyclic” refers to heteroaromatic rings (also known as heteroaryls) and heterocycloallcyl rings (also known as heteroalicyclic groups) that includes at least one heteroatom selected from nitrogen, oxygen and sulfur, wherein each heterocyclic group has from 3 to 12 atoms in its ring system, and with the proviso that any ring does not contain two adjacent O or S atoms. A “heterocyclyl” is a univalent group formed by removing a hydrogen atom from any ring atoms of a heterocyclic compound. In some embodiments, heterocycles are monocyclic, bicyclic, polycyclic, spirocyclic or bridged compounds. Non-aromatic heterocyclic groups (also known as heterocycloalkyls) include rings having 3 to 12 atoms in its ring system and aromatic heterocyclic groups include rings having 5 to 12 atoms in its ring system. The heterocyclic groups include benzofused ring systems. Examples of non-aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, oxazolidinonyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl. thiomorpholinyl, thioxanyl, piperazinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, pyrrolin-2-yl, pyrrolin-3-yl, indolinyl, 2H-pyranyl, 4Hpyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazoli diny I, 3-az.abicy c1 o[3. 1.0]hexany 1,3- azabicyclo[4.1.0]heptanyl, 3 h-indolyl, indolin-2-onyl, isoindolin-1-onyl, isoindoline-1,3-dionyl, 3,4- dihydroisoquinolin-1(2H)-onyl, 3,4-dihydroquinolin-2(1H)-onyl, isoindoline-1,3-dithionyl, benzo[d]oxazol-2(3H)-onyl, 1H-benzo[d]imidazol-2(3H)-onyl, benzo[d]thiazol-2(3H)-onyl, and quinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, futyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furaz.anyl, benzofuraz.anyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The foregoing groups are either C-attached (or Clinked) or N-attached where such is possible. For instance, a group derived from pyrrole includes both pyrrol-1-yl (N-attached) or pyrrol-3-yl (C-attached). Further, a group derived from imidazole includes imidazol-1-yl or imidazol-3-yl (both N-attached) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all C-attached). The heterocyclic groups include benzo-fused ring systems. Non-aromatic heterocycles are optionally substituted with one or two oxo (═O) moieties, such as pyrrolidin-2-one. In some embodiments, at least one of the two rings of a bicyclic heterocycle is aromatic. In some embodiments, both rings of a bicyclic heterocycle are aromatic. 
     The term “heterocycloalkyl” refers to a cycloalkyl group that includes at least one heteroatom selected from nitrogen, oxygen, and sulfur. Unless stated otherwise specifically in the specification, the heterocycloalkyl radical may be a monocyclic, or bicyclic ring system, which may include fused (when fused with an aryl or a heterowyl ring, the heterocycloalkyl is bonded through a non-aromatic ring atom) or bridged ring systems. The nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized. The nitrogen atom may be optionally quaternized. The heterocycloalkyl radical is partially or fully saturated. Examples of heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl, 1,1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes all ring forms of carbohydrates, including but not limited to monosaccharides, disaccharides and oligosaccharides. Unless otherwise noted, heterocycloalkyls have from 2 to 12 carbons in the ring. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring and 1 or 2 N atoms. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring and 3 or 4 N atoms. In some embodiments, heterocycloalkyls have from 2 to 12 carbons, 0-2 N atoms, 0-2 O atoms, 0-2 P atoms, and 0-1 S atoms in the ring. In some embodiments, heterocycloalkyls have from 2 to 12 carbons, 1-3 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. It is understood that when referring to the number of carbon atoms in a heterocycloalkyl, the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including the heteroatoms) that make up the heterocycloalkyl (i.e. skeletal atoms of the heterocycloalkyl ring). Unless stated otherwise specifically in the specification, a heterocycloalkyl group may be optionally substituted. As used herein, the term “teterocycloalkylene” can refer to a divalent heterocycloalkyl group. 
     The term “heteroaryl” refers to an aryl group that includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur. The heteroaryl is monocyclic or bicyclic. Illustrative examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, furazanyl, indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. Illustrative examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl. Illustrative examples of bicyclic heteroaryls include indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. In some embodiments, heteroaryl is pyridinyl, pyrazinyl, pyrimidinyl, thiazolyl, thienyl, thiadiazolyl or furyl. In some embodiments, a heteroaryl contains 0-6 N atoms in the ring. In some embodiments, a heteroaryl contains 1-4 N atoms in the ring. In some embodiments, a heteroaryl contains 4-6 N atoms in the ring. In some embodiments, a heteroaryl contains 0-4 N atoms, 0-1 O atoms, 0-1 P atoms, and 0-1 S atoms in the ring. In some embodiments, a heteroaryl contains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In some embodiments, heteroaryl is a C 1 -C 9  heteroaryl. In some embodiments, monocyclic heteroaryl is a C 1 -C 5  heteroaryl. In some embodiments, monocyclic heteroaryl is a 5- membered or 6-membered heteroaryl. In some embodiments, a bicyclic heteroaryl is a C 6 -C 9  heteroaryl. In some embodiments, a heteroaryl group is partially reduced to form a heterocycloalkyl group defined herein. In some embodiments, a heteroaryl group is fully reduced to form a heterocycloalkyl group defined herein. 
     As used herein, the “N/P ratio” is the molar ratio of ionizable (e.g., in the physiological pH range) nitrogen atoms in a lipid (or lipids) to phosphate groups in a nucleic acid molecular entity (or nucleic acid molecular entities), e.g., in a nanoparticle composition comprising a lipid component and an RNA. Ionizable nitrogen atoms can include, for example, nitrogen atoms that can be protonated at about pH 1, about pH 2, about pH 3, about pH 4, about pH5, about pH 6, about pH 7, about pH 7.5, or about pH 8 or higher. The physiological pH range can include, for example, the pH range of different cellular compartments (such as organs, tissues, and cells) and bodily fluids (such as blood, CSF, gastric juice, milk, bile, saliva, tears, and urine). In certain specific embodiments, the physiological pH range refers to the pH range of blood in a mammal, for example, from about 7.35 to about 7.45. Similarly, for phosphate charge neutralizers that have one or more ionizable nitrogen atoms, the N/P ratio can refer to a molar ratio of ionizable nitrogen atoms in the phosphate charge neutralizer to the phosphate groups in a nucleic acid. In some embodiments, ionizable nitrogen atoms refer to those nitrogen atoms that are ionizable within a pH range between 5 and 14. 
     It is further contemplated that the LNP formulations that encapsulate the gRNAs and mRNAs drug substances described herein may be modified to include GalNac lipid formulations such as those disclosed in co-owned U.S. patent application Ser. No. 17/192,709 filed on Mar. 4, 2021 with a parallel PCT application being filed on same date having International Application Number PCT/US21/20955. Employment of such GalNac lipid formulations are understood to be capable of enhancing LNP uptake in LDL-R deficient cells, such as those associated with heterozygous and homozygous familial hypercholesterolemia patient populations. 
     For the payload that does not contain a phosphate group, the N/P ratio can refer to a molar ratio of ionizable nitrogen atoms in a lipid to the total negative charge in the payload. For example, the N/P ratio of an LNP composition can refer to a molar ratio of the total ionizable nitrogen atoms in the LNP composition to the total negative charge in the payload that is present in the composition. 
     As used herein, amino lipids can contain at least one primary, secondary or tertiary amine moiety that is protonatable (or ionizable) between pH range 4 and 14. In some embodiments, the amine moiety/moieties function as the hydrophilic headgroup of the amino lipids. When most of the amine moiety(ies) of an amino lipid (or amino lipids) in a nucleic acid- lipid nanoparticle formulation is protonated at physiological pH, then the nanoparticles can be termed as cationic lipid nanoparticle (cLNP). When most of the amine moiety(ies) of an amino lipid (or amino lipids) in a nucleic acid-lipid nanoparticle formulation is not protonated at physiological pH but can be protonated at acidic pH, endosomal pH for example, can be termed as ionizable lipid nanoparticle (iLNP). The amino lipids that constitute cLNPs can be generally called cationic amino lipids (cLipids). The amino lipids that constitute iLNPs can be called ionizable amino lipids (iLipids). The amino lipid can be an iLipid or a cLipid at physiological pH. 
     Kits 
     One aspect of the disclosure relates to kits including the compositions comprising a single guide RNA as provided herein, the base editor system and complex as provided herein, the composition as provided herein, or the lipid nanoparticle as provided herein for treating or preventing a condition. The kits can further include one or more additional therapeutic regimens or agents for treating or preventing a condition. 
     Also disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more methods described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic. 
     The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. 
     For example, the container(s) include the composition of the disclosure, and optionally in addition with therapeutic regimens or agents disclosed herein. Such kits optionally include an identifying description or label or instructions relating to its use in the methods described herein. 
     A kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included. 
     In embodiments, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein. 
     EXAMPLES 
     Introduction 
     The present disclosure will be described in greater detail by way of specific examples. These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein. Those of skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield alternative embodiments according to the present disclosure. 
     Gene-editing technologies, including CRISPR-Cas9 nucleases and CRISPR base editors, have the potential to precisely and permanently modify disease-causing genes in human patients. The demonstration of durable editing in target organs of non-human primates (NHPs) is an important and necessary step prior to in vivo administration of gene editors to patients in clinical trials. Herein provided is the first demonstration that CRISPR base editors delivered in vivo using lipid nanoparticles (LNP) can efficiently modify disease-related genes in living NHPs. In addition to providing proof-of-concept for a once-and-done approach to reduce cholesterol and treat coronary heart disease, the leading cause of death worldwide, these results demonstrate how CRISPR base editors can be productively applied to a variety of therapeutic target genes in the liver and potentially other organs. 
     In vivo gene editing is an emerging new therapeutic approach to make DNA modifications in a patient&#39;s own body in organs such as the liver. Gene-editing methods include CRISPR-Cas9 and -Cas12 nucleases, CRISPR cytosine base editors, CRISPR adenine base editors, and CRISPR prime editors. The human PCSK9 and ANGPTL3 gene are particularly attractive potential target(s) for in vivo gene editing. In principle, editing of PCSK9 could produce durable reductions in blood LDL-C levels and thereby dramatically lower one&#39;s cumulative exposure to LDL-C in contrast to all existing therapies (e.g., statins, ezetimibe, or PCSK9 inhibitors), which must be taken daily or every few weeks to months and can suffer from lack of patient adherence. 
     In vivo delivery of a gene editor that can introduce a PCSK9 or ANGPTL3 loss-of-function mutation in the liver has the potential to offer a new kind of once-and-done therapy that offers lifelong treatment of coronary heart disease. CRISPR base editors make an attractive gene-editing modality for this purpose because they function efficiently for introducing precise targeted alterations and, in contrast to CRISPR-Cas9 and other gene-editing nucleases, minimize any deleterious consequences of introducing double-strand DNA breaks. 
     CRISPR adenine base editors can induce targeted A→G edits in DNA (T→C on the opposing strand) and can be used to inactivate genes by disrupting start codons, splice donors (canonical GT sequence on the sense strand) or splice acceptors (canonical AG sequence on the sense strand) at exon-intron or intron-exon boundaries, or introducing missense mutations. Adenine 8.8-m (hereafter referred to as ABE8.8) uses its core  Streptococcus pyogenes  nickase Cas9 (nSpCas9) protein with a guide RNA (gRNA) to engage a double-strand protospacer DNA sequence, flanked by an NGG protospacer-adjacent motif (PAM) sequence on its 3′ end. The protospacer sequence is specified via hybridization of the first 20 bases of the gRNA with a complementary sequence on the “target” DNA strand, leaving part of the other (“non-target”) strand in exposed single-strand form structure called the R-loop. The ABE base editor uses an evolved deoxyadenosine deaminase domain—fused to nSpCas9—to chemically modify an adenosine nucleoside, contained in the single-stranded DNA portion of the R-loop, into inosine and nicks the target DNA strand within the DNA:RNA heteroduplex of the R-loop. This nick biases DNA repair machinery to use the freshly deaminated strand as a template, enabling highly efficient transition mutation at the targeted site. The activity window of ABE8.8 typically ranges from positions 3 to 9 in the protospacer DNA sequence specified by the gRNA, 12 to 18 base pairs 5′ of the NGG PAM (positions 21 to 23), with peak editing observed at position 6 of the protospacer. Although the mechanism of action of ABE8.8 does not involve double-strand DNA breaks, indel mutagenesis can occur low frequency. 
     The advantages of base editing compared to CRISPR-Cas9 genome editing for gene inactivation or other types of gene alteration is noted. Standard CRISPR-Cas9 editing carries higher risks of unwanted on-target effects and off-target effects emanating from double-strand breaks (large deletions, integration of vector DNA sequences, chromosomal rearrangements, induction of p53 activity, etc.). Even the intended on-target effects-small indel mutations—have an element of unpredictability, as they can result in frameshift mutations that add varied strings of anomalous amino acids at the end of the truncated protein product, or in-frame mutations that add or remove amino acids from the protein without knocking out its function. In contrast, base editing offers a means to efficiently make precise, stereotyped changes in the genome, allowing for more reproducible alteration of gene function. Base editing mitigates unintended on-target effects by virtue of not requiring double-strand breaks but instead acting through enzymatic modification of DNA bases via the deaminase domain. 
     While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. 
     Protospacer Selection and Editing 
     Example 1. gRNA Design for Targeting PCSK9 and ANGPTL3 
     All protospacers shown in Table 1 and Table 24 were chosen by two criteria. First, they matched (or very closely matched) sequences in the human, cynomolgus monkey, and/or mouse orthologs of the genes. Second, they had favorable predicted off-target profiles, as judged by the MIT Specificity Score (calculated by http://crispor.tefor.net/; minimum score of 50). 
     Where the goal is to disrupt a gene in vivo for a therapeutic purpose, cytosine base editors have the advantage of being able to directly introduce stop codons into the coding sequence of the gene (nonsense mutations) by altering specific codons for glutamine (CAG→TAG, CAA→TAA), arginine (CGA→TGA), and tryptophan (TGG→TAG/TAA/TGA, with editing of cytosines on the antisense strand). In contrast, adenine base editors cannot directly introduce stop codons, as there are no A→G changes that result in nonsense mutations. Nonetheless, the more favorable off-target profile of adenine base editors, particularly with respect to gRNA-independent off-target DNA base editing, recommend the use of adenine base editors over cytosine base editors for therapeutic purposes. Similar approaches were followed to identify and select mouse/rodent specific protospacers listed in Tables 1 and 24. 
     One strategy by which adenine base editors might be used to disrupt gene function is to edit the start codon, such as from ATG→GTG or ATG→ACG. Therefore, the resultant translation into protein will not initiate at the canonical ATG site. A second strategy by which adenine base editors might be used to disrupt gene function is to edit splice sites, whether splice donors at the 5′ ends of introns or splice acceptors at the 3′ ends of introns. Splice site disruption can result in the inclusion of intronic sequences in messenger RNA (mRNA) potentially introducing nonsense, frameshift, or in-frame indel mutations that result in premature stop codons or in insertion/deletion of amino acids that disrupt protein activity—or in the exclusion of exonic sequences, which can also introduce nonsense, frameshift, or in-frame indel mutations. Canonical splice donors comprise the DNA sequence GT on the sense strand, whereas canonical splice acceptors comprise the DNA sequence AG. Alteration of the sequence disrupts normal splicing. Splice donors can be disrupted by adenine base editing of the complementary base in the second position in the antisense strand (GT→GC), and splice acceptors can be disrupted by adenine base editing of the first position in the sense strand (AG→GG). A third strategy by which adenine base editors might be used to disrupt gene function is to introduce a missense mutation(s) into the coding region of the gene that results in production of a less functional, or non-functional protein. 
     All gRNA spacer sequences that would permit ABE8.8 (and other ABE variants containing  Streptococcus pyogenes  Cas9, such as ABE7.10, or another Cas protein that can use the NGG PAMN) to disrupt splice sites were identified, whether donors or acceptors, via A→G editing within its editing window (roughly positions 3 to 9 in the 20-nt protospacer region of DNA) were identified. Guide RNAs matching each of the protospacer sequences and otherwise conforming to the standard 100-nt  Streptococcus pyogenes  CRISPR gRNA sequence were synthesized, with each gRNA molecule having a modest degree of chemical modifications (e.g., in Table 1). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Guide RNAs (SgRNA/gRNA) for Cas9 nuclease and ABE editors 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 Proto- 
                 SEQ 
                   
                 SEQ 
                 Editor 
               
               
                 Gene 
                 gRNA 
                 spacer 
                 ID 
                   
                 ID 
                 associated 
               
               
                 Target 
                 ID 
                 (5′-3′) 
                 NO 
                 Sequence (5′-3′) 
                 NO 
                 data 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 hcPCSK9 
                 GA001 
                 GGTGCTAG 
                 66 
                 GsGsUsGCUAGCCUUGCGUUCCG 
                 253 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 GUUUUAGAgcuagaaauagcAAGUU 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 AAAAUAAGGCUAGUCCGUUAU 
                   
                   
               
               
                   
                   
                   
                   
                 CAacuugaaaaaguggcaccgagucggugcu 
                   
                   
               
               
                   
                   
                   
                   
                 sususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA002 
                 GGTGCTAG 
                 66 
                 gsgsusGCUAGCCUUGCGUUCCGG 
                 253 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 UUUUAGAgcuagaaauagcAAGUUA 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 AAAUAAGGCUAGUCCGUUAUC 
                   
                   
               
               
                   
                   
                   
                   
                 Aacuugaaaaaguggcaccgagucggugcusu 
                   
                   
               
               
                   
                   
                   
                   
                 susu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA003 
                 GGTGCTAG 
                 66 
                 gsgsusGCUAGCCUUGCGUUCCGG 
                 254 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 UUUUsAGAgcuagaaauagcAAGUUs 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 AAAAUsAAGGCUsAGUCCGUUs 
                   
                   
               
               
                   
                   
                   
                   
                 AUCsAacuugaaaaaguggcaccgagucgg 
                   
                   
               
               
                   
                   
                   
                   
                 ugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA004 
                 GGTGCTAG 
                 66 
                 gsgsusGCUAGCCUUGCGUUCCGg 
                 255 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 AuAaggcuaGUccGUUAucAAcuugaa 
                   
                   
               
               
                   
                   
                   
                   
                 aaagugGcaccgagucggugcuuuuu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA005 
                 GGTGCTAG 
                 66 
                 GsGsUsGCUAGCCUUGCGUUCCG 
                 255 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 aAuAaggcuaGUccGUUAucAAcuuga 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcuuuuu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA006 
                 GGTGCTAG 
                 66 
                 GsGsUsGCUAGCCUUGCGUUCCG 
                 253 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 aAuAaggcuaGUccGUUAucAAcuuga 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA007 
                 GGTGCTAG 
                 66 
                 gsgsusGCUAGCCUUGCGUUCCGg 
                 253 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 AuAaggcuaGUccGUUAucAAcuugaa 
                   
                   
               
               
                   
                   
                   
                   
                 aaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA007/ 
                 GGTGCTAG 
                 66 
                 gsgsusGCUAGCCUUGCGUUCCGg 
                 253 
                 SpCas9 
               
               
                   
                 GA252 
                 CCTTGCGT 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 AuAaggcuaGUccGUUAucAAcuugaa 
                   
                   
               
               
                   
                   
                   
                   
                 aaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA008 
                 GGTGCTAG 
                 66 
                 gsgsusGCUAGCCUUGCGUUCCGg 
                 256 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 UUUUAGagcuagaaauagcaaGUUsaA 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 aAuAaggcuaGUccGUUsAucAAcuug 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA009 
                 GGTGCTAG 
                 66 
                 gsgsusGCUAGCCUUGCGUUCCGG 
                 253 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 mcPCSK9 
                 GA052 
                 CAGGTTCC 
                 81 
                 csasgsGUUCCAUGGGAUGCUCUG 
                 55 
                 SpCas9 
               
               
                   
                   
                 ATGGGATG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CTCT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 mcPCSK9 
                 GA053 
                 CAGGTTCC 
                 81 
                 csasgsGUUCCAUGGGAUGCUCUG 
                 55 
                 SpCas9 
               
               
                   
                   
                 ATGGGATG 
                   
                 UUUUAGAgcuagaaauagcAAGUUA 
                   
                   
               
               
                   
                   
                 CTCT 
                   
                 AAAUAAGGCUAGUCCGUUAUC 
                   
                   
               
               
                   
                   
                   
                   
                 Aacuugaaaaaguggcaccgagucggugcusu 
                   
                   
               
               
                   
                   
                   
                   
                 susu 
                   
                   
               
               
                   
               
               
                 mcPCSK9 
                 GA054 
                 CAGGTTCC 
                 81 
                 csasgsGUUCCAUGGGAUGCUCUg 
                 55 
                 SpCas9 
               
               
                   
                   
                 ATGGGATG 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 CTCT 
                   
                 AuAaggCUaGUCcGUUAucAAcuuG 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaaguGgcaccgAgUCggugcusususu 
                   
                   
               
               
                   
               
               
                 mcPCSK9 
                 GA055 
                 CAGGTTCC 
                 81 
                 csasgsGUUCCAUGGGAUGCUCUg 
                 55 
                 SpCas9 
               
               
                   
                   
                 ATGGGATG 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 CTCT 
                   
                 AuAaggcuaGUccGUUAucAAcuugaa 
                   
                   
               
               
                   
                   
                   
                   
                 aaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA066/ 
                 CCCGCACC 
                 13 
                 cscscsGCACCUUGGCGCAGCGGG 
                 9 
                 ABE/ 
               
               
                   
                 GA095 
                 TTGGCGCA 
                   
                 UUUUAGAgcuagaaauagcAAGUUA 
                   
                 SpCas9 
               
               
                   
                   
                 GCGG 
                   
                 AAAUAAGGCUAGUCCGUUAUC 
                   
                   
               
               
                   
                   
                   
                   
                 Aacuugaaaaaguggcaccgagucggugcusu 
                   
                   
               
               
                   
                   
                   
                   
                 susu 
                   
                   
               
               
                   
               
               
                 cANGPTL3 
                 GA067/ 
                 AAGATACC 
                 14 
                 asasgsAUACCUGAAUAACUCUCG 
                 257 
                 ABE/ 
               
               
                   
                 GA101 
                 TGAATAAC 
                   
                 UUUUAGAgcuagaaauagcAAGUUA 
                   
                 SpCas9 
               
               
                   
                   
                 TCTC 
                   
                 AAAUAAGGCUAGUCCGUUAUC 
                   
                   
               
               
                   
                   
                   
                   
                 Aacuugaaaaaguggcaccgagucggugcusu 
                   
                   
               
               
                   
                   
                   
                   
                 susu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA072 
                 GGTGGCTC 
                 82 
                 gsgsusGGCUCACCAGCUCCAGCG 
                 258 
                 ABE 
               
               
                   
                   
                 ACCAGCTC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CAGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA073 
                 GCTTACCT 
                 67 
                 gscsusUACCUGUCUGUGGAAGC 
                 259 
                 ABE 
               
               
                   
                   
                 GTCTGTGG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 AAGC 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA074 
                 TGCTTACC 
                 68 
                 usgscsUUACCUGUCUGUGGAAG 
                 260 
                 ABE 
               
               
                   
                   
                 TGTCTGTG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 GAAG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hPCSK9 
                 GA075 
                 TTGGAAAG 
                 83 
                 ususgsGAAAGACGGAGGCAGCC 
                 261 
                 ABE 
               
               
                   
                   
                 ACGGAGG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 CAGCC 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hPCSK9 
                 GA076 
                 GAAAGAC 
                 84 
                 gsasasAGACGGAGGCAGCCUGGG 
                 262 
                 ABE 
               
               
                   
                   
                 GGAGGCA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GCCTGG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA077 
                 TCCCAGGC 
                 85 
                 uscscsCAGGCCUGGAGUUUAUU 
                 263 
                 ABE 
               
               
                   
                   
                 CTGGAGTT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 TATT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hPCSK9 
                 GA078 
                 AGCACCTA 
                 86 
                 asgscsACCUACCUCGGGAGCTGG 
                 264 
                 ABE 
               
               
                   
                   
                 CCTCGGGA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GCTG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA079 
                 CTTTCCAG 
                 87 
                 csususUCCAGGUCAUCACAGUUG 
                 265 
                 ABE 
               
               
                   
                   
                 GTCATCAC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 AGTT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA080 
                 CCTTTCCA 
                 88 
                 cscsusUUCCAGGUCAUCACAGUG 
                 266 
                 ABE 
               
               
                   
                   
                 GGTCATCA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CAGT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA081 
                 TTTCCAGG 
                 89 
                 usususCCAGGUCAUCACAGUUG 
                 267 
                 ABE 
               
               
                   
                   
                 TCATCACA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 GTTG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hPCSK9 
                 GA082 
                 CTTACCTG 
                 90 
                 csususACCUGCCCCAUGGGUGCG 
                 268 
                 ABE 
               
               
                   
                   
                 CCCCATGG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GTGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hPCSK9 
                 GA083 
                 TAAGGCCC 
                 91 
                 usasasGGCCCAAGGGGGCAAGCG 
                 269 
                 ABE 
               
               
                   
                   
                 AAGGGGG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CAAGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hPCSK9 
                 GA084 
                 CCTCTTCA 
                 92 
                 cscsusCUUCACCUGCUCCUGAGG 
                 270 
                 ABE 
               
               
                   
                   
                 CCTGCTCC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 TGAG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hPCSK9 
                 GA085 
                 GCCTCTTC 
                 93 
                 gscscsUCUUCACCUGCUCCUGAG 
                 271 
                 ABE 
               
               
                   
                   
                 ACCTGCTC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CTGA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hPCSK9 
                 GA086 
                 TTCACCTG 
                 94 
                 ususcsACCUGCUCCUGAGGGGCG 
                 272 
                 ABE 
               
               
                   
                   
                 CTCCTGAG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GGGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA087 
                 TCACCTGC 
                 95 
                 uscsasCCUGCUCCUGAGGGGCCG 
                 273 
                 ABE 
               
               
                   
                   
                 TCCTGAGG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GGCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA088 
                 CCCAGGCT 
                 96 
                 cscscsAGGCUGCAGCUCCCACUG 
                 274 
                 ABE 
               
               
                   
                   
                 GCAGCTCC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CACT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hPCSK9 
                 GA089 
                 CCCCAGGC 
                 97 
                 cscscsCAGGCUGCAGCUCCCACG 
                 275 
                 ABE 
               
               
                   
                   
                 TGCAGCTC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CCAC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hPCSK9 
                 GA090 
                 GCAGGTG 
                 98 
                 gscsasGGUGACCGUGGCCUGCGG 
                 276 
                 ABE 
               
               
                   
                   
                 ACCGTGGC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CTGCG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA091 
                 AAGATACC 
                 15 
                 asasgsAUACCUGAAUAACCCUCG 
                 59 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CCTC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA092 
                 CTCCTTTA 
                 99 
                 csuscsCUUUAGGAGGCUGGUGG 
                 277 
                 ABE 
               
               
                   
                   
                 GGAGGCT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 GGTGG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA093 
                 TTTTCAGG 
                 100 
                 usususUCAGGAGAAUUUUGGUU 
                 278 
                 ABE 
               
               
                   
                   
                 AGAATTTT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 GGTT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA094/ 
                 CTTTTCAG 
                 101 
                 csususUUCAGGAGAAUUUUGGU 
                 279 
                 ABE 
               
               
                   
                 GA153 
                 GAGAATTT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 TGGT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA095/ 
                 CCCGCACC 
                 13 
                 cscscsGCACCUUGGCGCAGCGGG 
                 9 
                 ABE 
               
               
                   
                 GA066 
                 TTGGCGCA 
                   
                 UUUUAGAgcuagaaauagcAAGUUA 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 AAAUAAGGCUAGUCCGUUAUC 
                   
                   
               
               
                   
                   
                   
                   
                 Aacuugaaaaaguggcaccgagucggugcusu 
                   
                   
               
               
                   
                   
                   
                   
                 susu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA096 
                 CCCGCACC 
                 13 
                 cscscsGCACCUUGGCGCAGCGGg 
                 9 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 AuAaggCUaGUCcGUUAucAAcuuG 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaaguGgcaccgAgUCggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA097 
                 CCCGCACC 
                 13 
                 cscscsGCACCUUGGCGCAGCGGg 
                 9 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 AuAaggcuaGUccGUUAucAAcuugaa 
                   
                   
               
               
                   
                   
                   
                   
                 aaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA098 
                 AAGATACC 
                 15 
                 asasgsAUACCUGAAUAACCCUCG 
                 59 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUUAGAgcuagaaauagcAAGUUA 
                   
                   
               
               
                   
                   
                 CCTC 
                   
                 AAAUAAGGCUAGUCCGUUAUC 
                   
                   
               
               
                   
                   
                   
                   
                 Aacuugaaaaaguggcaccgagucggugcusu 
                   
                   
               
               
                   
                   
                   
                   
                 susu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA099 
                 AAGATACC 
                 15 
                 asasgsAUACCUGAAUAACCCUCg 
                 59 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 CCTC 
                   
                 AuAaggCUaGUCcGUUAucAAcuuG 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaaguGgcaccgAgUCggugcusususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA100 
                 AAGATACC 
                 15 
                 asasgsAUACCUGAAUAACCCUCg 
                 59 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 CCTC 
                   
                 AuAaggcuaGUccGUUAucAAcuugaa 
                   
                   
               
               
                   
                   
                   
                   
                 aaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 cANGPTL3 
                 GA101/ 
                 AAGATACC 
                 14 
                 asasgsAUACCUGAAUAACUCUCG 
                 257 
                 ABE 
               
               
                   
                 GA067 
                 TGAATAAC 
                   
                 UUUUAGAgcuagaaauagcAAGUUA 
                   
                   
               
               
                   
                   
                 TCTC 
                   
                 AAAUAAGGCUAGUCCGUUAUC 
                   
                   
               
               
                   
                   
                   
                   
                 Aacuugaaaaaguggcaccgagucggugcusu 
                   
                   
               
               
                   
                   
                   
                   
                 susu 
                   
                   
               
               
                   
               
               
                 cANGPTL3 
                 GA102 
                 AAGATACC 
                 14 
                 asasgsAUACCUGAAUAACUCUCg 
                 257 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 TCTC 
                   
                 AuAaggCUaGUCcGUUAucAAcuuG 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaaguGgcaccgAgUCggugcusususu 
                   
                   
               
               
                   
               
               
                 cANGPTL3 
                 GA103 
                 AAGATACC 
                 14 
                 asasgsAUACCUGAAUAACUCUCg 
                 257 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 TCTC 
                   
                 AuAaggcuaGUccGUUAucAAcuugaa 
                   
                   
               
               
                   
                   
                   
                   
                 aaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA104 
                 TAATTTGG 
                 102 
                 usasasUUUGGCCCUUCGUCUUAG 
                 280 
                 ABE 
               
               
                   
                   
                 CCCTTCGT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CTTA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA105 
                 AGACTTTG 
                 103 
                 asgsasCUUUGUCCAUAAGACGAG 
                 281 
                 ABE 
               
               
                   
                   
                 TCCATAAG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 ACGA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA106 
                 GACTTTGT 
                 104 
                 gsascsUUUGUCCAUAAGACGAA 
                 282 
                 ABE 
               
               
                   
                   
                 CCATAAGA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 CGAA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA107 
                 AGCCAATG 
                 105 
                 asgscsCAAUGGCCUCCUUCAGUG 
                 283 
                 SpCas9 
               
               
                   
                   
                 GCCTCCTT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CAGT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA108 
                 TCCCAACT 
                 106 
                 uscscsCAACUGAAGGAGGCCAUG 
                 284 
                 SpCas9 
               
               
                   
                   
                 GAAGGAG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GCCAT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA109 
                 GGCCTCCT 
                 107 
                 gsgscsCUCCUUCAGUUGGGACAG 
                 285 
                 SpCas9 
               
               
                   
                   
                 TCAGTTGG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GACA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA110 
                 GACCATGT 
                 108 
                 gsascsCAUGUCCCAACUGAAGGG 
                 286 
                 SpCas9 
               
               
                   
                   
                 CCCAACTG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 AAGG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA111 
                 GCCAATGG 
                 109 
                 gscscsAAUGGCCUCCUUCAGUUG 
                 287 
                 SpCas9 
               
               
                   
                   
                 CCTCCTTC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 AGTT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA112 
                 ATTGTCTT 
                 110 
                 asususGUCUUGAUCAAUUCUGG 
                 288 
                 ABE/ 
               
               
                   
                   
                 GATCAATT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 CTGG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA113 
                 ATTCTGGA 
                 111 
                 asususCUGGAGGAAAUAACUAG 
                 289 
                 ABE/ 
               
               
                   
                   
                 GGAAATA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 ACTAG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA114 
                 TCTGGGTG 
                 112 
                 uscsusGGGUGUUCUGGAGUUUC 
                 290 
                 SpCas9 
               
               
                   
                   
                 TTCTGGAG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 TTTC 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA115 
                 AACATAGC 
                 113 
                 asascsAUAGCAAAUCUUGAUUU 
                 291 
                 ABE/ 
               
               
                   
                   
                 AAATCTTG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 ATTT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA116 
                 GTAGAATT 
                 114 
                 gsusasGAAUUUUUUCUUCUAGG 
                 292 
                 ABE/ 
               
               
                   
                   
                 TTTTCTTC 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 TAGG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA117 
                 ACTACAAG 
                 115 
                 ascsusACAAGUCAAAAAUGAAG 
                 293 
                 ABE/ 
               
               
                   
                   
                 TCAAAAAT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 GAAG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA118 
                 TATATTGG 
                 116 
                 usasusAUUGGUCUUCCACGGUCG 
                 294 
                 ABE/ 
               
               
                   
                   
                 TCTTCCAC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                 SpCas9 
               
               
                   
                   
                 GGTC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA119 
                 CAAAGAC 
                 117 
                 csasasAGACCUUCUCCAGACCGG 
                 295 
                 ABE/ 
               
               
                   
                   
                 CTTCTCCA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                 SpCas9 
               
               
                   
                   
                 GACCG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA120 
                 GGTCTTCC 
                 118 
                 gsgsusCUUCCACGGUCUGGAGA 
                 296 
                 ABE/ 
               
               
                   
                   
                 ACGGTCTG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 GAGA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA121 
                 TTGTTTAT 
                 119 
                 ususgsUUUAUAUUGGUCUUCCA 
                 297 
                 ABE/ 
               
               
                   
                   
                 ATTGGTCT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 TCCA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA122 
                 CTTTTATT 
                 120 
                 csususUUAUUUGACUAUGCUGU 
                 298 
                 ABE/ 
               
               
                   
                   
                 TGACTATG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 CTGT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA123 
                 AAAGTCTG 
                 121 
                 asasasGUCUGGAUAUAGAGAGU 
                 299 
                 ABE/ 
               
               
                   
                   
                 GATATAGA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 GAGT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA124 
                 GTTGGTTT 
                 122 
                 gsususGGUUUAAUUGUUUAUAU 
                 300 
                 ABE/ 
               
               
                   
                   
                 AATTGTTT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 ATAT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA125 
                 TGATGGTA 
                 123 
                 usgsasUGGUAAGACACUUUGGU 
                 301 
                 ABE/ 
               
               
                   
                   
                 AGACACTT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 TGGT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA126 
                 GGAGTAGT 
                 124 
                 gsgsasGUAGUUCUUGGUGCUCU 
                 302 
                 ABE/ 
               
               
                   
                   
                 TCTTGGTG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 CTCT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA127 
                 AACATGAT 
                 125 
                 asascsAUGAUGGUAAGACACUU 
                 303 
                 ABE/ 
               
               
                   
                   
                 GGTAAGA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 CACTT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA128 
                 TGAAGAA 
                 126 
                 usgsasAGAAAGGGAGUAGUUCU 
                 304 
                 ABE/ 
               
               
                   
                   
                 AGGGAGT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 AGTTCT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA129 
                 AGTTCTTG 
                 127 
                 asgsusUCUUGGUGCUCUUGGCU 
                 305 
                 ABE/ 
               
               
                   
                   
                 GTGCTCTT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 GGCT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA130 
                 ATGATGGT 
                 128 
                 asusgsAUGGUAAGACACUUUGG 
                 306 
                 ABE/ 
               
               
                   
                   
                 AAGACACT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 TTGG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA131 
                 GAAGATA 
                 129 
                 gsasasGAUAGAGAAAUUUCUGU 
                 307 
                 ABE/ 
               
               
                   
                   
                 GAGAAATT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 TCTGT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA132 
                 GGAAGAT 
                 130 
                 gsgsasAGAUAGAGAAAUUUCUG 
                 308 
                 ABE/ 
               
               
                   
                   
                 AGAGAAA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 TTTCTG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA133 
                 TATTTCAT 
                 131 
                 usasusUUCAUUCAACUGAAGAA 
                 309 
                 ABE/ 
               
               
                   
                   
                 TCAACTGA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 AGAA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA134 
                 ATTTCATT 
                 132 
                 asususUCAUUCAACUGAAGAAA 
                 310 
                 ABE/ 
               
               
                   
                   
                 CAACTGAA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 GAAA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA135 
                 GTCTACTG 
                 133 
                 gsuscsUACUGUGAUGUUAUAUC 
                 311 
                 ABE/ 
               
               
                   
                   
                 TGATGTTA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 TATC 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA136 
                 TAAATGGT 
                 134 
                 usasasAUGGUGGUACAUUCAGC 
                 312 
                 ABE/ 
               
               
                   
                   
                 GGTACATT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 CAGC 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA137 
                 TATCAGGT 
                 135 
                 usasusCAGGUAAAACCUGUCUA 
                 313 
                 ABE/ 
               
               
                   
                   
                 AAAACCTG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 TCTA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA138 
                 TGTACCAC 
                 136 
                 usgsusACCACCAUUUAUAACAG 
                 314 
                 ABE/ 
               
               
                   
                   
                 CATTTATA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 ACAG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA139 
                 TTCACCTC 
                 137 
                 ususcsACCUCUGUUAUAAAUGG 
                 315 
                 ABE/ 
               
               
                   
                   
                 TGTTATAA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 ATGG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA140 
                 AACAGAG 
                 138 
                 asascsAGAGGUGAACAUACAAG 
                 316 
                 ABE/ 
               
               
                   
                   
                 GTGAACAT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 ACAAG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA141 
                 TTGAGAGT 
                 139 
                 ususgsAGAGUUGCUGGGUCUGA 
                 317 
                 ABE/ 
               
               
                   
                   
                 TGCTGGGT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 CTGA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA142 
                 TGAAAAA 
                 140 
                 usgsasAAAACUUGAGAGUUGCU 
                 318 
                 SpCas9 
               
               
                   
                   
                 CTTGAGAG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 TTGCT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA143 
                 TTAATTCA 
                 141 
                 ususasAUUCAACAUCGAAUAGA 
                 319 
                 ABE/ 
               
               
                   
                   
                 ACATCGAA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 TAGA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA144 
                 TTTGGGAG 
                 142 
                 usususGGGAGGCUUGAUGGUAA 
                 320 
                 ABE/ 
               
               
                   
                   
                 GCTTGATG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 GTAA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA145 
                 CATTATAT 
                 143 
                 csasusUAUAUUCAGGUAGUCCA 
                 321 
                 ABE/ 
               
               
                   
                   
                 TCAGGTAG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 TCCA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA146 
                 TTGGGAGG 
                 144 
                 ususgsGGAGGCUUGAUGGUAAG 
                 322 
                 ABE/ 
               
               
                   
                   
                 CTTGATGG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 TAAG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA147 
                 TTTTGGGA 
                 145 
                 usususUGGGAGGCUUGAUGGUA 
                 323 
                 ABE/ 
               
               
                   
                   
                 GGCTTGAT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 GGTA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA148 
                 ACAAAACT 
                 146 
                 ascsasAAACUUCAAUGAAACGUG 
                 324 
                 ABE/ 
               
               
                   
                   
                 TCAATGAA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                 SpCas9 
               
               
                   
                   
                 ACGT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA149 
                 TATGGTTT 
                 147 
                 usasusGGUUUUGGGAGGCUUGA 
                 325 
                 ABE/ 
               
               
                   
                   
                 TGGGAGG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 CTTGA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA150 
                 ACTACAAA 
                 148 
                 ascsusACAAAUAUGGUUUUGGG 
                 326 
                 ABE/ 
               
               
                   
                   
                 TATGGTTT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 TGGG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA151 
                 GAGAACT 
                 149 
                 gsasgsAACUACAAAUAUGGUUU 
                 327 
                 ABE/ 
               
               
                   
                   
                 ACAAATAT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 GGTTT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA152 
                 AGGACACT 
                 150 
                 asgsgsACACUUCAACUGUCCAGG 
                 328 
                 ABE/ 
               
               
                   
                   
                 TCAACTGT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                 SpCas9 
               
               
                   
                   
                 CCAG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA094/ 
                 CTTTTCAG 
                 101 
                 csususUUCAGGAGAAUUUUGGU 
                 279 
                 ABE/ 
               
               
                   
                 GA153 
                 GAGAATTT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                 SpCas9 
               
               
                   
                   
                 TGGT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA156 
                 GGTGCTAG 
                 66 
                 gsgsusGCUAGCCUUGCGUUCCGG 
                 253 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA157 
                 GCCGTCCT 
                 151 
                 gscscsGUCCUCCUCGGAACGCAG 
                 329 
                 SpCas9 
               
               
                   
                   
                 CCTCGGAA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CGCA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA158 
                 GCTAGCCT 
                 152 
                 gscsusAGCCUUGCGUUCCGAGGG 
                 330 
                 SpCas9 
               
               
                   
                   
                 TGCGTTCC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GAGG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA159 
                 GCGTTCCG 
                 153 
                 gscsgsUUCCGAGGAGGACGGCCG 
                 331 
                 SpCas9 
               
               
                   
                   
                 AGGAGGA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CGGCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA160 
                 GCCTTGCG 
                 154 
                 gscscsUUGCGUUCCGAGGAGGA 
                 332 
                 SpCas9 
               
               
                   
                   
                 TTCCGAGG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 AGGA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA161 
                 GGACGAG 
                 155 
                 gsgsasCGAGGACGGCGACUACGG 
                 333 
                 SpCas9 
               
               
                   
                   
                 GACGGCG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 ACTACG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA162 
                 GGACGGC 
                 156 
                 gsgsasCGGCGACUACGAGGAGCG 
                 334 
                 SpCas9 
               
               
                   
                   
                 GACTACGA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GGAGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA163 
                 CGTCCTCG 
                 157 
                 csgsusCCUCGUCCUCCUGCGCAG 
                 335 
                 SpCas9 
               
               
                   
                   
                 TCCTCCTG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CGCA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA164 
                 GTCCTCGT 
                 158 
                 gsuscsCUCGUCCUCCUGCGCACG 
                 336 
                 SpCas9 
               
               
                   
                   
                 CCTCCTGC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GCAC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA165 
                 CCGTCAGC 
                 159 
                 cscsgsUCAGCUCCAGGCGGUCCG 
                 337 
                 SpCas9 
               
               
                   
                   
                 TCCAGGCG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GTCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA166 
                 CGCCCGTG 
                 160 
                 csgscsCCGUGCGCAGGAGGACGG 
                 338 
                 SpCas9 
               
               
                   
                   
                 CGCAGGA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GGACG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA167 
                 TCAGCTCC 
                 161 
                 uscsasGCUCCAGGCGGUCCUGGG 
                 339 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA168 
                 CTTGGCGC 
                 162 
                 csususGGCGCAGCGGUGGAAGG 
                 340 
                 SpCas9 
               
               
                   
                   
                 AGCGGTG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 GAAGG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA169 
                 CGTGCGCA 
                 163 
                 csgsusGCGCAGGAGGACGAGGA 
                 341 
                 SpCas9 
               
               
                   
                   
                 GGAGGAC 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 GAGGA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA170 
                 CCAGGACC 
                 164 
                 cscsasGGACCGCCUGGAGCUGAG 
                 342 
                 SpCas9 
               
               
                   
                   
                 GCCTGGAG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CTGA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA171 
                 TCTTGGTG 
                 165 
                 uscsusUGGUGAGGUAUCCCCGG 
                 343 
                 SpCas9 
               
               
                   
                   
                 AGGTATCC 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 CCGG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA172 
                 CAGTGCGC 
                 166 
                 csasgsUGCGCUCUGACUGCGAGG 
                 344 
                 SpCas9 
               
               
                   
                   
                 TCTGACTG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CGAG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA173 
                 CTTGGTGA 
                 167 
                 csususGGUGAGGUAUCCCCGGCG 
                 345 
                 SpCas9 
               
               
                   
                   
                 GGTATCCC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CGGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA174 
                 GGATCTTG 
                 168 
                 gsgsasUCUUGGUGAGGUAUCCC 
                 346 
                 SpCas9 
               
               
                   
                   
                 GTGAGGTA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 TCCC 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA175 
                 GAAGATG 
                 169 
                 gsasasGAUGAGUGGCGACCUGCG 
                 347 
                 SpCas9 
               
               
                   
                   
                 AGTGGCG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 ACCTGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA176 
                 AGCACCAC 
                 170 
                 asgscsACCACCACGUAGGUGCCG 
                 348 
                 SpCas9 
               
               
                   
                   
                 CACGTAGG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 TGCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA177 
                 GGTCGCCA 
                 171 
                 gsgsusCGCCACUCAUCUUCACCG 
                 349 
                 SpCas9 
               
               
                   
                   
                 CTCATCTT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CACC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA178 
                 CTCCTTCA 
                 172 
                 csuscsCUUCAGCACCACCACGUG 
                 350 
                 SpCas9 
               
               
                   
                   
                 GCACCACC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 ACGT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA179 
                 GAGTGGC 
                 173 
                 gsasgsUGGCGACCUGCUGGAGCG 
                 351 
                 SpCas9 
               
               
                   
                   
                 GACCTGCT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GGAGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA180 
                 GCGCACTG 
                 174 
                 gscsgsCACUGCCCGCCGCCUGCG 
                 352 
                 SpCas9 
               
               
                   
                   
                 CCCGCCGC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CTGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA181 
                 GGCTTCCT 
                 175 
                 gsgscsUUCCUGGUGAAGAUGAG 
                 353 
                 SpCas9 
               
               
                   
                   
                 GGTGAAG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 ATGAG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA182 
                 CACCTACG 
                 176 
                 csascsCUACGUGGUGGUGCUGAG 
                 354 
                 SpCas9 
               
               
                   
                   
                 TGGTGGTG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CTGA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA183 
                 CTACGTGG 
                 177 
                 csusasCGUGGUGGUGCUGAAGG 
                 355 
                 SpCas9 
               
               
                   
                   
                 TGGTGCTG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 AAGG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA184 
                 ATGGAAG 
                 178 
                 asusgsGAAGACAUGCAGGAUCU 
                 356 
                 SpCas9 
               
               
                   
                   
                 ACATGCAG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 GATCT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA185 
                 AGACATGC 
                 179 
                 asgsasCAUGCAGGAUCUUGGUG 
                 357 
                 SpCas9 
               
               
                   
                   
                 AGGATCTT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 GGTG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA186 
                 CTCATCTT 
                 180 
                 csuscsAUCUUCACCAGGAAGCCG 
                 358 
                 SpCas9 
               
               
                   
                   
                 CACCAGG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 AAGCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA187 
                 CTCCTCGA 
                 181 
                 csuscsCUCGAUGUAGUCGACAUG 
                 359 
                 SpCas9 
               
               
                   
                   
                 TGTAGTCG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 ACAT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA188 
                 TATTCATC 
                 182 
                 usasusUCAUCCGCCCGGUACCGG 
                 360 
                 SpCas9 
               
               
                   
                   
                 CGCCCGGT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 ACCG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA189 
                 TCCTCGAT 
                 183 
                 uscscsUCGAUGUAGUCGACAUG 
                 361 
                 SpCas9 
               
               
                   
                   
                 GTAGTCGA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 CATG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA190 
                 CCTCCTCG 
                 184 
                 cscsusCCUCGAUGUAGUCGACAG 
                 362 
                 SpCas9 
               
               
                   
                   
                 ATGTAGTC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GACA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA191 
                 GCCCCATG 
                 185 
                 gscscsCCAUGUCGACUACAUCGG 
                 363 
                 SpCas9 
               
               
                   
                   
                 TCGACTAC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 ATCG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA192 
                 CCATGTCG 
                 186 
                 cscsasUGUCGACUACAUCGAGGG 
                 364 
                 SpCas9 
               
               
                   
                   
                 ACTACATC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GAGG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA193 
                 GGGGCTG 
                 187 
                 gsgsgsGCUGGUAUUCAUCCGCCG 
                 365 
                 SpCas9 
               
               
                   
                   
                 GTATTCAT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CCGCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA194 
                 GTCGACAT 
                 188 
                 gsuscsGACAUGGGGCAACUUCA 
                 366 
                 SpCas9 
               
               
                   
                   
                 GGGGCAA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 CTTCA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA195 
                 ACCACCGG 
                 189 
                 ascscsACCGGGAAAUCGAGGGCG 
                 367 
                 SpCas9 
               
               
                   
                   
                 GAAATCG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 AGGGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA196 
                 GAGTGACC 
                 190 
                 gsasgsUGACCACCGGGAAAUCGG 
                 368 
                 SpCas9 
               
               
                   
                   
                 ACCGGGA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 AATCG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA197 
                 AGTGACCA 
                 191 
                 asgsusGACCACCGGGAAAUCGAG 
                 369 
                 SpCas9 
               
               
                   
                   
                 CCGGGAA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 ATCGA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA198 
                 CCACCGGG 
                 192 
                 cscsasCCGGGAAAUCGAGGGCAG 
                 370 
                 SpCas9 
               
               
                   
                   
                 AAATCGA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GGGCA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA199 
                 GAAGCGG 
                 193 
                 gsasasGCGGGUCCCGUCCUCCUG 
                 371 
                 SpCas9 
               
               
                   
                   
                 GTCCCGTC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CTCCT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA200 
                 CTAGGAG 
                 194 
                 csusasGGAGAUACACCUCCACCG 
                 372 
                 SpCas9 
               
               
                   
                   
                 ATACACCT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CCACC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA201 
                 CAGCATAC 
                 195 
                 csasgsCAUACAGAGUGACCACCG 
                 373 
                 SpCas9 
               
               
                   
                   
                 AGAGTGA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CCACC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA202 
                 AAGCGGG 
                 196 
                 asasgsCGGGUCCCGUCCUCCUCG 
                 374 
                 SpCas9 
               
               
                   
                   
                 TCCCGTCC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 TCCTC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA203 
                 TGACCCTG 
                 197 
                 usgsasCCCUGCCCUCGAUUUCCG 
                 375 
                 SpCas9 
               
               
                   
                   
                 CCCTCGAT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 TTCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA204 
                 CACTCTGT 
                 198 
                 csascsUCUGUAUGCUGGUGUCUG 
                 376 
                 SpCas9 
               
               
                   
                   
                 ATGCTGGT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GTCT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA205 
                 CCCTGCCC 
                 199 
                 cscscsUGCCCUCGAUUUCCCGGG 
                 377 
                 SpCas9 
               
               
                   
                   
                 TCGATTTC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CCGG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA206 
                 CCGGTGGT 
                 200 
                 cscsgsGUGGUCACUCUGUAUGCG 
                 378 
                 SpCas9 
               
               
                   
                   
                 CACTCTGT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 ATGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA207 
                 GGAAATC 
                 201 
                 gsgsasAAUCGAGGGCAGGGUCA 
                 379 
                 SpCas9 
               
               
                   
                   
                 GAGGGCA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 GGGTCA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA208 
                 CCAGCATA 
                 202 
                 cscsasGCAUACAGAGUGACCACG 
                 380 
                 SpCas9 
               
               
                   
                   
                 CAGAGTG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 ACCAC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA209 
                 CTTGGCAG 
                 203 
                 csususGGCAGUUGAGCACGCGCG 
                 381 
                 SpCas9 
               
               
                   
                   
                 TTGAGCAC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GCGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA210 
                 CTGCGCGT 
                 204 
                 csusgsCGCGUGCUCAACUGCCAG 
                 382 
                 SpCas9 
               
               
                   
                   
                 GCTCAACT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GCCA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA211 
                 TGCGCGTG 
                 205 
                 usgscsGCGUGCUCAACUGCCAAG 
                 383 
                 SpCas9 
               
               
                   
                   
                 CTCAACTG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CCAA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA212 
                 CGGGATGC 
                 206 
                 csgsgsGAUGCCGGCGUGGCCAAG 
                 384 
                 SpCas9 
               
               
                   
                   
                 CGGCGTGG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CCAA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA213 
                 CGTGCTCA 
                 207 
                 csgsusGCUCAACUGCCAAGGGAG 
                 385 
                 SpCas9 
               
               
                   
                   
                 ACTGCCAA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GGGA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA214 
                 CCTTGGCC 
                 208 
                 cscsusUGGCCACGCCGGCAUCCG 
                 386 
                 SpCas9 
               
               
                   
                   
                 ACGCCGGC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 ATCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA215 
                 CAGCGGCC 
                 209 
                 csasgsCGGCCGGGAUGCCGGCGG 
                 387 
                 SpCas9 
               
               
                   
                   
                 GGGATGCC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GGCG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA216 
                 CCGGGATG 
                 210 
                 cscsgsGGAUGCCGGCGUGGCCAG 
                 388 
                 SpCas9 
               
               
                   
                   
                 CCGGCGTG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GCCA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA217 
                 GTGGTCAG 
                 211 
                 gsusgsGUCAGCGGCCGGGAUGC 
                 389 
                 SpCas9 
               
               
                   
                   
                 CGGCCGG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 GATGC 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA218 
                 CGCTGACC 
                 212 
                 csgscsUGACCACCCCUGCCAGGG 
                 390 
                 SpCas9 
               
               
                   
                   
                 ACCCCTGC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CAGG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA219 
                 GGCAGGG 
                 213 
                 gsgscsAGGGGUGGUCAGCGGCC 
                 391 
                 SpCas9 
               
               
                   
                   
                 GTGGTCAG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 CGGCC 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA220 
                 GTGCTCAA 
                 214 
                 gsusgsCUCAACUGCCAAGGGAA 
                 392 
                 SpCas9 
               
               
                   
                   
                 CTGCCAAG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 GGAA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hPCSK9 
                 GA221 
                 TCATGGCA 
                 215 
                 uscsasUGGCACCCACCUGGCAGG 
                 393 
                 SpCas9 
               
               
                   
                   
                 CCCACCTG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GCAG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA222 
                 TGGCAGG 
                 216 
                 usgsgsCAGGGGUGGUCAGCGGC 
                 394 
                 SpCas9 
               
               
                   
                   
                 GGTGGTCA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 GCGGC 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA223 
                 GCTGACCA 
                 217 
                 gscsusGACCACCCCUGCCAGGUG 
                 395 
                 SpCas9 
               
               
                   
                   
                 CCCCTGCC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 AGGT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA224 
                 GGCCGCTG 
                 218 
                 gsgscsCGCUGACCACCCCUGCCG 
                 396 
                 SpCas9 
               
               
                   
                   
                 ACCACCCC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 TGCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA225 
                 GGCATCGT 
                 219 
                 gsgscsAUCGUCCCGGAAGUUGCG 
                 397 
                 SpCas9 
               
               
                   
                   
                 CCCGGAA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GTTGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA226 
                 GGCTTTTC 
                 220 
                 gsgscsUUUUCCGAAUAAACUCCG 
                 398 
                 SpCas9 
               
               
                   
                   
                 CGAATAA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 ACTCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA227 
                 GTCCCGGA 
                 221 
                 gsuscsCCGGAAGUUGCCGGCAGG 
                 399 
                 SpCas9 
               
               
                   
                   
                 AGTTGCCG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GCAG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA228 
                 CGGCTGTA 
                 222 
                 csgsgsCUGUACCCACCCGCCAGG 
                 400 
                 SpCas9 
               
               
                   
                   
                 CCCACCCG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CCAG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA229 
                 GTCGTGCT 
                 223 
                 gsuscsGUGCUGGUCACCGCUGCG 
                 401 
                 SpCas9 
               
               
                   
                   
                 GGTCACCG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CTGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA230 
                 AGTAGAG 
                 224 
                 asgsusAGAGGCAGGCAUCGUCCG 
                 402 
                 SpCas9 
               
               
                   
                   
                 GCAGGCAT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CGTCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA231 
                 TCCGAATA 
                 225 
                 uscscsGAAUAAACUCCAGGCCUG 
                 403 
                 SpCas9 
               
               
                   
                   
                 AACTCCAG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GCCT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA232 
                 GTTTATTC 
                 226 
                 gsususUAUUCGGAAAAGCCAGC 
                 404 
                 SpCas9 
               
               
                   
                   
                 GGAAAAG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 CCAGC 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hPCSK9 
                 GA233 
                 GCGGCTGT 
                 227 
                 gscsgsGCUGUACCCACCCGCCAG 
                 405 
                 SpCas9 
               
               
                   
                   
                 ACCCACCC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GCCA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA235 
                 CACCGCTG 
                 228 
                 csascsCGCUGCCGGCAACUUCCG 
                 406 
                 SpCas9 
               
               
                   
                   
                 CCGGCAAC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 TTCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA236 
                 AGCCCTCG 
                 229 
                 asgscsCCUCGCCAGGCGCUGGCG 
                 407 
                 SpCas9 
               
               
                   
                   
                 CCAGGCGC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 TGGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA237 
                 CAACGCCG 
                 230 
                 csasasCGCCGCCUGCCAGCGCCG 
                 408 
                 SpCas9 
               
               
                   
                   
                 CCTGCCAG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CGCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA238 
                 GCACGACC 
                 231 
                 gscsasCGACCCCAGCCCUCGCCG 
                 409 
                 SpCas9 
               
               
                   
                   
                 CCAGCCCT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CGCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA239 
                 CCGCCTGC 
                 232 
                 cscsgsCCUGCCAGCGCCUGGCGG 
                 410 
                 SpCas9 
               
               
                   
                   
                 CAGCGCCT 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GGCG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA240 
                 TGCCAGCG 
                 233 
                 usgscsCAGCGCCUGGCGAGGGCG 
                 411 
                 SpCas9 
               
               
                   
                   
                 CCTGGCGA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GGGC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA241 
                 TGCTGCTG 
                 234 
                 usgscsUGCUGCCCCUGGCGGGUG 
                 412 
                 SpCas9 
               
               
                   
                   
                 CCCCTGGC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GGGT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA242 
                 TCACCGCT 
                 235 
                 uscsasCCGCUGCCGGCAACUUCG 
                 413 
                 SpCas9 
               
               
                   
                   
                 GCCGGCA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 ACTTC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA243 
                 GGCGAGG 
                 236 
                 gsgscsGAGGGCUGGGGUCGUGC 
                 414 
                 SpCas9 
               
               
                   
                   
                 GCTGGGGT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 CGTGC 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA244 
                 TTCCGAAT 
                 237 
                 ususcsCGAAUAAACUCCAGGCCG 
                 415 
                 SpCas9 
               
               
                   
                   
                 AAACTCCA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GGCC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA245 
                 GTGCTGCT 
                 238 
                 gsusgsCUGCUGCCCCUGGCGGGG 
                 416 
                 SpCas9 
               
               
                   
                   
                 GCCCCTGG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CGGG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA246 
                 GCCAGCGC 
                 239 
                 gscscsAGCGCCUGGCGAGGGCUG 
                 417 
                 SpCas9 
               
               
                   
                   
                 CTGGCGAG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GGCT 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA247 
                 CCTCGCCA 
                 240 
                 cscsusCGCCAGGCGCUGGCAGGG 
                 418 
                 SpCas9 
               
               
                   
                   
                 GGCGCTGG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 CAGG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA248 
                 GGTGCTAG 
                 66 
                 gsgsusG(cPACE)UAGCCUUGCGU 
                 419 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 UCCGGUUUUAGAgcuagaaauagcA 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAacuugaaaaaguggcaccgaguc 
                   
                   
               
               
                   
                   
                   
                   
                 ggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA249 
                 GGTGCTAG 
                 66 
                 gsgsusGCUAGCC(uPACE)UGCGU 
                 420 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 UCCGGUUUUAGAgcuagaaauagcA 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAacuugaaaaaguggcaccgaguc 
                   
                   
               
               
                   
                   
                   
                   
                 ggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA250 
                 GGTGCTAG 
                 66 
                 gsgsusGcUAGCCUUGCGUUCCGG 
                 253 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 UUUUAGAgcuagaaauagcAAGUUA 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 AAAUAAGGCUAGUCCGUUAUC 
                   
                   
               
               
                   
                   
                   
                   
                 Aacuugaaaaaguggcaccgagucggugcusu 
                   
                   
               
               
                   
                   
                   
                   
                 susu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA251 
                 GGTGCTAG 
                 66 
                 gsgsusGCUAGCCuUGCGUUCCGG 
                 253 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 UUUUAGAgcuagaaauagcAAGUUA 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 AAAUAAGGCUAGUCCGUUAUC 
                   
                   
               
               
                   
                   
                   
                   
                 Aacuugaaaaaguggcaccgagucggugcusu 
                   
                   
               
               
                   
                   
                   
                   
                 susu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA253 
                 GGTGCTAG 
                 66 
                 gsgsusG(cPACE)UAGCCUUGCGU 
                 419 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 UCCGgUUUUAGagcuagaaauagcaaG 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 UUaAaAuAaggcuaGUccGUUAucA 
                   
                   
               
               
                   
                   
                   
                   
                 AcuugaaaaagugGcaccgagucggugcusu 
                   
                   
               
               
                   
                   
                   
                   
                 susu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA254 
                 GGTGCTAG 
                 66 
                 gsgsusGCUAGCC(uPACE)UGCGU 
                 420 
                 SpCas9 
               
               
                   
                   
                 CCTTGCGT 
                   
                 UCCGgUUUUAGagcuagaaauagcaaG 
                   
                   
               
               
                   
                   
                 TCCG 
                   
                 UUaAaAuAaggcuaGUccGUUAucA 
                   
                   
               
               
                   
                   
                   
                   
                 AcuugaaaaagugGcaccgagucggugcusu 
                   
                   
               
               
                   
                   
                   
                   
                 susu 
                   
                   
               
               
                   
               
               
                 mPCSK9 
                 GA255 
                 CCCATACC 
                 69 
                 cscscsAUACCUUGGAGCAACGGg 
                 421 
                 ABE 
               
               
                   
                   
                 TTGGAGCA 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 ACGG 
                   
                 AuAaggCUaGUCcGUUAucAAcuuG 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaaguGgcaccgAgUCggugcusususu 
                   
                   
               
               
                   
               
               
                 mPCSK9 
                 GA256 
                 CCCATACC 
                 69 
                 cscscsAUACCUUGGAGCAACGGg 
                 421 
                 ABE 
               
               
                   
                   
                 TTGGAGCA 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 ACGG 
                   
                 AuAaggcuaGUccGUUAucAAcuugaa 
                   
                   
               
               
                   
                   
                   
                   
                 aaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 mPCSK9 
                 GA257 
                 CCCATACC 
                 69 
                 cscscsAUACCUUGGAGCAACGGg 
                 421 
                 ABE 
               
               
                   
                   
                 TTGGAGCA 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 ACGG 
                   
                 AuAaggcuaGUccGUUAucAAcuuGa 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 mANGPTL3 
                 GA258 
                 GAGATACC 
                 241 
                 gsasgsAUACCUGAGUAACUUUCg 
                 422 
                 ABE 
               
               
                   
                   
                 TGAGTAAC 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 TTTC 
                   
                 AuAaggCUaGUCcGUUAucAAcuuG 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaaguGgcaccgAgUCggugcusususu 
                   
                   
               
               
                   
               
               
                 mANGPTL3 
                 GA259 
                 GAGATACC 
                 241 
                 gsasgsAUACCUGAGUAACUUUCg 
                 422 
                 ABE 
               
               
                   
                   
                 TGAGTAAC 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 TTTC 
                   
                 AuAaggcuaGUccGUUAucAAcuugaa 
                   
                   
               
               
                   
                   
                   
                   
                 aaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 mANGPTL3 
                 GA260 
                 GAGATACC 
                 241 
                 gsasgsAUACCUGAGUAACUUUCg 
                 422 
                 ABE 
               
               
                   
                   
                 TGAGTAAC 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 TTTC 
                   
                 AuAaggcuaGUccGUUAucAAcuuGa 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA276 
                 ACGTGGG 
                 242 
                 ascsgsUGGGAGAACUACAAAUA 
                 423 
                 ABE 
               
               
                   
                   
                 AGAACTAC 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 AAATA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA277 
                 CGATGTTG 
                 243 
                 csgsasUGUUGAAUUAAUGUCCA 
                 424 
                 ABE 
               
               
                   
                   
                 AATTAATG 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 TCCA 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA278 
                 CACAAAA 
                 244 
                 csascsAAAACUUCAAUGAAACGG 
                 425 
                 ABE 
               
               
                   
                   
                 CTTCAATG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 AAACG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA279 
                 TACGAATT 
                 245 
                 usascsGAAUUGAGUUGGAAGAC 
                 426 
                 ABE 
               
               
                   
                   
                 GAGTTGGA 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 AGAC 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA284 
                 CTATGGAG 
                 246 
                 csusasUGGAGUAUAUCUUCUCU 
                 427 
                 ABE 
               
               
                   
                   
                 TATATCTT 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 CTCT 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA343 
                 CCCGCACC 
                 13 
                 cscscsGCACCUUGGCGCAGCGGg 
                 9 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 cANGPTL3 
                 GA344 
                 AAGATACC 
                 14 
                 asasgsAUACCUGAAUAACUCUCG 
                 257 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 TCTC 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA346 
                 CCCGCACC 
                 13 
                 cscscsGCACCUUGGCGCAGCGGg 
                 9 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 AuAaggcuaGUccGUUAucAAcuuGa 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 cANGPTL3 
                 GA347 
                 AAGATACC 
                 14 
                 asasgsAUACCUGAAUAACUCUCg 
                 257 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 TCTC 
                   
                 AuAaggcuaGUccGUUAucAAcuuGa 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 mANGPTL3 
                 GA349 
                 GAGATACC 
                 241 
                 gsasgsAUACCUGAGUAACUUUC 
                 422 
                 ABE 
               
               
                   
                   
                 TGAGTAAC 
                   
                 GUUUUAGAgcuagaaauagcAAGUU 
                   
                   
               
               
                   
                   
                 TTTC 
                   
                 AAAAUAAGGCUAGUCCGUUAU 
                   
                   
               
               
                   
                   
                   
                   
                 CAacuugaaaaaguggcaccgagucggugcu 
                   
                   
               
               
                   
                   
                   
                   
                 sususu 
                   
                   
               
               
                   
               
               
                 mANGPTL3 
                 GA353 
                 GAGATACC 
                 241 
                 gsasgsAUACCUGAGUAACUUUC 
                 422 
                 ABE 
               
               
                   
                   
                 TGAGTAAC 
                   
                 GUUUUAGAGCUAGAAAUAGCA 
                   
                   
               
               
                   
                   
                 TTTC 
                   
                 AGUUAAAAUAAGGCUAGUCCG 
                   
                   
               
               
                   
                   
                   
                   
                 UUAUCAACUUGAAAAAGUGGC 
                   
                   
               
               
                   
                   
                   
                   
                 ACCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA375 
                 CCCGCACC 
                 13 
                 cscscsGCACCUUGGCGCAGCGGg 
                 428 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 AuAaggcuaGUccGUUAacAAcgggaa 
                   
                   
               
               
                   
                   
                   
                   
                 accgugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA376 
                 CCCGCACC 
                 13 
                 cscscsdGCdACdCUdUGdGCdGCdA 
                 429 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 GdCGdGgUUUUAGagcuaGaaauagc 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 aaGUUaAaAuAaggcuaGUccGUUAu 
                   
                   
               
               
                   
                   
                   
                   
                 cAAcuuGaaaaagugGcaccgagucggugc 
                   
                   
               
               
                   
                   
                   
                   
                 usususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA377 
                 CCCGCACC 
                 13 
                 cscscsGdCAdCCdUUdGGdCGdCAd 
                 430 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 GCdGGgUUUUAGagcuaGaaauagcaa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 GUUaAaAuAaggcuaGUccGUUAuc 
                   
                   
               
               
                   
                   
                   
                   
                 AAcuuGaaaaagugGcaccgagucggugcu 
                   
                   
               
               
                   
                   
                   
                   
                 sususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA380 
                 CCCGCACC 
                 13 
                 cscscsGdCACCUUGGCGdCAGCG 
                 431 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 GgUUUUAGagcuaGaaauagcaaGUUa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 AaAuAaggcuaGUccGUUAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 GaaaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA381 
                 CCCGCACC 
                 13 
                 cscscsGCdACCUUGGCGCdAGCG 
                 432 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 GgUUUUAGagcuaGaaauagcaaGUUa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 AaAuAaggcuaGUccGUUAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 GaaaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA382 
                 CCCGCACC 
                 13 
                 cscscsGCACCUdUGGdCGCAGdCG 
                 433 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 GgUUUUAGagcuaGaaauagcaaGUUa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 AaAuAaggcuaGUccGUUAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 GaaaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA383 
                 CCCGCACC 
                 13 
                 cscscsGCACCUUdGGCdGCAGCdG 
                 434 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 GgUUUUAGagcuaGaaauagcaaGUUa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 AaAuAaggcuaGUccGUUAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 GaaaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA384 
                 CCCGCACC 
                 13 
                 cscscsdGdCdACCdUdUGGdCGCAd 
                 435 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 GCGdGgUUUUAGagcuaGaaauagcaa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 GUUaAaAuAaggcuaGUccGUUAuc 
                   
                   
               
               
                   
                   
                   
                   
                 AAcuuGaaaaagugGcaccgagucggugcu 
                   
                   
               
               
                   
                   
                   
                   
                 sususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA385 
                 CCCGCACC 
                 13 
                 cscscsGCACCUUGGCGCAGCGGg 
                 11 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 AuAaggcuaGUccGUUAucAAcuugaa 
                   
                   
               
               
                   
                   
                   
                   
                 aaagugGcaccgagucggugcusususuuuu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA386 
                 CCCGCACC 
                 13 
                 cscscsGCACCUUGGCGCAGCGGg 
                 11 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 AuAaggcuaGUccGUUAucAAcuugaa 
                   
                   
               
               
                   
                   
                   
                   
                 aaagugGcaccgagucggugcusususuuUu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA387 
                 CCCGCACC 
                 13 
                 cscscsGCACCUUGGCGCAGCGgU 
                 436 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 UUUAGagcuaGaaauagcaaGUUaAaA 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 uAaggcuaGUccGUUAucAAcuuGaaa 
                   
                   
               
               
                   
                   
                   
                   
                 aagugGcaccgagucggugcuususuuuu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA388 
                 CCCGCACC 
                 13 
                 cscscsGCACCUUGGCGCAGCGGg 
                 9 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 AuAaggcuaGUCcGUUAucAAcuuga 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaguggcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA389 
                 CCCGCACC 
                 13 
                 cscscsGCACCUUGGCGCAGCGGg 
                 9 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 UUUUAGagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 AuAaggcuaGuCcGUUaucAAcuugaa 
                   
                   
               
               
                   
                   
                   
                   
                 aaaguggcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA391 
                 CCGCACCT 
                 247 
                 cscsGCACCUUGGCGCAGCGGgU 
                 437 
                 ABE 
               
               
                   
                   
                 TGGCGCAG 
                   
                 UUUAGagcuaGaaauagcaaGUUaAaA 
                   
                   
               
               
                   
                   
                 CGG 
                   
                 uAaggcuaGUccGUUAucAAcuuGaaa 
                   
                   
               
               
                   
                   
                   
                   
                 aagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA441 
                 AAGATACC 
                 15 
                 asasgsAUACCUGAAUAACCCUCg 
                 59 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 CCTC 
                   
                 AuAaggcuaGUccGUUAucAAcuuGa 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA442 
                 AAGATACC 
                 15 
                 asasgsAUACCUGAAUAACCCUCg 
                 438 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 CCTC 
                   
                 AuAaggcuaGUccGUUAucAAcuuGa 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususuUU 
                   
                   
               
               
                   
                   
                   
                   
                 u 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA472 
                 AAGATACC 
                 15 
                 asasisAUACCUGAAUAACCCUCg 
                 439 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 CCTC 
                   
                 AuAaggcuaGUccGUUAucAAcuuGa 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA473 
                 AAGATACC 
                 15 
                 asasisAUACCUIAAUAACCCUCgU 
                 440 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUAGagcuaGaaauagcaaGUUaAaA 
                   
                   
               
               
                   
                   
                 CCTC 
                   
                 uAaggcuaGUccGUUAucAAcuuGaaa 
                   
                   
               
               
                   
                   
                   
                   
                 aagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA474 
                 AAGATACC 
                 15 
                 asasgsAUACCUIAAUAACCCUCgU 
                 441 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUAGagcuaGaaauagcaaGUUaAaA 
                   
                   
               
               
                   
                   
                 CCTC 
                   
                 uAaggcuaGUccGUUAucAAcuuGaaa 
                   
                   
               
               
                   
                   
                   
                   
                 aagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA475 
                 AGATACCT 
                 248 
                 asgsasUACCUGAAUAACCCUCgU 
                 442 
                 ABE 
               
               
                   
                   
                 GAATAACC 
                   
                 UUUAGagcuaGaaauagcaaGUUaAaA 
                   
                   
               
               
                   
                   
                 CTC 
                   
                 uAaggcuaGUccGUUAucAAcuuGaaa 
                   
                   
               
               
                   
                   
                   
                   
                 aagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA476 
                 GATACCTG 
                 249 
                 gsasUACCUGAAUAACCCUCgUU 
                 443 
                 ABE 
               
               
                   
                   
                 AATAACCC 
                   
                 UUAGagcuaGaaauagcaaGUUaAaAu 
                   
                   
               
               
                   
                   
                 TC 
                   
                 AaggcuaGUccGUUAucAAcuuGaaaa 
                   
                   
               
               
                   
                   
                   
                   
                 agugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA477 
                 ATACCTGA 
                 250 
                 asusACCUGAAUAACCCUCgUUU 
                 444 
                 ABE 
               
               
                   
                   
                 ATAACCCT 
                   
                 UAGagcuaGaaauagcaaGUUaAaAuA 
                   
                   
               
               
                   
                   
                 C 
                   
                 aggcuaGUccGUUAucAAcuuGaaaaag 
                   
                   
               
               
                   
                   
                   
                   
                 ugGcaccgagucggugcusususu 
                   
                   
               
               
                   
               
               
                 hANGPTL3 
                 GA547 
                 AAGATACC 
                 14 
                 asasgsAUACCUGAAUAACUCUCg 
                 445 
                 ABE 
               
               
                   
                   
                 TGAATAAC 
                   
                 UUUUAGagcuaGaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 TCTC 
                   
                 AuAaggcuaGUccGUUAucAAcuuGa 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaguggcaccgagucggugcusususuUU 
                   
                   
               
               
                   
                   
                   
                   
                 u 
                   
                   
               
               
                   
               
               
                 mPCSK9 
                 GA010 
                 GGCTGATG 
                 251 
                 5′gsgscsUGAUGAGGCCGCACAU 
                 446 
                 SpCas9 
               
               
                   
                   
                 AGGCCGC 
                   
                 GGUUUUAGAgcuagaaauagcAAGU 
                   
                   
               
               
                   
                   
                 ACATG 
                   
                 UAAAAUAAGGCUAGUCCGUUA 
                   
                   
               
               
                   
                   
                   
                   
                 UCAacuugaaaaaguggcaccgagucggugc 
                   
                   
               
               
                   
                   
                   
                   
                 usususu-3′ 
                   
                   
               
               
                   
               
               
                 mPCSK9 
                 GA011 
                 GGCTGATG 
                 251 
                 5′gsgscsUGAUGAGGCCGCACAU 
                 446 
                 SpCas9 
               
               
                   
                   
                 AGGCCGC 
                   
                 GGUUUUAGAgcuagaaauagcAAGU 
                   
                   
               
               
                   
                   
                 ACATG 
                   
                 UAAAAUAAGGCUAGUCCGUUA 
                   
                   
               
               
                   
                   
                   
                   
                 UCAacuugaaaaaguggcaccgagucggugc 
                   
                   
               
               
                   
                   
                   
                   
                 usususu-3′ 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA016 
                 GGCCTCCT 
                 107 
                 5′gsgscsCUCCUUCAGUUGGGAC 
                 285 
                 SpCas9 
               
               
                   
                   
                 TCAGTTGG 
                   
                 AGUUUUAGAgcuagaaauagcAAGU 
                   
                   
               
               
                   
                   
                 GACA 
                   
                 UAAAAUAAGGCUAGUCCGUUA 
                   
                   
               
               
                   
                   
                   
                   
                 UCAacuugaaaaaguggcaccgagucggugc 
                   
                   
               
               
                   
                   
                   
                   
                 usususu-3′ 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA017 
                 GCCAATGG 
                 109 
                 5′gscscsAAUGGCCUCCUUCAGU 
                 287 
                 SpCas9 
               
               
                   
                   
                 CCTCCTTC 
                   
                 UGUUUUAGAgcuagaaauagcAAGU 
                   
                   
               
               
                   
                   
                 AGTT 
                   
                 UAAAAUAAGGCUAGUCCGUUA 
                   
                   
               
               
                   
                   
                   
                   
                 UCAacuugaaaaaguggcaccgagucggugc 
                   
                   
               
               
                   
                   
                   
                   
                 usususu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA395 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 447 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUUAGagcuaGaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 aAuAaggcuaGUccGUUAucAAcuuG 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaagugGcaccgagucggugcusususuuu 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA396 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 448 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUUAGagcuaGaaauagcaaGUUsa 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 AaAuAaggcuaGUccGUUAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 GaaaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
                   
                   
                   
                 uuu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA397 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 449 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUUAGagcuaGaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 aAuAaggcuaGUccGUUsAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 GaaaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
                   
                   
                   
                 uuu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA398 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 447 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 aAuAaggcuaGUCcGUUAucAAcuug 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaaguggcaccgagucggugcusususuuu 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA399 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 447 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 aAuAaggcuaGuCcGUUaucAAcuuga 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaguggcaccgagucggugcusususuuu 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA400 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 447 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUuagagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 AuaaggcuaGuccgUUaucaacuugaaaaa 
                   
                   
               
               
                   
                   
                   
                   
                 guggcaccgagucggugcusususuuuu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA401 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 447 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 aAuAaggcuaGUccGUUAucAAcuuga 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususuuu 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA402 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 447 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUUAGagcuaGaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 aAuAaggcuaGUccGUUAucAAcuuG 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaagugGcaccgagucggugcusususuu 
                   
                   
               
               
                   
                   
                   
                   
                 Uu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA403 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 448 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUUAGagcuaGaaauagcaaGUUsa 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 AaAuAaggcuaGUccGUUAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 GaaaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
                   
                   
                   
                 uUu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA404 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 449 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUUAGagcuaGaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 aAuAaggcuaGUccGUUsAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 GaaaaagugGcaccgagucggugcusususu 
                   
                   
               
               
                   
                   
                   
                   
                 uUu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA405 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 447 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 aAuAaggcuaGUCcGUUAucAAcuug 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaaguggcaccgagucggugcusususuuU 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA406 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 447 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 aAuAaggcuaGuCcGUUaucAAcuuga 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaguggcaccgagucggugcusususuuU 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA407 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 447 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUuagagcuagaaauagcaaGUUaAa 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 AuaaggcuaGuccgUUaucaacuugaaaaa 
                   
                   
               
               
                   
                   
                   
                   
                 guggcaccgagucggugcusususuuUu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA408 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 447 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 aAuAaggcuaGUccGUUAucAAcuuga 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususuuU 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA409 
                 TCAGCTCC 
                 161 
                 5′uscsasG(C- 
                 450 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 PACE)UCCAGGCGGUCCUGGgU 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 UUUAGagcuagaaauagcaaGUUaAaA 
                   
                   
               
               
                   
                   
                   
                   
                 uAaggcuaGUccGUUAucAAcuugaaa 
                   
                   
               
               
                   
                   
                   
                   
                 aagugGcaccgagucggugcusususu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA410 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAG(G- 
                 451 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 PACE)CGGUCCUGGgUUUUAGag 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 cuagaaauagcaaGUUaAaAuAaggcuaG 
                   
                   
               
               
                   
                   
                   
                   
                 UccGUUAucAAcuugaaaaagugGcacc 
                   
                   
               
               
                   
                   
                   
                   
                 gagucggugcusususu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA439 
                 CCCGCACC 
                 13 
                 5cscscsGCACCUUGGCGCAGCGG 
                 11 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 gUUUUAGagcuaGaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 aAuAaggcuaGUccGUUAucAAcuuG 
                   
                   
               
               
                   
                   
                   
                   
                 aaaaagugGcaccgagucggugcusususuU 
                   
                   
               
               
                   
                   
                   
                   
                 Uu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA440 
                 CCCGCACC 
                 13 
                 5′cscscsGCACCUUGGCGCAGCGG 
                 11 
                 ABE 
               
               
                   
                   
                 TTGGCGCA 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 GCGG 
                   
                 aAuAaggcuaGUccGUUAucAAcuuga 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususuUU 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA234 
                 CGCCTGCC 
                 252 
                 csgscsCUGCCAGCGCCUGGCGAG 
                 452 
                 SpCas9 
               
               
                   
                   
                 AGCGCCTG 
                   
                 UUUUAGAGCUAGAAAUAGCAA 
                   
                   
               
               
                   
                   
                 GCGA 
                   
                 GUUAAAAUAAGGCUAGUCCGU 
                   
                   
               
               
                   
                   
                   
                   
                 UAUCAACUUGAAAAAGUGGCA 
                   
                   
               
               
                   
                   
                   
                   
                 CCGAGUCGGUGCUsususu 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA261 
                 TGAAGAA 
                 126 
                 5′usgsasAGAAAGGGAGUAGUUC 
                 304 
                 SpCas9 
               
               
                   
                   
                 AGGGAGT 
                   
                 UgUUUUAGagcuagaaauagcaaGUUa 
                   
                   
               
               
                   
                   
                 AGTTCT 
                   
                 AaAuAaggcuaGUccGUUAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 gaaaaagugGcaccgagucggugcususus 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA262 
                 CATTATAT 
                 143 
                 5′csasusUAUAUUCAGGUAGUCC 
                 321 
                 SpCas9 
               
               
                   
                   
                 TCAGGTAG 
                   
                 AgUUUUAGagcuagaaauagcaaGUUa 
                   
                   
               
               
                   
                   
                 TCCA 
                   
                 AaAuAaggcuaGUccGUUAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 gaaaaagugGcaccgagucggugcususus 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA263 
                 ACAAAACT 
                 146 
                 5′ascsasAAACUUCAAUGAAACG 
                 324 
                 SpCas9 
               
               
                   
                   
                 TCAATGAA 
                   
                 UgUUUUAGagcuagaaauagcaaGUUa 
                   
                   
               
               
                   
                   
                 ACGT 
                   
                 AaAuAaggcuaGUccGUUAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 gaaaaagugGcaccgagucggugcususus 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA264 
                 GCCAATGG 
                 109 
                 5′gscscsAAUGGCCUCCUUCAGU 
                 287 
                 SpCas9 
               
               
                   
                   
                 CCTCCTTC 
                   
                 UgUUUUAGagcuagaaauagcaaGUUa 
                   
                   
               
               
                   
                   
                 AGTT 
                   
                 AaAuAaggcuaGUccGUUAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 gaaaaagugGcaccgagucggugcususus 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcANGPTL3 
                 GA265 
                 GCCAATGG 
                 109 
                 5′gscscsAAUGGCCUCCUUCAGU 
                 287 
                 SpCas9 
               
               
                   
                   
                 CCTCCTTC 
                   
                 UgUUUUAGagcuaGaaauagcaaGUUa 
                   
                   
               
               
                   
                   
                 AGTT 
                   
                 AaAuAaggCUaGUCcGUUAucAAcu 
                   
                   
               
               
                   
                   
                   
                   
                 uGaaaaaguGgcaccgAgUCggugcususu 
                   
                   
               
               
                   
                   
                   
                   
                 su-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA266 
                 TCAGCTCC 
                 161 
                 5′uscsasGCUCCAGGCGGUCCUGG 
                 339 
                 SpCas9 
               
               
                   
                   
                 AGGCGGTC 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 CTGG 
                   
                 aAuAaggcuaGUccGUUAucAAcuuga 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA267 
                 GCCCCATG 
                 185 
                 5′gscscsCCAUGUCGACUACAUCG 
                 363 
                 SpCas9 
               
               
                   
                   
                 TCGACTAC 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 ATCG 
                   
                 aAuAaggcuaGUccGUUAucAAcuuga 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA268 
                 GGGGCTG 
                 187 
                 5′gsgsgsGCUGGUAUUCAUCCGC 
                 365 
                 SpCas9 
               
               
                   
                   
                 GTATTCAT 
                   
                 UgUUUUAGagcuagaaauagcaaGUUa 
                   
                   
               
               
                   
                   
                 CCGCC 
                   
                 AaAuAaggcuaGUccGUUAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 gaaaaagugGcaccgagucggugcususus 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA269 
                 CTAGGAG 
                 194 
                 5′csusasGGAGAUACACCUCCACC 
                 372 
                 SpCas9 
               
               
                   
                   
                 ATACACCT 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 CCACC 
                   
                 aAuAaggcuaGUccGUUAucAAcuuga 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA270 
                 CTGCGCGT 
                 204 
                 5′csusgsCGCGUGCUCAACUGCCA 
                 382 
                 SpCas9 
               
               
                   
                   
                 GCTCAACT 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 GCCA 
                   
                 aAuAaggcuaGUccGUUAucAAcuuga 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususu-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA271 
                 CGGGATGC 
                 206 
                 5′csgsgsGAUGCCGGCGUGGCCA 
                 384 
                 SpCas9 
               
               
                   
                   
                 CGGCGTGG 
                   
                 AgUUUUAGagcuagaaauagcaaGUUa 
                   
                   
               
               
                   
                   
                 CCAA 
                   
                 AaAuAaggcuaGUccGUUAucAAcuu 
                   
                   
               
               
                   
                   
                   
                   
                 gaaaaagugGcaccgagucggugcususus 
                   
                   
               
               
                   
                   
                   
                   
                 u-3′ 
                   
                   
               
               
                   
               
               
                 hcPCSK9 
                 GA272 
                 CGCTGACC 
                 212 
                 5′csgscsUGACCACCCCUGCCAGG 
                 390 
                 SpCas9 
               
               
                   
                   
                 ACCCCTGC 
                   
                 gUUUUAGagcuagaaauagcaaGUUaA 
                   
                   
               
               
                   
                   
                 CAGG 
                   
                 aAuAaggcuaGUccGUUAucAAcuuga 
                   
                   
               
               
                   
                   
                   
                   
                 aaaagugGcaccgagucggugcusususu-3′ 
                   
                   
               
               
                   
               
               
                 mPCSK9 
                 GA292 
                 cccatacC 
                 69 
                 5′cscscsAUACCUUGGAGCAACG 
                 421 
                 ABE 
               
               
                   
                   
                 TTGGAGCA 
                   
                 GGUUUUAGAGCUAGAAAUAGC 
                   
                   
               
               
                   
                   
                 ACGG 
                   
                 AAGUUAAAAUAAGGCUAGUCC 
                   
                   
               
               
                   
                   
                   
                   
                 GUUAUCAACUUGAAAAAGUGG 
                   
                   
               
               
                   
                   
                   
                   
                 CACCGAGUCGGUGCUsususu-3′ 
               
               
                   
               
            
           
         
       
     
     As used in Table 1 above, Table 23, and Table 24, in the Protospacer: uppercase nucleotides (A, G, C, I and T) indicate 2′-deoxyribonucleotides, adenine, guanine, cytosine, inosine, and thymine, respectively; in Guide RNA sequence: uppercase nucleotides (A, C, G, I and U) indicate ribonucleotides, adenine, guanine, cytosine, inosine, and uracil, respectively and lowercase nucleotides (a, g, c, i and u) indicate 2′-O-methylribonucleotide (2′-OMe) unless otherwise specified. It is understood that the DNA protospacer is converted to RNA or RNA-equivalent in the guide RNA design except when other modifications including 2′-deoxyribonucleotides are introduced into the spacer section of the single guide RNA; s: phosphorothioate (PS), X=ribonebularine; x=2′-O-methylnebularine; dX=2′-deoxynebularine; 5′-NNN-3′ indicates uniform A, C, G, I, U, dA, dG, dC, dI, T, X, x, dX, and combinations thereof. 5′-nnn-3′ indicates uniform a, c, g, i, u, dA, dG, dC or T, x, dX, and combinations thereof. mANGPTL3: mouse ANGPTL3; hANGPTL3: human ANGPTL3; cANGPTL3: cynomolgus ANGPTL3; mcANGPTL3: mouse, cynomolgus cross-reactive ANGPTL3; hcANGPTL3: human, cynomolgus cross-reactive ANGPTL3; mPCSK9: mouse PCSK9; hPCSK9: human PCSK9; cPCSK9: cynomolgus PCSK9; mcPCSK9: mouse, cynomolgus cross-reactive PCSK9; hcPCSK9: human, cynomolgus cross-reactive PCSK9; hcAPOC3: human, cynomolgus cross-reactive APOC3. As disclosed herein, the nucleotide sequences and modification patterns encompass all length, structure, and type of RNAs or fragments thereof, CRISPR guide RNAs, e.g. sgRNAs, dual guide RNAs, or mRNAs. For example, nucleotide sequences and modification patterns as described in the Table above may indicate RNA sequences and modification patterns in a single guide RNA, a dual guide RNA, a nuclease mRNA, or any fragment or segment thereof. In Table 23, u′ indicates N 1 -methylpseudouridine. 
     Example 2. PCSK9 and ANGPTL3 Cas9 Editing In Vitro 
     In set of experiments, PCSK9 gRNAs were co-transfected with an equivalent amount (1:1 ratio by weight) of in vitro transcribed commercially available SpCas9 mRNA MS002 purchased from TriLink Biotechnologies into primary human hepatocytes at 2500, 500, or 100 ng/RNA/mL and processed as described in detailed methods. Extracted genomic DNA was analyzed for gene editing at the target site with next-generation sequencing of PCR amplicons generated around the target sites. Samples were prepared using the Nextera XT DNA library preparation kit (Illumina) according to the manufacturer&#39;s protocol. Briefly, two rounds of PCR were performed first to amplify the region of interest and second to add DNA sequences required for next generation sequencing and sample identification to the initial product. The final amplicon was sequenced on the Illumina MiSeq instrument according to the manufacturer&#39;s protocol. A wide range of editing activities (Table 2) were observed. In a second set of experiments, ANGPTL3 gRNAs were co-transfected with an equivalent amount of in vitro transcribed SpCas9 mRNA (1:1 ratio by weight) into primary human hepatocytes and processed and analyzed similarly (Table 3). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 PCSK9 gRNA/SpCas9 editing in primary hepatocytes 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                 Human Primary 
                 Cyno Primary Hepatocyte 
               
               
                   
                 Protospacer 
                 ID 
                 Hepatocyte Editing % 
                 Editing % 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 gRNA 
                 (5′-3′) 
                 NO: 
                 2500 
                 500 
                 100 
                 2500 
                 500 
                 100 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 GA15 
                 GGTGCTAGCCT 
                 66 
                 26.34 
                 6.22 
                 1.48 
                 33.94 
                 14.31 
                 2.34 
               
               
                 6 
                 TGCGTTCCG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA15 
                 GCCGTCCTCCT 
                 151 
                 10.03 
                 2.82 
                 0.62 
                 13.78 
                 2.93 
                 0.78 
               
               
                 7 
                 CGGAACGCA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA15 
                 GCTAGCCTTGC 
                 152 
                 37.94 
                 6.55 
                 2 
                 18.27 
                 4.42 
                 1.04 
               
               
                 8 
                 GTTCCGAGG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA15 
                 GCGTTCCGAGG 
                 153 
                 14.68 
                 3.74 
                 1.35 
                 12.21 
                 3.69 
                 1.19 
               
               
                 9 
                 AGGACGGCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA16 
                 GCCTTGCGTTC 
                 154 
                 23.3 
                 5.58 
                 1.19 
                 23.15 
                 6.60 
                 1.38 
               
               
                 0 
                 CGAGGAGGA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA16 
                 GGACGAGGAC 
                 155 
                 35.08 
                 8.74 
                 3.65 
                 48.24 
                 14.98 
                 4.52 
               
               
                 1 
                 GGCGACTACG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA16 
                 GGACGGCGAC 
                 156 
                 42.8 
                 12.97 
                 2.14 
                 28.26 
                 11.18 
                 1.62 
               
               
                 2 
                 TACGAGGAGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA16 
                 CGTCCTCGTCC 
                 157 
                 29.32 
                 9.2 
                 2.31 
                 49.50 
                 17.69 
                 5.99 
               
               
                 3 
                 TCCTGCGCA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA16 
                 GTCCTCGTCCT 
                 158 
                 25.77 
                 11.12 
                 2.68 
                 34.89 
                 13.96 
                 2.75 
               
               
                 4 
                 CCTGCGCAC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA16 
                 CCGTCAGCTCC 
                 159 
                 40.27 
                 22.3 
                 4.99 
                 35.57 
                 18.00 
                 5.43 
               
               
                 5 
                 AGGCGGTCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA16 
                 CGCCCGTGCGC 
                 160 
                 36.89 
                 10.99 
                 2.89 
                 44.77 
                 14.39 
                 3.93 
               
               
                 6 
                 AGGAGGACG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA16 
                 TCAGCTCCAGG 
                 161 
                 53.71 
                 25.87 
                 8.2 
                 66.07 
                 35.98 
                 11.98 
               
               
                 7 
                 CGGTCCTGG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA16 
                 CTTGGCGCAGC 
                 162 
                 54.99 
                 25.77 
                 ND 
                 41.49 
                 15.90 
                 4.75 
               
               
                 8 
                 GGTGGAAGG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA16 
                 CGTGCGCAGG 
                 163 
                 46.65 
                 15.15 
                 5.49 
                 42.38 
                 18.28 
                 3.45 
               
               
                 9 
                 AGGACGAGGA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA17 
                 CCAGGACCGC 
                 164 
                 31.35 
                 15.84 
                 2.9 
                 23.17 
                 11.48 
                 4.03 
               
               
                 0 
                 CTGGAGCTGA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA17 
                 TCTTGGTGAGG 
                 165 
                 31.75 
                 9.38 
                 2.37 
                 27.79 
                 9.06 
                 1.29 
               
               
                 1 
                 TATCCCCGG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA17 
                 CAGTGCGCTCT 
                 166 
                 14.01 
                 3.52 
                 0.32 
                 15.58 
                 2.11 
                 0.51 
               
               
                 2 
                 GACTGCGAG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA17 
                 CTTGGTGAGGT 
                 167 
                 23.38 
                 3.94 
                 0.69 
                 35.03 
                 8.70 
                 2.06 
               
               
                 3 
                 ATCCCCGGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA17 
                 GGATCTTGGTG 
                 168 
                 14.74 
                 5.24 
                 1.18 
                 18.65 
                 4.88 
                 1.60 
               
               
                 4 
                 AGGTATCCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA17 
                 GAAGATGAGT 
                 169 
                 24.81 
                 7.53 
                 1.55 
                 44.77 
                 12.37 
                 1.45 
               
               
                 5 
                 GGCGACCTGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA17 
                 AGCACCACCA 
                 170 
                 4.98 
                 1.26 
                 0.36 
                 22.65 
                 8.11 
                 2.11 
               
               
                 6 
                 CGTAGGTGCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA17 
                 GGTCGCCACTC 
                 171 
                 12.57 
                 2.67 
                 0.65 
                 15.91 
                 4.68 
                 0.38 
               
               
                 7 
                 ATCTTCACC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA17 
                 CTCCTTCAGCA 
                 172 
                 18.29 
                 5.43 
                 1.26 
                 25.22 
                 5.78 
                 2.21 
               
               
                 8 
                 CCACCACGT 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA17 
                 GAGTGGCGAC 
                 173 
                 21.87 
                 6 
                 1.29 
                 12.94 
                 4.21 
                 1.06 
               
               
                 9 
                 CTGCTGGAGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA18 
                 GCGCACTGCCC 
                 174 
                 17.16 
                 2.22 
                 0.22 
                 14.12 
                 2.45 
                 0.91 
               
               
                 0 
                 GCCGCCTGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA18 
                 GGCTTCCTGGT 
                 175 
                 ND 
                 ND 
                 28.87 
                 45.12 
                 15.43 
                 3.66 
               
               
                 1 
                 GAAGATGAG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA18 
                 CACCTACGTGG 
                 176 
                 17.44 
                 4.49 
                 0.95 
                 22.89 
                 6.14 
                 1.55 
               
               
                 2 
                 TGGTGCTGA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA18 
                 CTACGTGGTGG 
                 177 
                 32.35 
                 9.89 
                 2.21 
                 39.56 
                 15.99 
                 2.87 
               
               
                 3 
                 TGCTGAAGG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA18 
                 ATGGAAGACA 
                 178 
                 29.55 
                 8.29 
                 2.65 
                 31.87 
                 10.54 
                 2.90 
               
               
                 4 
                 TGCAGGATCT 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA18 
                 AGACATGCAG 
                 179 
                 27.98 
                 10.91 
                 1.6 
                 20.29 
                 7.75 
                 1.14 
               
               
                 5 
                 GATCTTGGTG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA18 
                 CTCATCTTCAC 
                 180 
                 12.01 
                 3.57 
                 0.79 
                 33.48 
                 7.59 
                 1.96 
               
               
                 6 
                 CAGGAAGCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA18 
                 CTCCTCGATGT 
                 181 
                 ND 
                 6.47 
                 2.44 
                 29.24 
                 12.30 
                 3.77 
               
               
                 7 
                 AGTCGACAT 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA18 
                 TATTCATCCGC 
                 182 
                 41.02 
                 15.38 
                 4.21 
                 0.06 
                 0.01 
                 0.01 
               
               
                 8 
                 CCGGTACCG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA18 
                 TCCTCGATGTA 
                 183 
                 31.23 
                 8.94 
                 1.76 
                 41.70 
                 15.58 
                 3.30 
               
               
                 9 
                 GTCGACATG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA19 
                 CCTCCTCGATG 
                 184 
                 25.39 
                 9.73 
                 2.78 
                 26.78 
                 5.53 
                 1.17 
               
               
                 0 
                 TAGTCGACA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA19 
                 GCCCCATGTCG 
                 185 
                 34.6 
                 12.2 
                 3 
                 51.79 
                 26.79 
                 5.46 
               
               
                 1 
                 ACTACATCG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA19 
                 CCATGTCGACT 
                 186 
                 16.7 
                 6.02 
                 1.14 
                 10.26 
                 1.50 
                 0.60 
               
               
                 2 
                 ACATCGAGG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA19 
                 GGGGCTGGTAT 
                 187 
                 35.77 
                 15.68 
                 3.92 
                 52.89 
                 32.02 
                 8.70 
               
               
                 3 
                 TCATCCGCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA19 
                 GTCGACATGG 
                 188 
                 32.97 
                 12.39 
                 2.32 
                 30.91 
                 9.49 
                 2.38 
               
               
                 4 
                 GGCAACTTCA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA19 
                 ACCACCGGGA 
                 189 
                 12.57 
                 13.3 
                 3.58 
                 18.78 
                 4.77 
                 0.69 
               
               
                 5 
                 AATCGAGGGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA19 
                 GAGTGACCAC 
                 190 
                 17.19 
                 2.9 
                 0.75 
                 27.79 
                 3.4 
                 1.09 
               
               
                 6 
                 CGGGAAATCG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA19 
                 AGTGACCACC 
                 191 
                 15.03 
                 3.29 
                 1.22 
                 18.21 
                 3.89 
                 0.9 
               
               
                 7 
                 GGGAAATCGA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA19 
                 CCACCGGGAA 
                 192 
                 30.93 
                 10.58 
                 0.04 
                 49 
                 16.15 
                 3.53 
               
               
                 8 
                 ATCGAGGGCA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA19 
                 GAAGCGGGTC 
                 193 
                 29.95 
                 6.89 
                 1.17 
                 45.32 
                 15.11 
                 3.07 
               
               
                 9 
                 CCGTCCTCCT 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA20 
                 CTAGGAGATA 
                 194 
                 5.31 
                 8.68 
                 3.8 
                 54.36 
                 20.51 
                 5.15 
               
               
                 0 
                 CACCTCCACC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA20 
                 CAGCATACAG 
                 195 
                 31.51 
                 12.92 
                 3.29 
                 44.01 
                 14.37 
                 4.33 
               
               
                 1 
                 AGTGACCACC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA20 
                 AAGCGGGTCC 
                 196 
                 34.75 
                 10.75 
                 2.54 
                 31.13 
                 8.91 
                 1.54 
               
               
                 2 
                 CGTCCTCCTC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA20 
                 TGACCCTGCCC 
                 197 
                 37.02 
                 12.76 
                 2.57 
                 32.85 
                 10.49 
                 3.66 
               
               
                 3 
                 TCGATTTCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA20 
                 CACTCTGTATG 
                 198 
                 24.98 
                 8.98 
                 1.27 
                 19.37 
                 5.27 
                 0.91 
               
               
                 4 
                 CTGGTGTCT 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA20 
                 CCCTGCCCTCG 
                 199 
                 38.04 
                 12.42 
                 3.74 
                 36.43 
                 12.04 
                 3.01 
               
               
                 5 
                 ATTTCCCGG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA20 
                 CCGGTGGTCAC 
                 200 
                 20.73 
                 2.7 
                 0.85 
                 16.9 
                 2.73 
                 0.84 
               
               
                 6 
                 TCTGTATGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA20 
                 GGAAATCGAG 
                 201 
                 20.79 
                 3.46 
                 0.84 
                 19.16 
                 2.72 
                 1.05 
               
               
                 7 
                 GGCAGGGTCA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA20 
                 CCAGCATACA 
                 202 
                 25.76 
                 5.72 
                 1.38 
                 25.07 
                 6.01 
                 1.15 
               
               
                 8 
                 GAGTGACCAC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA20 
                 CTTGGCAGTTG 
                 203 
                 33.23 
                 8.76 
                 0.02 
                 40.78 
                 13.96 
                 3.59 
               
               
                 9 
                 AGCACGCGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA21 
                 CTGCGCGTGCT 
                 204 
                 14.3 
                 5.86 
                 0.04 
                 54.88 
                 19.72 
                 4.5 
               
               
                 0 
                 CAACTGCCA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA21 
                 TGCGCGTGCTC 
                 205 
                 38.07 
                 10.29 
                 1.31 
                 28.63 
                 8.3 
                 1.01 
               
               
                 1 
                 AACTGCCAA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA21 
                 CGGGATGCCG 
                 206 
                 12.45 
                 5.59 
                 1.17 
                 54.89 
                 16.53 
                 1.96 
               
               
                 2 
                 GCGTGGCCAA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA21 
                 CGTGCTCAACT 
                 207 
                 24.28 
                 8.77 
                 2.17 
                 45.22 
                 16.35 
                 2.73 
               
               
                 3 
                 GCCAAGGGA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA21 
                 CCTTGGCCACG 
                 208 
                 22.92 
                 6.28 
                 1.94 
                 50.35 
                 14.84 
                 3.06 
               
               
                 4 
                 CCGGCATCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA21 
                 CAGCGGCCGG 
                 209 
                 4.94 
                 1.01 
                 0.13 
                 7.52 
                 1.07 
                   
               
               
                 5 
                 GATGCCGGCG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA21 
                 CCGGGATGCC 
                 210 
                 14.65 
                 3.7 
                 0.95 
                 37.79 
                 4.97 
                 1.77 
               
               
                 6 
                 GGCGTGGCCA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA21 
                 GTGGTCAGCG 
                 211 
                 15.31 
                 2.75 
                 0.77 
                 39.21 
                 10.2 
                 2.6 
               
               
                 7 
                 GCCGGGATGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA21 
                 CGCTGACCACC 
                 212 
                 31.71 
                 8.96 
                 3.81 
                 58.66 
                 19.95 
                 5.42 
               
               
                 8 
                 CCTGCCAGG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA21 
                 GGCAGGGGTG 
                 213 
                 15.97 
                 4.57 
                 1.28 
                 48.67 
                 11.89 
                 0.35 
               
               
                 9 
                 GTCAGCGGCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA22 
                 GTGCTCAACTG 
                 214 
                 21.87 
                 9.12 
                 2.17 
                 45.17 
                 12.39 
                 3.13 
               
               
                 0 
                 CCAAGGGAA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA22 
                 TCATGGCACCC 
                 215 
                 29.35 
                 7.48 
                 1.11 
                 57.4 
                 20.27 
                 2.82 
               
               
                 1 
                 ACCTGGCAG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA22 
                 TGGCAGGGGT 
                 216 
                 2.04 
                 0.66 
                 0.5 
                 16.7 
                 3.32 
                 0.88 
               
               
                 2 
                 GGTCAGCGGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA22 
                 GCTGACCACCC 
                 217 
                 17.5 
                 7.28 
                 1.44 
                 57.58 
                 21.4 
                 3.46 
               
               
                 3 
                 CTGCCAGGT 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA22 
                 GGCCGCTGACC 
                 218 
                 13.02 
                 2.19 
                 1.01 
                 47.5 
                 12.34 
                 2.61 
               
               
                 4 
                 ACCCCTGCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA22 
                 GGCATCGTCCC 
                 219 
                 18.82 
                 6.39 
                 0.89 
                 8.57 
                 3.11 
                 2.03 
               
               
                 5 
                 GGAAGTTGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA22 
                 GGCTTTTCCGA 
                 220 
                 7.61 
                 2.99 
                 0.4 
                 ND 
                 ND 
                 ND 
               
               
                 6 
                 ATAAACTCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA22 
                 GTCCCGGAAGT 
                 221 
                 32.09 
                 8.29 
                 0.02 
                 32.41 
                 4.58 
                 0.56 
               
               
                 7 
                 TGCCGGCAG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA22 
                 CGGCTGTACCC 
                 222 
                 37.1 
                 10.96 
                 5.9 
                 31.36 
                 7.89 
                 1.95 
               
               
                 8 
                 ACCCGCCAG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA22 
                 GTCGTGCTGGT 
                 223 
                 30.41 
                 6.85 
                 0.47 
                 34.28 
                 5.39 
                 0.75 
               
               
                 9 
                 CACCGCTGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA23 
                 AGTAGAGGCA 
                 224 
                 29.31 
                 12.83 
                 ND 
                 35.86 
                 ND 
                 ND 
               
               
                 0 
                 GGCATCGTCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA23 
                 TCCGAATAAAC 
                 225 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 1 
                 TCCAGGCCT 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA23 
                 GTTTATTCGGA 
                 226 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 2 
                 AAAGCCAGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA23 
                 GCGGCTGTACC 
                 227 
                 19.97 
                 5.39 
                 3.08 
                 37.8 
                 10.84 
                 2.38 
               
               
                 3 
                 CACCCGCCA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA23 
                 CGCCTGCCAGC 
                 252 
                 5.45 
                 2.29 
                 0.06 
                 6.44 
                 1.53 
                 0.21 
               
               
                 4 
                 GCCTGGCGA 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA23 
                 CACCGCTGCCG 
                 228 
                 ND 
                 ND 
                 ND 
                 50.03 
                 17.51 
                 2.51 
               
               
                 5 
                 GCAACTTCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA23 
                 AGCCCTCGCCA 
                 229 
                 22.41 
                 3.13 
                 6.92 
                 37.44 
                 11.62 
                 2.31 
               
               
                 6 
                 GGCGCTGGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA23 
                 CAACGCCGCCT 
                 230 
                 36.78 
                 15.83 
                 4.53 
                 46.01 
                 13.46 
                 2.17 
               
               
                 7 
                 GCCAGCGCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA23 
                 GCACGACCCC 
                 231 
                 ND 
                 ND 
                 ND 
                 17.92 
                 4.01 
                 0.94 
               
               
                 8 
                 AGCCCTCGCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA23 
                 CCGCCTGCCAG 
                 232 
                 3.49 
                 1.73 
                 0.06 
                 4.55 
                 0.55 
                 0.02 
               
               
                 9 
                 CGCCTGGCG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA24 
                 TGCCAGCGCCT 
                 233 
                 7.94 
                 0.24 
                 1.05 
                 10.62 
                 1.9 
                 0.52 
               
               
                 0 
                 GGCGAGGGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA24 
                 TGCTGCTGCCC 
                 234 
                 5.39 
                 1.49 
                 0.61 
                 16.11 
                 2.94 
                 0.97 
               
               
                 1 
                 CTGGCGGGT 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA24 
                 TCACCGCTGCC 
                 235 
                 ND 
                 ND 
                 ND 
                 31.71 
                 7.13 
                 1.79 
               
               
                 2 
                 GGCAACTTC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA24 
                 GGCGAGGGCT 
                 236 
                 ND 
                 ND 
                 ND 
                 10.18 
                 1.84 
                 0.51 
               
               
                 3 
                 GGGGTCGTGC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA24 
                 TTCCGAATAAA 
                 237 
                 ND 
                 ND 
                 ND 
                 28.16 
                 7.89 
                 1.63 
               
               
                 4 
                 CTCCAGGCC 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA24 
                 GTGCTGCTGCC 
                 238 
                 2.9 
                 2.97 
                 1.68 
                 7.07 
                 0.88 
                 0.19 
               
               
                 5 
                 CCTGGCGGG 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA24 
                 GCCAGCGCCTG 
                 239 
                 17.95 
                 2.17 
                 0.08 
                 24.63 
                 5.78 
                 1.11 
               
               
                 6 
                 GCGAGGGCT 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA24 
                 CCTCGCCAGGC 
                 240 
                 6.76 
                 0.17 
                 0.25 
                 11.48 
                 2.62 
                 0.71 
               
               
                 7 
                 GCTGGCAGG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 ANGPTL3 gRNA/SpCas9 editing in primary hepatocytes 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                 Human Primary Hepatocyte 
                 Cyno Primary Hepatocyte 
               
               
                   
                 Protospacer 
                 ID 
                 Editing % 
                 Editing % 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 gRNA 
                 (5′-3′) 
                 NO: 
                 500 ng/mL 
                 100 ng/mL 
                 500 ng/mL 
                 100 ng/mL 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 GA107 
                 AGCCAATGGCC 
                 105 
                 5.09 
                 3.72 
                 ND 
                 32.58 
               
               
                   
                 TCCTTCAGT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA108 
                 TCCCAACTGAA 
                 106 
                 19.43 
                 4.95 
                 15.49 
                 4.11 
               
               
                   
                 GGAGGCCAT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA109 
                 GGCCTCCTTCA 
                 107 
                 18.07 
                 6.60 
                 15.11 
                 4.15 
               
               
                   
                 GTTGGGACA 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA110 
                 GACCATGTCCC 
                 108 
                 13.63 
                 5.37 
                 15.99 
                 8.59 
               
               
                   
                 AACTGAAGG 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA111 
                 GCCAATGGCCT 
                 109 
                 24.08 
                 10.72 
                 28.98 
                 22.61 
               
               
                   
                 CCTTCAGTT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA113 
                 ATTCTGGAGGA 
                 111 
                 19.97 
                 6.00 
                 26.67 
                 12.07 
               
               
                   
                 AATAACTAG 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA115 
                 AACATAGCAAA 
                 113 
                 ND 
                 ND 
                 25.51 
                 12.58 
               
               
                   
                 TCTTGATTT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA116 
                 GTAGAATTTTTT 
                 114 
                 16.33 
                 3.78 
                 19.83 
                 10.84 
               
               
                   
                 CTTCTAGG 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA117 
                 ACTACAAGTCA 
                 115 
                 16.55 
                 7.08 
                 26.11 
                 10.01 
               
               
                   
                 AAAATGAAG 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA122 
                 CTTTTATTTGAC 
                 120 
                 13.82 
                 4.45 
                 16.76 
                 7.28 
               
               
                   
                 TATGCTGT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA125 
                 TGATGGTAAGA 
                 123 
                 16.27 
                 5.04 
                 19.72 
                 12.91 
               
               
                   
                 CACTTTGGT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA126 
                 GGAGTAGTTCT 
                 124 
                 15.21 
                 6.73 
                 17.57 
                 8.29 
               
               
                   
                 TGGTGCTCT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA127 
                 AACATGATGGT 
                 125 
                 18.74 
                 5.77 
                 33.67 
                 19.89 
               
               
                   
                 AAGACACTT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA128 
                 TGAAGAAAGGG 
                 126 
                 25.39 
                 10.29 
                 32.55 
                 0.19 
               
               
                   
                 AGTAGTTCT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA129 
                 AGTTCTTGGTGC 
                 127 
                 ND 
                 ND 
                 20.06 
                 5.36 
               
               
                   
                 TCTTGGCT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA130 
                 ATGATGGTAAG 
                 128 
                 17.09 
                 7.51 
                 25.90 
                 14.60 
               
               
                   
                 ACACTTTGG 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA131 
                 GAAGATAGAGA 
                 129 
                 18.64 
                 ND 
                 27.57 
                 10.10 
               
               
                   
                 AATTTCTGT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA132 
                 GGAAGATAGAG 
                 130 
                 28.39 
                 15.25 
                 26 
                 15 
               
               
                   
                 AAATTTCTG 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA137 
                 TATCAGGTAAA 
                 135 
                 29.70 
                 11.44 
                 30.57 
                 28.01 
               
               
                   
                 ACCTGTCTA 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA141 
                 TTGAGAGTTGC 
                 139 
                 14.87 
                 5.80 
                 10.32 
                 5.49 
               
               
                   
                 TGGGTCTGA 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA142 
                 TGAAAAACTTG 
                 140 
                 24.90 
                 10.50 
                 27.59 
                 20.09 
               
               
                   
                 AGAGTTGCT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA143 
                 TTAATTCAACAT 
                 141 
                 19.36 
                 9.30 
                 21.16 
                 14.84 
               
               
                   
                 CGAATAGA 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA145 
                 CATTATATTCAG 
                 143 
                 26.33 
                 12.55 
                 30.34 
                 18.77 
               
               
                   
                 GTAGTCCA 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA148 
                 ACAAAACTTCA 
                 146 
                 31.67 
                 12.01 
                 69.75 
                 28.57 
               
               
                   
                 ATGAAACGT 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA151 
                 GAGAACTACAA 
                 149 
                 31.88 
                 10.56 
                 38.91 
                 11.32 
               
               
                   
                 ATATGGTTT 
               
               
                   
               
            
           
         
       
     
     The modes of operation of Cas9, cytidine base editors (CBE), and adenine base editors (ABE) respectively, along with relevant terminology used in this application including “protospacer”, “PAM”, “spacer” are illustrated ( FIGS.  1 A- 1 C ). For one of the guides that performed well, GA156, with spacer sequence 5′-GGTGCTAGCCTTGCGTTCCG-3′ (SEQ ID NO: 66), additional guides were synthesized with modifications to the tracrRNA sequence. Additionally, an X-ray crystal structure-guided approach was used ( FIG.  1 D ). Structure-guided spacer and tracr designs were informed by the crystal structures of  S. pyogenes  Cas9 in complex with sgRNA (Jiang et al., 2015, PDB ID 4ZT0) and  S. pyogenes  Cas9 in a pre-catalytic ternary complex (Jiang et al., 2016, PDB ID 5F9R) ( FIG.  2   ). Any positions in the crystal structures where there appeared to be a hydrogen bond between the 2′-OH of a given nucleotide and the Cas9 protein (or another part of the sgRNA) were left unmodified. Additionally, any positions where a steric clash was predicted to occur between a 2′-OMe and the protein were left unmodified. At sites where the 2′-OH was solvent-exposed or otherwise distant from protein residues, a 2′-OMe substitution was made. In cases where the two crystal structures disagreed, no modification was made. This strategy was applied to the entire sgRNA or just the tracr region. Exemplary structures are shown in  FIGS.  3 - 7   . 
     PCSK9 gRNAs were co-transfected with an equivalent amount of in vitro transcribed SpCas9 mRNA MS002 (1:1 ratio by weight) into primary human hepatocytes and processed as described in detailed methods. A wide range of editing activities were observed (Table 4; ND=Not determined). The guides with the highest editing activity at five days post transfection were GA001, GA002, GA007, and GA008. The structure-guided 2′-OMe heavy guide RNA (gRNA) designs GA007 and GA008 showed improved editing over minimally modified control gRNA GA009. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 In vitro evaluation of modified gRNA via SpCas9 
               
               
                 editing in primary human hepatocytes 
               
            
           
           
               
               
               
               
            
               
                   
                 gRNA 
                 % Editing, day 3 
                 % Editing, day 5 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 GA001 
                 16 
                 27 
               
               
                   
                 GA002 
                 28 
                 23 
               
               
                   
                 GA003 
                 ND 
                 7 
               
               
                   
                 GA004 
                  7 
                 12 
               
               
                   
                 GA005 
                  1 
                 15 
               
               
                   
                 GA006 
                 ND 
                 12 
               
               
                   
                 GA007 
                 30 
                 27 
               
               
                   
                 GA008 
                 20 
                 28 
               
               
                   
                 GA009 
                  4 
                 19 
               
               
                   
                   
               
            
           
         
       
     
     Example 3. PCSK9 and ANGPTL3 Base Editing In Vitro 
     Editing of PCSK9 by ABE8.8 was observed in primary hepatocytes. PCSK9 gRNAs were co-transfected with an equivalent amount of in vitro transcribed ABE8.8 mRNA MA002 (1:1 ratio by weight) into primary hepatocytes and processed as described in detailed methods. In one set of experiments, extracted genomic DNA was analyzed for base editing of the target splice site with next-generation sequencing of PCR amplicons generated around the target sites. Samples were prepared using the Nextera XT DNA library preparation kit (Illumina) according to the manufacturer&#39;s protocol. Briefly, two rounds of PCR were performed first to amplify the region of interest and second to add DNA sequences required for next generation sequencing and sample identification to the initial product. The final amplicon was sequenced on the Illumina MiSeq instrument according to the manufacturer&#39;s protocol. A wide range of editing activities (Table 5;  FIG.  8   ) were observed, with guides GA066, GA073, and GA074 performing the best. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 ABE8.8/PCSK9 gRNA editing in primary hepatocytes 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Human Primary 
                 Cynomolgous Primary 
               
               
                   
                   
                 Hepatocytes- 
                 Hepatocytes- 
               
               
                   
                   
                 splice site 
                 splice site 
               
               
                   
                   
                 editing % 
                 editing % 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 5000 
                 2500 
                 1250 
                 5000 
                 2500 
                 1250 
               
               
                 gRNA 
                 Species 
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 GA066 
                 Human/ 
                 25.9 
                 18.7 
                 13.6 
                 24.3 
                 29.1 
                 21.4 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA072 
                 Human/ 
                 2.5 
                 2.2 
                 1.5 
                 2.2 
                 1.7 
                 2.3 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA073 
                 Human/ 
                 25.4 
                 17.5 
                 11.2 
                 29.2 
                 23.2 
                 24.7 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA074 
                 Human/ 
                 23.8 
                 18.8 
                 11.6 
                 18.7 
                 20.1 
                 17.5 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA075 
                 Human/ 
                 5.5 
                 3.1 
                 1.8 
                 ND 
                 ND 
                 ND 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA076 
                 Human/ 
                 4.9 
                 3.9 
                 1.6 
                 ND 
                 ND 
                 ND 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA077 
                 Human/ 
                 6.0 
                 5.7 
                 2.8 
                 5.4 
                 5.7 
                 4.1 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA078 
                 Human/ 
                 12.6 
                 10.0 
                 6.5 
                 ND 
                 ND 
                 ND 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA079 
                 Human/ 
                 6.5 
                 4.9 
                 2.2 
                 0.5 
                 0.5 
                 0.6 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA080 
                 Human/ 
                 0.6 
                 0.5 
                 0.4 
                 7.0 
                 8.8 
                 7.1 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA081 
                 Human/ 
                 8.3 
                 6.1 
                 4.3 
                 0.3 
                 0.3 
                 0.3 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA082 
                 Human/ 
                 9.8 
                 9.6 
                 5.6 
                 ND 
                 ND 
                 ND 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA083 
                 Human/ 
                 0.2 
                 0.2 
                 0.2 
                 ND 
                 ND 
                 ND 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA084 
                 Human/ 
                 8.6 
                 7.6 
                 4.9 
                 ND 
                 ND 
                 ND 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA085 
                 Human/ 
                 2.4 
                 1.8 
                 1.2 
                 ND 
                 ND 
                 ND 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA086 
                 Human/ 
                 5.7 
                 4.9 
                 2.9 
                 ND 
                 ND 
                 ND 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA087 
                 Human/ 
                 1.9 
                 1.4 
                 1.0 
                 0.2 
                 0.2 
                 0.2 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA088 
                 Human/ 
                 7.2 
                 6.2 
                 3.6 
                 24.8 
                 23.0 
                 17.0 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA089 
                 Human/ 
                 18.1 
                 14.1 
                 5.4 
                 ND 
                 ND 
                 ND 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
               
               
                 GA090 
                 Human/ 
                 0.4 
                 0.5 
                 0.3 
                 ND 
                 ND 
                 ND 
               
               
                   
                 Cyno 
               
               
                   
               
            
           
         
       
     
     When considering the data from primary human hepatocytes, PCSK9-targeting gRNAs were identified that matched the following three protospacer sequences as having the best cross-species activity between both primary human hepatocytes and primary cynomolgous hepatocytes: 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13) (GA066), 5′-GCTTACCTGTCTGTGGAAGC-3′ (SEQ ID NO: 67) (GA073), and 5′-TGCTTACCTGTCTGTGGAAG-3′ (SEQ ID NO: 68) (GA074). The GA066 sequence targets the splice donor at the 5′ end of human PCSK9 intron 1 and is predicted to result in an aberrant PCSK9 protein translated from exon 1, followed by several amino acids translated from the beginning of intron 1 (read through from exon 1 into intron 1), followed by a premature stop codon ( FIG.  9   ). The GA073 and GA074 sequences each target the splice donor at the 5′ end of human PCSK9 intron 4 and is predicted to result in an aberrant PCSK9 protein translated from exons 1, 2, 3, and 4, followed by several amino acids translated from the beginning of intron 4 (readthrough from exon 4 into intron 4), followed by a premature stop codon. The predicted prematurely truncated protein in each of these cases is likely to have complete loss of function as well as having minimal immunogenicity due to the near-identical match to part of the naturally occurring wild-type human PCSK9 amino acid sequence. The same protospacer sequence, 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13) (GA066), is found in the cynomolgus monkey genome, similarly targeting the splice donor at the 5′ end of cyno PCSK9 intron 1. 
     Editing of ANGPTL3 by ABE8.8 was observed in primary hepatocytes. ANGPTL3 gRNAs were co-transfected with an equivalent amount of in vitro transcribed ABE8.8 mRNA MA004 (1:1 ratio by weight) into primary hepatocytes and processed as described in detailed methods. A human ANGPTL3-targeting gRNA matching the following protospacer sequence 5′- AAGATACCTGAATAACCCTC-3′ (SEQ ID NO: 15) (GA091) was identified. The GA091 sequence targets the splice donor at the 5′ end of human ANGPTL3 intron 6 and is predicted to result in an aberrant ANGPTL3 protein translated from exons 1, 2, 3, 4, 5, and 6, followed by several amino acids translated from the beginning of intron 6 (readthrough from exon 6 into intron 6), followed by a premature stop codon ( FIG.  10   ). Notably, there is a naturally occurring rare human DNA variant that disrupts the same splice donor and bears the designation rs398122985. A gRNA was synthesized with the orthologous cynomolgous spacer sequence, differing by 1 nucleotide: 5′-AAGATACCTGAATAACTCTC-3′ (SEQ ID NO: 14) (GA067) and bearing modest chemical modifications (Table 1). Both GA091 in human primary hepatocytes, and GA067 in cynomolgus primary hepatocytes, lead to substantial splice editing when co-transfected with ABE8.8 mRNA (protocol described in the detailed methods section) (Table 6). RNA was transfected at 2500, 1250, 625, and 312.5 ng/test article/mL, with two replicates reported (rep 1 and rep 2). 
     
       
         
           
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 ABE8.8/ANGPTL3 gRNA editing in primary hepatocytes 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                   
                 Human Primary Hepatocytes-splice site editing % (Dose, Replicate #) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 2500, 
                 2500, 
                 1250, 
                 1250, 
                 625, 
                 625, 
                 312.5, 
                 312.5, 
               
               
                 gRNA 
                 Species 
                 rep 1 
                 rep 2 
                 rep 1 
                 rep 2 
                 rep 1 
                 rep 2 
                 rep 1 
                 rep 2 
               
               
                   
               
               
                 GA091 
                 Human 
                 72.23 
                 72.04 
                 62.01 
                 65.15 
                 47.85 
                 47.3 
                 30.71 
                 29.62 
               
               
                 GA092 
                 Human/ 
                 38.39 
                 41.71 
                 33.38 
                 29.83 
                 20.73 
                 19.94 
                 8.54 
                 10.92 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 GA093 
                 Human 
                 63.53 
                 66.9 
                 55.54 
                 56.2 
                 39.22 
                 39.8 
                 20.7 
                 17.32 
               
               
                 GA094 
                 Human 
                 44.84 
                 46.65 
                 39.62 
                 46.8 
                 28.75 
                 14.74 
                 14.12 
                 26.7 
               
               
                   
               
            
           
           
               
               
               
            
               
                   
                   
                 Cyno Primary Hepatocytes-splice site editing (Dose, Replicate #) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 2500, 
                 2500, 
                 1250, 
                 1250, 
                 625, 
                 625, 
                 312.5, 
                 312.5, 
               
               
                   
                   
                 rep 1 
                 rep 2 
                 rep 1 
                 rep 2 
                 rep 1 
                 rep 2 
                 rep 1 
                 rep 2 
               
               
                   
               
               
                 GA067 
                 Cyno 
                 66.06 
                 64.84 
                 64.92 
                 63.78 
                 53.56 
                 53.66 
                 37.05 
                 39.36 
               
               
                 GA092 
                 Human/ 
                 45.76 
                 43.27 
                 43 
                 44.28 
                 32.42 
                 32.56 
                 25.72 
                 19.87 
               
               
                   
                 Cyno 
               
               
                   
               
            
           
         
       
     
     Another strategy by which adenine base editors might be used to disrupt gene function is to introduce a missense mutation(s) into the coding region of the gene that results in production of a less functional, or non-functional protein. Guide RNAs targeting protospacers in ANGPTL3 exons were assessed for base editing activity to introduce missense mutation(s). Each of the gRNAs with an equivalent amount of in vitro transcribed ABE8.8 mRNA MA004 (1:1 ratio by weight) were co- transfected in two replicates (rep 1 and rep 2) at 2500, 1250, 625, and 312.5 ng/RNA/mL into primary human hepatocytes and processed as described in detailed methods. The resulting base editing efficiency, as well as one potential amino acid substitution, is listed in Table 7. 
     In some embodiments, adenine base editors disrupts gene function by replacing any one amino acid selected from A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V with a non-identical amin acid A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. In some embodiments, adenine base editors disrupt gene function by substituting I with T or V. In some embodiments, adenine base editors disrupt gene function by substituting D with G. In some embodiments, adenine base editors disrupt gene function by substituting E with G. In some embodiments, adenine base editors disrupt gene function by substituting F with L or P. In some embodiments, adenine base editors disrupt gene function by substituting H with R. In some embodiments, adenine base editors disrupt gene function by substituting L with P. In some embodiments, adenine base editors disrupt gene function by substituting S with P. In some embodiments, adenine base editors disrupt gene function by substituting M with T. In some embodiments, adenine base editors disrupt gene function by substituting N with G. In some embodiments, adenine base editors disrupt gene function by substituting Q with R. In some embodiments, adenine base editors disrupt gene function by substituting R with G. In some embodiments, adenine base editors disrupt gene function by substituting T with A. In some embodiments, adenine base editors disrupt gene function by substituting V with A. In some embodiments, adenine base editors disrupt gene function by substituting Y with C or H. In some embodiments, adenine base editors disrupt gene function by substituting K with G. In some embodiments, adenine base editors disrupt gene function by introducing an amino acid substitute as listed in Table 7. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 ANGPTL3 gRNA ABE editing can introduce missense mutations 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Example 
                   
               
               
                   
                   
                   
                 Amino 
                 Human Primary Hepatocytes-Editing % (Dose,Replicate #) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Proto- 
                 SEQ 
                 Acid 
                 2500, 
                 2500, 
                 1250, 
                 1250, 
                 6259 
                 6259 
                 312.5, 
                 312.5, 
               
               
                   
                 spacer  
                 ID 
                 Substi- 
                 rep 
                 rep 
                 rep 
                 rep 
                 rep 
                 rep 
                 rep 
                 rep 
               
               
                 gRNA 
                 (5′-3′) 
                 NO 
                 tution 
                 1 
                 2 
                 1 
                 2 
                 1 
                 2 
                 1 
                 2 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 GA 
                 TAATTT 
                 102 
                 I68T 
                 0.27 
                 0.31 
                 0.17 
                 0.29 
                 0.27 
                 0.31 
                 0.21 
                 0.27 
               
               
                 104 
                 GGCCCT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TCGTCT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 AGACTT 
                 103 
                 D59G 
                 1.12 
                 1.06 
                 0.98 
                 1 
                 0.95 
                 1.28 
                 1.07 
                 1.01 
               
               
                 105 
                 TGTCCA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TAAGAC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 GACTTT 
                 104 
                 N/A (D59G) 
                 1.24 
                 1.08 
                 1.15 
                 1.13 
                 1.28 
                 1.24 
                 1.07 
                 1.34 
               
               
                 106 
                 GTCCAT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AAGACG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 ATTGTC 
                 110 
                 N/A 
                 0.53 
                 0.44 
                 0.46 
                 0.54 
                 0.49 
                 0.35 
                 0.39 
                 0.5 
               
               
                 112 
                 TTGATC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AATTCT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 ATTCTG 
                 ill 
                 S17P 
                 1.22 
                 1.31 
                 1.17 
                 1.26 
                 1.23 
                 1.19 
                 1.21 
                 1.13 
               
               
                 113 
                 GAGGAA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 ATAACT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 AACATA 
                 113 
                 M39T 
                 58.3 
                 ND 
                 50.19 
                 ND 
                 29.1 
                 ND 
                 16.55 
                 ND 
               
               
                 115 
                 GCAAAT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CTTGAT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 GTAGAA 
                 114 
                 I132T 
                 54.78 
                 ND 
                 65.37 
                 ND 
                 33.93 
                 ND 
                 19.8 
                 ND 
               
               
                 116 
                 TTTTTTC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TTCTAG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 G 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 ACTACA 
                 115 
                 Q107R 
                 56.95 
                 57.99 
                 42.71 
                 48.24 
                 25.63 
                 27.9 
                 13.13 
                 12.8 
               
               
                 117 
                 AGTCAA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AAATGA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TATATT 
                 116 
                 Y186C 
                 37.26 
                 37.2 
                 30.38 
                 30.76 
                 20.85 
                 20.44 
                 9.23 
                 11.01 
               
               
                 118 
                 GGTCTT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CCACGG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 CAAAGA 
                 117 
                 D177G 
                 54.92 
                 55.55 
                 14.06 
                 42.4 
                 25.96 
                 25.9 
                 10.84 
                 12.43 
               
               
                 119 
                 CCTTCT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CCAGAC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 GGTCTT 
                 118 
                 V182A 
                 1.79 
                 2.04 
                 1.41 
                 1.59 
                 1.21 
                 0.89 
                 0.73 
                 0.76 
               
               
                 120 
                 CCACGG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TCTGGA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TTGTTT 
                 119 
                 Y186H 
                 57.04 
                 65.91 
                 45.78 
                 45.61 
                 30.15 
                 33.83 
                 15.68 
                 13.37 
               
               
                 121 
                 ATATTG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GTCTTC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 CTTTTA 
                 120 
                 I196T 
                 40.76 
                 37.69 
                 37.03 
                 34.27 
                 21.08 
                 18.83 
                 11.25 
                 7.49 
               
               
                 122 
                 TTTGAC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TATGCT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 AAAGTC 
                 121 
                 F167L 
                 12.51 
                 13.68 
                 8.79 
                 12.05 
                 8.1 
                 5.76 
                 3.63 
                 2.83 
               
               
                 123 
                 TGGATA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TAGAGA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 GTTGGT 
                 122 
                 L189P 
                 4.43 
                 4.46 
                 4.13 
                 4.34 
                 2.5 
                 2.4 
                 1.2 
                 0.71 
               
               
                 124 
                 TTAATT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GTTTAT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TGATGG 
                 123 
                 N/A 
                 23.46 
                 26.03 
                 20.98 
                 24.59 
                 13.15 
                 15.36 
                 6.44 
                 5.84 
               
               
                 125 
                 TAAGAC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 ACTTTG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 GGAGTA 
                 124 
                 N/A 
                 30.93 
                 32.19 
                 18.11 
                 28.57 
                 15.14 
                 14.56 
                 7.37 
                 5.22 
               
               
                 126 
                 GTTCTT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GGTGCT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 AACATG 
                 125 
                 H239R 
                 43.8 
                 45.03 
                 35.09 
                 34.98 
                 23.07 
                 24.46 
                 12.12 
                 12.5 
               
               
                 127 
                 ATGGTA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AGACAC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TGAAGA 
                 126 
                 F228P, 
                 57.41 
                 74.82 
                 54.67 
                 60.9 
                 34.35 
                 48.55 
                 22.73 
                 17.55 
               
               
                 128 
                 AAGGGA 
                   
                 L229P 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GTAGTT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 AGTTCT 
                 127 
                 N/A 
                 0.19 
                 0.07 
                 0.07 
                 0.13 
                 0.05 
                 0.11 
                 0.1 
                 0.08 
               
               
                 129 
                 TGGTGC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TCTTGG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 ATGATG 
                 128 
                 D240G 
                 22.16 
                 22.73 
                 18.57 
                 20.93 
                 10.79 
                 10.52 
                 5.93 
                 5.09 
               
               
                 130 
                 GTAAGA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CACTTT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 GAAGAT 
                 129 
                 L216P, 
                 73.63 
                 82.24 
                 65.4 
                 74.19 
                 42.42 
                 47.99 
                 25.24 
                 27.14 
               
               
                 131 
                 AGAGAA 
                   
                 S217P 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 ATTTCT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 GGAAGA 
                 130 
                 S217P 
                 76.43 
                 88.77 
                 69.65 
                 70.55 
                 49.49 
                 46.32 
                 22.36 
                 23.83 
               
               
                 132 
                 TAGAGA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AATTTC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TATTTC 
                 131 
                 I234T 
                 33.9 
                 31.49 
                 26.95 
                 30.48 
                 14.09 
                 21.88 
                 12.1 
                 11.31 
               
               
                 133 
                 ATTCAA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CTGAAG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 ATTTCA 
                 132 
                 N/A 
                 41.07 
                 47.04 
                 38.09 
                 38.78 
                 24.71 
                 21 
                 15.48 
                 15.68 
               
               
                 134 
                 TTCAAC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TGAAGA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 GTCTAC 
                 133 
                 Y273C 
                 34.5 
                 36.73 
                 31.33 
                 31.88 
                 19.77 
                 19.85 
                 12.12 
                 10.61 
               
               
                 135 
                 TGTGAT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GTTATA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TAAATG 
                 134 
                 I249T 
                 31.45 
                 31.46 
                 22.08 
                 22.42 
                 12.96 
                 13.18 
                 6.23 
                 5 
               
               
                 136 
                 GTGGTA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CATTCA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TATCAG 
                 135 
                 N/A 
                 39.23 
                 45.5 
                 35.54 
                 35.24 
                 20.93 
                 15.07 
                 10.3 
                 9.13 
               
               
                 137 
                 GTAAAA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CCTGTC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TGTACC 
                 136 
                 T247A 
                 38.65 
                 38.24 
                 29.16 
                 29.42 
                 19.7 
                 17.09 
                 10.32 
                 8.59 
               
               
                 138 
                 ACCATT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TATAAC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TTCACC 
                 137 
                 N/A 
                 13 
                 11.96 
                 10.47 
                 9.45 
                 6.86 
                 4.95 
                 3.51 
                 3.78 
               
               
                 139 
                 TCTGTT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 ATAAAT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 AACAGA 
                 138 
                 R252G 
                 30.27 
                 41.96 
                 32.49 
                 33.65 
                 18.78 
                 17.9 
                 9.48 
                 7.7 
               
               
                 140 
                 GGTGAA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CATACA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TTGAGA 
                 139 
                 S267P 
                 0.4 
                 0.41 
                 0.37 
                 0.51 
                 0.4 
                 0.67 
                 0.44 
                 0.45 
               
               
                 141 
                 GTTGCT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GGGTCT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TTAATT 
                 141 
                 I285V 
                 12.33 
                 10.68 
                 9.23 
                 8.81 
                 5.47 
                 4.63 
                 3.05 
                 2.94 
               
               
                 143 
                 CAACAT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CGAATA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TTTGGG 
                 142 
                 R308G 
                 15.05 
                 15.17 
                 10.82 
                 12.69 
                 7.52 
                 8.04 
                 4.16 
                 4.56 
               
               
                 144 
                 AGGCTT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GATGGT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 CATTAT 
                 143 
                 N/A 
                 66.32 
                 62.98 
                 55.96 
                 58.5 
                 37.77 
                 37.26 
                 20.55 
                 17.46 
               
               
                 145 
                 ATTCAG 
                   
                 (intron) 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GTAGTC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TTGGGA 
                 144 
                 R308G 
                 22.26 
                 23.4 
                 16.22 
                 18.81 
                 9.91 
                 10.61 
                 4.57 
                 4.62 
               
               
                 146 
                 GGCTTG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 ATGGTA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TTTTGG 
                 145 
                 R308G 
                 3.99 
                 3.67 
                 3.46 
                 2.72 
                 1.96 
                 2.41 
                 1.03 
                 1.2 
               
               
                 147 
                 GAGGCT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TGATGG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 ACAAAA 
                 146 
                 N294G 
                 25.08 
                 21.83 
                 22.57 
                 21.33 
                 12.96 
                 16.13 
                 8.62 
                 7.59 
               
               
                 148 
                 CTTCAA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TGAAAC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TATGGT 
                 147 
                 Y304C 
                 0.47 
                 0.43 
                 0.42 
                 0.48 
                 0.52 
                 0.47 
                 0.45 
                 0.47 
               
               
                 149 
                 TTTGGG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AGGCTT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 ACTACA 
                 148 
                 Y302C, 
                 51.08 
                 51.83 
                 40.09 
                 43.73 
                 23.76 
                 22.55 
                 10.14 
                 9.52 
               
               
                 150 
                 AATATG 
                   
                 K303G 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GTTTTG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 GAGAAC 
                 149 
                 N301G 
                 45.3 
                 44.63 
                 31.68 
                 35.7 
                 22.72 
                 20.85 
                 10.83 
                 12.4 
               
               
                 151 
                 TACAAA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TATGGT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 AGGACA 
                 150 
                 H391R 
                 15.32 
                 14.78 
                 9.37 
                 11.5 
                 6.33 
                 6.89 
                 4.95 
                 4.38 
               
               
                 152 
                 CTTCAA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CTGTCC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 CTTTTC 
                 101 
                 N/A 
                 22.82 
                 23.57 
                 18.25 
                 16.17 
                 8.41 
                 7.17 
                 4.79 
                 ND 
               
               
                 153 
                 AGGAGA 
                   
                 (intron) 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 ATTTTG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 GT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 ACGTGG 
                 242 
                 E300G 
                 4.85 
                 5.62 
                 3.78 
                 4 
                 2.98 
                 2.75 
                 2.38 
                 1.96 
               
               
                 276 
                 GAGAAC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TACAAA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 CGATGT 
                 243 
                 N/A 
                 2.36 
                 2.43 
                 1.69 
                 1.87 
                 1.53 
                 1.72 
                 1.16 
                 1.17 
               
               
                 277 
                 TGAATT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AATGTC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 CACAAA 
                 244 
                 Q293R 
                 4.09 
                 4.48 
                 2.43 
                 2.81 
                 2.2 
                 2.01 
                 1.68 
                 1.74 
               
               
                 278 
                 ACTTCA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 ATGAAA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 TACGAA 
                 245 
                 1333V 
                 22.69 
                 29.48 
                 19.35 
                 21.19 
                 12.41 
                 15.31 
                 7.23 
                 7.37 
               
               
                 279 
                 TTGAGT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TGGAAG 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 AC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA 
                 CTATGG 
                 246 
                 S322P 
                 22.78 
                 30.95 
                 22.27 
                 19.12 
                 16.01 
                 16.29 
                 11.29 
                 5.95 
               
               
                 284 
                 AGTATA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 TCTTCT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 CT 
               
               
                   
               
            
           
         
       
     
     The gRNAs GA066/GA095, GA096, G097, GA346, matching the 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13) (GA066) protospacer sequence but with more extensive chemical modifications of various kinds (Table 1) were synthesized. Similarly, three gRNAs (GA098, GA099, GA100) were synthesized matching the 5′-AAGATACCTGAATAACCCTC-3′ (SEQ ID NO: 15) (GA091) human protospacer sequence, as well as four gRNAs (GA067/GA101, GA102, GA103, GA347) matching the 5′-AAGATACCTGAATAACTCTC-3′ (SEQ ID NO: 14) (GA067) cynomolgus protospacer sequence, but with more extensive chemical modifications of various kinds (Table 1). GA066 and GA095 contain the same chemical composition and modification pattern, as do GA067 and GA101 with one another. It is contemplated that such gRNAs may improve stability against nucleases and gRNA-base editor complex, and reduce or suppress gRNA-triggered immune reaction. Each of the gRNAs described in Table 8, with an equivalent amount of in vitro transcribed ABE8.8 mRNA MA002 (1:1 ratio by weight), were co-transfected into primary human hepatocytes and primary cynomolgus hepatocytes via MessengerMax reagent, using various dilutions to assess for editing activity at different concentrations of test article. Three days after transfection, genomic DNA was harvested from the hepatocytes, and then assessed for base editing of the target splice site with next-generation sequencing. In both human and cynomolgus hepatocytes, as high as 60%-70% editing of the target splice site (PCSK9 intron 1 splice donor; ANGPTL3 intron 6 splice donor) were observed (Table 8). 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Chemically modified gRNAs have high editing efficiency in  
               
               
                 human and cynomolgus primary hepatocytes 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Human Primary Hepatocytes- 
                 Cynomolgus Primary Hepatocytes- 
               
               
                   
                   
                 splice site editing % 
                 splice site editing % 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 2500 
                 500 
                 100 
                 20 
                 2500 
                 500 
                 100 
                 20 
               
               
                 gRNA 
                 Species 
                 ng/mL  
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL  
                 ng/mL 
                 ng/mL 
                 ng/mL 
               
               
                   
               
               
                 GA066 
                 Human/ 
                 67.7 
                 55.1 
                 38.9 
                 13.3 
                 48.2 
                 55.3 
                 43.8 
                 19.0 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 GA096 
                 Human/ 
                 62.8 
                 49.9 
                 41.2 
                 15.6 
                 65.9 
                 51.4 
                 48.0 
                 19.6 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 GA097 
                 Human/ 
                 67.3 
                 68.1 
                 54.1 
                 22.6 
                 62.2 
                 58.1 
                 56.8 
                 28.0 
               
               
                   
                 Cyno 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 GA098 
                 Human 
                 70.7 
                 51.3 
                 36.3 
                 10.9 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 GA099 
                 Human 
                 70.3 
                 47.0 
                 34.4 
                 11.5 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 GA100 
                 Human 
                 69.2 
                 48.6 
                 38.5 
                 12.4 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 GA066/ 
                 Cyno 
                 ND 
                 ND 
                 ND 
                 ND 
                 61.4 
                 49.0 
                 45.9 
                 16.0 
               
               
                 GA101 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 GA102 
                 Cyno 
                 ND 
                 ND 
                 ND 
                 ND 
                 62.5 
                 51.7 
                 58.1 
                 25.9 
               
               
                 GA103 
                 Cyno 
                 ND 
                 ND 
                 ND 
                 ND 
                 62.7 
                 44.2 
                 44.6 
                 17.2 
               
               
                   
               
            
           
         
       
     
     The method by which classical CRISPR/Cas9 disrupts a gene by ultimately introducing an indel, is distinctly and significantly different than base editing. Specifically, base editing is used to introduce a base mutation(s) within a target window closer to the 5′ region of the protospacer, as opposed to 3-4 bp away from the PAM, as routinely seen with CRISPR/Cas9. Further, a target region that is highly amenable to CRISPR/Cas9 editing does not necessarily mean base editing at that location will occur, and vice versa. LNPs containing either: (1) Cas9 mRNA MS010 and a gRNA matching 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13) (GA097) protospacer sequence; or (2) ABE8.8 mRNA MA004 and gRNA matching 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13) (GA097) protospacer sequence, were transfected in human primary hepatocytes. Sanger sequencing of the edited genomic DNA was performed and illustrated the difference in gene editing that occurred from CRISPR/Cas9 compared to base editing ( FIG.  11   ). The arrow highlights the main base editing position, while the scissors highlight the general region where Cas9 cuts. 
     To demonstrate that base editing of a splice site disrupts splicing, reverse transcription-PCR of mRNA from treated primary human hepatocytes, using primers in exon 1 and exon 2, was performed. Results confirmed that splice site disruption resulted in the use of alternative splice donor sites within intron 1, well downstream of the in-frame TAG stop codon ( FIG.  12   , Table 9). The table reports the number of mapped reads for each splice site donor as determined from next generation sequencing. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Alternative splice donor sites within PCSK9 intron 1 resulting from editing of 
               
               
                 PCSK9 exon 1 splice-donor adenine base in primary human hepatocytes. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Number of aligned reads corresponding to: 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 Intron 
                 Intron 
                 Intron 
                 Intron 
                 Intron 
                 Intron 
               
               
                   
                   
                 length 
                 length 
                 length 
                 length 
                 length 
                 length 
               
               
                   
                 Mapped 
                 0 
                 83 
                 87 
                 135 
                 153 
                 154 
               
               
                 Sample* 
                 reads 
                 (GT donor) 
                 (CC donor) 
                 (GT donor) 
                 (CG donor) 
                 (GC donor) 
                 (CA donor) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 control 
                 7827 
                 3187 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 (1) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 control 
                 10584 
                 5956 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 (2) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 control 
                 8575 
                 2307 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 (3) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 control 
                 6721 
                 5153 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 (4) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 treated 
                 8278 
                 2368 
                 0 
                 243 
                 184 
                 984 
                 0 
               
               
                 (1) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 treated 
                 4127 
                 1851 
                 19 
                 235 
                 0 
                 785 
                 0 
               
               
                 (2) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 treated 
                 5694 
                 1096 
                 21 
                 563 
                 0 
                 392 
                 130 
               
               
                 (3) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 treated 
                 4214 
                 2036 
                 0 
                 70 
                 0 
                 1203 
                 0 
               
               
                 (4) 
               
               
                   
               
               
                 *Four different primer pairs were used for RT-PCR of the contro /treated samples. 
               
            
           
         
       
     
     Example 4. PCSK9 and ANGPTL3 Off-Target Validation In Vitro 
     With a view towards establishing the safety of a base-editing therapy knocking down PCSK9 in the human liver in vivo, off-target mutagenesis analysis was assessed. A list of candidate sites in the human genome for off-target mutagenesis was assembled using two different methods. The first method used bioinformatic analysis of the human genome, identifying all sites with a PAM sequence compatible with  Streptococcus pyogenes  Cas9 (and therefore ABE 8.8) and a protospacer sequence with up to 4 single-nucleotide mismatches with the GA066 spacer sequence 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13). The second method to generate candidate sites used an in vitro biochemical assay, ONE-seq, that determined the propensity of a ribonucleoprotein comprising the ABE8.8 base editor protein and PCSK9 gRNA (5′-CCCGCACCTTGGCGCAGCGG-3′(SEQ ID NO: 13)) to cleave oligonucleotides in a library. The reference human genome (GRCh38) was searched for sites with up to 6 mismatches to the protospacer sequence specified by the PCSK9 gRNA (5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13)), and sites with up to 4 mismatches plus up to 2 DNA or RNA bulges using Cas-Designer were identified. More specifics on ONE-seq library preparation, experimental protocol, and bioinformatic analysis are described in the additional detailed methods section. 
     Any cleaved oligonucleotides are PCR-amplified and undergo next-generation sequencing. Oligonucleotides with higher sequence counts reflect a higher propensity for Cas9/gRNA cleavage in vitro and represent the sites most likely to suffer off-target mutagenesis in cells. The top site identified by the ONE-seq assay was the on-target PCSK9 site. The list of candidate off-target sites comprises more than 250 sites, and warranted further investigation (Table 10). 
     Primary human hepatocytes were treated with lipid nanoparticle (LNP) encapasulating ABE8.8 mRNA MA004 and PCSK9 gRNA (GA097 or GA346) at 1:1 weight ratio (see Lipid Nanoparticle Formulation and Analysis for details of LNP preparation) Upon next-generation sequencing using the Agilent SureSelect technology (Table 11), when the observed base editing rates in control cells were subtracted from the observed base editing rates in LNP-treated cells across the on-target site and candidate off-target sites (to account for background sequencing errors inherent in next-generation sequencing), appreciable base editing was observed at the on-target PCSK9 target site. These results were replicated in three different primary hepatocyte lots (STL, HLY, and JLP) from two male and one female patients. 
     Additionally, using hepatocytes from four individual donors (including lot TLY from a female patient, in addition to the three lots listed above), more than 50 off-target sites were assessed by performing next-generation sequencing of targeted PCR amplicons from LNP-treated versus untreated hepatocytes. Editing at none of these potential off-target sites, and only on-target editing at the PCSK9 target site, was observed ( FIG.  13   ). 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 PCSK9 gRNA (Protospacer 5′-CCCGCACCTTGGCGCAGCGG-3′(SEQ ID 
               
               
                 NO: 13)) candidate off-target sites as determined by ONE-seq 
               
            
           
           
               
               
               
               
               
            
               
                 Chromo- 
                   
                 Potential Off-target sequence 
                 SEQ 
                   
               
               
                 some 
                 Location 
                 (5′-3′) 
                 ID NO: 
                 Alignment 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 55040029- 
                 CCCGCACCTTGGCGCAGCGGTGG 
                 80 
                 X00 
               
               
                   
                 55040051 
                   
                   
                   
               
               
                   
               
               
                 1 
                 6888039- 
                 CTAGCACC-TGCCCCAGCGGTGG 
                 453 
                 RNA41 
               
               
                   
                 6888061 
                   
                   
                   
               
               
                   
               
               
                 1 
                 10640222- 
                 TCCCTACCCTGGCACAGCAGGGG 
                 454 
                 X60 
               
               
                   
                 10640244 
                   
                   
                   
               
               
                   
               
               
                 1 
                 15101074- 
                 CCCACACTCAGACGCAGCGAGGG 
                 455 
                 X60 
               
               
                   
                 15101096 
                   
                   
                   
               
               
                   
               
               
                 1 
                 20712184- 
                 CCCGCAC--TGGAGCAGCAGGGA 
                 456 
                 RNA32 
               
               
                   
                 20712206 
                   
                   
                   
               
               
                   
               
               
                 1 
                 22256342- 
                 CTTGCACCTTGGTAGCAGCTGGGG 
                 457 
                 DNA41 
               
               
                   
                 22256365 
                   
                   
                   
               
               
                   
               
               
                 1 
                 25103342- 
                 CTGGCACCATGGCCCAGCAGTGG 
                 458 
                 X50 
               
               
                   
                 25103364 
                   
                   
                   
               
               
                   
               
               
                 1 
                 29882027- 
                 ACTGCATCTTAGTGCAGAGGTGG 
                 459 
                 X60 
               
               
                   
                 29882049 
                   
                   
                   
               
               
                   
               
               
                 1 
                 40866872- 
                 TCCACACCTTGGCGCACTGGAAG 
                 460 
                 X50 
               
               
                   
                 40866894 
                   
                   
                   
               
               
                   
               
               
                 1 
                 43692630- 
                 CATTCACCTTGTCACAGCTGGGG 
                 461 
                 X60 
               
               
                   
                 43692652 
                   
                   
                   
               
               
                   
               
               
                 1 
                 44163492- 
                 ACCGCACCATTGCACAGCCTAGG 
                 462 
                 X60 
               
               
                   
                 44163514 
                   
                   
                   
               
               
                   
               
               
                 1 
                 54256943- 
                 GCCGCACCATGGGCACAGCGGGGA 
                 463 
                 DNA41 
               
               
                   
                 54256966 
                   
                   
                   
               
               
                   
               
               
                 1 
                 60335313- 
                 CCCATACC-TGGCACAGCGGTGG 
                 464 
                 RNA31 
               
               
                   
                 60335335 
                   
                   
                   
               
               
                   
               
               
                 1 
                 64198527- 
                 CAAGCACC-TGGCTCAGAGGTGG 
                 465 
                 RNA41 
               
               
                   
                 64198549 
                   
                   
                   
               
               
                   
               
               
                 1 
                 64266524- 
                 ACCGCACC-TGGCACAGTGGAAG 
                 466 
                 RNA41 
               
               
                   
                 64266546 
                   
                   
                   
               
               
                   
               
               
                 1 
                 110096163- 
                 CCCCACCCTTGGCACAGAGCAGG 
                 467 
                 X60 
               
               
                   
                 110096185 
                   
                   
                   
               
               
                   
               
               
                 1 
                 121262583- 
                 CCAGCACC-TAGCGCAGAGCTGG 
                 468 
                 RNA41 
               
               
                   
                 121262605 
                   
                   
                   
               
               
                   
               
               
                 1 
                 145017699- 
                 CCAGCACC-TAGCGCAGAGCTGG 
                 468 
                 RNA41 
               
               
                   
                 145017721 
                   
                   
                   
               
               
                   
               
               
                 1 
                 155615561- 
                 CCCGCACC-TACCTCAGCAGGGG 
                 469 
                 RNA41 
               
               
                   
                 155615583 
                   
                   
                   
               
               
                   
               
               
                 1 
                 155751756- 
                 CCCGCACC-TACCTCAGCAGGGG 
                 469 
                 RNA41 
               
               
                   
                 155751778 
                   
                   
                   
               
               
                   
               
               
                 1 
                 157194781- 
                 CCCGCACCCCACCGCAGCGGGGG 
                 470 
                 X40 
               
               
                   
                 157194803 
                   
                   
                   
               
               
                   
               
               
                 1 
                 163263975- 
                 TCTTTACCTTGGAGCAGCTGAGG 
                 471 
                 X60 
               
               
                   
                 163263997 
                   
                   
                   
               
               
                   
               
               
                 1 
                 175575740- 
                 CCCGCACCACAGTGCACCGGGGG 
                 472 
                 X50 
               
               
                   
                 175575762 
                   
                   
                   
               
               
                   
               
               
                 1 
                 204314754- 
                 CCTCCACAATGGCGCAGGGGCGG 
                 473 
                 X50 
               
               
                   
                 204314776 
                   
                   
                   
               
               
                   
               
               
                 1 
                 206281058- 
                 CCAGCACC-TAGCGCAGAGCTGG 
                 468 
                 RNA41 
               
               
                   
                 206281080 
                   
                   
                   
               
               
                   
               
               
                 1 
                 208574946- 
                 CCAGCACC-TGGCACAGAAGAGG 
                 474 
                 RNA41 
               
               
                   
                 208574968 
                   
                   
                   
               
               
                   
               
               
                 1 
                 228337504- 
                 ACCGCACCAAGGTGCATAGGAGG 
                 475 
                 X60 
               
               
                   
                 228337526 
                   
                   
                   
               
               
                   
               
               
                 1 
                 240808285- 
                 CCAGCA-CTTGCCCCAGCAGAGG 
                 476 
                 RNA41 
               
               
                   
                 240808307 
                   
                   
                   
               
               
                   
               
               
                 2 
                 9138986-9139008 
                 CTCCCACCCTGGCCCAGCGGGGG 
                 477 
                 X40 
               
               
                   
               
               
                 2 
                 11670027- 
                 ACAGCACCTTGGCCCAGAGGCGA 
                 478 
                 X50 
               
               
                   
                 11670049 
                   
                   
                   
               
               
                   
               
               
                 2 
                 15561025- 
                 ACGGCACCTTGGGCTAGCGGGGG 
                 479 
                 X50 
               
               
                   
                 15561047 
                   
                   
                   
               
               
                   
               
               
                 2 
                 42969964- 
                 CCCGCACCCAGCTGCAGCCGAGG 
                 480 
                 X50 
               
               
                   
                 42969986 
                   
                   
                   
               
               
                   
               
               
                 2 
                 69112187- 
                 CCAGCACC-TGGTGCAGAGTAGG 
                 481 
                 RNA41 
               
               
                   
                 69112209 
                   
                   
                   
               
               
                   
               
               
                 2 
                 71667395- 
                 CCCACACCTGGGCCCAGTGGAGG 
                 482 
                 X40 
               
               
                   
                 71667417 
                   
                   
                   
               
               
                   
               
               
                 2 
                 72985325- 
                 CCAGCACC-TGGTGCAGCTTAGG 
                 483 
                 RNA41 
               
               
                   
                 72985347 
                   
                   
                   
               
               
                   
               
               
                 2 
                 85317392- 
                 TCCACACAATGGCACAGCAGAGG 
                 484 
                 X60 
               
               
                   
                 85317414 
                   
                   
                   
               
               
                   
               
               
                 2 
                 90356123- 
                 CCCGCACC-TGGCTCAGAGGGTC 
                 485 
                 RNA41 
               
               
                   
                 90356145 
                   
                   
                   
               
               
                   
               
               
                 2 
                 91447288- 
                 CCCGCACC-TGGCTCAGAGGGTC 
                 485 
                 RNA41 
               
               
                   
                 91447310 
                   
                   
                   
               
               
                   
               
               
                 2 
                 107914896- 
                 CGGGCACCATGGCAGCAGCGGAGG 
                 486 
                 DNA31 
               
               
                   
                 107914919 
                   
                   
                   
               
               
                   
               
               
                 2 
                 110633933- 
                 CGGGCACCATGGCAGCAGCGGAGG 
                 486 
                 DNA31 
               
               
                   
                 110633956 
                   
                   
                   
               
               
                   
               
               
                 2 
                 120922089- 
                 CCCACACCATGGCCCAGTGAGAG 
                 487 
                 X60 
               
               
                   
                 120922111 
                   
                   
                   
               
               
                   
               
               
                 2 
                 127571237- 
                 CCAGCACC-TGGCACAGTGAAGG 
                 488 
                 RNA41 
               
               
                   
                 127571259 
                   
                   
                   
               
               
                   
               
               
                 2 
                 132524147- 
                 CCCACACATGGCCACAGCAGTGG 
                 489 
                 X60 
               
               
                   
                 132524169 
                   
                   
                   
               
               
                   
               
               
                 2 
                 179801496- 
                 CTGGCACCTTAGCACAGAGGAGG 
                 490 
                 X50 
               
               
                   
                 179801518 
                   
                   
                   
               
               
                   
               
               
                 2 
                 182608809- 
                 CCCGCACCAGAGCGCAGGGGGAG 
                 491 
                 X50 
               
               
                   
                 182608831 
                   
                   
                   
               
               
                   
               
               
                 2 
                 197360047- 
                 GCCACACC-TGGCCCAGCAGGGG 
                 492 
                 RNA41 
               
               
                   
                 197360069 
                   
                   
                   
               
               
                   
               
               
                 2 
                 203642814- 
                 CCCGCACCAGGGCGCAGATGGAG 
                 493 
                 X50 
               
               
                   
                 203642836 
                   
                   
                   
               
               
                   
               
               
                 2 
                 203866756- 
                 TCCACACCTCAGCACAGCAGAGG 
                 494 
                 X60 
               
               
                   
                 203866778 
                   
                   
                   
               
               
                   
               
               
                 2 
                 219451287- 
                 ACCGCACCTTGGCCCAGTGCTGG 
                 495 
                 X40 
               
               
                   
                 219451309 
                   
                   
                   
               
               
                   
               
               
                 2 
                 231686140- 
                 ACCGCAGC-TGGCGCAGCATGGG 
                 496 
                 RNA41 
               
               
                   
                 231686162 
                   
                   
                   
               
               
                   
               
               
                 3 
                 3719723-3719745 
                 CCCGCACC-TGGCTCAGAGGGTC 
                 485 
                 RNA41 
               
               
                   
               
               
                 3 
                 10287991- 
                 CCCGCA-TTTGCCTCAGCGGTGG 
                 497 
                 RNA31 
               
               
                   
                 10288013 
                   
                   
                   
               
               
                   
               
               
                 3 
                 13178217- 
                 CCAGCA-CTTGGCACAGCAGTGG 
                 498 
                 RNA31 
               
               
                   
                 13178239 
                   
                   
                   
               
               
                   
               
               
                 3 
                 14242673- 
                 CCCACACCTTGGTGTCAGCGGAGG 
                 499 
                 DNA21 
               
               
                   
                 14242696 
                   
                   
                   
               
               
                   
               
               
                 3 
                 14819436- 
                 CCTGCACC-CGGAGCAGCAGGGG 
                 500 
                 RNA41 
               
               
                   
                 14819458 
                   
                   
                   
               
               
                   
               
               
                 3 
                 38369509- 
                 CCTCCACCTTGGCCCAGTGGAGG 
                 501 
                 X40 
               
               
                   
                 38369531 
                   
                   
                   
               
               
                   
               
               
                 3 
                 52005791- 
                 CTCCCACCCTGGCGCAGAGGAGG 
                 502 
                 X40 
               
               
                   
                 52005813 
                   
                   
                   
               
               
                   
               
               
                 3 
                 54016522- 
                 TCAGCACC-TGGTGCAGAGGAGG 
                 503 
                 RNA41 
               
               
                   
                 54016544 
                   
                   
                   
               
               
                   
               
               
                 3 
                 118171416- 
                 CGAGCACCATGGCGCAGCCCCGG 
                 504 
                 X50 
               
               
                   
                 118171438 
                   
                   
                   
               
               
                   
               
               
                 3 
                 118171480- 
                 CCCGCA-CTTGGAGCAGCCGGCG 
                 505 
                 RNA31 
               
               
                   
                 118171502 
                   
                   
                   
               
               
                   
               
               
                 3 
                 139288546- 
                 GGAGCACCTTGACCCAGCAGAGG 
                 506 
                 X60 
               
               
                   
                 139288568 
                   
                   
                   
               
               
                   
               
               
                 3 
                 139677330- 
                 CCAGCA-CTTAGCCCAGCAGCGG 
                 507 
                 RNA41 
               
               
                   
                 139677352 
                   
                   
                   
               
               
                   
               
               
                 3 
                 139731008- 
                 CCCGCACCTCGGCACAGCTAGGG 
                 508 
                 X40 
               
               
                   
                 139731030 
                   
                   
                   
               
               
                   
               
               
                 3 
                 150408362- 
                 GCCCCATCTTGGCCCAGCGGAGG 
                 509 
                 X40 
               
               
                   
                 150408384 
                   
                   
                   
               
               
                   
               
               
                 3 
                 182806878- 
                 CTAGAACCATGGTGCAGAGGGGG 
                 510 
                 X60 
               
               
                   
                 182806900 
                   
                   
                   
               
               
                   
               
               
                 4 
                 1221653-1221675 
                 ATCCCACCTCGGCACAGCTGGGG 
                 511 
                 X60 
               
               
                   
               
               
                 4 
                 1308120-1308142 
                 CCCGCACCGTGGTACAGCCTGTG 
                 512 
                 X60 
               
               
                   
               
               
                 4 
                 6008382-6008404 
                 CCCGCACC-AGGCCCAGCTGGCG 
                 513 
                 RNA41 
               
               
                   
               
               
                 4 
                 6747615-6747637 
                 CCCACACCTTGGTGCAGCTCTGT 
                 514 
                 X50 
               
               
                   
               
               
                 4 
                 7054192-7054214 
                 ACCACACC-TGTCCCAGCGGAGG 
                 515 
                 RNA41 
               
               
                   
               
               
                 4 
                 37229056- 
                 CACACTACTTGGCACAGAGGAGG 
                 516 
                 X60 
               
               
                   
                 37229078 
                   
                   
                   
               
               
                   
               
               
                 4 
                 84244376- 
                 ACCGCA-CTCAGAGCAGCGGAGG 
                 517 
                 RNA41 
               
               
                   
                 84244398 
                   
                   
                   
               
               
                   
               
               
                 4 
                 114598629- 
                 TCCGCA-CTTGGCTCAGCGGGGC 
                 518 
                 RNA31 
               
               
                   
                 114598651 
                   
                   
                   
               
               
                   
               
               
                 4 
                 115179695- 
                 CCCCGCACTCGGAGCAGCGGCGG 
                 519 
                 X60 
               
               
                   
                 115179717 
                   
                   
                   
               
               
                   
               
               
                 4 
                 139511785- 
                 TCCGCACCCTGAGAGCAGTGGAGG 
                 520 
                 DNA41 
               
               
                   
                 139511808 
                   
                   
                   
               
               
                   
               
               
                 4 
                 141615591- 
                 CCCTGCACTTGGTGCAGCTGGGG 
                 521 
                 X60 
               
               
                   
                 141615613 
                   
                   
                   
               
               
                   
               
               
                 4 
                 158623208- 
                 ACCGCACC-TGACCCAGTGGTGG 
                 522 
                 RNA41 
               
               
                   
                 158623230 
                   
                   
                   
               
               
                   
               
               
                 4 
                 182110224- 
                 CTCCTACCATGGCACAGCTGAGG 
                 523 
                 X60 
               
               
                   
                 182110246 
                   
                   
                   
               
               
                   
               
               
                 5 
                 767330-767352 
                 CCCACACCAGGGCGCAGCTGAAG 
                 524 
                 X50 
               
               
                   
               
               
                 5 
                 1516550-1516572 
                 CCAGTACCTTGGCCCAGCTTTGG 
                 525 
                 X50 
               
               
                   
               
               
                 5 
                 77203454- 
                 ACAGCACC-TGGCACAGTGGTGG 
                 526 
                 RNA41 
               
               
                   
                 77203476 
                   
                   
                   
               
               
                   
               
               
                 5 
                 126364896- 
                 CCCACACC-TGGAGCACCAGGGG 
                 527 
                 RNA41 
               
               
                   
                 126364918 
                   
                   
                   
               
               
                   
               
               
                 5 
                 126628420- 
                 CAAGCACCTTGGCAGCAGCTGAGG 
                 528 
                 DNA31 
               
               
                   
                 126628443 
                   
                   
                   
               
               
                   
               
               
                 5 
                 173667321- 
                 CCCATGCCTTAGCACAGCGATGG 
                 529 
                 X60 
               
               
                   
                 173667343 
                   
                   
                   
               
               
                   
               
               
                 5 
                 178526685- 
                 CTCTATCCTTGGCCCAGCAGTGG 
                 530 
                 X60 
               
               
                   
                 178526707 
                   
                   
                   
               
               
                   
               
               
                 6 
                 3751269-3751291 
                 CCCGCACCTTGCCGCAGCGGCCC 
                 531 
                 X30 
               
               
                   
               
               
                 6 
                 15092621- 
                 ATGGCACCTTGGCACATCAGAGG 
                 532 
                 X60 
               
               
                   
                 15092643 
                   
                   
                   
               
               
                   
               
               
                 6 
                 16346963- 
                 CCCCGCACTCGGAGCAGCGGCGG 
                 519 
                 X60 
               
               
                   
                 16346985 
                   
                   
                   
               
               
                   
               
               
                 6 
                 29016770- 
                 TCCCCACACTGTCGCAGAGGAGG 
                 533 
                 X60 
               
               
                   
                 29016792 
                   
                   
                   
               
               
                   
               
               
                 6 
                 36310604- 
                 CCAGCACCATGGCACAGAGAGGT 
                 534 
                 X60 
               
               
                   
                 36310626 
                   
                   
                   
               
               
                   
               
               
                 6 
                 44047493- 
                 ACAGCACC-TGGCACAGAGGAGG 
                 535 
                 RNA41 
               
               
                   
                 44047515 
                   
                   
                   
               
               
                   
               
               
                 6 
                 47477658- 
                 ACCGCACCTGGCAGCAGCCGTGG 
                 536 
                 X50 
               
               
                   
                 47477680 
                   
                   
                   
               
               
                   
               
               
                 6 
                 73315113- 
                 CCAGCACC-TGGAGCAGCCGAGG 
                 537 
                 RNA31 
               
               
                   
                 73315135 
                   
                   
                   
               
               
                   
               
               
                 6 
                 138138382- 
                 CCAGCACC-TGGCACAAAGGAGG 
                 538 
                 RNA41 
               
               
                   
                 138138404 
                   
                   
                   
               
               
                   
               
               
                 6 
                 139537177- 
                 GAGGCACCTTGGACCAGCAGCGG 
                 539 
                 X60 
               
               
                   
                 139537199 
                   
                   
                   
               
               
                   
               
               
                 6 
                 148698322- 
                 CCCGCA-CTTGGAGCAGCCGGTG 
                 540 
                 RNA31 
               
               
                   
                 148698344 
                   
                   
                   
               
               
                   
               
               
                 6 
                 153756217- 
                 CCAGCA-CTTAGTGCAGCGCAGG 
                 541 
                 RNA41 
               
               
                   
                 153756239 
                   
                   
                   
               
               
                   
               
               
                 6 
                 165843913- 
                 CCCACACC-TGGAGCAGCAGGAG 
                 542 
                 RNA41 
               
               
                   
                 165843935 
                   
                   
                   
               
               
                   
               
               
                 7 
                 93102-93124 
                 CCCACACC-AGGCACGGCGGGGG 
                 543 
                 RNA41 
               
               
                   
               
               
                 7 
                 5360719-5360741 
                 CCCTCACC-AGCCGCAGCAGGGG 
                 544 
                 RNA41 
               
               
                   
               
               
                 7 
                 24849083- 
                 CACCCGACTTGGCGACAGCGGGGG 
                 545 
                 DNA41 
               
               
                   
                 24849106 
                   
                   
                   
               
               
                   
               
               
                 7 
                 74043103- 
                 ACCGCACC-TGGCACAGAAGGGG 
                 546 
                 RNA41 
               
               
                   
                 74043125 
                   
                   
                   
               
               
                   
               
               
                 7 
                 84150566- 
                 CCCGCACC-TGGCTCAGAGGGTC 
                 485 
                 RNA41 
               
               
                   
                 84150588 
                   
                   
                   
               
               
                   
               
               
                 7 
                 96041009- 
                 ACCACACC-TGGCCCAGCAGTGG 
                 547 
                 RNA41 
               
               
                   
                 96041031 
                   
                   
                   
               
               
                   
               
               
                 7 
                 98765214- 
                 CCCTCACC-GGGCACAGAGGAGG 
                 548 
                 RNA41 
               
               
                   
                 98765236 
                   
                   
                   
               
               
                   
               
               
                 7 
                 106882973- 
                 CCAGTACC-TGGCCCAGAGGAGG 
                 549 
                 RNA41 
               
               
                   
                 106882995 
                   
                   
                   
               
               
                   
               
               
                 7 
                 123763164- 
                 CCCGCACC-AGGCGCAGGTGGAG 
                 550 
                 RNA41 
               
               
                   
                 123763186 
                   
                   
                   
               
               
                   
               
               
                 7 
                 129225099- 
                 CAGGCACCTTGGCGGCAGCCGCGG 
                 551 
                 DNA31 
               
               
                   
                 129225122 
                   
                   
                   
               
               
                   
               
               
                 7 
                 139596895- 
                 TGTGCACCATGGCACAGCGGGGA 
                 552 
                 X60 
               
               
                   
                 139596917 
                   
                   
                   
               
               
                   
               
               
                 7 
                 147737806- 
                 CCCGCACC-TGGCTCAGAGGGTC 
                 485 
                 RNA41 
               
               
                   
                 147737828 
                   
                   
                   
               
               
                   
               
               
                 7 
                 150737589- 
                 CCCGCACACAGACGCAGAGCAGG 
                 553 
                 X60 
               
               
                   
                 150737611 
                   
                   
                   
               
               
                   
               
               
                 7 
                 153256176- 
                 TCCACACC-TGGCCCAGCTGTGG 
                 554 
                 RNA41 
               
               
                   
                 153256198 
                   
                   
                   
               
               
                   
               
               
                 7 
                 155527865- 
                 CCTGCAC--TGGCCCAGCAGCGG 
                 555 
                 RNA32 
               
               
                   
                 155527887 
                   
                   
                   
               
               
                   
               
               
                 8 
                 2485153-2485175 
                 CCTGCA-CTAGCCGCAACGGGGG 
                 556 
                 RNA41 
               
               
                   
               
               
                 8 
                 26191893- 
                 TGAGCACCTTGGGGCAGCTGGGG 
                 557 
                 X50 
               
               
                   
                 26191915 
                   
                   
                   
               
               
                   
               
               
                 8 
                 100957388- 
                 CCCGCA-CTTGGCGCAGCTGGCC 
                 558 
                 RNA31 
               
               
                   
                 100957410 
                   
                   
                   
               
               
                   
               
               
                 8 
                 139813559- 
                 CCAGCACCTTGGCAGCAGCACCGG 
                 559 
                 DNA31 
               
               
                   
                 139813582 
                   
                   
                   
               
               
                   
               
               
                 8 
                 142499068- 
                 TCCCCCACTTGGCACAGCTGGGG 
                 560 
                 X60 
               
               
                   
                 142499090 
                   
                   
                   
               
               
                   
               
               
                 8 
                 143383198- 
                 CCAGCA-CAGGGCGCAGAGGTGG 
                 561 
                 RNA41 
               
               
                   
                 143383220 
                   
                   
                   
               
               
                   
               
               
                 9 
                 33873295- 
                 CCCACCCCTTAGCACAGCAATGG 
                 562 
                 X60 
               
               
                   
                 33873317 
                   
                   
                   
               
               
                   
               
               
                 9 
                 34958979- 
                 CCCACACCGTGGCCCAGAGGTGG 
                 563 
                 X40 
               
               
                   
                 34959001 
                   
                   
                   
               
               
                   
               
               
                 9 
                 109123571- 
                 CCAGCACC-TGGCACAGCATAGG 
                 564 
                 RNA41 
               
               
                   
                 109123593 
                   
                   
                   
               
               
                   
               
               
                 9 
                 137012994- 
                 ACCACTACCTGGCCCAGCGGTGG 
                 565 
                 X60 
               
               
                   
                 137013016 
                   
                   
                   
               
               
                   
               
               
                 9 
                 137167999- 
                 TCCGCACCTTGGTCCAGCAGGGG 
                 566 
                 X40 
               
               
                   
                 137168021 
                   
                   
                   
               
               
                   
               
               
                 9 
                 137477162- 
                 TACGCACC-TGGCTCAGCAGAGG 
                 567 
                 RNA41 
               
               
                   
                 137477184 
                   
                   
                   
               
               
                   
               
               
                 10 
                 1572578-1572600 
                 AGCCCAGCATGGCGCAGAGGTGG 
                 568 
                 X60 
               
               
                   
               
               
                 10 
                 5824416-5824438 
                 CCAGCACC-TGGCACAGAGGAGA 
                 569 
                 RNA41 
               
               
                   
               
               
                 10 
                 16285410- 
                 ACCGCACC-TGGCCCAGATGGGG 
                 570 
                 RNA41 
               
               
                   
                 16285432 
                   
                   
                   
               
               
                   
               
               
                 10 
                 20297215- 
                 CCCGCACC-TGGCTCAGAGGGGT 
                 571 
                 RNA31 
               
               
                   
                 20297237 
                   
                   
                   
               
               
                   
               
               
                 10 
                 21126683- 
                 ACCGCACC-TGGCCCAGTGGAGG 
                 572 
                 RNA31 
               
               
                   
                 21126705 
                   
                   
                   
               
               
                   
               
               
                 10 
                 28839554- 
                 CCCACATCCTGGCCCAGCAGGGG 
                 573 
                 X50 
               
               
                   
                 28839576 
                   
                   
                   
               
               
                   
               
               
                 10 
                 47223533- 
                 CCTGCACC-AGGCTCAGTGGGGG 
                 574 
                 RNA41 
               
               
                   
                 47223555 
                   
                   
                   
               
               
                   
               
               
                 10 
                 59089166- 
                 CGAGCACCTTGGCAGCAGCTGAGG 
                 575 
                 DNA31 
               
               
                   
                 59089189 
                   
                   
                   
               
               
                   
               
               
                 10 
                 60958888- 
                 ACCGCACC-TGGCCCAGAGTAGG 
                 576 
                 RNA41 
               
               
                   
                 60958910 
                   
                   
                   
               
               
                   
               
               
                 10 
                 88036335- 
                 ACAGCACC-TGGCGCAGAGCGGG 
                 577 
                 RNA41 
               
               
                   
                 88036357 
                   
                   
                   
               
               
                   
               
               
                 10 
                 97031490- 
                 ACTCCACCATGGCACAGCGTGGG 
                 578 
                 X60 
               
               
                   
                 97031512 
                   
                   
                   
               
               
                   
               
               
                 10 
                 111259337- 
                 ACCACACC-TGGCCCAGAGGAGG 
                 579 
                 RNA41 
               
               
                   
                 111259359 
                   
                   
                   
               
               
                   
               
               
                 10 
                 122572126- 
                 GCGGCACCTTGGCCCAGCGTGGG 
                 580 
                 X40 
               
               
                   
                 122572148 
                   
                   
                   
               
               
                   
               
               
                 10 
                 130560773- 
                 CCTGCACCTGAGCACAGAGGAGG 
                 581 
                 X50 
               
               
                   
                 130560795 
                   
                   
                   
               
               
                   
               
               
                 11 
                 2916587-2916609 
                 CCCGCACCCT-GTGCAGCCGAGG 
                 582 
                 RNA31 
               
               
                   
               
               
                 11 
                 10630862- 
                 CCCAC-TCTTGGCACAGGGGAGG 
                 583 
                 RNA41 
               
               
                   
                 10630884 
                   
                   
                   
               
               
                   
               
               
                 11 
                 18729719- 
                 CCCGCACC-AGGTGCAGGAGTGG 
                 584 
                 RNA41 
               
               
                   
                 18729741 
                   
                   
                   
               
               
                   
               
               
                 11 
                 47350413- 
                 ACCGCACC-TGGCCCAGCAAAGG 
                 585 
                 RNA41 
               
               
                   
                 47350435 
                   
                   
                   
               
               
                   
               
               
                 11 
                 47941203- 
                 GCTGCACCTTAGCACAGTGAAGG 
                 586 
                 X60 
               
               
                   
                 47941225 
                   
                   
                   
               
               
                   
               
               
                 11 
                 57761981- 
                 TCAGCACCTTGGCGAAGCCTTGG 
                 587 
                 X50 
               
               
                   
                 57762003 
                   
                   
                   
               
               
                   
               
               
                 11 
                 72870610- 
                 ACCGCACC-TGGCCCAAAGGGGG 
                 588 
                 RNA41 
               
               
                   
                 72870632 
                   
                   
                   
               
               
                   
               
               
                 11 
                 76659109- 
                 CCCTCACCCTGGCACAGCCGGGG 
                 589 
                 X40 
               
               
                   
                 76659131 
                   
                   
                   
               
               
                   
               
               
                 11 
                 117692097- 
                 TTCACACCTTGGTGCAGAGGTGA 
                 590 
                 X60 
               
               
                   
                 117692119 
                   
                   
                   
               
               
                   
               
               
                 11 
                 121325368- 
                 CCAGCACC-TAGCGCAGCGGCTG 
                 591 
                 RNA31 
               
               
                   
                 121325390 
                   
                   
                   
               
               
                   
               
               
                 11 
                 129981605- 
                 ACCGCACC-TGGCCCATCAGGGG 
                 592 
                 RNA41 
               
               
                   
                 129981627 
                   
                   
                   
               
               
                   
               
               
                 12 
                 3851459-3851481 
                 CCCGCACC-TGGCTCAGCGGGTC 
                 593 
                 RNA31 
               
               
                   
               
               
                 12 
                 32850805- 
                 TCAGCACC-TGGAGCAGCCGAGG 
                 594 
                 RNA41 
               
               
                   
                 32850827 
                   
                   
                   
               
               
                   
               
               
                 12 
                 48000120- 
                 CCCCACCCTTGGTGCAGAGGAGG 
                 595 
                 X50 
               
               
                   
                 48000142 
                   
                   
                   
               
               
                   
               
               
                 12 
                 53328526- 
                 CATACACCTTGCCGCAGCCAGGG 
                 596 
                 X60 
               
               
                   
                 53328548 
                   
                   
                   
               
               
                   
               
               
                 12 
                 54190321- 
                 CACACACCTTAGCACAGCCAAGG 
                 597 
                 X60 
               
               
                   
                 54190343 
                   
                   
                   
               
               
                   
               
               
                 12 
                 65971780- 
                 CTAGCACC-TGGCACAGTGGTGG 
                 598 
                 RNA41 
               
               
                   
                 65971802 
                   
                   
                   
               
               
                   
               
               
                 12 
                 95590363- 
                 ACCGTACC-TGGCCCAGAGGAGG 
                 599 
                 RNA41 
               
               
                   
                 95590385 
                   
                   
                   
               
               
                   
               
               
                 12 
                 106690762- 
                 GCCCCACCTTAATGCAGCGGGGG 
                 600 
                 X50 
               
               
                   
                 106690784 
                   
                   
                   
               
               
                   
               
               
                 12 
                 114491014- 
                 CCAGCTACATGGCCCAGAGGAGG 
                 601 
                 X60 
               
               
                   
                 114491036 
                   
                   
                   
               
               
                   
               
               
                 12 
                 127431260- 
                 CCCACACCAT-GCGCAGAGTGGG 
                 602 
                 RNA41 
               
               
                   
                 127431282 
                   
                   
                   
               
               
                   
               
               
                 13 
                 25788073- 
                 CCCGCACCCAGGC-CAGCTGTGG 
                 603 
                 RNA31 
               
               
                   
                 25788095 
                   
                   
                   
               
               
                   
               
               
                 13 
                 69424400- 
                 CCAGCACC-TGCTGCAGCGATGG 
                 604 
                 RNA41 
               
               
                   
                 69424422 
                   
                   
                   
               
               
                   
               
               
                 13 
                 85917572- 
                 CCCGCACC-TGGCTCAGAGGGTC 
                 485 
                 RNA41 
               
               
                   
                 85917594 
                   
                   
                   
               
               
                   
               
               
                 13 
                 98388403- 
                 TCCGCACCTTGGCCCAGTTGAAG 
                 605 
                 X50 
               
               
                   
                 98388425 
                   
                   
                   
               
               
                   
               
               
                 14 
                 20813343- 
                 ACAGCACCTTGGCAGCAGAGGTGG 
                 606 
                 DNA31 
               
               
                   
                 20813366 
                   
                   
                   
               
               
                   
               
               
                 14 
                 24408232- 
                 CCCGCACC-AGGCTCAGCAGCAG 
                 607 
                 RNA41 
               
               
                   
                 24408254 
                   
                   
                   
               
               
                   
               
               
                 14 
                 38663021- 
                 CCAGCACC-TGGCACAGCCAGGG 
                 608 
                 RNA41 
               
               
                   
                 38663043 
                   
                   
                   
               
               
                   
               
               
                 14 
                 65250553- 
                 GCTGCACCCTGCCACAGAGGAGG 
                 609 
                 X60 
               
               
                   
                 65250575 
                   
                   
                   
               
               
                   
               
               
                 14 
                 89310097- 
                 CCAGCACCCTAGTGCAGAGTGGG 
                 610 
                 X60 
               
               
                   
                 89310119 
                   
                   
                   
               
               
                   
               
               
                 14 
                 90554536- 
                 CCCGCACC-TGGCACAGCACCAG 
                 611 
                 RNA41 
               
               
                   
                 90554558 
                   
                   
                   
               
               
                   
               
               
                 14 
                 100213915- 
                 CCCG-ACCCTGACGCAGCCTGGG 
                 612 
                 RNA41 
               
               
                   
                 100213937 
                   
                   
                   
               
               
                   
               
               
                 14 
                 101153383- 
                 CCCGCACCCTATCACAGCAGAGG 
                 613 
                 X50 
               
               
                   
                 101153405 
                   
                   
                   
               
               
                   
               
               
                 14 
                 102929418- 
                 CTCGCACC-AGGCGCAGCCGTGG 
                 614 
                 RNA31 
               
               
                   
                 102929440 
                   
                   
                   
               
               
                   
               
               
                 15 
                 42252041- 
                 CCCGCACC-TGGCTCAGCGGGTC 
                 593 
                 RNA31 
               
               
                   
                 42252063 
                   
                   
                   
               
               
                   
               
               
                 15 
                 47827093- 
                 CATGTACCTTGCCACAGCAGGGG 
                 615 
                 X60 
               
               
                   
                 47827115 
                   
                   
                   
               
               
                   
               
               
                 15 
                 69452825- 
                 CGAGCACCTTGGCGGCAGCTGAGG 
                 616 
                 DNA31 
               
               
                   
                 69452848 
                   
                   
                   
               
               
                   
               
               
                 15 
                 86059010- 
                 TCCACACCTAGGCACAGCCTAGG 
                 617 
                 X60 
               
               
                   
                 86059032 
                   
                   
                   
               
               
                   
               
               
                 15 
                 94089230- 
                 CCAGCACCAAGGCTCAGCTGAGG 
                 618 
                 X50 
               
               
                   
                 94089252 
                   
                   
                   
               
               
                   
               
               
                 15 
                 99993270- 
                 ACCGCACC-TGGCACATTGGAGG 
                 619 
                 RNA41 
               
               
                   
                 99993292 
                   
                   
                   
               
               
                   
               
               
                 16 
                 1003178-1003200 
                 CCCACACCTTGGCCCAGCCCTGG 
                 620 
                 X40 
               
               
                   
               
               
                 16 
                 2092489-2092511 
                 GCCGCACC-TGCCGCAGCCGTGG 
                 621 
                 RNA31 
               
               
                   
               
               
                 16 
                 3089598-3089620 
                 CCCGCA-CTGGGTGCAGTGGTGG 
                 622 
                 RNA31 
               
               
                   
               
               
                 16 
                 14882693- 
                 CCCACACCCAGGTGCAGCGGCAG 
                 623 
                 X50 
               
               
                   
                 14882715 
                   
                   
                   
               
               
                   
               
               
                 16 
                 16097875- 
                 CCAGCACC-TGGCACAGCAGGTG 
                 624 
                 RNA41 
               
               
                   
                 16097897 
                   
                   
                   
               
               
                   
               
               
                 16 
                 16281338- 
                 CCCACACCCAGGTGCAGCGGCAG 
                 623 
                 X50 
               
               
                   
                 16281360 
                   
                   
                   
               
               
                   
               
               
                 16 
                 18430777- 
                 CCCACACCCAGGTGCAGCGGCAG 
                 623 
                 X50 
               
               
                   
                 18430799 
                   
                   
                   
               
               
                   
               
               
                 16 
                 18513312- 
                 CCCACACCCAGGTGCAGCGGCAG 
                 623 
                 X50 
               
               
                   
                 18513334 
                   
                   
                   
               
               
                   
               
               
                 16 
                 21358246- 
                 CCCGCATCTTGGTGCAGCTGCTT 
                 625 
                 X50 
               
               
                   
                 21358268 
                   
                   
                   
               
               
                   
               
               
                 16 
                 28383449- 
                 ACCGCACCATGGTGCAGCTGGGC 
                 626 
                 X50 
               
               
                   
                 28383471 
                   
                   
                   
               
               
                   
               
               
                 16 
                 28731839- 
                 ACCGCACCATGGTGCAGCTGGGC 
                 626 
                 X50 
               
               
                   
                 28731861 
                   
                   
                   
               
               
                   
               
               
                 16 
                 29792483- 
                 CCAACACC-TGGCGCAGAGTAGG 
                 627 
                 RNA41 
               
               
                   
                 29792505 
                   
                   
                   
               
               
                   
               
               
                 16 
                 29863301- 
                 CTCGTACCAAAGCGCAGAGGAGG 
                 628 
                 X60 
               
               
                   
                 29863323 
                   
                   
                   
               
               
                   
               
               
                 16 
                 30783356- 
                 CCCGCA-CTCGGTGCAGCGGTAT 
                 629 
                 RNA41 
               
               
                   
                 30783378 
                   
                   
                   
               
               
                   
               
               
                 16 
                 31081352- 
                 CCCGCA-CTGGGTGCAGCGGAAG 
                 630 
                 RNA31 
               
               
                   
                 31081374 
                   
                   
                   
               
               
                   
               
               
                 16 
                 31436901- 
                 GCCGCA-CTTGGCGCAGCTGTGG 
                 631 
                 RNA21 
               
               
                   
                 31436923 
                   
                   
                   
               
               
                   
               
               
                 16 
                 67748649- 
                 CTAACACCTTGGTGCAGAGGTGG 
                 632 
                 X50 
               
               
                   
                 67748671 
                   
                   
                   
               
               
                   
               
               
                 16 
                 78768901- 
                 CCTACACCATGGCGCAGCGTCAG 
                 633 
                 X50 
               
               
                   
                 78768923 
                   
                   
                   
               
               
                   
               
               
                 16 
                 81278358- 
                 CCAGCACC-TGGCACATCGGTGG 
                 634 
                 RNA31 
               
               
                   
                 81278380 
                   
                   
                   
               
               
                   
               
               
                 16 
                 88044071- 
                 CCCGCATCTTGGTGCAGCTGCTT 
                 625 
                 X50 
               
               
                   
                 88044093 
                   
                   
                   
               
               
                   
               
               
                 16 
                 88839686- 
                 CCCGCA-CATGGTGCAGTGGCTG 
                 635 
                 RNA41 
               
               
                   
                 88839708 
                   
                   
                   
               
               
                   
               
               
                 17 
                 7223899-7223921 
                 CCCAC-CCCTGGCCCAGCCGAGG 
                 636 
                 RNA41 
               
               
                   
               
               
                 17 
                 7462659-7462681 
                 TGCACACCTTGGCGCAGTGGGGG 
                 637 
                 X40 
               
               
                   
               
               
                 17 
                 7511796-7511818 
                 CCAGCACC-TGGCACAGGGGAGG 
                 638 
                 RNA31 
               
               
                   
               
               
                 17 
                 19391599- 
                 CCCACACC-AGGCACAGAGGAGG 
                 639 
                 RNA41 
               
               
                   
                 19391621 
                   
                   
                   
               
               
                   
               
               
                 17 
                 26736056- 
                 CCCGCACC-TGGCTCAGAGGGTC 
                 485 
                 RNA41 
               
               
                   
                 26736078 
                   
                   
                   
               
               
                   
               
               
                 17 
                 28662198- 
                 GTAGCACGTTGACGCAGCAGCGG 
                 640 
                 X60 
               
               
                   
                 28662220 
                   
                   
                   
               
               
                   
               
               
                 17 
                 38870195- 
                 GCCGCACC-GGGCGCAGTTGGGG 
                 641 
                 RNA41 
               
               
                   
                 38870217 
                   
                   
                   
               
               
                   
               
               
                 17 
                 45753295- 
                 CCCACACC-TGCCCCAGAGGTGG 
                 642 
                 RNA41 
               
               
                   
                 45753317 
                   
                   
                   
               
               
                   
               
               
                 17 
                 45993823- 
                 CCCACAGC-TGGCCCAGCAGGGG 
                 643 
                 RNA41 
               
               
                   
                 45993845 
                   
                   
                   
               
               
                   
               
               
                 17 
                 63929863- 
                 TCAGCACCTTGGCACAGCTGAAG 
                 644 
                 X50 
               
               
                   
                 63929885 
                   
                   
                   
               
               
                   
               
               
                 17 
                 79154410- 
                 ACAGCACCTCGGTGCAGCAGAGA 
                 645 
                 X60 
               
               
                   
                 79154432 
                   
                   
                   
               
               
                   
               
               
                 17 
                 81131740- 
                 CCCTC-CCATGGCGCAGCTTCGG 
                 646 
                 RNA41 
               
               
                   
                 81131762 
                   
                   
                   
               
               
                   
               
               
                 18 
                 3448410-3448432 
                 CCCGCACC-GGGCGCAGCAGCTG 
                 647 
                 RNA31 
               
               
                   
               
               
                 18 
                 39380982- 
                 CATGCTACATGGCGCAGCGGTAG 
                 648 
                 X60 
               
               
                   
                 39381004 
                   
                   
                   
               
               
                   
               
               
                 18 
                 58247634- 
                 CTCGCACCCGAACGCAGCAGAGG 
                 649 
                 X60 
               
               
                   
                 58247656 
                   
                   
                   
               
               
                   
               
               
                 18 
                 59917417- 
                 TCCCCACCCCAGCGCAGCAGAGG 
                 650 
                 X60 
               
               
                   
                 59917439 
                   
                   
                   
               
               
                   
               
               
                 18 
                 78993257- 
                 CGAAGACCTTGGCGCAGAAGCGG 
                 651 
                 X60 
               
               
                   
                 78993279 
                   
                   
                   
               
               
                   
               
               
                 19 
                 1104611-1104633 
                 CCCGCACCTTGGCCTAGCGCGGT 
                 652 
                 X40 
               
               
                   
               
               
                 19 
                 1274125-1274147 
                 TCTGCACCTTGGCGCAGCTGGAG 
                 653 
                 X40 
               
               
                   
               
               
                 19 
                 2209581-2209603 
                 CTCCCACCTTAAAGCAGCGGTGG 
                 654 
                 X50 
               
               
                   
               
               
                 19 
                 5543277-5543299 
                 ACCGCACC-TGGCCCAGAGTGGG 
                 655 
                 RNA41 
               
               
                   
               
               
                 19 
                 6431799-6431821 
                 CCCCCACCTTGGCCCAGCGTTGG 
                 656 
                 X30 
               
               
                   
               
               
                 19 
                 10872441- 
                 GCCACACC-TCGCACAGCGGTGG 
                 657 
                 RNA41 
               
               
                   
                 10872463 
                   
                   
                   
               
               
                   
               
               
                 19 
                 11391283- 
                 ACCACACCTTGCTGCAGAGGAGG 
                 658 
                 X50 
               
               
                   
                 11391305 
                   
                   
                   
               
               
                   
               
               
                 19 
                 11406443- 
                 CCCGCA-GAGGGCGCAGCGGCTG 
                 659 
                 RNA41 
               
               
                   
                 11406465 
                   
                   
                   
               
               
                   
               
               
                 19 
                 12747607- 
                 CCAGCAC-TGGCTCAGCAGAGG 
                 660 
                 RNA32 
               
               
                   
                 12747629 
                   
                   
                   
               
               
                   
               
               
                 19 
                 36110180- 
                 ACCGCACC-TGGCCCAAAGGGGG 
                 588 
                 RNA41 
               
               
                   
                 36110202 
                   
                   
                   
               
               
                   
               
               
                 19 
                 46709648- 
                 ACCGCACC-CGGCCCAGTGGAGG 
                 661 
                 RNA41 
               
               
                   
                 46709670 
                   
                   
                   
               
               
                   
               
               
                 19 
                 49720095- 
                 CCTGCACTCAGGCGCAGACGGGG 
                 662 
                 X60 
               
               
                   
                 49720117 
                   
                   
                   
               
               
                   
               
               
                 19 
                 50490618- 
                 GCTGCACCTTGGCACAGTGGAGG 
                 663 
                 X40 
               
               
                   
                 50490640 
                   
                   
                   
               
               
                   
               
               
                 19 
                 51068476- 
                 CCCACACCCAGGCCCAGAGGAGG 
                 664 
                 X50 
               
               
                   
                 51068498 
                   
                   
                   
               
               
                   
               
               
                 19 
                 51606728- 
                 CCTGCACCT--GCACAGCAGGGG 
                 665 
                 RNA32 
               
               
                   
                 51606750 
                   
                   
                   
               
               
                   
               
               
                 19 
                 53775475- 
                 CGAGCACCTTGGCAGCAGCTGAGG 
                 575 
                 DNA31 
               
               
                   
                 53775498 
                   
                   
                   
               
               
                   
               
               
                 19 
                 56088833- 
                 CTCGCACC-TGGTGCAGCACCGG 
                 666 
                 RNA41 
               
               
                   
                 56088855 
                   
                   
                   
               
               
                   
               
               
                 20 
                 13900645- 
                 GCCGCAC--AGGCACAGCGGCGG 
                 667 
                 RNA32 
               
               
                   
                 13900667 
                   
                   
                   
               
               
                   
               
               
                 20 
                 23109029- 
                 TCAGCACCTTGGCACATCCAGGG 
                 668 
                 X60 
               
               
                   
                 23109051 
                   
                   
                   
               
               
                   
               
               
                 20 
                 31859435- 
                 CCTGCACCTTTGCACAGCACTGG 
                 669 
                 X50 
               
               
                   
                 31859457 
                   
                   
                   
               
               
                   
               
               
                 20 
                 45967752- 
                 CCCGCA-TGTGGCGCAGCAGTGT 
                 670 
                 RNA41 
               
               
                   
                 45967774 
                   
                   
                   
               
               
                   
               
               
                 20 
                 49558961- 
                 CCCAGCACTTGGTGAAGCGGAGG 
                 671 
                 X60 
               
               
                   
                 49558983 
                   
                   
                   
               
               
                   
               
               
                 21 
                 37176555- 
                 CCCACAC-TGGTGCAGAGGTGG 
                 672 
                 RNA32 
               
               
                   
                 37176577 
                   
                   
                   
               
               
                   
               
               
                 22 
                 22912001- 
                 CCTGCACCATGGTGCACCGGCAG 
                 673 
                 X50 
               
               
                   
                 22912023 
                   
                   
                   
               
               
                   
               
               
                 22 
                 25849024- 
                 CCCGCAC-AGCCGCAGAGGAGG 
                 674 
                 RNA32 
               
               
                   
                 25849046 
                   
                   
                   
               
               
                   
               
               
                 22 
                 27735501- 
                 ACCGCACC-TGGCACAGCTTGGG 
                 675 
                 RNA41 
               
               
                   
                 27735523 
                   
                   
                   
               
               
                   
               
               
                 22 
                 49971621- 
                 CCCACACCCTGGCCCAGCCTCGG 
                 676 
                 X50 
               
               
                   
                 49971643 
                   
                   
                   
               
               
                   
               
               
                 X 
                 1842130- 
                 CCAGCACTTTGGTACAGCATAGG 
                 677 
                 X60 
               
               
                   
                 1842152 
                   
                   
                   
               
               
                   
               
               
                 X 
                 23566446- 
                 CCCGCACC-TGGCTCAGAGGGGT 
                 571 
                 RNA31 
               
               
                   
                 23566468 
                   
                   
                   
               
               
                   
               
               
                 X 
                 50814798- 
                 CCCGCACCCTGGCTCAGTGTTGG 
                 678 
                 X40 
               
               
                   
                 50814820 
                   
                   
                   
               
               
                   
               
               
                 X 
                 76172984- 
                 CCCGCAC-AGGCGCAGAGGTGG 
                 679 
                 RNA22 
               
               
                   
                 76173006 
                   
                   
                   
               
               
                   
               
               
                 X 
                 83385071- 
                 CCAGCACC-TGGTCCAGTGGAGG 
                 680 
                 RNA41 
               
               
                   
                 83385093 
                   
                   
                   
               
               
                   
               
               
                 X 
                 107536875- 
                 CCAGCACC-TGGCACAGAGTAGG 
                 681 
                 RNA41 
               
               
                   
                 107536897 
                   
                   
                   
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 PCSK9 gRNA GA346 off-target site validation in human primary hepatocytes 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 STL 
                 HLY 
                 JLP 
                 STL 
                 HLY 
                 JLP 
                   
                   
                   
               
               
                 Chro- 
                 Lot #: 
                 Treat- 
                 Treat- 
                 Treat- 
                 Con- 
                 Con- 
                 Con- 
                 Average 
                 Average 
                 Net 
               
               
                 mosome 
                 Location 
                 ed 
                 ed 
                 ed 
                 trol 
                 trol 
                 trol 
                 treated 
                 untreated 
                 Editing 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                  55040029- 
                 42.18 
                 49.76 
                 78.54 
                 0.13 
                 0.19 
                 0.03 
                 56.83 
                 0.12 
                 56.71 
               
               
                   
                 55040051 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                  54256943- 
                 0 
                 0.07 
                 0.04 
                 0.1 
                 0 
                 0.03 
                 0.04 
                 0.04 
                 −0.01 
               
               
                   
                 54256966 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                  22256342- 
                 0 
                 0 
                 0.02 
                 0 
                 0.07 
                 0.02 
                 0.01 
                 0.03 
                 −0.02 
               
               
                   
                 22256365 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                  10640222- 
                 0 
                 0 
                 0 
                 0 
                 0.04 
                 0 
                 0.00 
                 0.01 
                 −0.01 
               
               
                   
                 10640244 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                  6888039- 
                 0 
                 0 
                 0.03 
                 0.03 
                 0.05 
                 0.03 
                 0.01 
                 0.04 
                 −0.03 
               
               
                   
                 6888061 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                  60335313- 
                 0 
                 0.02 
                 0.01 
                 0.03 
                 0 
                 0.02 
                 0.01 
                 0.02 
                 −0.01 
               
               
                   
                 60335335 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                  64198527- 
                 0 
                 0.07 
                 0 
                 0.02 
                 0.06 
                 0 
                 0.02 
                 0.03 
                 0.00 
               
               
                   
                 64198549 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                  64266524- 
                 0 
                 0.04 
                 0.05 
                 0.02 
                 0 
                 0.05 
                 0.03 
                 0.02 
                 0.01 
               
               
                   
                 64266546 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                 121262583- 
                 0.02 
                 0.04 
                 0.05 
                 0.02 
                 0 
                 0.04 
                 0.04 
                 0.02 
                 0.02 
               
               
                   
                 121262605 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                  20712184- 
                 0.05 
                 0.05 
                 0.03 
                 0.03 
                 0.06 
                 0.06 
                 0.04 
                 0.05 
                 −0.01 
               
               
                   
                 20712206 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                 240808285- 
                 0.06 
                 0.03 
                 0.01 
                 0 
                 0.06 
                 0.02 
                 0.03 
                 0.03 
                 0.01 
               
               
                   
                 240808307 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                 145017699- 
                 0.05 
                 0.05 
                 0.08 
                 0.07 
                 0.14 
                 0.05 
                 0.06 
                 0.09 
                 −0.03 
               
               
                   
                 145017721 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                 155615561- 
                 0 
                 0.04 
                 0.01 
                 0 
                 0.05 
                 0.03 
                 0.02 
                 0.03 
                 −0.01 
               
               
                   
                 155615583 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 1 
                 155751756- 
                 0.02 
                 0 
                 0.06 
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                 0.02 
               
               
                   
                 14882715 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 16 
                  16281338- 
                 0.02 
                 0.04 
                 0.06 
                 0.06 
                 0 
                 0.06 
                 0.04 
                 0.04 
                 0.00 
               
               
                   
                 16281360 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 16 
                  18430777- 
                 0.08 
                 0 
                 0.09 
                 0.07 
                 0.05 
                 0.02 
                 0.06 
                 0.05 
                 0.01 
               
               
                   
                 18430799 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 16 
                  18513312- 
                 0.04 
                 0 
                 0 
                 0.02 
                 0 
                 0.07 
                 0.01 
                 0.03 
                 −0.02 
               
               
                   
                 18513334 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 16 
                  21358246- 
                 0 
                 0 
                 0 
                 0.02 
                 0 
                 0.02 
                 0.00 
                 0.01 
                 −0.01 
               
               
                   
                 21358268 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 16 
                  28383449- 
                 0.02 
                 0.1 
                 0.03 
                 0.03 
                 0.04 
                 0.01 
                 0.05 
                 0.03 
                 0.02 
               
               
                   
                 28383471 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 16 
                  28731839- 
                 0.05 
                 0.02 
                 0.05 
                 0.02 
                 0 
                 0.04 
                 0.04 
                 0.02 
                 0.02 
               
               
                   
                 28731861 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 16 
                  29863301- 
                 0 
                 0 
                 0 
                 0.03 
                 0 
                 0 
                 0.00 
                 0.01 
                 −0.01 
               
               
                   
                 29863323 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 16 
                  67748649- 
                 0 
                 0 
                 0.03 
                 0 
                 0 
                 0 
                 0.01 
                 0.00 
                 0.01 
               
               
                   
                 67748671 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 16 
                  78768901- 
                 0.04 
                 0 
                 0.01 
                 0 
                 0.02 
                 0.03 
                 0.02 
                 0.02 
                 0.00 
               
               
                   
                 78768923 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 16 
                  88044071- 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0.00 
                 0.00 
                 0.00 
               
               
                   
                 88044093 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 17 
                  7223899- 
                 0 
                 0 
                 0 
                 0.02 
                 0 
                 0.06 
                 0.00 
                 0.03 
                 −0.03 
               
               
                   
                 7223921 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 17 
                  7511796- 
                 0 
                 0.02 
                 0.05 
                 0.04 
                 0.09 
                 0 
                 0.02 
                 0.04 
                 −0.02 
               
               
                   
                 7511818 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 17 
                  19391599- 
                 0.03 
                 0.03 
                 0.1 
                 0.05 
                 0.06 
                 0.06 
                 0.05 
                 0.06 
                 0.00 
               
               
                   
                 19391621 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 17 
                  26736056- 
                 0.03 
                 0.08 
                 0 
                 0.04 
                 0 
                 0 
                 0.04 
                 0.01 
                 0.02 
               
               
                   
                 26736078 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 17 
                  38870195- 
                 0 
                 0.03 
                 0.05 
                 0.03 
                 0.07 
                 0 
                 0.03 
                 0.03 
                 −0.01 
               
               
                   
                 38870217 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 17 
                  45753295- 
                 0.03 
                 0 
                 0.02 
                 0.04 
                 0 
                 0.04 
                 0.02 
                 0.03 
                 −0.01 
               
               
                   
                 45753317 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 17 
                  45993823- 
                 0 
                 0.06 
                 0.02 
                 0.05 
                 0.07 
                 0 
                 0.03 
                 0.04 
                 −0.01 
               
               
                   
                 45993845 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 17 
                  81131740- 
                 0 
                 0 
                 0 
                 0.01 
                 0 
                 0.03 
                 0.00 
                 0.01 
                 −0.01 
               
               
                   
                 81131762 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 17 
                  7462659- 
                 0.05 
                 0 
                 0.04 
                 0.01 
                 0.06 
                 0 
                 0.03 
                 0.02 
                 0.01 
               
               
                   
                 7462681 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 17 
                  28662198- 
                 0 
                 0 
                 0 
                 0.07 
                 0 
                 0.03 
                 0.00 
                 0.03 
                 −0.03 
               
               
                   
                 28662220 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 17 
                  63929863- 
                 0.07 
                 0.02 
                 0.03 
                 0.01 
                 0.02 
                 0.03 
                 0.04 
                 0.02 
                 0.02 
               
               
                   
                 63929885 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 17 
                  79154410- 
                 0 
                 0 
                 0.03 
                 0.02 
                 0 
                 0 
                 0.01 
                 0.01 
                 0.00 
               
               
                   
                 79154432 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 18 
                  3448410- 
                 0.03 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0.01 
                 0.00 
                 0.01 
               
               
                   
                 3448432 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 18 
                  39380982- 
                 0 
                 0.04 
                 0.03 
                 0.01 
                 0 
                 0.05 
                 0.02 
                 0.02 
                 0.00 
               
               
                   
                 39381004 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 18 
                  58247634- 
                 0.08 
                 0.02 
                 0 
                 0 
                 0.05 
                 0.03 
                 0.03 
                 0.03 
                 0.01 
               
               
                   
                 58247656 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 18 
                  59917417- 
                 0.06 
                 0 
                 0 
                 0.02 
                 0 
                 0 
                 0.02 
                 0.01 
                 0.01 
               
               
                   
                 59917439 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 18 
                  78993257- 
                 0 
                 0 
                 0.04 
                 0 
                 0.11 
                 0.02 
                 0.01 
                 0.04 
                 −0.03 
               
               
                   
                 78993279 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  53775475- 
                 0.03 
                 0 
                 0.02 
                 0.02 
                 0 
                 0 
                 0.02 
                 0.01 
                 0.01 
               
               
                   
                 53775498 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  11406443- 
                 0.02 
                 0 
                 0.08 
                 0.02 
                 0 
                 0.1 
                 0.03 
                 0.04 
                 −0.01 
               
               
                   
                 11406465 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  10872441- 
                 0.06 
                 0 
                 0 
                 0.03 
                 0 
                 0.03 
                 0.02 
                 0.02 
                 0.00 
               
               
                   
                 10872463 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  36110180- 
                 0 
                 0 
                 0 
                 0.13 
                 0 
                 0 
                 0.00 
                 0.04 
                 −0.04 
               
               
                   
                 36110202 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  46709648- 
                 0 
                 0 
                 0.09 
                 0.03 
                 0 
                 0.12 
                 0.03 
                 0.05 
                 −0.02 
               
               
                   
                 46709670 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  12747607- 
                 0.03 
                 0 
                 0.06 
                 0.04 
                 0.05 
                 0.02 
                 0.03 
                 0.04 
                 −0.01 
               
               
                   
                 12747629 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  56088833- 
                 0 
                 0.07 
                 0 
                 0.05 
                 0.14 
                 0 
                 0.02 
                 0.06 
                 −0.04 
               
               
                   
                 56088855 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  51606728- 
                 0.02 
                 0 
                 0.01 
                 0.01 
                 0 
                 0.06 
                 0.01 
                 0.02 
                 −0.01 
               
               
                   
                 51606750 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  1104611- 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0.00 
                 0.00 
                 0.00 
               
               
                   
                 1104633 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  1274125- 
                 0 
                 0.05 
                 0.02 
                 0.05 
                 0 
                 0 
                 0.02 
                 0.02 
                 0.01 
               
               
                   
                 1274147 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  2209581- 
                 0 
                 0 
                 0 
                 0.02 
                 0.07 
                 0 
                 0.00 
                 0.03 
                 −0.03 
               
               
                   
                 2209603 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  6431799- 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0.03 
                 0.00 
                 0.01 
                 −0.01 
               
               
                   
                 6431821 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  11391283- 
                 0.03 
                 0.04 
                 0.03 
                 0.07 
                 0.1 
                 0.03 
                 0.03 
                 0.07 
                 −0.03 
               
               
                   
                 11391305 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  49720095- 
                 0.04 
                 0 
                 0 
                 0 
                 0 
                 0.1 
                 0.01 
                 0.03 
                 −0.02 
               
               
                   
                 49720117 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  50490618- 
                 0 
                 0.05 
                 0.02 
                 0 
                 0.03 
                 0 
                 0.02 
                 0.01 
                 0.01 
               
               
                   
                 50490640 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 19 
                  51068476- 
                 0.07 
                 0.06 
                 0.1 
                 0.08 
                 0 
                 0.02 
                 0.08 
                 0.03 
                 0.04 
               
               
                   
                 51068498 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 20 
                  45967752- 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0.03 
                 0.00 
                 0.01 
                 −0.01 
               
               
                   
                 45967774 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 20 
                  13900645- 
                 0.03 
                 0 
                 0.03 
                 0.04 
                 0.05 
                 0.06 
                 0.02 
                 0.05 
                 −0.03 
               
               
                   
                 13900667 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 20 
                  23109029- 
                 0 
                 0 
                 0.01 
                 0 
                 0 
                 0.01 
                 0.00 
                 0.00 
                 0.00 
               
               
                   
                 23109051 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 20 
                  31859435- 
                 0 
                 0.02 
                 0 
                 0 
                 0 
                 0.03 
                 0.01 
                 0.01 
                 0.00 
               
               
                   
                 31859457 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 20 
                  49558961- 
                 0.04 
                 0.09 
                 0 
                 0 
                 0.07 
                 0 
                 0.04 
                 0.02 
                 0.02 
               
               
                   
                 49558983 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 21 
                  37176555- 
                 0 
                 0 
                 0.03 
                 0.07 
                 0.05 
                 0 
                 0.01 
                 0.04 
                 −0.03 
               
               
                   
                 37176577 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 22 
                  27735501- 
                 0 
                 0.08 
                 0 
                 0 
                 0.04 
                 0.05 
                 0.03 
                 0.03 
                 0.00 
               
               
                   
                 27735523 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 22 
                  25849024- 
                 0.03 
                 0.04 
                 0.01 
                 0 
                 0 
                 0.02 
                 0.03 
                 0.01 
                 0.02 
               
               
                   
                 25849046 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 22 
                  22912001- 
                 0 
                 0 
                 0.01 
                 0 
                 0.05 
                 0 
                 0.00 
                 0.02 
                 −0.01 
               
               
                   
                 22912023 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 22 
                  49971621- 
                 0 
                 0.15 
                 0.06 
                 0 
                 0.11 
                 0.03 
                 0.07 
                 0.05 
                 0.02 
               
               
                   
                 49971643 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 X 
                  1842130- 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0.00 
                 0.00 
                 0.00 
               
               
                   
                 1842152 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 X 
                  50814798- 
                 0 
                 0 
                 0 
                 0.06 
                 0 
                 0 
                 0.00 
                 0.02 
                 −0.02 
               
               
                   
                 50814820 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 X 
                  23566446- 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0.00 
                 0.00 
                 0.00 
               
               
                   
                 23566468 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 X 
                  83385071- 
                 0 
                 0.08 
                 0 
                 0 
                 0 
                 0 
                 0.03 
                 0.00 
                 0.03 
               
               
                   
                 83385093 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 X 
                  76172984- 
                 0 
                 0 
                 0.03 
                 0 
                 0 
                 0.05 
                 0.01 
                 0.02 
                 −0.01 
               
               
                   
                 76173006 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 X 
                 107536875- 
                 0 
                 0 
                 0 
                 0.03 
                 0 
                 0 
                 0.00 
                 0.01 
                 −0.01 
               
               
                   
                 107536897 
               
               
                   
               
            
           
         
       
     
     Adenine base editors have been reported to induce gRNA-independent RNA editing via the deoxyadenosine deaminase domain. To assess for gRNA-independent RNA editing, primary human hepatocytes were treated with mRNA and PCSK9 gRNA (n=4 biological replicates), SpCas9 mRNA and gRNA (n=4), or were untreated (n=4). RNA was extracted after 2 days, and processed as described in the additional detailed methods section. Comparing the RNA profiles of the ABE8.8- and SpCas9-treated hepatocytes with untreated hepatocytes, no substantial RNA edits in the ABE8.8-treated hepatocytes was observed ( FIG.  14   ). Each replicate was compared against each of four untreated hepatocyte samples to eliminate any positions with editing that were common to both conditions. The jitter plots portray transcriptomic loci with editing in the treated sample (number indicates total edited loci identified in the treated sample, boxplot indicates median ±interquartile range of proportion of edited reads across all edited loci in the sample). 
     Modifications and/or truncations to either the spacer or tracr portion of the gRNA was assessed by alteration of the GA066 spacer (5′-CCCGCACCTTGGCGCAGCGG-3′(SEQ TD NO: 13)) (Table 12). The modifications to the guide can serve to improve on-target editing efficiency and/or improve off-target editing efficiency. Each of the gRNAs with an equivalent amount of in vitro transcribed ABE8.8 mRNA (1:1 ratio by weight) were co-transfected into primary human hepatocytes and primary cynomolgus hepatocytes and processed as described in detailed methods. 
     
       
         
           
               
             
               
                 TABLE 12 
               
               
                   
               
               
                 Modifications and/or truncations of the PCSK9 guide GA346 results in high- 
               
               
                 level editing in human primary hepatocytes 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 Human Prirmary Hepatocytes-Editing % at Position 6 (Dose, Replicate #) 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2500 
                 2500 
                 1250 
                 1250 
                 625 
                 625 
                 312.5 
                 312.5 
               
               
                   
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL 
               
               
                 gRNA 
                 rep1 
                 rep2 
                 rep1 
                 rep2 
                 rep1 
                 rep2 
                 rep1 
                 rep2 
               
               
                   
               
               
                 GA346 
                 30.69 
                 35.58 
                 19.99 
                 34.67 
                 27.13 
                 16.93 
                 12.56 
                 9.92 
               
               
                 GA376 
                 38.15 
                 33.98 
                 31.43 
                 25.28 
                 24.02 
                 22.03 
                 14.81 
                 12.02 
               
               
                 GA377 
                 35.11 
                 37.31 
                 26.39 
                 31.48 
                 21.39 
                 23.19 
                 15.34 
                 14.9 
               
               
                 GA380 
                 38.5 
                 35.38 
                 32.41 
                 32.43 
                 25.28 
                 20.36 
                 12.85 
                 ND 
               
               
                 GA381 
                 29.7 
                 31.2 
                 24.12 
                 29.23 
                 17.32 
                 18.43 
                 11.44 
                 11 
               
               
                 GA382 
                 34.56 
                 30.02 
                 22.5 
                 24.45 
                 18.48 
                 15.64 
                 8.45 
                 8.86 
               
               
                 GA383 
                 42.2 
                 34.95 
                 32.76 
                 31.79 
                 22.24 
                 24.17 
                 13.27 
                 13.26 
               
               
                 GA384 
                 8.49 
                 8.5 
                 7.07 
                 6.51 
                 5.03 
                 4.25 
                 2.61 
                 2.7 
               
               
                 GA385 
                 40.2 
                 29.05 
                 26.39 
                 21.43 
                 18.88 
                 16.66 
                 11.76 
                 7.63 
               
               
                 GA386 
                 43.73 
                 30.67 
                 31.57 
                 26.5 
                 27.51 
                 19.19 
                 16.94 
                 13.41 
               
               
                 GA387 
                 22.61 
                 16.64 
                 17.25 
                 12.78 
                 11.55 
                 8.26 
                 5.28 
                 4.24 
               
               
                 GA388 
                 28.77 
                 23.84 
                 20.46 
                 20.33 
                 13.73 
                 ND 
                 7.26 
                 ND 
               
               
                 GA389 
                 28.45 
                 23.46 
                 21.98 
                 17.88 
                 12.62 
                 12.78 
                 8.06 
                 8.54 
               
               
                 GA391 
                 25.58 
                 30.06 
                 25.43 
                 27.3 
                 27.12 
                 18.67 
                 16.86 
                 15.78 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 Cyno Primary Hepatocytes-Editing % at Position 6 (Dose, Replicate #) 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 2500 
                 2500 
                 1250 
                 1250 
                 625 
                 625 
                 312.5 
                 312.5 
               
               
                   
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL 
                 ng/mL 
               
               
                 gRNA 
                 rep1 
                 rep2 
                 rep1 
                 rep2 
                 rep1 
                 rep2 
                 rep1 
                 rep2 
               
               
                   
               
               
                 GA346 
                 40.1 
                 37.48 
                 36.97 
                 34.08 
                 25.91 
                 25.59 
                 14.83 
                 15.25 
               
               
                 GA376 
                 29.57 
                 29.57 
                 27.62 
                 31.98 
                 23.08 
                 22.29 
                 15.12 
                 15.75 
               
               
                 GA377 
                 35.36 
                 42.67 
                 35.66 
                 43.35 
                 30.68 
                 34.73 
                 21.24 
                 21.79 
               
               
                 GA380 
                 43.27 
                 44.85 
                 39.03 
                 38.76 
                 33.31 
                 34.1 
                 21.83 
                 21.51 
               
               
                 GA381 
                 39.45 
                 47.85 
                 35.82 
                 41.03 
                 28.42 
                 31.87 
                 15.51 
                 18.36 
               
               
                 GA382 
                 36.34 
                 39.97 
                 32.74 
                 37.41 
                 26.15 
                 25.68 
                 14.08 
                 12.74 
               
               
                 GA383 
                 39.02 
                 53.37 
                 35.44 
                 44.23 
                 28 
                 33.42 
                 16 
                 20.03 
               
               
                 GA384 
                 26.71 
                 22.52 
                 20.97 
                 21.53 
                 14.38 
                 16.21 
                 8.31 
                 8.03 
               
               
                 GA385 
                 38.44 
                 37.36 
                 38.31 
                 31.84 
                 31.97 
                 28.48 
                 18.23 
                 17.68 
               
               
                 GA386 
                 48.11 
                 43.61 
                 40.09 
                 40.44 
                 37.59 
                 37.42 
                 23.07 
                 25.27 
               
               
                 GA387 
                 27.3 
                 21.08 
                 23.71 
                 21.53 
                 16.82 
                 13.62 
                 7.34 
                 6.03 
               
               
                 GA388 
                 41.95 
                 36.03 
                 32.87 
                 29.9 
                 23.65 
                 25.43 
                 12.13 
                 13.19 
               
               
                 GA389 
                 34.62 
                 33.47 
                 32.15 
                 29.86 
                 25.87 
                 23.29 
                 11.17 
                 12.33 
               
               
                   
               
            
           
         
       
     
     With a view towards establishing the safety of a base-editing therapy knocking down ANGPTL3 in the human liver in vivo, a list of candidate sites in the human genome for off-target mutagenesis was assembled using two different methods. The first method used bioinformatic analysis of the human genome, identifying all sites with a PAM sequence compatible with  Streptococcus pyogenes  Cas9 (and therefore ABE 8.8) and a protospacer sequence with up to 4 single-nucleotide mismatches with the GA100 spacer sequence 5′-AAGATACCTGAATAACTCTC-3′ (SEQ ID NO: 14). 
     The second method to generate candidate sites used an in vitro biochemical assay, ONE-seq, that determined the propensity of a ribonucleoprotein comprising the ABE8.8 base editor protein and gRNA GA441 to cleave oligonucleotides in a library. The reference human genome (GRCh38) was searched for sites with up to 6 mismatches to the protospacer sequence specified by the ANGPTL3 gRNA (5′-AAGATACCTGAATAACTCTC-3′ (SEQ ID NO: 14)), and sites with up to 4 mismatches plus up to 2 DNA or RNA bulges using Cas-Designer were identified. More specifics on ONE-seq library preparation, experimental protocol, and bioinformatic analysis are described in the additional detailed methods section. 
     Oligonucleotides with higher sequence counts reflect a higher propensity for Cas9/gRNA cleavage in vitro and represent the sites most likely to suffer off-target mutagenesis in cells. The top candidate sites are identified in Table 13. Validation of candidate sites was performed using ANGPTL3 edited, and untreated human primary hepatocytes. Upon next-generation sequencing (Table 14), when the observed base editing rates in control cells were subtracted from the observed base editing rates in LNP-treated cells across the on-target site and candidate off-target sites (to account for background sequencing errors inherent in next-generation sequencing), appreciable base editing were observed at the on-target ANGPTL3 target site. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 ANGPTL3 gRNA (Protospacer 5′-AAGATACCTGAATAACCCTC-3′ (SEQ ID NO: 15)) 
               
               
                 candidate off-target sites identified by ONE-seq 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
                   
               
               
                   
                   
                 Potential Off-target 
                 ID 
                   
               
               
                 chromosome 
                 location 
                 Sequence (5′-3′) 
                 NO: 
                 alignment 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 4546642 
                 CAGATACCATAAAAATCATCTGG 
                 682 
                 X60 
               
               
                   
               
               
                 1 
                 6955991 
                 AAGATACTGAAAAACCCTGCAA 
                 683 
                 RNA41 
               
               
                   
               
               
                 1 
                 16544549 
                 CAGAGAGCAGAGTAAACCTCTGG 
                 684 
                 X60 
               
               
                   
               
               
                 1 
                 23996891 
                 CAGAAAACTATAACCCTCAGG 
                 685 
                 RNA32 
               
               
                   
               
               
                 1 
                 26088616 
                 AGAATGGCTGCATAACCCTTGGG 
                 686 
                 X60 
               
               
                   
               
               
                 1 
                 27386898 
                 AAGGTTACTTAATAAACATCTGG 
                 687 
                 X60 
               
               
                   
               
               
                 1 
                 28115884 
                 AAGATACCTGAATTAAATCTCAAA 
                 688 
                 DNA41 
               
               
                   
               
               
                 1 
                 30795252 
                 AAGTATCTTGAATAACCCTCCTG 
                 689 
                 X50 
               
               
                   
               
               
                 1 
                 31542787 
                 CAAATAACTAAATGACCCTCTTG 
                 690 
                 X60 
               
               
                   
               
               
                 1 
                 32826604 
                 AAGATACTGAAAAACTCTCTTC 
                 691 
                 RNA41 
               
               
                   
               
               
                 1 
                 33418548 
                 TAGATAACCTGAATAGCCCTATAG 
                 692 
                 DNA41 
               
               
                   
               
               
                 1 
                 45361452 
                 TAGATACTTCAATACCTCTCAGT 
                 693 
                 X60 
               
               
                   
               
               
                 1 
                 57486127 
                 AGCAATCCTGAAAAATCCTCTGG 
                 694 
                 X60 
               
               
                   
               
               
                 1 
                 59422147 
                 ACGATACTTGGATAACCCTCTGG 
                 695 
                 X30 
               
               
                   
               
               
                 1 
                 59572755 
                 GAGATACCTAATAAGCCTCTAT 
                 696 
                 RNA41 
               
               
                   
               
               
                 1 
                 62604219 
                 AAGATACCTGAATAACCCTCTGG 
                 697 
                 XOO 
               
               
                   
               
               
                 1 
                 66687653 
                 AAGTTACTGAATATCTCTCAGC 
                 698 
                 RNA41 
               
               
                   
               
               
                 1 
                 76436411 
                 AGTATACCTCAATAACCACCTGG 
                 699 
                 X50 
               
               
                   
               
               
                 1 
                 77254313 
                 AAGATCCAGAAGAATCCCCTGG 
                 700 
                 RNA41 
               
               
                   
               
               
                 1 
                 78181978 
                 AGGATACCTGGAAACCCTACTGG 
                 701 
                 X60 
               
               
                   
               
               
                 1 
                 83689742 
                 AAGATAACTAGCTAGCTCTCTGG 
                 702 
                 X60 
               
               
                   
               
               
                 1 
                 89670639 
                 GAGATACTTGAATAAACTTCTAT 
                 703 
                 X60 
               
               
                   
               
               
                 1 
                 93001069 
                 AAGATACCTGACTAACCTGTTTT 
                 704 
                 X60 
               
               
                   
               
               
                 1 
                 97782174 
                 TAGATACATGATTAAACCACTGC 
                 705 
                 X60 
               
               
                   
               
               
                 1 
                 110981606 
                 TATATACATGAATAACTCCGG 
                 706 
                 RNA32 
               
               
                   
               
               
                 1 
                 114341782 
                 ATTATTCTTGAATAACCCCAGGG 
                 707 
                 X60 
               
               
                   
               
               
                 1 
                 114403243 
                 AAGATACCTGAGAAACTCTGCAA 
                 708 
                 X60 
               
               
                   
               
               
                 1 
                 114800896 
                 ATGACTGCTGAGAAACCCTCTGG 
                 709 
                 X60 
               
               
                   
               
               
                 1 
                 114851709 
                 AAGAATCCTAGATATCCCTCAGT 
                 710 
                 X60 
               
               
                   
               
               
                 1 
                 116440140 
                 AAAATACCAAAAAAACCCTATAG 
                 711 
                 X60 
               
               
                   
               
               
                 1 
                 118751265 
                 AAGAATTTGAATAACCCTCAAT 
                 712 
                 RNA41 
               
               
                   
               
               
                 1 
                 119001156 
                 AGGCTAACAGAATCACCCTCGGG 
                 713 
                 X50 
               
               
                   
               
               
                 1 
                 150002706 
                 GTGATCCTGAGTAAACCTCAGG 
                 714 
                 RNA41 
               
               
                   
               
               
                 1 
                 156332931 
                 TAGATACCTATAAGCCTCTGG 
                 715 
                 RNA22 
               
               
                   
               
               
                 1 
                 162745410 
                 AAGATACATGAAAAAAACATTGG 
                 716 
                 X60 
               
               
                   
               
               
                 1 
                 164962899 
                 AAGATACCTTAAAACCCTTCAGC 
                 717 
                 X50 
               
               
                   
               
               
                 1 
                 165289582 
                 AATTTACCTGAATAGTCATTAGG 
                 718 
                 X60 
               
               
                   
               
               
                 1 
                 167577809 
                 AAGATACACAAATAGCCATCAAG 
                 719 
                 X60 
               
               
                   
               
               
                 1 
                 170883188 
                 AACAAACCTGAATAGTCCTACTG 
                 720 
                 X60 
               
               
                   
               
               
                 1 
                 175232700 
                 CAGATATCTGAATCACTCTTCTG 
                 721 
                 X60 
               
               
                   
               
               
                 1 
                 176788495 
                 AAAATATGAATAACCCTTTGG 
                 722 
                 RNA22 
               
               
                   
               
               
                 1 
                 177848832 
                 CAGATACTGAGTCACCCTCAGC 
                 723 
                 RNA41 
               
               
                   
               
               
                 1 
                 178104014 
                 AAGTACCTAAATACCCTTGGGG 
                 724 
                 RNA41 
               
               
                   
               
               
                 1 
                 179507899 
                 AAGATACCATTCTAACCCTCTGA 
                 725 
                 X50 
               
               
                   
               
               
                 1 
                 180840385 
                 AAGGTAACTAAGAAACCCTCAGA 
                 726 
                 X60 
               
               
                   
               
               
                 1 
                 183484429 
                 CTCATACCACAATAACCCTCATG 
                 727 
                 X60 
               
               
                   
               
               
                 1 
                 183743474 
                 AAGATAATGAATATCACCCAGG 
                 728 
                 RNA41 
               
               
                   
               
               
                 1 
                 185824500 
                 AAGATACTAATAAACACTCAGG 
                 729 
                 RNA41 
               
               
                   
               
               
                 1 
                 188687384 
                 CAGATACCTGAATAAGCAGAAGC 
                 730 
                 X60 
               
               
                   
               
               
                 1 
                 194817380 
                 ATAATACCTGAATACTCTCAGA 
                 731 
                 RNA41 
               
               
                   
               
               
                 1 
                 199542263 
                 TCTATACCTGCATAACCCTAAGG 
                 732 
                 X50 
               
               
                   
               
               
                 1 
                 201739039 
                 AAGTTACCAGGAGGACCCTCAGG 
                 733 
                 X50 
               
               
                   
               
               
                 1 
                 203127991 
                 AAGAGACCGGAATGCCCCCTTGG 
                 734 
                 X60 
               
               
                   
               
               
                 1 
                 209196372 
                 AACAGGCCATAAGAACCCTCTGG 
                 735 
                 X60 
               
               
                   
               
               
                 1 
                 209428278 
                 TAAATGCCAGGATAAGCCTCTGG 
                 736 
                 X60 
               
               
                   
               
               
                 1 
                 217518556 
                 TACATAACTGAATAACTCTCCAA 
                 737 
                 X60 
               
               
                   
               
               
                 1 
                 222014072 
                 AAGAAAGCTGAGTAAGCCCGGG 
                 738 
                 RNA41 
               
               
                   
               
               
                 1 
                 228023082 
                 GGGATACCCGAATAACCCCACAG 
                 739 
                 X60 
               
               
                   
               
               
                 1 
                 228546593 
                 GAGACACCTGGGTAACCCCAGG 
                 740 
                 RNA41 
               
               
                   
               
               
                 1 
                 228724320 
                 GTTATACCTGAATAAATCTGGGG 
                 741 
                 X60 
               
               
                   
               
               
                 1 
                 230479071 
                 AAAATACCTGCATAACCCTGAGA 
                 742 
                 X40 
               
               
                   
               
               
                 1 
                 230750818 
                 CAGTTCCCTGAGTCACCCTCAGT 
                 743 
                 X60 
               
               
                   
               
               
                 1 
                 231757688 
                 AAGATACATGAACTTCCCACAGG 
                 744 
                 X50 
               
               
                   
               
               
                 1 
                 232190288 
                 AAAATAAGTGAATAACCCACTAA 
                 745 
                 X60 
               
               
                   
               
               
                 1 
                 233265568 
                 TAGGTACCTGAGTAACTCTGCGG 
                 746 
                 X50 
               
               
                   
               
               
                 1 
                 235139684 
                 AAGAAACCAGATAACCCACAGC 
                 747 
                 RNA41 
               
               
                   
               
               
                 1 
                 238895801 
                 AAGATACCTGCATAATCTAGGA 
                 748 
                 RNA41 
               
               
                   
               
               
                 1 
                 244048003 
                 AGGATACCTGCGTAACTCGCAGC 
                 749 
                 X60 
               
               
                   
               
               
                 1 
                 245228289 
                 AAGATACCTGGAGATGCCCCTTG 
                 750 
                 X60 
               
               
                   
               
               
                 2 
                 2541015 
                 AAGATATCAGAATAACTCTGGCT 
                 751 
                 X60 
               
               
                   
               
               
                 2 
                 5866267 
                 CCTGTACCTGCATAACCTTCTGG 
                 752 
                 X60 
               
               
                   
               
               
                 2 
                 11192249 
                 AAGATACCTAAAAAGCCCCCAGC 
                 753 
                 X50 
               
               
                   
               
               
                 2 
                 14318310 
                 TAGATACCCAAATAGCCCCTCAGG 
                 754 
                 DNA41 
               
               
                   
               
               
                 2 
                 17555591 
                 TCTATACCTAAATAGTCCTCAGG 
                 755 
                 X60 
               
               
                   
               
               
                 2 
                 18083124 
                 AAAATACCAGAATAACCCATTTA 
                 756 
                 X60 
               
               
                   
               
               
                 2 
                 18570047 
                 AAGATATCTGAATAAACTCCAAA 
                 757 
                 X60 
               
               
                   
               
               
                 2 
                 23315873 
                 ATGATTATTAAATAACCCTCAAG 
                 758 
                 X60 
               
               
                   
               
               
                 2 
                 24850333 
                 AAGATCCCTGATTACCATTCCAG 
                 759 
                 X60 
               
               
                   
               
               
                 2 
                 28691746 
                 AAGATACCTGGCTCAACCTCATC 
                 760 
                 X60 
               
               
                   
               
               
                 2 
                 29326137 
                 AATGTGGCTGAAAAAGCCTCTGG 
                 761 
                 X60 
               
               
                   
               
               
                 2 
                 30453369 
                 AAGATACCTAAACATTCCAGTGG 
                 762 
                 X60 
               
               
                   
               
               
                 2 
                 31504056 
                 AAGAGAGCTGGACAACTCTAAGG 
                 763 
                 X60 
               
               
                   
               
               
                 2 
                 33271364 
                 AAGATAGGTCAATAACCACCCGG 
                 764 
                 X50 
               
               
                   
               
               
                 2 
                 33829479 
                 TAGATAACTGAATAAACTCTATGG 
                 765 
                 DNA41 
               
               
                   
               
               
                 2 
                 35540632 
                 AAGATGTATTATTGACCCTCTGG 
                 766 
                 X60 
               
               
                   
               
               
                 2 
                 38119450 
                 TAGAAAACAGAATGACTCTCAGG 
                 767 
                 X60 
               
               
                   
               
               
                 2 
                 42166900 
                 AATAACCAAAATAACCCTCCAG 
                 768 
                 RNA41 
               
               
                   
               
               
                 2 
                 46398173 
                 TATGTACCTAATAAGCCTCAGG 
                 769 
                 RNA41 
               
               
                   
               
               
                 2 
                 49841412 
                 CATATATCTGAATAGCCTTCTGG 
                 770 
                 X50 
               
               
                   
               
               
                 2 
                 56159084 
                 AAGATACAGAATTCCCCTCTGA 
                 771 
                 RNA41 
               
               
                   
               
               
                 2 
                 56257553 
                 AAGTTACATGAAAAACATCTGG 
                 772 
                 RNA41 
               
               
                   
               
               
                 2 
                 57130463 
                 AAAATACCTGAATAATCCTCGAA 
                 773 
                 X40 
               
               
                   
               
               
                 2 
                 59482506 
                 AAGATACCAGAGAAAGTCTCAGA 
                 774 
                 X60 
               
               
                   
               
               
                 2 
                 60290320 
                 CAAATACCAGAATACCCTACAGG 
                 775 
                 X60 
               
               
                   
               
               
                 2 
                 64169496 
                 ATGATCCTAAATAACCTTCCAG 
                 776 
                 RNA41 
               
               
                   
               
               
                 2 
                 68986545 
                 AACAACATGAATGACTCTCTGG 
                 777 
                 RNA41 
               
               
                   
               
               
                 2 
                 71741885 
                 AAGATAAAGGTATCTCCCTCTGG 
                 778 
                 X60 
               
               
                   
               
               
                 2 
                 72415642 
                 AAGCCAACTGAATAACACTGTTG 
                 779 
                 X60 
               
               
                   
               
               
                 2 
                 72619690 
                 ATGATACCTGAATACCTCTAATG 
                 780 
                 X50 
               
               
                   
               
               
                 2 
                 75266724 
                 AAGCCAACTGAAGAGCCCTTGGG 
                 781 
                 X60 
               
               
                   
               
               
                 2 
                 80321182 
                 AAGAACTTGATAAACCCTCAAG 
                 782 
                 RNA41 
               
               
                   
               
               
                 2 
                 87443936 
                 TTGACACCTAAATAACCCTTTGG 
                 783 
                 X50 
               
               
                   
               
               
                 2 
                 98349660 
                 AAGAGACCAGAAGCAACCTCTGT 
                 784 
                 X60 
               
               
                   
               
               
                 2 
                 100696913 
                 AAGAAACATGAACAGAGCCTCAGG 
                 785 
                 DNA41 
               
               
                   
               
               
                 2 
                 104408132 
                 GAGATACCTGTATAAGTCACTAG 
                 786 
                 X60 
               
               
                   
               
               
                 2 
                 108664444 
                 AAAATGCCTTAAGAACCCTGGGG 
                 787 
                 X50 
               
               
                   
               
               
                 2 
                 111506561 
                 TTGACACCTAAATAACCCTTTGG 
                 783 
                 X50 
               
               
                   
               
               
                 2 
                 112735695 
                 TAGATACTCAGTAACCCTCTTG 
                 788 
                 RNA41 
               
               
                   
               
               
                 2 
                 115162717 
                 AAGAGACAGGAATAACCCTCAGC 
                 789 
                 X40 
               
               
                   
               
               
                 2 
                 121516112 
                 AAAATACCATGTAAAACACTCTGG 
                 790 
                 DNA41 
               
               
                   
               
               
                 2 
                 122492850 
                 GAGATACAGAATAGCCCTGGGG 
                 791 
                 RNA41 
               
               
                   
               
               
                 2 
                 124401576 
                 AAGAACCTAAATCACCATTGGG 
                 792 
                 RNA41 
               
               
                   
               
               
                 2 
                 128856709 
                 AAGATATCTGGATAATCCACCAA 
                 793 
                 X60 
               
               
                   
               
               
                 2 
                 141714040 
                 AAGATACTGGAATAAACATAAAG 
                 794 
                 X60 
               
               
                   
               
               
                 2 
                 143719680 
                 AAAATGCCTCAATATCTCTGTGG 
                 795 
                 X60 
               
               
                   
               
               
                 2 
                 144191771 
                 AAAATACCAAAATAACCCTAAAG 
                 796 
                 X50 
               
               
                   
               
               
                 2 
                 149423627 
                 TAGATACCTGAAATACTCTATGC 
                 797 
                 X60 
               
               
                   
               
               
                 2 
                 153591411 
                 AAAATACCATAATTTCCCTCAGA 
                 798 
                 X60 
               
               
                   
               
               
                 2 
                 154142624 
                 AAGATACCAAATAAACCTCTGA 
                 799 
                 RNA31 
               
               
                   
               
               
                 2 
                 155791011 
                 AAGATACCTAAAATACCTATTGG 
                 800 
                 X60 
               
               
                   
               
               
                 2 
                 155965426 
                 CAGATACCTGAAAAACTCTCTCT 
                 801 
                 X50 
               
               
                   
               
               
                 2 
                 157734996 
                 AATAAACACTGAATAACCCTTTGT 
                 802 
                 DNA41 
               
               
                   
               
               
                 2 
                 163699397 
                 CAGCTACCTAATTACCCTCTGG 
                 803 
                 RNA31 
               
               
                   
               
               
                 2 
                 163782922 
                 TAGGTACCTGATTTACCCTGATG 
                 804 
                 X60 
               
               
                   
               
               
                 2 
                 166944919 
                 AAGATACCTCAGGAACCAGCAGC 
                 805 
                 X60 
               
               
                   
               
               
                 2 
                 168821195 
                 AAAAGACCTAAAAAGCCCTCTGG 
                 806 
                 X50 
               
               
                   
               
               
                 2 
                 169944031 
                 TCAATTACTGAATAACCCTCTTG 
                 807 
                 X60 
               
               
                   
               
               
                 2 
                 170941666 
                 AATATACATGAATAAATCTCACA 
                 808 
                 X60 
               
               
                   
               
               
                 2 
                 173215359 
                 AAAAAACGTGAAAAACCTTAGGG 
                 809 
                 X60 
               
               
                   
               
               
                 2 
                 174371138 
                 AAGATACCTGTTAGCCCTCACC 
                 810 
                 RNA41 
               
               
                   
               
               
                 2 
                 176698280 
                 AAGTTTCATGCATAAAACTCTGG 
                 811 
                 X60 
               
               
                   
               
               
                 2 
                 180424100 
                 AAGGTACCTGAAGAACTCAACTG 
                 812 
                 X60 
               
               
                   
               
               
                 2 
                 181934742 
                 AAGATGTATGAATAACTCTCGGG 
                 813 
                 X40 
               
               
                   
               
               
                 2 
                 183685747 
                 AAGCAACCTAAATGTCCATCAGG 
                 814 
                 X60 
               
               
                   
               
               
                 2 
                 183737398 
                 GAGATACCTGGATAACCACTTGG 
                 815 
                 X50 
               
               
                   
               
               
                 2 
                 184251994 
                 AAGACACCTGGAGAACTCTCGGG 
                 816 
                 X40 
               
               
                   
               
               
                 2 
                 188364288 
                 AAGTAACCCAATAACCCTGAGG 
                 817 
                 RNA41 
               
               
                   
               
               
                 2 
                 188883471 
                 ACAATACGTGAATGAATCCTCTGG 
                 818 
                 DNA41 
               
               
                   
               
               
                 2 
                 191075654 
                 AAGATTCCAGACATACACTCTGG 
                 819 
                 X60 
               
               
                   
               
               
                 2 
                 191371557 
                 TAAGTAGCTGTATAACCCTGGGG 
                 820 
                 X60 
               
               
                   
               
               
                 2 
                 195665474 
                 TAAATACTTGAAAAACCCACAGA 
                 821 
                 X60 
               
               
                   
               
               
                 2 
                 196276026 
                 AAGTTATCTGACTAACAATCTGT 
                 822 
                 X60 
               
               
                   
               
               
                 2 
                 196963744 
                 AATATACATGAATTTCTCTTAGG 
                 823 
                 X60 
               
               
                   
               
               
                 2 
                 197268832 
                 AAGATGCCTCCCTAACACTGGGG 
                 824 
                 X60 
               
               
                   
               
               
                 2 
                 197475333 
                 TACATACCAACAACCCTCTGG 
                 825 
                 RNA32 
               
               
                   
               
               
                 2 
                 198342120 
                 AATAGAGCTGAAAAACCCTCACT 
                 826 
                 X60 
               
               
                   
               
               
                 2 
                 198435701 
                 AAATTAGATGAATAACCCTTAGT 
                 827 
                 X60 
               
               
                   
               
               
                 2 
                 202129138 
                 AAAATACCTGATACCCCCTGG 
                 828 
                 RNA32 
               
               
                   
               
               
                 2 
                 202797188 
                 AGAGTACCTGAATACCTCTGG 
                 829 
                 RNA32 
               
               
                   
               
               
                 2 
                 203165696 
                 AGAATATCTGAATTACCCTCAAG 
                 830 
                 X50 
               
               
                   
               
               
                 2 
                 204928058 
                 AAGATACATGAGTAAACCCAGC 
                 831 
                 RNA41 
               
               
                   
               
               
                 2 
                 206764957 
                 AGGAGACCTGCACAGGCCTCTGG 
                 832 
                 X60 
               
               
                   
               
               
                 2 
                 216172775 
                 AATTTATATGAAAAACCCACTGG 
                 833 
                 X60 
               
               
                   
               
               
                 2 
                 217541307 
                 AAGATACCTGATCAATCCTCTTC 
                 834 
                 X50 
               
               
                   
               
               
                 2 
                 217737466 
                 AAGATACCAGACCAAAGCTCAGG 
                 835 
                 X50 
               
               
                   
               
               
                 2 
                 220751309 
                 TAGAGACATGAAAAACCCTCCAA 
                 836 
                 X60 
               
               
                   
               
               
                 2 
                 221762944 
                 AATATTCCTGAATCCCTCGGG 
                 837 
                 RNA22 
               
               
                   
               
               
                 2 
                 226128392 
                 CTCATACCTGAATAACCTTCTCA 
                 838 
                 X60 
               
               
                   
               
               
                 2 
                 226932531 
                 CAGATACCGAGAAAAACCCTCTGG 
                 839 
                 DNA31 
               
               
                   
               
               
                 2 
                 227560446 
                 AAGATATATGAAAAAGCCTATGC 
                 840 
                 X60 
               
               
                   
               
               
                 2 
                 228586979 
                 CAGATACCTCACCAACACTCCAG 
                 841 
                 X60 
               
               
                   
               
               
                 2 
                 232730167 
                 CAGAGACCTGAAAAGCCCTGCCG 
                 842 
                 X60 
               
               
                   
               
               
                 2 
                 240208622 
                 AAAAGGCCTGCAGAAACCTCTGG 
                 843 
                 X60 
               
               
                   
               
               
                 2 
                 241728292 
                 CAAATCCTTGAATCACCCCCAGG 
                 844 
                 X60 
               
               
                   
               
               
                 2 
                 241778506 
                 AAGATACCTGGCCACACCCCAGG 
                 845 
                 X60 
               
               
                   
               
               
                 3 
                 539550 
                 GAGATAATGAATAGCCCTCCGG 
                 846 
                 RNA31 
               
               
                   
               
               
                 3 
                 7611430 
                 AAGATACCTGTCATCCCTCCGT 
                 847 
                 RNA41 
               
               
                   
               
               
                 3 
                 8738135 
                 GGGATACCTGCATAACTCTCAGG 
                 848 
                 X40 
               
               
                   
               
               
                 3 
                 11156153 
                 GAAATGCCTGAATCACACTCGAG 
                 849 
                 X60 
               
               
                   
               
               
                 3 
                 11683514 
                 AAGATACTGAATCAACTTCAGT 
                 850 
                 RNA41 
               
               
                   
               
               
                 3 
                 16474484 
                 AAGATACCAGTACACCCCCAGG 
                 851 
                 RNA41 
               
               
                   
               
               
                 3 
                 19871463 
                 ATGATACCTCAATAACCCTATTT 
                 852 
                 X50 
               
               
                   
               
               
                 3 
                 25070750 
                 AAAATACCTGAACATACCTTGAG 
                 853 
                 X60 
               
               
                   
               
               
                 3 
                 35461847 
                 TACATACCTAACAACCCCTCAGG 
                 854 
                 X60 
               
               
                   
               
               
                 3 
                 35699317 
                 AAGAAACAAGGGAAACCCTCAGG 
                 855 
                 X60 
               
               
                   
               
               
                 3 
                 36482333 
                 AAGATACCAGGATGGCTCTCTGC 
                 856 
                 X60 
               
               
                   
               
               
                 3 
                 37570248 
                 CAACAACCAGAATAACCCACAGG 
                 857 
                 X60 
               
               
                   
               
               
                 3 
                 37680767 
                 AAGATTAAAAGAATAAACCTCAGG 
                 858 
                 DNA41 
               
               
                   
               
               
                 3 
                 38875399 
                 AACTTACCTGCAAAACCATCTGG 
                 859 
                 X50 
               
               
                   
               
               
                 3 
                 56933123 
                 AAGATCTGAATTACCATCAGT 
                 860 
                 RNA32 
               
               
                   
               
               
                 3 
                 61401423 
                 GAGATCCTTAACAACCCTCAAG 
                 861 
                 RNA41 
               
               
                   
               
               
                 3 
                 64532843 
                 AAGATATCTGAGCCACACTCTCG 
                 862 
                 X60 
               
               
                   
               
               
                 3 
                 68532737 
                 AAGCTACCTGAAAACCTTCAGG 
                 863 
                 RNA21 
               
               
                   
               
               
                 3 
                 70442317 
                 AAGATGACTGAAGAAACCTTGGG 
                 864 
                 X50 
               
               
                   
               
               
                 3 
                 70620477 
                 AAGATCCCTGCAAACCCTTTTGG 
                 865 
                 X60 
               
               
                   
               
               
                 3 
                 71429771 
                 AAGACAGCTAAATCACCCCACGG 
                 866 
                 X60 
               
               
                   
               
               
                 3 
                 71714408 
                 AAGATCACAGAATCACCCTCCTC 
                 867 
                 X60 
               
               
                   
               
               
                 3 
                 74054464 
                 AATAGACATGAATAACCCTAAGA 
                 868 
                 X50 
               
               
                   
               
               
                 3 
                 76238395 
                 CAGACACCTTACTAACTCTCCTG 
                 869 
                 X60 
               
               
                   
               
               
                 3 
                 82942470 
                 AAGATACACAAATAAACTTCTAG 
                 870 
                 X60 
               
               
                   
               
               
                 3 
                 83311768 
                 AAGATATCTGAAAAACTCTCAAA 
                 871 
                 X50 
               
               
                   
               
               
                 3 
                 84371314 
                 AAGACACTGAATAACCCACAGA 
                 872 
                 RNA31 
               
               
                   
               
               
                 3 
                 86668690 
                 TAGATAGCAAATAACCCTAGGG 
                 873 
                 RNA41 
               
               
                   
               
               
                 3 
                 98032158 
                 AATAGACCTAAATAACCTCAAG 
                 874 
                 RNA41 
               
               
                   
               
               
                 3 
                 99319950 
                 AAGTTACCTGACTAACCCTGAGG 
                 875 
                 X30 
               
               
                   
               
               
                 3 
                 100809940 
                 AAGATATCTGAATAACTACAAGG 
                 876 
                 X50 
               
               
                   
               
               
                 3 
                 107069791 
                 AAGATACTTGGATAACTCTGCTA 
                 877 
                 X60 
               
               
                   
               
               
                 3 
                 107509328 
                 AAGAGGCTGGAATAAGCCACGGG 
                 878 
                 X60 
               
               
                   
               
               
                 3 
                 111016912 
                 AAGATACATAAATATCACTGCAG 
                 879 
                 X60 
               
               
                   
               
               
                 3 
                 118950239 
                 CACTAACCTGAATAGCCCTAAGG 
                 880 
                 X60 
               
               
                   
               
               
                 3 
                 120988045 
                 AATATACCTGAAAAACCATCAGG 
                 881 
                 X30 
               
               
                   
               
               
                 3 
                 121667776 
                 AAAATACCTAAAGAAATTTCAGG 
                 882 
                 X60 
               
               
                   
               
               
                 3 
                 122037269 
                 CGTGTACCTGAAAAAGCCTCAGG 
                 883 
                 X60 
               
               
                   
               
               
                 3 
                 126759212 
                 GTGAGGCCTGAAGAACACTCTGG 
                 884 
                 X60 
               
               
                   
               
               
                 3 
                 133985020 
                 AAGTTACTGAATATCCCTCCAG 
                 885 
                 RNA31 
               
               
                   
               
               
                 3 
                 140863741 
                 AAGAAACCGGAATGAGCCCCCAG 
                 886 
                 X60 
               
               
                   
               
               
                 3 
                 141050688 
                 CAGATACCAGAAGCACCCTCGAG 
                 887 
                 X50 
               
               
                   
               
               
                 3 
                 142685343 
                 TAGATAACTGAAATATCCCTCTGG 
                 888 
                 DNA31 
               
               
                   
               
               
                 3 
                 143875802 
                 GAGATACCCAAATAAACCTCAAA 
                 889 
                 X60 
               
               
                   
               
               
                 3 
                 151175376 
                 AAGATACTGAATAAAGCTCTTA 
                 890 
                 RNA41 
               
               
                   
               
               
                 3 
                 151226712 
                 AAGATAACTGAAGAAACTGAAGG 
                 891 
                 X60 
               
               
                   
               
               
                 3 
                 151395707 
                 AAGATACCAGGGTAACTCTATGA 
                 892 
                 X60 
               
               
                   
               
               
                 3 
                 155083487 
                 AAGATACCTGAATATCCAACCTA 
                 893 
                 X50 
               
               
                   
               
               
                 3 
                 161319971 
                 AAGCTGACTGAAGAGCCCTTGGG 
                 894 
                 X60 
               
               
                   
               
               
                 3 
                 168443226 
                 AGTATTTCTGACAAACCCTCAGG 
                 895 
                 X60 
               
               
                   
               
               
                 3 
                 169534358 
                 GAGATAGCTAATAACCCCCTGG 
                 896 
                 RNA31 
               
               
                   
               
               
                 3 
                 171933625 
                 AAGACAACTGAAAAACGCTGTGG 
                 897 
                 X50 
               
               
                   
               
               
                 3 
                 173995167 
                 AAGACACCTGAATAATCTCTGGA 
                 898 
                 X60 
               
               
                   
               
               
                 3 
                 176890998 
                 ATGATAAGTGAATAAGTCTCATG 
                 899 
                 X60 
               
               
                   
               
               
                 3 
                 177768689 
                 AGGATATATGAAAAAGCCTGCGG 
                 900 
                 X60 
               
               
                   
               
               
                 3 
                 187379619 
                 AAGATACCCTAAAAGCCTTTGG 
                 901 
                 RNA41 
               
               
                   
               
               
                 3 
                 188484179 
                 AAGATGCATGAATAACCTCTGGA 
                 902 
                 X60 
               
               
                   
               
               
                 3 
                 190699564 
                 ATGACACCAGAAAGACCCTAAGG 
                 903 
                 X60 
               
               
                   
               
               
                 3 
                 192710228 
                 AAAATACTAAATAACCCCATGG 
                 904 
                 RNA41 
               
               
                   
               
               
                 4 
                 723653 
                 CAGATTCCTGGAAGACCCTCAAG 
                 905 
                 X60 
               
               
                   
               
               
                 4 
                 11334933 
                 AAGAGACCTCAAGGTCTCTCTGG 
                 906 
                 X60 
               
               
                   
               
               
                 4 
                 12971711 
                 AAGACTAAAGAATAACCCTCTGG 
                 907 
                 X50 
               
               
                   
               
               
                 4 
                 15327544 
                 AATATGCCTACATTACACTCAGG 
                 908 
                 X60 
               
               
                   
               
               
                 4 
                 16411507 
                 AAGACACTGAATAACCCTTGGC 
                 909 
                 RNA31 
               
               
                   
               
               
                 4 
                 20157051 
                 AAGATAGTGAGTAAGTCTCAGG 
                 910 
                 RNA41 
               
               
                   
               
               
                 4 
                 23244775 
                 TAGATAAGAATAGCCCTCAGG 
                 911 
                 RNA32 
               
               
                   
               
               
                 4 
                 26200297 
                 TCTATACCTCAATAACCTCCTGG 
                 912 
                 X60 
               
               
                   
               
               
                 4 
                 28742386 
                 AAGATACCTAAGTGCTCCACAGG 
                 913 
                 X60 
               
               
                   
               
               
                 4 
                 29008961 
                 AAGAAACCTACAATAACCTTCATG 
                 914 
                 DNA41 
               
               
                   
               
               
                 4 
                 33579022 
                 CCGATATCAGAATATCCCTTAGG 
                 915 
                 X60 
               
               
                   
               
               
                 4 
                 44748785 
                 AAGATGCATGAAAAAGCTTCAGT 
                 916 
                 X60 
               
               
                   
               
               
                 4 
                 44812393 
                 TAGATATCTGCAGAACTCTCCGC 
                 917 
                 X60 
               
               
                   
               
               
                 4 
                 45258647 
                 AACATACATCAATAACTTTCTTG 
                 918 
                 X60 
               
               
                   
               
               
                 4 
                 46495164 
                 AAGATACTTGAATACCAATCAAG 
                 919 
                 X50 
               
               
                   
               
               
                 4 
                 47192935 
                 TAAAGACCTGAATAACCTCAAG 
                 920 
                 RNA41 
               
               
                   
               
               
                 4 
                 47840228 
                 AAGATATTGAATATACCTCAGG 
                 921 
                 RNA31 
               
               
                   
               
               
                 4 
                 51965380 
                 TAGTTACCTGAATACACCTCTGG 
                 922 
                 X40 
               
               
                   
               
               
                 4 
                 66291743 
                 TATATACCTGGAAAACCCTAAAG 
                 923 
                 X60 
               
               
                   
               
               
                 4 
                 74170265 
                 TAGATACATAATAACCAACTGG 
                 924 
                 RNA41 
               
               
                   
               
               
                 4 
                 84374124 
                 GAGATACTTGAATCCCTCAGG 
                 925 
                 RNA22 
               
               
                   
               
               
                 4 
                 87976264 
                 AAGATACCTGTATGACACTGTGT 
                 926 
                 X50 
               
               
                   
               
               
                 4 
                 88117912 
                 AAGATACCTAAATAACAAGCTGG 
                 927 
                 X40 
               
               
                   
               
               
                 4 
                 90779595 
                 AGGATACATGCATAACCCATGAG 
                 928 
                 X60 
               
               
                   
               
               
                 4 
                 91244542 
                 AAGATCCAGAATAATTCTCTGG 
                 929 
                 RNA31 
               
               
                   
               
               
                 4 
                 91936467 
                 AAGATACATGAAAAATTCTCTCA 
                 930 
                 X60 
               
               
                   
               
               
                 4 
                 93373949 
                 AATATACCCTAATAAGCCTTCTG 
                 931 
                 X60 
               
               
                   
               
               
                 4 
                 93711948 
                 AAGATGGCTGAATAGAACCTTCCAG 
                 932 
                 DNA42 
               
               
                   
               
               
                 4 
                 96659665 
                 AATATACCTTATTATCCTTCAGA 
                 933 
                 X60 
               
               
                   
               
               
                 4 
                 100450684 
                 CAGATACAAGAGTACTCCTCAGG 
                 934 
                 X60 
               
               
                   
               
               
                 4 
                 101268626 
                 TCTATACCTAAATAAACCTAAGG 
                 935 
                 X60 
               
               
                   
               
               
                 4 
                 104738004 
                 TAGATACCTCAATAATTCTCTCT 
                 936 
                 X60 
               
               
                   
               
               
                 4 
                 115433239 
                 AAATAACATAAATAACCCTCAGA 
                 937 
                 X60 
               
               
                   
               
               
                 4 
                 115953135 
                 CAGAAGCTGGATAGCCCTCGGG 
                 938 
                 RNA41 
               
               
                   
               
               
                 4 
                 136101368 
                 AAGAACATGAGTAACCCTAAGG 
                 939 
                 RNA31 
               
               
                   
               
               
                 4 
                 136644335 
                 AAGATACTTGAAAAACACCAGGT 
                 940 
                 X60 
               
               
                   
               
               
                 4 
                 136853658 
                 AAGAGAACTGAAAAGCCTCAAG 
                 941 
                 RNA41 
               
               
                   
               
               
                 4 
                 144234750 
                 AAGATACCAAATAACTCTCTGG 
                 942 
                 RNA21 
               
               
                   
               
               
                 4 
                 146835606 
                 AATATGCCTGTAACCCTCAAG 
                 943 
                 RNA32 
               
               
                   
               
               
                 4 
                 149064984 
                 AAGCTGACTGAAGAGCCCTTAGG 
                 944 
                 X60 
               
               
                   
               
               
                 4 
                 155932126 
                 AAAATTCATGAAGAACCCTGGGT 
                 945 
                 X60 
               
               
                   
               
               
                 4 
                 157312624 
                 AAGATACGTGAATAACTCAGGAT 
                 946 
                 X60 
               
               
                   
               
               
                 4 
                 159332385 
                 AAGATATCTGAACAATTCTAAGG 
                 947 
                 X50 
               
               
                   
               
               
                 4 
                 160192361 
                 AAGATACTTGAATCACTGTCAGT 
                 948 
                 X50 
               
               
                   
               
               
                 4 
                 168412887 
                 AAGATCCTGAATAATTCTCTGG 
                 949 
                 RNA21 
               
               
                   
               
               
                 4 
                 170886954 
                 AAGAAGCCTGGATAGAACTCTGG 
                 950 
                 X60 
               
               
                   
               
               
                 4 
                 172543878 
                 TTGATACCTGAATAACACTCCAG 
                 951 
                 X40 
               
               
                   
               
               
                 4 
                 172763007 
                 AAAAAAAATGAATTACCCTCATG 
                 952 
                 X60 
               
               
                   
               
               
                 4 
                 174561024 
                 GAGATCCAGAATTACCCTTGGG 
                 953 
                 RNA41 
               
               
                   
               
               
                 4 
                 181671436 
                 AACATACCTAAATAACTCCTGGC 
                 954 
                 X60 
               
               
                   
               
               
                 4 
                 184625030 
                 AATCTACCTGATGACCCTCGGC 
                 955 
                 RNA41 
               
               
                   
               
               
                 4 
                 186075007 
                 AAGTTAACTGTAAAACAGCCTCAGG 
                 956 
                 DNA42 
               
               
                   
               
               
                 5 
                 6252615 
                 AAGAAATCTGAAAGCCCTGGGG 
                 957 
                 RNA41 
               
               
                   
               
               
                 5 
                 7102627 
                 GAGATACCTGCATAACACTTAAT 
                 958 
                 X60 
               
               
                   
               
               
                 5 
                 7274614 
                 CAGATAACTGAACAACCAGATGG 
                 959 
                 X60 
               
               
                   
               
               
                 5 
                 7657678 
                 AACTTAGGTGATTAACCCTCACG 
                 960 
                 X60 
               
               
                   
               
               
                 5 
                 8704833 
                 ATGATACTGAATAAGTCTCAGG 
                 961 
                 RNA31 
               
               
                   
               
               
                 5 
                 9774717 
                 AAGATCCAGAATAATTCTCTGG 
                 929 
                 RNA31 
               
               
                   
               
               
                 5 
                 13575913 
                 AGAATACCTAGTGACCCTCAGG 
                 962 
                 RNA41 
               
               
                   
               
               
                 5 
                 16123391 
                 AACAGACCTTCATAGCCCTCAGA 
                 963 
                 X60 
               
               
                   
               
               
                 5 
                 16404802 
                 TATATACCTAAATAACCTAGGG 
                 964 
                 RNA41 
               
               
                   
               
               
                 5 
                 20733472 
                 AAGATCTCTGAAATGCCCTGAGG 
                 965 
                 X60 
               
               
                   
               
               
                 5 
                 23248793 
                 AACTTAACATAATAACCTTCAGG 
                 966 
                 X60 
               
               
                   
               
               
                 5 
                 34351741 
                 ATGATAGTGAATAATCCTCATG 
                 967 
                 RNA41 
               
               
                   
               
               
                 5 
                 35773444 
                 AAGATACGAGAATAACCTCAAGA 
                 968 
                 X60 
               
               
                   
               
               
                 5 
                 35946321 
                 AAACTACCTGGATGACCCTCTGA 
                 969 
                 X50 
               
               
                   
               
               
                 5 
                 36662538 
                 AAGAAGATGAATTACTCTCTGG 
                 970 
                 RNA41 
               
               
                   
               
               
                 5 
                 42904833 
                 AAGATACAGAGTAACTCTTGGG 
                 971 
                 RNA41 
               
               
                   
               
               
                 5 
                 43693099 
                 GAGATAGCTGAATAATCCCTCAG 
                 972 
                 X60 
               
               
                   
               
               
                 5 
                 45541710 
                 AAGATACATCAGTAAGCCACAGT 
                 973 
                 X60 
               
               
                   
               
               
                 5 
                 55453226 
                 AAAATACATTAATAAAACTAAGG 
                 974 
                 X60 
               
               
                   
               
               
                 5 
                 58609559 
                 AGTAGAGGTGAATAACCCTCAGC 
                 975 
                 X60 
               
               
                   
               
               
                 5 
                 60295257 
                 CAGATACTTAAATAACCATAAGG 
                 976 
                 X50 
               
               
                   
               
               
                 5 
                 61555769 
                 AAGAACCTGACAAGCCCTCAGA 
                 977 
                 RNA41 
               
               
                   
               
               
                 5 
                 66999062 
                 AAGATCCATCAATAAACCACGTG 
                 978 
                 X60 
               
               
                   
               
               
                 5 
                 69412657 
                 AAGATACTTTTAAAACCCAGTGG 
                 979 
                 X60 
               
               
                   
               
               
                 5 
                 73013090 
                 AAGATCCTGAATTACTCTCAAG 
                 980 
                 RNA31 
               
               
                   
               
               
                 5 
                 73504738 
                 AAGATACCTGAACAACTCCACAC 
                 981 
                 X60 
               
               
                   
               
               
                 5 
                 75225969 
                 AAGCTGACTGAAGAGCCCTAGGG 
                 982 
                 X60 
               
               
                   
               
               
                 5 
                 78444765 
                 TTGATACCTGTATTACACTCATG 
                 983 
                 X60 
               
               
                   
               
               
                 5 
                 82799696 
                 AAGATACAGAGAATAACCCTCACA 
                 984 
                 DNA41 
               
               
                   
               
               
                 5 
                 83300276 
                 AAAATACCTCACTAGCTCTCCAG 
                 985 
                 X60 
               
               
                   
               
               
                 5 
                 85150663 
                 AAAATAATTGAAAAACTCTCTGT 
                 986 
                 X60 
               
               
                   
               
               
                 5 
                 87098398 
                 ATAAGAACAGAATAACCTTCTGG 
                 987 
                 X60 
               
               
                   
               
               
                 5 
                 91842929 
                 AAGAGTCATGCATAGCCCTCCAG 
                 988 
                 X60 
               
               
                   
               
               
                 5 
                 105774459 
                 AAGATACTGAATAAACTCCTGC 
                 989 
                 RNA41 
               
               
                   
               
               
                 5 
                 106733933 
                 AAGAAACACAAATAACCATCAGA 
                 990 
                 X60 
               
               
                   
               
               
                 5 
                 108874504 
                 GAGATGCTGAATAACTCTCTGG 
                 991 
                 RNA31 
               
               
                   
               
               
                 5 
                 112311434 
                 AAGATACTGAGTATCTCACAGG 
                 992 
                 RNA41 
               
               
                   
               
               
                 5 
                 112422645 
                 AAGACACCAAGATAGCACCCTCGGG 
                 993 
                 DNA42 
               
               
                   
               
               
                 5 
                 113833519 
                 AGTATACCTTAATACCCCCTGG 
                 994 
                 RNA41 
               
               
                   
               
               
                 5 
                 114129926 
                 ATTATACCTAATAACCCTATGG 
                 995 
                 RNA31 
               
               
                   
               
               
                 5 
                 117372593 
                 AATATAGCCCAATATCCCTCATG 
                 996 
                 X60 
               
               
                   
               
               
                 5 
                 118452920 
                 CAGATACCTGAATGATCCTTTTC 
                 997 
                 X60 
               
               
                   
               
               
                 5 
                 119760952 
                 AAGAAAGCTGAGTAGTCCTCAGA 
                 998 
                 X60 
               
               
                   
               
               
                 5 
                 132792147 
                 AAGGGACAGGAAGAACCCTGAGG 
                 999 
                 X60 
               
               
                   
               
               
                 5 
                 132904762 
                 AAGATAGCTGTATAACCTTTATC 
                 1000 
                 X60 
               
               
                   
               
               
                 5 
                 137163670 
                 AAGTTAGCGGAACTCACCCTCAGG 
                 1001 
                 DNA41 
               
               
                   
               
               
                 5 
                 137764269 
                 AAGAGAGCTAGCTAGCCCTCTGG 
                 1002 
                 X60 
               
               
                   
               
               
                 5 
                 142640990 
                 AGGATTTCTGGATAACACTTTGG 
                 1003 
                 X60 
               
               
                   
               
               
                 5 
                 143734240 
                 GAAGTACCTGAAAAACCCTCTGC 
                 1004 
                 X50 
               
               
                   
               
               
                 5 
                 148112086 
                 GAGGTACTTGAATCACCATCAAG 
                 1005 
                 X60 
               
               
                   
               
               
                 5 
                 151722432 
                 TAGCTTACTGAATAACCCTAAGG 
                 1006 
                 X50 
               
               
                   
               
               
                 5 
                 152708508 
                 AAGAGGCATGAATAACCCTGTTC 
                 1007 
                 X60 
               
               
                   
               
               
                 5 
                 153529703 
                 CAGTTACGAATAACCCTCAGA 
                 1008 
                 RNA32 
               
               
                   
               
               
                 5 
                 156251323 
                 AAGATACTGAAAAACCCTCTGA 
                 1009 
                 RNA21 
               
               
                   
               
               
                 5 
                 160330029 
                 AACAGACATGAAGAAGGCTCAGG 
                 1010 
                 X60 
               
               
                   
               
               
                 5 
                 163829739 
                 ACCATACCTGAAGGACCATCTTG 
                 1011 
                 X60 
               
               
                   
               
               
                 5 
                 164401431 
                 AAGAGACCTTAAAACTCTCTGT 
                 1012 
                 RNA41 
               
               
                   
               
               
                 5 
                 165782539 
                 ACAAGACCAAAATAACCCACAGG 
                 1013 
                 X60 
               
               
                   
               
               
                 5 
                 172047699 
                 ACGATGGCAGAACAAACCTCAGG 
                 1014 
                 X60 
               
               
                   
               
               
                 5 
                 173042104 
                 AAGATACCTCAAACACCAGCCGT 
                 1015 
                 X60 
               
               
                   
               
               
                 5 
                 175751428 
                 AAGACACCTAAATATCCCTGGGA 
                 1016 
                 X50 
               
               
                   
               
               
                 5 
                 177550455 
                 AAGATACTGACTAATCATCTGC 
                 1017 
                 RNA41 
               
               
                   
               
               
                 6 
                 2810238 
                 TAGACACCTGAGTCACCCTCTTA 
                 1018 
                 X60 
               
               
                   
               
               
                 6 
                 3633347 
                 GTGATACCAGATTAACCTCTGG 
                 1019 
                 RNA41 
               
               
                   
               
               
                 6 
                 3993855 
                 AGAGTAACTGAATAACCCTACAG 
                 1020 
                 X60 
               
               
                   
               
               
                 6 
                 6543792 
                 TTTGTACCAGAATAACCCTCCTG 
                 1021 
                 X60 
               
               
                   
               
               
                 6 
                 8845584 
                 AAATTAACTGCATAACCCTGGGT 
                 1022 
                 X60 
               
               
                   
               
               
                 6 
                 12146644 
                 AAAAAACTTGAATTACCTTCTAG 
                 1023 
                 X60 
               
               
                   
               
               
                 6 
                 13274573 
                 CTGAAACCTAAATAATCCCCCGG 
                 1024 
                 X60 
               
               
                   
               
               
                 6 
                 15494007 
                 AAGACCCAGGAATCACACTCTGG 
                 1025 
                 X60 
               
               
                   
               
               
                 6 
                 16159742 
                 AGAAGACCTGAATACCCCCAGG 
                 1026 
                 RNA41 
               
               
                   
               
               
                 6 
                 17837365 
                 CAAATACCTAAAGAACTCTCCTG 
                 1027 
                 X60 
               
               
                   
               
               
                 6 
                 18241159 
                 CCTATACCTAATAACCTTCAGG 
                 1028 
                 RNA41 
               
               
                   
               
               
                 6 
                 20501819 
                 AAGATACCTGGAAAACTCCCAAA 
                 1029 
                 X60 
               
               
                   
               
               
                 6 
                 22811364 
                 TAGACACCTAATAATCCCCAGG 
                 1030 
                 RNA41 
               
               
                   
               
               
                 6 
                 24130572 
                 GTGATACCTGTCAGACCCTCGGG 
                 1031 
                 X60 
               
               
                   
               
               
                 6 
                 25968782 
                 AAGATACCAGAATTCCCCTCTTC 
                 1032 
                 X50 
               
               
                   
               
               
                 6 
                 26726581 
                 AAGATGGCTGAGAAAAACTCAGG 
                 1033 
                 X60 
               
               
                   
               
               
                 6 
                 26775081 
                 AAGATGGCTGAGAAAAACTCAGG 
                 1033 
                 X60 
               
               
                   
               
               
                 6 
                 28642665 
                 CGTGTACCAGAATCACCCTCAGG 
                 1034 
                 X60 
               
               
                   
               
               
                 6 
                 28976992 
                 AAGAACTTGTAGAACCATCAGG 
                 1035 
                 RNA41 
               
               
                   
               
               
                 6 
                 31728324 
                 AAGATACCCCATCACCCCTCCCG 
                 1036 
                 X60 
               
               
                   
               
               
                 6 
                 31925117 
                 AAGATCAGCAGAAGAACCTTCTGG 
                 1037 
                 DNA41 
               
               
                   
               
               
                 6 
                 32569518 
                 AGTATACCTAAACAAACCTCAGA 
                 1038 
                 X60 
               
               
                   
               
               
                 6 
                 35414348 
                 AAGATCCTGCATACCCTTCTGT 
                 1039 
                 RNA41 
               
               
                   
               
               
                 6 
                 39979448 
                 AAGATCCAGATTAACCCTCTAG 
                 1040 
                 RNA31 
               
               
                   
               
               
                 6 
                 40805097 
                 AACATCCCTGAAGACCCTTGAGG 
                 1041 
                 X60 
               
               
                   
               
               
                 6 
                 41733389 
                 AAGATACCTGGGTCCCCATTTGG 
                 1042 
                 X60 
               
               
                   
               
               
                 6 
                 42264231 
                 AAGATCCTTAAATACCCTCTGG 
                 1043 
                 RNA31 
               
               
                   
               
               
                 6 
                 42676866 
                 AAGATAACTAACTCACCTTCCCG 
                 1044 
                 X60 
               
               
                   
               
               
                 6 
                 45506487 
                 ATGATATATGAACAACCCTCTGG 
                 1045 
                 X40 
               
               
                   
               
               
                 6 
                 45565147 
                 AACATACTCGAATACCCCTCAAA 
                 1046 
                 X60 
               
               
                   
               
               
                 6 
                 45812353 
                 AGCTTACCTGCTTAACCCTCAGG 
                 1047 
                 X50 
               
               
                   
               
               
                 6 
                 45989719 
                 AAGATACCAAAATTCCCCAATGG 
                 1048 
                 X60 
               
               
                   
               
               
                 6 
                 46206185 
                 ACGATACTGGAATAAGCCTTTGA 
                 1049 
                 X60 
               
               
                   
               
               
                 6 
                 51499203 
                 CAGTACCTCAACAACCCCCTGG 
                 1050 
                 RNA41 
               
               
                   
               
               
                 6 
                 51857715 
                 AATATCCTGAATAAACCTCTAG 
                 1051 
                 RNA31 
               
               
                   
               
               
                 6 
                 57915493 
                 AAGACACCTGATAAAAACTCTGT 
                 1052 
                 X60 
               
               
                   
               
               
                 6 
                 62452292 
                 AAGATACAGAATAAACCTAAGG 
                 1053 
                 RNA31 
               
               
                   
               
               
                 6 
                 66308858 
                 GAGATACTTGAACAACTCACTGG 
                 1054 
                 X50 
               
               
                   
               
               
                 6 
                 66899926 
                 AAGAAACCAGAGAAACCTTCGAG 
                 1055 
                 X60 
               
               
                   
               
               
                 6 
                 71004311 
                 AAAGTACCTAAATAACCAACCAG 
                 1056 
                 X60 
               
               
                   
               
               
                 6 
                 76467643 
                 AAGAAGGTGAATAACACTTAGG 
                 1057 
                 RNA41 
               
               
                   
               
               
                 6 
                 78402401 
                 AAGATACCTGGCTAGCCATATGC 
                 1058 
                 X60 
               
               
                   
               
               
                 6 
                 79368236 
                 AAGCTACCAGAGGAACAATCAGG 
                 1059 
                 X60 
               
               
                   
               
               
                 6 
                 84651006 
                 CATTTACTGAATAACCCTTTGG 
                 1060 
                 RNA41 
               
               
                   
               
               
                 6 
                 84877021 
                 GATAAACATGAAGGACCCTCAGG 
                 1061 
                 X60 
               
               
                   
               
               
                 6 
                 89636030 
                 AACGTACCTGAACAACCCTCAGG 
                 1062 
                 X30 
               
               
                   
               
               
                 6 
                 90026983 
                 ACACTACCTGAATGACCCTCAAG 
                 1063 
                 X50 
               
               
                   
               
               
                 6 
                 91316610 
                 AATATACATGAAACACACTTAGG 
                 1064 
                 X60 
               
               
                   
               
               
                 6 
                 100261928 
                 AAGATACCTAAATGTCCATCAAC 
                 1065 
                 X60 
               
               
                   
               
               
                 6 
                 101477606 
                 CAGATATGAATAAACCTCTAG 
                 1066 
                 RNA32 
               
               
                   
               
               
                 6 
                 104427293 
                 AATAGTACTCAATAAACCTCTGG 
                 1067 
                 X60 
               
               
                   
               
               
                 6 
                 105452361 
                 AGTATACATGAATAGCCCTCAGG 
                 1068 
                 X40 
               
               
                   
               
               
                 6 
                 113288211 
                 TTGCTACCTGAATAACCCTCTTG 
                 1069 
                 X40 
               
               
                   
               
               
                 6 
                 125169983 
                 AAGATATCTGAGGAAGCGTCTTG 
                 1070 
                 X60 
               
               
                   
               
               
                 6 
                 125494646 
                 AAGATACCTGAAACCCTCTGG 
                 1071 
                 RNA02 
               
               
                   
               
               
                 6 
                 126401280 
                 CAGATATTGAAAAACCCTCTGG 
                 1072 
                 RNA31 
               
               
                   
               
               
                 6 
                 127951944 
                 AAGGTATCCAAATAACCCTTTGG 
                 1073 
                 X50 
               
               
                   
               
               
                 6 
                 133766716 
                 TAAATACCTGAACAACTCTAGGG 
                 1074 
                 X50 
               
               
                   
               
               
                 6 
                 134020048 
                 AAGATTACATGAGTACCCCTCCTG 
                 1075 
                 DNA41 
               
               
                   
               
               
                 6 
                 141080346 
                 ATGATAGTGAATAACTCTCTTG 
                 1076 
                 RNA41 
               
               
                   
               
               
                 6 
                 141657228 
                 AAGATATCTGAAAAATCCACAAA 
                 1077 
                 X60 
               
               
                   
               
               
                 6 
                 142866995 
                 AAGATTTCTTATAACCCTCTGG 
                 1078 
                 RNA31 
               
               
                   
               
               
                 6 
                 146355335 
                 AAGATACTCTGAATAACTTTCTGG 
                 1079 
                 DNA21 
               
               
                   
               
               
                 6 
                 147553748 
                 AAGTCATCTGAATAACCCTCAAG 
                 1080 
                 X40 
               
               
                   
               
               
                 6 
                 152311384 
                 AAGATACGTGCAATAACTATCAAG 
                 1081 
                 DNA41 
               
               
                   
               
               
                 6 
                 158194025 
                 AAGATACATTAGTAACATTTTGG 
                 1082 
                 X60 
               
               
                   
               
               
                 6 
                 159249441 
                 AAGAAACACTTAAAAACCATCTGG 
                 1083 
                 DNA41 
               
               
                   
               
               
                 6 
                 159877092 
                 TAGATACCTGTATAACCACCACC 
                 1084 
                 X60 
               
               
                   
               
               
                 6 
                 163714704 
                 AACTTACCTGAACATGCCCCTGG 
                 1085 
                 X60 
               
               
                   
               
               
                 6 
                 166082374 
                 TAGATACATGGATTAACTTCTGG 
                 1086 
                 X60 
               
               
                   
               
               
                 6 
                 168015970 
                 AAGACAGCAGTAAAACACTCTGG 
                 1087 
                 X60 
               
               
                   
               
               
                 6 
                 168145686 
                 AAGATCTGGAAGAACTCTCTGG 
                 1088 
                 RNA41 
               
               
                   
               
               
                 7 
                 5143278 
                 AAGAAAGGTGAGCAACACTCGGG 
                 1089 
                 X60 
               
               
                   
               
               
                 7 
                 5180429 
                 AAGATACTTTAAAAAACCCCTGC 
                 1090 
                 X60 
               
               
                   
               
               
                 7 
                 12098480 
                 AATATACCTAGCTAAGCCTCAGA 
                 1091 
                 X60 
               
               
                   
               
               
                 7 
                 16189181 
                 AAGATAGCCTGAGTCACACTCTTG 
                 1092 
                 DNA41 
               
               
                   
               
               
                 7 
                 17175165 
                 AAGAAAACAGCGTAAGCCTCAGG 
                 1093 
                 X60 
               
               
                   
               
               
                 7 
                 18301478 
                 ATGAAACCTAAATAAGTCTCTGG 
                 1094 
                 X50 
               
               
                   
               
               
                 7 
                 18326887 
                 TGTTTACCTGAATAACCTTCAGG 
                 1095 
                 X50 
               
               
                   
               
               
                 7 
                 20063992 
                 AAGATATCTGAAAAGCCCACTTT 
                 1096 
                 X60 
               
               
                   
               
               
                 7 
                 21393775 
                 TGTAAACCTGGATAACCCTCTAG 
                 1097 
                 X60 
               
               
                   
               
               
                 7 
                 22673635 
                 AAGTATCATGAATACCCTCAGG 
                 1098 
                 RNA41 
               
               
                   
               
               
                 7 
                 25237966 
                 AAGATCCTATATAACCCTCTGA 
                 1099 
                 RNA31 
               
               
                   
               
               
                 7 
                 28451369 
                 AAAATACCTGAATAACCTGGGCC 
                 1100 
                 X60 
               
               
                   
               
               
                 7 
                 36309411 
                 AAGTTACCAGAACAACCCTTAGG 
                 1101 
                 X40 
               
               
                   
               
               
                 7 
                 43099959 
                 AAGAATTTGAATAACTCTCTAG 
                 1102 
                 RNA41 
               
               
                   
               
               
                 7 
                 45063943 
                 AGGATACCTGAATCCCTCCAG 
                 1103 
                 RNA22 
               
               
                   
               
               
                 7 
                 46108303 
                 GAGACACCTGAATAAGTGTCTGG 
                 1104 
                 X50 
               
               
                   
               
               
                 7 
                 46457470 
                 AAGCTAGTGAAAAACCATCAGG 
                 1105 
                 RNA41 
               
               
                   
               
               
                 7 
                 47819173 
                 AGGATACTTGAATATCCCTGCCT 
                 1106 
                 X60 
               
               
                   
               
               
                 7 
                 49066063 
                 CAGATACTGAAAAACACTCTGG 
                 1107 
                 RNA31 
               
               
                   
               
               
                 7 
                 49306954 
                 AAGAAATCTGGATAACCCCCAAT 
                 1108 
                 X60 
               
               
                   
               
               
                 7 
                 60892285 
                 AACATACCCAAAAAAACCTCTGA 
                 1109 
                 X60 
               
               
                   
               
               
                 7 
                 61597579 
                 AACATACCCAAAAAAACCTCTGA 
                 1109 
                 X60 
               
               
                   
               
               
                 7 
                 62026418 
                 AACATACCCAAAAAAACCTCTGA 
                 1109 
                 X60 
               
               
                   
               
               
                 7 
                 78658625 
                 AAAATATCCAAATAACCCCCTGG 
                 1110 
                 X50 
               
               
                   
               
               
                 7 
                 81886792 
                 AAGATATCTGAATAATCATCAGG 
                 1111 
                 X30 
               
               
                   
               
               
                 7 
                 88664778 
                 AAGATACTGAGTAACACTGAAG 
                 1112 
                 RNA41 
               
               
                   
               
               
                 7 
                 89093321 
                 AAGTTACCTAAATAACTTTGG 
                 1113 
                 RNA32 
               
               
                   
               
               
                 7 
                 89117479 
                 ACCATAATTGATTAACTCTCTGG 
                 1114 
                 X60 
               
               
                   
               
               
                 7 
                 94742185 
                 AAGAAACCTAAGTAAACCTCTAA 
                 1115 
                 X60 
               
               
                   
               
               
                 7 
                 96159184 
                 AAGATACATCATCACCCTCCTG 
                 1116 
                 RNA41 
               
               
                   
               
               
                 7 
                 96499495 
                 AAGACACATAAGGAACCCTCATG 
                 1117 
                 X60 
               
               
                   
               
               
                 7 
                 101395824 
                 GAGATACCTGAGCTGCCCTCAGC 
                 1118 
                 X60 
               
               
                   
               
               
                 7 
                 104765414 
                 GAGATACCTAAATAACCTTCCAG 
                 1119 
                 X40 
               
               
                   
               
               
                 7 
                 107527319 
                 AAGATACGAATAATCCTCCGA 
                 1120 
                 RNA22 
               
               
                   
               
               
                 7 
                 114126788 
                 ATTAAACCAAAATAACCCTCGGG 
                 1121 
                 X50 
               
               
                   
               
               
                 7 
                 117090821 
                 CACAAACCTTAATAACCCTCAAG 
                 1122 
                 X50 
               
               
                   
               
               
                 7 
                 117393513 
                 AAGATAAAAAAATACCCCTAGGG 
                 1123 
                 X60 
               
               
                   
               
               
                 7 
                 123985680 
                 AAGATATCTGAAAAAGTCTTGAG 
                 1124 
                 X60 
               
               
                   
               
               
                 7 
                 124105262 
                 GAGATAACTGAATAACCATTTGG 
                 1125 
                 X40 
               
               
                   
               
               
                 7 
                 125205185 
                 AAGATATCTGTAAAACCCTTCAG 
                 1126 
                 X50 
               
               
                   
               
               
                 7 
                 125899502 
                 AACATACATGAAAAATCCTCAAC 
                 1127 
                 X60 
               
               
                   
               
               
                 7 
                 127268120 
                 AAGGTAACTGGATAGCCATCTGC 
                 1128 
                 X60 
               
               
                   
               
               
                 7 
                 132695120 
                 AAGAGACCTGAGTCACCCTGTCT 
                 1129 
                 X60 
               
               
                   
               
               
                 7 
                 134060777 
                 AAGATAAATGAATTTCCTTCTGT 
                 1130 
                 X60 
               
               
                   
               
               
                 7 
                 140628498 
                 AAGATACGGGAAATCCCCTTTGG 
                 1131 
                 X60 
               
               
                   
               
               
                 7 
                 146763269 
                 CAGATATCAGATAACCCTATGG 
                 1132 
                 RNA41 
               
               
                   
               
               
                 7 
                 153436410 
                 CAGATGCTTGTAAAACCATCAGG 
                 1133 
                 X60 
               
               
                   
               
               
                 7 
                 153479243 
                 ACAGTAGTTGCATAACCCTCTGG 
                 1134 
                 X60 
               
               
                   
               
               
                 7 
                 155749430 
                 AAGATTCAGCACTAACCCACAGG 
                 1135 
                 X60 
               
               
                   
               
               
                 7 
                 157980250 
                 GAGATACTGAATTACCCTCTGG 
                 1136 
                 RNA21 
               
               
                   
               
               
                 8 
                 4038549 
                 AAGAGACATGCATAATCTTCAGG 
                 1137 
                 X50 
               
               
                   
               
               
                 8 
                 5643554 
                 AAGTAAACTGAATACCTTCTGG 
                 1138 
                 RNA41 
               
               
                   
               
               
                 8 
                 7420667 
                 AAGAAACCACTATAACCATCAGT 
                 1139 
                 X60 
               
               
                   
               
               
                 8 
                 7890852 
                 AAGAAACCACTATAACCATCAGT 
                 1139 
                 X60 
               
               
                   
               
               
                 8 
                 19687879 
                 CAGATACTGTATAACCCTCAGA 
                 1140 
                 RNA31 
               
               
                   
               
               
                 8 
                 21016005 
                 AATGTAAATGTATAACCCTCTGT 
                 1141 
                 X60 
               
               
                   
               
               
                 8 
                 29936242 
                 AAGTTAACTGTAAAACAGCCTCAGG 
                 956 
                 DNA42 
               
               
                   
               
               
                 8 
                 36495615 
                 AGGATACCATAATAACCAGCATG 
                 1142 
                 X60 
               
               
                   
               
               
                 8 
                 43072291 
                 GCAATACCTGAATAACCCATTGT 
                 1143 
                 X60 
               
               
                   
               
               
                 8 
                 47579895 
                 AAGATACCAGACCATCTCCCAGG 
                 1144 
                 X60 
               
               
                   
               
               
                 8 
                 49597827 
                 AAGATCTGAATAGCACCCTGG 
                 1145 
                 RNA32 
               
               
                   
               
               
                 8 
                 55071567 
                 AAGATCACAGGACAACCCTCCGC 
                 1146 
                 X60 
               
               
                   
               
               
                 8 
                 55401326 
                 AAGATTCATGATAACCATCATG 
                 1147 
                 RNA41 
               
               
                   
               
               
                 8 
                 60813307 
                 CAGGTACCTGGAGAACCCTAGAG 
                 1148 
                 X60 
               
               
                   
               
               
                 8 
                 61779630 
                 AAGATACCTGAACACCCTACTGG 
                 1149 
                 X40 
               
               
                   
               
               
                 8 
                 64267393 
                 TAGCTAGCTGATAACACTCTGG 
                 1150 
                 RNA41 
               
               
                   
               
               
                 8 
                 65828227 
                 ATTATACTGAATAACCTTCCAG 
                 1151 
                 RNA41 
               
               
                   
               
               
                 8 
                 66202960 
                 AAGTTACCTGCATCAAACTCATG 
                 1152 
                 X60 
               
               
                   
               
               
                 8 
                 66947451 
                 AAGATACAGAATAACCCTCTAG 
                 1153 
                 RNA21 
               
               
                   
               
               
                 8 
                 67331610 
                 GATATACTCTAATAACCCTCAAG 
                 1154 
                 X60 
               
               
                   
               
               
                 8 
                 67691390 
                 AAGATACATAAATCACTTTCCAG 
                 1155 
                 X60 
               
               
                   
               
               
                 8 
                 67694023 
                 AAAATGCCTAATAGACCTCTGG 
                 1156 
                 RNA41 
               
               
                   
               
               
                 8 
                 68072298 
                 AAAACACCTGAAAAATCCTCCAC 
                 1157 
                 X60 
               
               
                   
               
               
                 8 
                 73416526 
                 AAGAAACACTGAATGTCCCTCTTG 
                 1158 
                 DNA41 
               
               
                   
               
               
                 8 
                 74109927 
                 GTGAAACCTAAATAACCCTCAGA 
                 1159 
                 X50 
               
               
                   
               
               
                 8 
                 75900398 
                 AATATACCAGAATAGTTCTCAGT 
                 1160 
                 X60 
               
               
                   
               
               
                 8 
                 76479079 
                 AAGATACTGAATATTCCTCAGG 
                 1161 
                 RNA21 
               
               
                   
               
               
                 8 
                 78222169 
                 GAGATACTTGAATAACCATCTCA 
                 1162 
                 X50 
               
               
                   
               
               
                 8 
                 80098340 
                 AAAGTACATTAAAAACCTTCTGG 
                 1163 
                 X60 
               
               
                   
               
               
                 8 
                 80176030 
                 AAGATAATTGAATAGCCATCTTA 
                 1164 
                 X60 
               
               
                   
               
               
                 8 
                 80682169 
                 AACAAACCCGTAAAACCCTCACG 
                 1165 
                 X60 
               
               
                   
               
               
                 8 
                 83187784 
                 AAGATAGTTAAATATCCCCCTGT 
                 1166 
                 X60 
               
               
                   
               
               
                 8 
                 88225407 
                 AAGATAATAAATAACCCTCAAG 
                 1167 
                 RNA31 
               
               
                   
               
               
                 8 
                 91354172 
                 AAGCTACTTGATAACCCTATGG 
                 1168 
                 RNA31 
               
               
                   
               
               
                 8 
                 102629148 
                 ATTATACCTGAATCCCTCAGG 
                 1169 
                 RNA22 
               
               
                   
               
               
                 8 
                 106844104 
                 AAGTTATCCTAATAACCCTATGG 
                 1170 
                 X50 
               
               
                   
               
               
                 8 
                 106983770 
                 ATGTTTACTGAATACCCCTCTGG 
                 1171 
                 X50 
               
               
                   
               
               
                 8 
                 108954887 
                 AAGGGACCTGAAATTCCCTGAGG 
                 1172 
                 X60 
               
               
                   
               
               
                 8 
                 115458181 
                 TAGATAACAGAATGACTCACAGG 
                 1173 
                 X60 
               
               
                   
               
               
                 8 
                 119894846 
                 AAGATTCTGAATAGACCCCAGG 
                 1174 
                 RNA41 
               
               
                   
               
               
                 8 
                 120688940 
                 AACATACCCAAATACTCTCAGG 
                 1175 
                 RNA41 
               
               
                   
               
               
                 8 
                 121544316 
                 AAGATACCTCAATAAGACTCGGG 
                 1176 
                 X30 
               
               
                   
               
               
                 8 
                 124209633 
                 TAGATACTGAAAATCTCTCAGG 
                 1177 
                 RNA41 
               
               
                   
               
               
                 8 
                 124951793 
                 AGAATACAAGAATAACCCTGGGG 
                 1178 
                 X50 
               
               
                   
               
               
                 8 
                 125028680 
                 AAGATACCTGAAATTCCTGCAGA 
                 1179 
                 X60 
               
               
                   
               
               
                 8 
                 125768967 
                 CTGGTACTTAATAACCCTCAGG 
                 1180 
                 RNA41 
               
               
                   
               
               
                 8 
                 130261595 
                 GAGATACCTAACACACACTCTGG 
                 1181 
                 X60 
               
               
                   
               
               
                 8 
                 139484036 
                 ACAATACCTGAAAAACTCCCTTG 
                 1182 
                 X60 
               
               
                   
               
               
                 8 
                 140142948 
                 CAGATACATTAAAACCCTCTGG 
                 1183 
                 RNA31 
               
               
                   
               
               
                 8 
                 141381027 
                 CAGAACCCACAATAAGCCTCTGG 
                 1184 
                 X60 
               
               
                   
               
               
                 9 
                 1548217 
                 AATAACATGAATAACACTCAAG 
                 1185 
                 RNA41 
               
               
                   
               
               
                 9 
                 2362898 
                 AAGTAACCCAAATAACCCCAGG 
                 1186 
                 RNA41 
               
               
                   
               
               
                 9 
                 3085581 
                 AAGATACAGAATTTCCCTTTGG 
                 1187 
                 RNA41 
               
               
                   
               
               
                 9 
                 7499375 
                 AAGGTCCCTGAGCCACCCTCCAG 
                 1188 
                 X60 
               
               
                   
               
               
                 9 
                 7701793 
                 AAGATCTGGAACAACTCTCTGG 
                 1189 
                 RNA41 
               
               
                   
               
               
                 9 
                 9668247 
                 AACACTATTGAATAACCATCTGG 
                 1190 
                 X60 
               
               
                   
               
               
                 9 
                 13025629 
                 GGGATACCTGAATATTCTCTGG 
                 1191 
                 RNA41 
               
               
                   
               
               
                 9 
                 13825298 
                 AAGATGCCTGCACATCCCTCCTT 
                 1192 
                 X60 
               
               
                   
               
               
                 9 
                 15566128 
                 AAGATACATGAATTACCAAAAAG 
                 1193 
                 X60 
               
               
                   
               
               
                 9 
                 18157881 
                 AAGATGCTTGAAGTAGCTTCAGG 
                 1194 
                 X60 
               
               
                   
               
               
                 9 
                 21582692 
                 TAGATACCTCTTATAACCCTCCAG 
                 1195 
                 DNA41 
               
               
                   
               
               
                 9 
                 22799849 
                 AAGATACATCAATAAACCTGATT 
                 1196 
                 X60 
               
               
                   
               
               
                 9 
                 22914887 
                 AAGATAGTCCTATAACTCTCTGG 
                 1197 
                 X60 
               
               
                   
               
               
                 9 
                 27875283 
                 AAAATTAGTGGATAAACCTCTGG 
                 1198 
                 X60 
               
               
                   
               
               
                 9 
                 28235239 
                 AAGATCCTGAAGAATTCTCTGA 
                 1199 
                 RNA41 
               
               
                   
               
               
                 9 
                 29142022 
                 AGAATACCTGAACAACACTCTAG 
                 1200 
                 X50 
               
               
                   
               
               
                 9 
                 33293321 
                 ATTAAACCTGAAACCCTCAGG 
                 1201 
                 RNA32 
               
               
                   
               
               
                 9 
                 33907178 
                 AAGATAGCTAAATAACCTCTGA 
                 1202 
                 RNA31 
               
               
                   
               
               
                 9 
                 36483605 
                 GAGACAACTGATTATCCCTCAAG 
                 1203 
                 X60 
               
               
                   
               
               
                 9 
                 39480043 
                 AATTTACCAGAATAGCCCTCTGA 
                 1204 
                 X50 
               
               
                   
               
               
                 9 
                 66885197 
                 AATTTACCAGAATAGCCCTCTGA 
                 1204 
                 X50 
               
               
                   
               
               
                 9 
                 74963383 
                 AAGATACTGAAATACCCCTATGG 
                 1205 
                 X50 
               
               
                   
               
               
                 9 
                 81338482 
                 AAGGTAAATGAGTAATCCTTTGG 
                 1206 
                 X60 
               
               
                   
               
               
                 9 
                 82995745 
                 AAGATTACTTGAAGAAGCCTCTGA 
                 1207 
                 DNA41 
               
               
                   
               
               
                 9 
                 85121255 
                 AAAGTAGCTGAATAAACCTCAAG 
                 1208 
                 X50 
               
               
                   
               
               
                 9 
                 88846233 
                 AAGATAACTGAAATGCCTTCGAG 
                 1209 
                 X60 
               
               
                   
               
               
                 9 
                 89929285 
                 ACTATTCCTGAATAACCCTTTGG 
                 1210 
                 X40 
               
               
                   
               
               
                 9 
                 94468528 
                 AACATCCTGAATCAACCTCTGC 
                 1211 
                 RNA41 
               
               
                   
               
               
                 9 
                 96771534 
                 GAAATACTGAGTACCCCTCAGG 
                 1212 
                 RNA41 
               
               
                   
               
               
                 9 
                 97299066 
                 TAGATCTAGAATAAGCCTCTGG 
                 1213 
                 RNA41 
               
               
                   
               
               
                 9 
                 100767516 
                 AAGATACCCAAATAACCATCAGA 
                 1214 
                 X40 
               
               
                   
               
               
                 9 
                 107058386 
                 AAGACAGCTAACTAACTCTCGGG 
                 1215 
                 X50 
               
               
                   
               
               
                 9 
                 107981569 
                 AAAGTAGCTGAACAGCCCTCAGG 
                 1216 
                 X50 
               
               
                   
               
               
                 9 
                 109732394 
                 AAGATACCTCATTAATCATCACA 
                 1217 
                 X60 
               
               
                   
               
               
                 9 
                 110545175 
                 AAGATATGGAATCAACCTCAGG 
                 1218 
                 RNA41 
               
               
                   
               
               
                 9 
                 112078020 
                 AAGATACCTAATAACCACAGGG 
                 1219 
                 RNA31 
               
               
                   
               
               
                 9 
                 114633862 
                 ACAGCACCTGAATCACACTCTGG 
                 1220 
                 X60 
               
               
                   
               
               
                 9 
                 114864429 
                 AAGATACAAAAACAACCGACTGG 
                 1221 
                 X60 
               
               
                   
               
               
                 9 
                 114970717 
                 AAGATACTCAATAAACCTCAAG 
                 1222 
                 RNA31 
               
               
                   
               
               
                 9 
                 116496855 
                 ATGATAGTGAATAATTCTCAGG 
                 1223 
                 RNA41 
               
               
                   
               
               
                 9 
                 123598985 
                 AAGACACATAAACCACTCTCAGG 
                 1224 
                 X60 
               
               
                   
               
               
                 9 
                 125297490 
                 AAGATACTGGATAACCTTCCAG 
                 1225 
                 RNA31 
               
               
                   
               
               
                 9 
                 125378327 
                 ATGATAATGAATAACCCTTGGG 
                 1226 
                 RNA31 
               
               
                   
               
               
                 9 
                 125911093 
                 AAGACACTGAAAAACCCTCAAG 
                 1227 
                 RNA31 
               
               
                   
               
               
                 9 
                 127691320 
                 AAGATAGCAGATTAGTCCTCAGT 
                 1228 
                 X60 
               
               
                   
               
               
                 9 
                 127974450 
                 AAGAGATCGGGATAAGCCTTTGG 
                 1229 
                 X60 
               
               
                   
               
               
                 10 
                 7147046 
                 TAAGTACCCAAATAAGCCTCTGG 
                 1230 
                 X60 
               
               
                   
               
               
                 10 
                 7993040 
                 AAGATATCTGAATATGGACCAGG 
                 1231 
                 X60 
               
               
                   
               
               
                 10 
                 11705444 
                 AAGATAGCTAAGAATCCCTCCAG 
                 1232 
                 X60 
               
               
                   
               
               
                 10 
                 13360457 
                 TAGTTACAAGAATAACCCCAGG 
                 1233 
                 RNA41 
               
               
                   
               
               
                 10 
                 14735911 
                 TAGATACATAATAGCCCTTTGG 
                 1234 
                 RNA41 
               
               
                   
               
               
                 10 
                 15941531 
                 AAGAGCCCTGAATAAGCTTTAGG 
                 1235 
                 X50 
               
               
                   
               
               
                 10 
                 16541599 
                 AAGATACTGAATAAACCCGAAG 
                 1236 
                 RNA41 
               
               
                   
               
               
                 10 
                 21114219 
                 AAGATATCTGGATAATCCCCAAA 
                 1237 
                 X60 
               
               
                   
               
               
                 10 
                 27972145 
                 AATATACCTTGAATACCCCTCCCT 
                 1238 
                 DNA41 
               
               
                   
               
               
                 10 
                 32570562 
                 GGGATACCTTAACAACCCTGTGG 
                 1239 
                 X50 
               
               
                   
               
               
                 10 
                 35982871 
                 AAAATACCTGACAGAACCACTGG 
                 1240 
                 X60 
               
               
                   
               
               
                 10 
                 38157649 
                 AAAATACCTGAGTGACCCTAGAA 
                 1241 
                 X60 
               
               
                   
               
               
                 10 
                 47125548 
                 AAGATTTGTGAATGACCCTAATG 
                 1242 
                 X60 
               
               
                   
               
               
                 10 
                 50305665 
                 AAGATACCAGTATACTCTCTGG 
                 1243 
                 RNA31 
               
               
                   
               
               
                 10 
                 51383143 
                 AAGATCCCTGAACAACACCAAAG 
                 1244 
                 X60 
               
               
                   
               
               
                 10 
                 54571271 
                 AAGATACCAGTCCCACCCTCTTG 
                 1245 
                 X60 
               
               
                   
               
               
                 10 
                 55277105 
                 TAGATACTCTCAATGACCCTCTGA 
                 1246 
                 DNA41 
               
               
                   
               
               
                 10 
                 59699528 
                 AAGTTACTTAAATAGCTCTCTGC 
                 1247 
                 X60 
               
               
                   
               
               
                 10 
                 62667353 
                 AAGATGCTTGAATTTCCCTAATG 
                 1248 
                 X60 
               
               
                   
               
               
                 10 
                 82421522 
                 AAGAGATCTGGATCACCCCCAGC 
                 1249 
                 X60 
               
               
                   
               
               
                 10 
                 83225274 
                 AAGATATTTGAATATTCCCTAGG 
                 1250 
                 X60 
               
               
                   
               
               
                 10 
                 84218985 
                 TAATTACCAGAATAACCCTATGA 
                 1251 
                 X60 
               
               
                   
               
               
                 10 
                 90306612 
                 AAGATACCTGAAATACCTGGGT 
                 1252 
                 RNA41 
               
               
                   
               
               
                 10 
                 94280573 
                 AAGGTACCTGAGTAAACATTCTG 
                 1253 
                 X60 
               
               
                   
               
               
                 10 
                 102517636 
                 AAGATACATGAATGAATTCAGG 
                 1254 
                 RNA41 
               
               
                   
               
               
                 10 
                 102734271 
                 CAGATACCTAAGAACCCTGACG 
                 1255 
                 RNA41 
               
               
                   
               
               
                 10 
                 106248382 
                 AGTATAACAGAATAACCCTATGT 
                 1256 
                 X60 
               
               
                   
               
               
                 10 
                 118171466 
                 AACATATCTGAATATCTCTCTAA 
                 1257 
                 X60 
               
               
                   
               
               
                 10 
                 119789592 
                 AAGATCCTGCCTCAACCTCTGG 
                 1258 
                 RNA41 
               
               
                   
               
               
                 10 
                 120632845 
                 AAGATACCAAGCTAATCCTCTGC 
                 1259 
                 X60 
               
               
                   
               
               
                 10 
                 121862898 
                 GAGATTTCTGAATAACCCACCCT 
                 1260 
                 X60 
               
               
                   
               
               
                 10 
                 121868681 
                 GAGAGGCCTGAGTAGCCCACGGG 
                 1261 
                 X60 
               
               
                   
               
               
                 10 
                 122274421 
                 AAGGAAACTGGAGAACTCTCCGG 
                 1262 
                 X60 
               
               
                   
               
               
                 10 
                 130321175 
                 AAGATACTTGAATAACCATTTCA 
                 1263 
                 X50 
               
               
                   
               
               
                 10 
                 131549720 
                 TGCTTACATGAATAACCCTCCAG 
                 1264 
                 X60 
               
               
                   
               
               
                 11 
                 2376222 
                 TCTGTACCTGGATAACCCCCTGG 
                 1265 
                 X60 
               
               
                   
               
               
                 11 
                 3277015 
                 AAGGTAACTGACAAACCATCTGA 
                 1266 
                 X60 
               
               
                   
               
               
                 11 
                 3322831 
                 AAGGTAACTGACAAACCATCTGA 
                 1266 
                 X60 
               
               
                   
               
               
                 11 
                 3502236 
                 AAAATAAATGAATAAAACTGTGG 
                 1267 
                 X60 
               
               
                   
               
               
                 11 
                 4864723 
                 AAGTCACCAGTTTAACACTCAGG 
                 1268 
                 X60 
               
               
                   
               
               
                 11 
                 7440757 
                 AAGATACCAGAATAAATCATTCTAG 
                 1269 
                 DNA42 
               
               
                   
               
               
                 11 
                 12052022 
                 TAAATACATGAATAACCATTAGG 
                 1270 
                 X50 
               
               
                   
               
               
                 11 
                 12138824 
                 AAAAGCCCTGATCAAACCTCAGG 
                 1271 
                 X60 
               
               
                   
               
               
                 11 
                 12410906 
                 CACTTACCTGTATAACCTCAGG 
                 1272 
                 RNA41 
               
               
                   
               
               
                 11 
                 14162519 
                 AAGATACTTGCAGAACCATCTGA 
                 1273 
                 X50 
               
               
                   
               
               
                 11 
                 15458757 
                 AAGATCTGAATTTCCATCAGG 
                 1274 
                 RNA32 
               
               
                   
               
               
                 11 
                 22689538 
                 AAGATATACATACTAACCCTCTGG 
                 1275 
                 DNA41 
               
               
                   
               
               
                 11 
                 22781693 
                 GAGTACCATAATAAACCTCAGG 
                 1276 
                 RNA41 
               
               
                   
               
               
                 11 
                 22831675 
                 AAAGAACCTAAATAAGCCTCTTG 
                 1277 
                 X60 
               
               
                   
               
               
                 11 
                 25615424 
                 AAGACATTAAAATAACCCACAGG 
                 1278 
                 X60 
               
               
                   
               
               
                 11 
                 25752459 
                 AATATACCTGGAAAACCCACACC 
                 1279 
                 X60 
               
               
                   
               
               
                 11 
                 26574272 
                 AAGACACATGAATATCACTAATG 
                 1280 
                 X60 
               
               
                   
               
               
                 11 
                 26949218 
                 AAGATATTGAATAACCCACATT 
                 1281 
                 RNA41 
               
               
                   
               
               
                 11 
                 26997914 
                 AACATCCCAGAATGACCCACAGC 
                 1282 
                 X60 
               
               
                   
               
               
                 11 
                 28826023 
                 AAGAAAGCTGGCTAGCTCTCTGG 
                 1283 
                 X60 
               
               
                   
               
               
                 11 
                 32956605 
                 AAGATAACTGACTTGACCCTGTGG 
                 1284 
                 DNA41 
               
               
                   
               
               
                 11 
                 36609914 
                 AAGATATGAATAAAACTCTGG 
                 1285 
                 RNA22 
               
               
                   
               
               
                 11 
                 50574461 
                 AAGCTATCTGAGAAACCCTTTTG 
                 1286 
                 X60 
               
               
                   
               
               
                 11 
                 58705310 
                 CAGATACCTAATAACCACACGG 
                 1287 
                 RNA41 
               
               
                   
               
               
                 11 
                 63223967 
                 AAGATACCTGAAATTATTTCAGG 
                 1288 
                 X60 
               
               
                   
               
               
                 11 
                 79064018 
                 TTGATACCTAAATACCCTGTGG 
                 1289 
                 RNA41 
               
               
                   
               
               
                 11 
                 80186841 
                 TAAATACATGAATAATCCTCGGG 
                 1290 
                 X40 
               
               
                   
               
               
                 11 
                 81449300 
                 AATTAACCTACATAACCCTCAGT 
                 1291 
                 X60 
               
               
                   
               
               
                 11 
                 83977145 
                 AAGATTCTTAAATAACCCACTTT 
                 1292 
                 X60 
               
               
                   
               
               
                 11 
                 87592156 
                 TAGAGACATGAAAAACCCTCCAA 
                 836 
                 X60 
               
               
                   
               
               
                 11 
                 88339518 
                 AAGATTTAAGAATGAACCTCAGG 
                 1293 
                 X60 
               
               
                   
               
               
                 11 
                 88892935 
                 AAGATACAGAATTTCCCTTTGG 
                 1187 
                 RNA41 
               
               
                   
               
               
                 11 
                 92568878 
                 TGGATACCTGTCAACCCTCTGG 
                 1294 
                 RNA41 
               
               
                   
               
               
                 11 
                 101689415 
                 AAGATCCGTGAATAAACCACAGC 
                 1295 
                 X50 
               
               
                   
               
               
                 11 
                 104364397 
                 AACATACACGAATACCTCACAGG 
                 1296 
                 X60 
               
               
                   
               
               
                 11 
                 106039497 
                 AAGATGATAAACAACCCTCTGG 
                 1297 
                 RNA41 
               
               
                   
               
               
                 11 
                 109887670 
                 TAGATCCTAATTAACCCTCAGA 
                 1298 
                 RNA41 
               
               
                   
               
               
                 11 
                 114155888 
                 AAGATACAGGTGTAGCCCTCCTG 
                 1299 
                 X60 
               
               
                   
               
               
                 11 
                 117119305 
                 CTGATATGTGAAAAACCCTAAGG 
                 1300 
                 X60 
               
               
                   
               
               
                 12 
                 3649402 
                 AACATACTGAACCAGCCTCTGG 
                 1301 
                 RNA41 
               
               
                   
               
               
                 12 
                 3696467 
                 AAGACACGAGAAGAACCTTCTGA 
                 1302 
                 X60 
               
               
                   
               
               
                 12 
                 3830100 
                 AAGACACTTGAATACCACTCTTT 
                 1303 
                 X60 
               
               
                   
               
               
                 12 
                 4704350 
                 TACATACCTTGATAACCTTCAGG 
                 1304 
                 X50 
               
               
                   
               
               
                 12 
                 8120901 
                 AAGTTACCTGAACAAACCTGCAA 
                 1305 
                 X60 
               
               
                   
               
               
                 12 
                 9334202 
                 AAAAAACTAGAAGAACCCTTTGG 
                 1306 
                 X60 
               
               
                   
               
               
                 12 
                 10687002 
                 AAGATCCCTGAATTTCCATCAGA 
                 1307 
                 X50 
               
               
                   
               
               
                 12 
                 14689862 
                 AAAATACCTGCATACCCTCAGG 
                 1308 
                 RNA21 
               
               
                   
               
               
                 12 
                 20320965 
                 AACTTACATGAATTACTCTTAGG 
                 1309 
                 X60 
               
               
                   
               
               
                 12 
                 22372837 
                 AACATTTCTCAATATCCCTTTGG 
                 1310 
                 X60 
               
               
                   
               
               
                 12 
                 26607410 
                 CAGATAACTGAATAACCCTCTGT 
                 1311 
                 X30 
               
               
                   
               
               
                 12 
                 28330833 
                 TAAATACCTATAACCCACTGG 
                 1312 
                 RNA32 
               
               
                   
               
               
                 12 
                 28550781 
                 GAGATACCTGAATATTCTTCCAA 
                 1313 
                 X60 
               
               
                   
               
               
                 12 
                 32209572 
                 ATTATATCTGAATATCCTCTGG 
                 1314 
                 RNA41 
               
               
                   
               
               
                 12 
                 45545836 
                 ATGATACCCAATACCCCTCCAG 
                 1315 
                 RNA41 
               
               
                   
               
               
                 12 
                 47016633 
                 CAGATACCTGAATCTCCTTCTTT 
                 1316 
                 X60 
               
               
                   
               
               
                 12 
                 49068780 
                 CAGTTACCTGGATAACTCCCTGG 
                 1317 
                 X50 
               
               
                   
               
               
                 12 
                 54882194 
                 AAAATACTAGAATAACCAGCTTG 
                 1318 
                 X60 
               
               
                   
               
               
                 12 
                 61361291 
                 AAGACTCAGGAATATCACTCAGG 
                 1319 
                 X60 
               
               
                   
               
               
                 12 
                 66409389 
                 AAGATCCAGAAGAATCATCTGG 
                 1320 
                 RNA41 
               
               
                   
               
               
                 12 
                 72361250 
                 AAAACACCTGAATAAATCCCATG 
                 1321 
                 X60 
               
               
                   
               
               
                 12 
                 73122159 
                 AACATCACTGAAGAACCCTGAAG 
                 1322 
                 X60 
               
               
                   
               
               
                 12 
                 74390201 
                 AAAATACCTCATAGCCCTCTGC 
                 1323 
                 RNA41 
               
               
                   
               
               
                 12 
                 77975069 
                 AAAATAGCTTAATAATGCTCAGT 
                 1324 
                 X60 
               
               
                   
               
               
                 12 
                 78463857 
                 AAGTTCCCTGAACACACCTCTGG 
                 1325 
                 X50 
               
               
                   
               
               
                 12 
                 79756510 
                 ACTATACTGACTAACCCTCTAG 
                 1326 
                 RNA41 
               
               
                   
               
               
                 12 
                 79913270 
                 AGAATATCTTCATAACCTTCGGG 
                 1327 
                 X60 
               
               
                   
               
               
                 12 
                 81976639 
                 AGAAAACCTGAATAACCTATGG 
                 1328 
                 RNA41 
               
               
                   
               
               
                 12 
                 87296671 
                 AAGATATTCAAATAACCCTGCTG 
                 1329 
                 X60 
               
               
                   
               
               
                 12 
                 98856708 
                 GAGATACTGAATAACCTCTGGG 
                 1330 
                 RNA41 
               
               
                   
               
               
                 12 
                 105221258 
                 AAGATAGCTGCTAACCCTGAGT 
                 1331 
                 RNA41 
               
               
                   
               
               
                 12 
                 106415644 
                 AAAATAAATGAATAACCATCAGA 
                 1332 
                 X50 
               
               
                   
               
               
                 12 
                 109568847 
                 AAGGTACTAGAAAACCCTGTGG 
                 1333 
                 RNA41 
               
               
                   
               
               
                 12 
                 124304548 
                 AAGACACTGCCAAACCCTCCGG 
                 1334 
                 RNA41 
               
               
                   
               
               
                 12 
                 128916634 
                 AAGATACATGAATGGCCAACGCG 
                 1335 
                 X60 
               
               
                   
               
               
                 12 
                 131173514 
                 CAGAGACCTGCATAGCACTGAGG 
                 1336 
                 X60 
               
               
                   
               
               
                 12 
                 131502294 
                 AAGATATCTGAAAATCCCTGCGC 
                 1337 
                 X50 
               
               
                   
               
               
                 13 
                 20297900 
                 AAGAACACTGAGAAAACCCTCTGG 
                 1338 
                 DNA41 
               
               
                   
               
               
                 13 
                 22964102 
                 AAGAAACCTCCATATCCATCGTG 
                 1339 
                 X60 
               
               
                   
               
               
                 13 
                 24389118 
                 AAGAAACCTCCATATCCATCGTG 
                 1339 
                 X60 
               
               
                   
               
               
                 13 
                 25266108 
                 AAGGTACCTTAACAACATTTTGG 
                 1340 
                 X60 
               
               
                   
               
               
                 13 
                 26448879 
                 AAGGTGACTGAAGGACCCTTGGG 
                 1341 
                 X60 
               
               
                   
               
               
                 13 
                 26553083 
                 ACGATAACTGAACAGCTCTCAAG 
                 1342 
                 X60 
               
               
                   
               
               
                 13 
                 34458374 
                 AAGAGGGCTTAATAACCTTCAAG 
                 1343 
                 X60 
               
               
                   
               
               
                 13 
                 39980150 
                 AAGATAACTGGATAGCTCTATGA 
                 1344 
                 X60 
               
               
                   
               
               
                 13 
                 43747762 
                 AAGATTCAGCAATACCCTCTGG 
                 1345 
                 RNA41 
               
               
                   
               
               
                 13 
                 50325631 
                 CAAAAACCAGAAAACCCTCAGG 
                 1346 
                 RNA41 
               
               
                   
               
               
                 13 
                 60126783 
                 CATATACCAAAATAACCCATGGG 
                 1347 
                 X60 
               
               
                   
               
               
                 13 
                 61481200 
                 AAAATACAAATAACCCTCAGA 
                 1348 
                 RNA32 
               
               
                   
               
               
                 13 
                 66207288 
                 AAAATACCTGAATAAACCTGAAG 
                 1349 
                 X40 
               
               
                   
               
               
                 13 
                 68049114 
                 AAGATAACTTAATAGGCCTCCAC 
                 1350 
                 X60 
               
               
                   
               
               
                 13 
                 68754577 
                 AAGATGGCTGAAAAAGCCTTTCG 
                 1351 
                 X60 
               
               
                   
               
               
                 13 
                 74802880 
                 AAGCTGACTGAAGAGCCCTTGGG 
                 894 
                 X60 
               
               
                   
               
               
                 13 
                 77274559 
                 AAGTTACCAGATAACCCACAGG 
                 1352 
                 RNA31 
               
               
                   
               
               
                 13 
                 84432474 
                 CAGATACCTGGTTAAACATCACG 
                 1353 
                 X60 
               
               
                   
               
               
                 13 
                 86737129 
                 AAGATACATGAATGGCCAACAGG 
                 1354 
                 X50 
               
               
                   
               
               
                 13 
                 92119936 
                 AAGCTACCAGCATAGCCCACTAG 
                 1355 
                 X60 
               
               
                   
               
               
                 13 
                 109759900 
                 AAGAAAGCTGGAAAACACTCCAG 
                 1356 
                 X60 
               
               
                   
               
               
                 13 
                 111418659 
                 AAGATGCCTGAGTCTCCCCCGTG 
                 1357 
                 X60 
               
               
                   
               
               
                 13 
                 112083112 
                 AAGGAAACTGAATAACCCTCAGA 
                 1358 
                 X40 
               
               
                   
               
               
                 13 
                 113351476 
                 CTGAGAACTGAATAAACCTCGAG 
                 1359 
                 X60 
               
               
                   
               
               
                 14 
                 18485266 
                 AAGGTTACAGAATAAAACTCTGG 
                 1360 
                 X60 
               
               
                   
               
               
                 14 
                 20357994 
                 CAGGATACTGAATAACCCTCAGA 
                 1361 
                 X60 
               
               
                   
               
               
                 14 
                 21233079 
                 CCATTACATGAATAACCTTCAGG 
                 1362 
                 X60 
               
               
                   
               
               
                 14 
                 22348176 
                 AAGACACCTGGATAACCATCAGG 
                 1363 
                 X30 
               
               
                   
               
               
                 14 
                 26263145 
                 AAATTATGAAAAACCCTCAGG 
                 1364 
                 RNA32 
               
               
                   
               
               
                 14 
                 30878091 
                 AAGATACTTGACAACTCTCTGA 
                 1365 
                 RNA41 
               
               
                   
               
               
                 14 
                 31153973 
                 AAGATAGATGAATGACCCACTAC 
                 1366 
                 X60 
               
               
                   
               
               
                 14 
                 35231283 
                 AAGATACTTAAATGATCCTTGGC 
                 1367 
                 X60 
               
               
                   
               
               
                 14 
                 37392538 
                 AATATACCTAAATAACTTACAGC 
                 1368 
                 X60 
               
               
                   
               
               
                 14 
                 40710593 
                 AAGATAACTGGATAGCCATATGC 
                 1369 
                 X60 
               
               
                   
               
               
                 14 
                 40860782 
                 AACAAACATGAAAAACACTCAGC 
                 1370 
                 X60 
               
               
                   
               
               
                 14 
                 41931495 
                 AAGATACCTAAATATATATCTGA 
                 1371 
                 X60 
               
               
                   
               
               
                 14 
                 42141012 
                 AGGATGACTGAAAAATCCCCAGG 
                 1372 
                 X60 
               
               
                   
               
               
                 14 
                 49513652 
                 AAGATGACTGCTTAACCCCCAGT 
                 1373 
                 X60 
               
               
                   
               
               
                 14 
                 51112581 
                 AAGATACCAAAATGTAACTCAGG 
                 1374 
                 X60 
               
               
                   
               
               
                 14 
                 52885591 
                 AAGATAACTTAAAAACTCTAAGG 
                 1375 
                 X50 
               
               
                   
               
               
                 14 
                 52900831 
                 ACAGAACCTGAATAGCCCTAGGG 
                 1376 
                 X60 
               
               
                   
               
               
                 14 
                 53020328 
                 AAGATGCCTGAACTAGTCCTTGGG 
                 1377 
                 DNA41 
               
               
                   
               
               
                 14 
                 56099999 
                 AAAATAACTGAAAACCCTCAAC 
                 1378 
                 RNA41 
               
               
                   
               
               
                 14 
                 59879739 
                 AACATACATCCACAACCATCAGG 
                 1379 
                 X60 
               
               
                   
               
               
                 14 
                 64921531 
                 AAGATACATGAATAAGCACCATG 
                 1380 
                 X50 
               
               
                   
               
               
                 14 
                 66348379 
                 CATATACTTGAATAACCAACTGG 
                 1381 
                 X50 
               
               
                   
               
               
                 14 
                 68160233 
                 AAGATCTTAATAATCATCTGG 
                 1382 
                 RNA32 
               
               
                   
               
               
                 14 
                 72604917 
                 AAGAAACCCGAGTCCCTCTGG 
                 1383 
                 RNA32 
               
               
                   
               
               
                 14 
                 74660460 
                 AAGATATCTCATATCACTCAGG 
                 1384 
                 RNA41 
               
               
                   
               
               
                 14 
                 74867740 
                 ACAATACCTTGAATAACCCACAAG 
                 1385 
                 DNA41 
               
               
                   
               
               
                 14 
                 77627074 
                 AAGATACATAAATAACCTCACAG 
                 1386 
                 X60 
               
               
                   
               
               
                 14 
                 80214772 
                 AAGTAACAGAATAATCCTAAGG 
                 1387 
                 RNA41 
               
               
                   
               
               
                 14 
                 81206628 
                 GAATAACCTGAATAACCCCCAGA 
                 1388 
                 X60 
               
               
                   
               
               
                 14 
                 82199597 
                 AAATTACCTAAATAACCCAAGGC 
                 1389 
                 X60 
               
               
                   
               
               
                 14 
                 82935226 
                 AAGGTACCTGAATATTGTTCTGT 
                 1390 
                 X60 
               
               
                   
               
               
                 14 
                 84727246 
                 AAGACAGCTGGGTATCTCTCTGG 
                 1391 
                 X60 
               
               
                   
               
               
                 14 
                 87353900 
                 CTGATAGTGAATAAGCCTCAGG 
                 1392 
                 RNA41 
               
               
                   
               
               
                 14 
                 87564479 
                 ATGATAGTGAATAAGTCTCAGG 
                 1393 
                 RNA41 
               
               
                   
               
               
                 14 
                 90316482 
                 TAGATATTTGAATAACCATTTAG 
                 1394 
                 X60 
               
               
                   
               
               
                 14 
                 94330884 
                 AAGATGCCCTGAGCAACCCTCCTG 
                 1395 
                 DNA41 
               
               
                   
               
               
                 14 
                 100626641 
                 ATGATCTCTGAATGGCCTTCAGG 
                 1396 
                 X60 
               
               
                   
               
               
                 14 
                 104781000 
                 CGTGTACCTGAATGACCCACGGG 
                 1397 
                 X60 
               
               
                   
               
               
                 14 
                 106194663 
                 TAGATACTGAGAAAGCCTCTGG 
                 1398 
                 RNA41 
               
               
                   
               
               
                 15 
                 26338242 
                 AAGTTATCTAAAAACCCTCTAG 
                 1399 
                 RNA41 
               
               
                   
               
               
                 15 
                 26400480 
                 AAGATATCTGAAAAATTCTCAAA 
                 1400 
                 X60 
               
               
                   
               
               
                 15 
                 36010542 
                 AAGTAAACTGAAAACCTTCTGG 
                 1401 
                 RNA41 
               
               
                   
               
               
                 15 
                 37986317 
                 AATATAACTGAATAAAACTCTTT 
                 1402 
                 X60 
               
               
                   
               
               
                 15 
                 38076935 
                 TAGCTACATGAAAACCCTGGGG 
                 1403 
                 RNA41 
               
               
                   
               
               
                 15 
                 40609022 
                 ATAATACTGAATAACCCTTTGA 
                 1404 
                 RNA41 
               
               
                   
               
               
                 15 
                 43079813 
                 CTTTTACCTGAATACCCTCAGG 
                 1405 
                 RNA41 
               
               
                   
               
               
                 15 
                 44348352 
                 AAGATGACTGAAGAGCCCTTGGG 
                 1406 
                 X50 
               
               
                   
               
               
                 15 
                 49430075 
                 ATGAGCCCAGAAGAACCCTCTTG 
                 1407 
                 X60 
               
               
                   
               
               
                 15 
                 57756788 
                 GAGATACCTGAAAAATCCTGGCT 
                 1408 
                 X60 
               
               
                   
               
               
                 15 
                 58607272 
                 TGTATACCAGAATCACCCTAGGG 
                 1409 
                 X60 
               
               
                   
               
               
                 15 
                 60876750 
                 CAGATACCTGGTAACTCTCAGG 
                 1410 
                 RNA31 
               
               
                   
               
               
                 15 
                 61064287 
                 AAATTACCTGGACAACCTTCGCG 
                 1411 
                 X60 
               
               
                   
               
               
                 15 
                 63004747 
                 AAGATACATGAATAACCTTGTCA 
                 1412 
                 X50 
               
               
                   
               
               
                 15 
                 63156318 
                 AAGATACATTAGAACCCTCTGT 
                 1413 
                 RNA41 
               
               
                   
               
               
                 15 
                 69510497 
                 AAGATATATGAATGGCCCTGAAG 
                 1414 
                 X60 
               
               
                   
               
               
                 15 
                 71127689 
                 AAGATACACAAACAGCCCACAGG 
                 1415 
                 X60 
               
               
                   
               
               
                 15 
                 73446678 
                 AACATACCTGAAAAAACCTCAGC 
                 1416 
                 X40 
               
               
                   
               
               
                 15 
                 74253079 
                 AGGATTCCAGAAGAAGCCTGTGG 
                 1417 
                 X60 
               
               
                   
               
               
                 15 
                 75470662 
                 AAGAAACCTGGATTTACCTCTGT 
                 1418 
                 X60 
               
               
                   
               
               
                 15 
                 77101670 
                 AGAATAGCTGAAGGACCCTGGGG 
                 1419 
                 X60 
               
               
                   
               
               
                 15 
                 84875080 
                 AGGATGCCAGGGTAACCCACTGG 
                 1420 
                 X60 
               
               
                   
               
               
                 15 
                 85440610 
                 TAGGTACCTGAATGACCTCAGG 
                 1421 
                 RNA31 
               
               
                   
               
               
                 15 
                 85452623 
                 AAGATACCTGACATACCCTCCCC 
                 1422 
                 X50 
               
               
                   
               
               
                 15 
                 90002805 
                 GAGATACATGAATGACCCTTTCA 
                 1423 
                 X60 
               
               
                   
               
               
                 15 
                 94867496 
                 GTGATAGTGAATAACTCTCAGG 
                 1424 
                 RNA41 
               
               
                   
               
               
                 15 
                 96462615 
                 AGGAAAGCTGAATAACTCTCTTA 
                 1425 
                 X60 
               
               
                   
               
               
                 15 
                 96700894 
                 AAGAAACGCTAATAAACCTCAGG 
                 1426 
                 X50 
               
               
                   
               
               
                 15 
                 99174006 
                 AACATTCCACACTAACCATCAGG 
                 1427 
                 X60 
               
               
                   
               
               
                 16 
                 2950487 
                 AAGATACGAGAGAAACGCCCGGG 
                 1428 
                 X60 
               
               
                   
               
               
                 16 
                 7926633 
                 CAATTACCAGAATAACCCACAGA 
                 1429 
                 X60 
               
               
                   
               
               
                 16 
                 11910562 
                 AAGAGATTTGATAACCTTCTGG 
                 1430 
                 RNA41 
               
               
                   
               
               
                 16 
                 14200710 
                 AAGATTCCTGATTCAGCCTCTGA 
                 1431 
                 X50 
               
               
                   
               
               
                 16 
                 15502916 
                 AAGAAGCCAGACTAACACTGGGG 
                 1432 
                 X60 
               
               
                   
               
               
                 16 
                 15969961 
                 CAAATAAATGAATAATCATCAGG 
                 1433 
                 X60 
               
               
                   
               
               
                 16 
                 16048075 
                 AAGAGGCCTGACTCTCCCTCCAG 
                 1434 
                 X60 
               
               
                   
               
               
                 16 
                 17024068 
                 AAGATACTTGTATAACCTCAAGA 
                 1435 
                 X60 
               
               
                   
               
               
                 16 
                 17094952 
                 AACATACCTGAACAACCCTCAGG 
                 1436 
                 X20 
               
               
                   
               
               
                 16 
                 22614186 
                 AAGATATTTATATACCCCTCCAG 
                 1437 
                 X60 
               
               
                   
               
               
                 16 
                 24555817 
                 TAGTTACATGAAGAAACCTCTAG 
                 1438 
                 X60 
               
               
                   
               
               
                 16 
                 25905845 
                 AAGATACATGCGTGAAGCCTCTGG 
                 1439 
                 DNA41 
               
               
                   
               
               
                 16 
                 26382131 
                 ATGGTACCTGTACAAACTTCAGG 
                 1440 
                 X60 
               
               
                   
               
               
                 16 
                 27966927 
                 AAGGCACCTGAAAGCTCTCTGG 
                 1441 
                 RNA41 
               
               
                   
               
               
                 16 
                 35693983 
                 GAGATACCTGGAAAACCCCAGGC 
                 1442 
                 X60 
               
               
                   
               
               
                 16 
                 46657396 
                 ATGTTAACAGAAGACCCCTCTGG 
                 1443 
                 X60 
               
               
                   
               
               
                 16 
                 47691992 
                 TAGATAACTGGATACCCCACTGC 
                 1444 
                 X60 
               
               
                   
               
               
                 16 
                 49245974 
                 AAAATTCCTGTCCAGCCCTCTGG 
                 1445 
                 X60 
               
               
                   
               
               
                 16 
                 49984669 
                 AGGATGCCTGAAAAAACCTCAAA 
                 1446 
                 X60 
               
               
                   
               
               
                 16 
                 51935176 
                 AAGATACATGAGAACCCTTTGC 
                 1447 
                 RNA41 
               
               
                   
               
               
                 16 
                 52525199 
                 AAGATACATGAATAGCCCTCCAC 
                 1448 
                 X40 
               
               
                   
               
               
                 17 
                 5340518 
                 TAAATACCTGAATAACCCATAGT 
                 1449 
                 X50 
               
               
                   
               
               
                 17 
                 5857462 
                 TATATACATGTATAACCCACAGG 
                 1450 
                 X50 
               
               
                   
               
               
                 17 
                 6194882 
                 AAGATACATTCTTAGCCCTCAAG 
                 1451 
                 X60 
               
               
                   
               
               
                 17 
                 7932420 
                 AAGAAACCTTGTGAACCCTCAGT 
                 1452 
                 X60 
               
               
                   
               
               
                 17 
                 14407307 
                 AAGAAATGGATAGCCCTCTGG 
                 1453 
                 RNA32 
               
               
                   
               
               
                 17 
                 21332996 
                 AAAATAGCTTAAAAAGCCTGTGG 
                 1454 
                 X60 
               
               
                   
               
               
                 17 
                 27457685 
                 AGGGTGCCCCAACAACCCTCGGG 
                 1455 
                 X60 
               
               
                   
               
               
                 17 
                 28706042 
                 AAGAAATGTGGGTAAGCCTCAGG 
                 1456 
                 X60 
               
               
                   
               
               
                 17 
                 29645114 
                 ATCATATCTGAATAACTCTCAAC 
                 1457 
                 X60 
               
               
                   
               
               
                 17 
                 32458400 
                 AACAAGCCTGTATAACCCTCATG 
                 1458 
                 X50 
               
               
                   
               
               
                 17 
                 34969762 
                 GTGATACTGAATAAGTCTCAGG 
                 1459 
                 RNA41 
               
               
                   
               
               
                 17 
                 37872545 
                 AACATACATGAATAATCTTCAAA 
                 1460 
                 X60 
               
               
                   
               
               
                 17 
                 39300993 
                 ATTATACCTCAATATCCCTCTGG 
                 1461 
                 X40 
               
               
                   
               
               
                 17 
                 44931191 
                 AAGATACATGAAAAACACTGGCT 
                 1462 
                 X60 
               
               
                   
               
               
                 17 
                 47455028 
                 AACATACATGAATAATCTTCAAA 
                 1460 
                 X60 
               
               
                   
               
               
                 17 
                 47541148 
                 AAGATACCTGTACAACACTATAC 
                 1463 
                 X60 
               
               
                   
               
               
                 17 
                 54308512 
                 CAGATACCTCATTTACCCTGATG 
                 1464 
                 X60 
               
               
                   
               
               
                 17 
                 65806019 
                 CAGATACTTATAACCCTCTGG 
                 1465 
                 RNA22 
               
               
                   
               
               
                 17 
                 67990176 
                 AAAATACTTGAAAAATCCCTCAGT 
                 1466 
                 DNA41 
               
               
                   
               
               
                 17 
                 70972434 
                 AAGATCAAGAATAACTGTCAGG 
                 1467 
                 RNA41 
               
               
                   
               
               
                 17 
                 81310828 
                 AAGACACTTGTCTAGTCCTCTGG 
                 1468 
                 X60 
               
               
                   
               
               
                 18 
                 8119646 
                 AAGAATCCGGAAAAACCCTATGC 
                 1469 
                 X60 
               
               
                   
               
               
                 18 
                 10478571 
                 AAGACAGCTGGAGAACTCTCAGG 
                 1470 
                 X50 
               
               
                   
               
               
                 18 
                 11443150 
                 AAGAAAAATGAACAAACCTCAGA 
                 1471 
                 X60 
               
               
                   
               
               
                 18 
                 11651294 
                 AAGTTACGTGAATAGGACTCAGT 
                 1472 
                 X60 
               
               
                   
               
               
                 18 
                 13336259 
                 AAGATAAAGAACAACTCTCTGG 
                 1473 
                 RNA41 
               
               
                   
               
               
                 18 
                 21942917 
                 CAGATACACAGATAACCCCCAGG 
                 1474 
                 X60 
               
               
                   
               
               
                 18 
                 24883127 
                 AAGAAACTTGAAAAATCCCAGG 
                 1475 
                 RNA41 
               
               
                   
               
               
                 18 
                 35027674 
                 CAGATAACAGAAAAACCCTGTTG 
                 1476 
                 X60 
               
               
                   
               
               
                 18 
                 36792668 
                 AAGATACCCAGATCCCCCTCCAG 
                 1477 
                 X60 
               
               
                   
               
               
                 18 
                 36915438 
                 TAGATATTCTGAATATACCTCTGG 
                 1478 
                 DNA41 
               
               
                   
               
               
                 18 
                 39891882 
                 CAGATACATGAAATAATCCTCCAG 
                 1479 
                 DNA41 
               
               
                   
               
               
                 18 
                 40348238 
                 AGTCATCCTGAATAACCCTCATG 
                 1480 
                 X60 
               
               
                   
               
               
                 18 
                 47612935 
                 GCCATACCAAATAACCCTCTGG 
                 1481 
                 RNA41 
               
               
                   
               
               
                 18 
                 49237013 
                 AAGATACCTGGATAAGCAACTGC 
                 1482 
                 X50 
               
               
                   
               
               
                 18 
                 49759577 
                 AAGAAACCTATAGAACCCTGAGT 
                 1483 
                 X60 
               
               
                   
               
               
                 18 
                 52354807 
                 AAGAAAACAGAATAAACCTCTAA 
                 1484 
                 X60 
               
               
                   
               
               
                 18 
                 53136651 
                 AAGATACCTTATCAACCCTAAAG 
                 1485 
                 X50 
               
               
                   
               
               
                 18 
                 54113085 
                 CAGAAATATAAAAAACCCTCAGG 
                 1486 
                 X60 
               
               
                   
               
               
                 18 
                 55667190 
                 AAGATTCAAGAAAGACCCTCTGC 
                 1487 
                 X60 
               
               
                   
               
               
                 18 
                 56622476 
                 TAAATACTGAATAACCCTGTGA 
                 1488 
                 RNA41 
               
               
                   
               
               
                 18 
                 58463120 
                 GAGATCTCAGCATAACTCTCTGG 
                 1489 
                 X60 
               
               
                   
               
               
                 18 
                 66313025 
                 AAGATACCTTATTTACCTATGG 
                 1490 
                 RNA41 
               
               
                   
               
               
                 18 
                 68281088 
                 TTCATACCTAAATAACCCTTAGA 
                 1491 
                 X60 
               
               
                   
               
               
                 18 
                 69322487 
                 AAGATACCTGAATAACCTAAGAC 
                 1492 
                 X50 
               
               
                   
               
               
                 18 
                 74469327 
                 AAGATACATAATAATCATCTGT 
                 1493 
                 RNA41 
               
               
                   
               
               
                 18 
                 77464690 
                 AAGAGCCCTTGCTAACCATCTGG 
                 1494 
                 X60 
               
               
                   
               
               
                 19 
                 11998269 
                 AAAGTATCTGAATAACACTTCTG 
                 1495 
                 X60 
               
               
                   
               
               
                 19 
                 12455264 
                 AGCCTACCTAAATAGCCCTAGGG 
                 1496 
                 X60 
               
               
                   
               
               
                 19 
                 27693648 
                 CAGACACCTGAATGACCCCAGGC 
                 1497 
                 X60 
               
               
                   
               
               
                 19 
                 28546228 
                 CACTGACCTGAATCACCCCCAGG 
                 1498 
                 X60 
               
               
                   
               
               
                 19 
                 37641018 
                 GAAATACTGAATAATCCTCAGG 
                 1499 
                 RNA31 
               
               
                   
               
               
                 19 
                 38143521 
                 AAGACACAGAAAGATCCCTCTGG 
                 1500 
                 X60 
               
               
                   
               
               
                 19 
                 42798163 
                 AAGGTACCAGATGAGCCCTTGGG 
                 1501 
                 X60 
               
               
                   
               
               
                 19 
                 45644796 
                 AGGATACCTCCCAAATCCTCTGG 
                 1502 
                 X60 
               
               
                   
               
               
                 19 
                 46147476 
                 CAAATACTTGAAAAGCCCTCGAG 
                 1503 
                 X60 
               
               
                   
               
               
                 19 
                 52963761 
                 TAAATATGAATGACCCTCAGG 
                 1504 
                 RNA32 
               
               
                   
               
               
                 19 
                 53485175 
                 CAGATATCTGAAGATCCCTCCAG 
                 1505 
                 X50 
               
               
                   
               
               
                 20 
                 1471262 
                 ACAGAACCTGTATAACCCTCCAG 
                 1506 
                 X60 
               
               
                   
               
               
                 20 
                 2545286 
                 AAGAAATCTAGAATACCCCTAGGG 
                 1507 
                 DNA41 
               
               
                   
               
               
                 20 
                 6093655 
                 AAGTTAAATGAATGACCCTGACCGG 
                 1508 
                 DNA42 
               
               
                   
               
               
                 20 
                 7232430 
                 AAGATATTTGATCAACCCTTGGA 
                 1509 
                 X60 
               
               
                   
               
               
                 20 
                 10350390 
                 AGGTAACATCAATATCCCTCCGG 
                 1510 
                 X60 
               
               
                   
               
               
                 20 
                 12411793 
                 AACGTACCTGAATAACTCATAAG 
                 1511 
                 X60 
               
               
                   
               
               
                 20 
                 13449167 
                 AAGATACCAGCATAGTCCTCCTG 
                 1512 
                 X50 
               
               
                   
               
               
                 20 
                 13559179 
                 AAGATATCTGAAGAGCCTTCCCA 
                 1513 
                 X60 
               
               
                   
               
               
                 20 
                 18449496 
                 AAGATACCTGAATTTACCATGAG 
                 1514 
                 X60 
               
               
                   
               
               
                 20 
                 19443923 
                 AAGATACTTTATTAACTCTCAAG 
                 1515 
                 X50 
               
               
                   
               
               
                 20 
                 22704701 
                 TAGATATGTGAATTAACTTCTGG 
                 1516 
                 X60 
               
               
                   
               
               
                 20 
                 23658111 
                 AAGATACCTGATCACACATCAGA 
                 1517 
                 X60 
               
               
                   
               
               
                 20 
                 37569891 
                 CAGATACCTCAATAACCCTGATG 
                 1518 
                 X40 
               
               
                   
               
               
                 20 
                 44858917 
                 AAGATACTTGGATAATGCTTGGT 
                 1519 
                 X60 
               
               
                   
               
               
                 20 
                 45794486 
                 AAGGTATCTGAATTACCCTCAAG 
                 1520 
                 X40 
               
               
                   
               
               
                 20 
                 52287275 
                 CTGAGACCTGAGTAACTCTCATG 
                 1521 
                 X60 
               
               
                   
               
               
                 20 
                 56292197 
                 AAGAAAATGAATAACCCTACAG 
                 1522 
                 RNA41 
               
               
                   
               
               
                 20 
                 60198378 
                 TAGATACATGAAAAACCCTTCAG 
                 1523 
                 X50 
               
               
                   
               
               
                 20 
                 60509505 
                 TAGATGCTTAAATGACCCTCTGC 
                 1524 
                 X60 
               
               
                   
               
               
                 21 
                 17021098 
                 TAGGATCCTGGATAACCCTCCAG 
                 1525 
                 X60 
               
               
                   
               
               
                 21 
                 17566032 
                 TTGATACTTGAATAACCATCTGA 
                 1526 
                 X50 
               
               
                   
               
               
                 21 
                 17907925 
                 AAGAAACCAATAACCCTCAGG 
                 1527 
                 RNA12 
               
               
                   
               
               
                 21 
                 19274949 
                 AAGGAACCAGAATTACTCTCAGT 
                 1528 
                 X60 
               
               
                   
               
               
                 21 
                 24464330 
                 CAGAAACCAGAATAACCTTCAGG 
                 1529 
                 X40 
               
               
                   
               
               
                 21 
                 24537041 
                 AAAATTCCTGAATAGCTCTCTGG 
                 1530 
                 X40 
               
               
                   
               
               
                 21 
                 26666529 
                 TAGAGACATGAAAAACCCTCCAA 
                 836 
                 X60 
               
               
                   
               
               
                 21 
                 30710727 
                 ATGATAGTGAATAACACTCATG 
                 1531 
                 RNA41 
               
               
                   
               
               
                 21 
                 30757521 
                 AGGATACAGAATATCCATCTGG 
                 1532 
                 RNA41 
               
               
                   
               
               
                 21 
                 31767749 
                 AATCTACCTGAAAAGCCCTCTGG 
                 1533 
                 X40 
               
               
                   
               
               
                 21 
                 33249604 
                 CTGATACCAGATAACCCTGAGG 
                 1534 
                 RNA41 
               
               
                   
               
               
                 21 
                 45566319 
                 CAGATATCTGAATAACCCACCAG 
                 1535 
                 X40 
               
               
                   
               
               
                 21 
                 46274280 
                 AAGATACATGGATAATGTTCAGG 
                 1536 
                 X50 
               
               
                   
               
               
                 21 
                 46379614 
                 AAGAAACATGAAGAAAACTCAGC 
                 1537 
                 X60 
               
               
                   
               
               
                 22 
                 15415612 
                 AAGGTTACAGAATAAAACTCTGG 
                 1360 
                 X60 
               
               
                   
               
               
                 22 
                 16210764 
                 AAAATACATGGAAAATCCTCATG 
                 1538 
                 X60 
               
               
                   
               
               
                 22 
                 18016369 
                 ATTATACCTGAATAAACCTGACT 
                 1539 
                 X60 
               
               
                   
               
               
                 22 
                 22356999 
                 GATTTACCAGAAAAACCCTCTGG 
                 1540 
                 X50 
               
               
                   
               
               
                 22 
                 22409053 
                 GATTGACCAGAATAACCCTCTGG 
                 1541 
                 X50 
               
               
                   
               
               
                 22 
                 26261319 
                 AAGATACATCGAATAACCCTTCTA 
                 1542 
                 DNA41 
               
               
                   
               
               
                 22 
                 31956781 
                 AAGGCAACTGAAGATCCTTCAGG 
                 1543 
                 X60 
               
               
                   
               
               
                 22 
                 48095647 
                 AAGATATCTGAATTCAGCTCTTG 
                 1544 
                 X60 
               
               
                   
               
               
                 X 
                 3121703 
                 AAGAAAACAGAATAAACCTCTCA 
                 1545 
                 X60 
               
               
                   
               
               
                 X 
                 3288787 
                 AAGATAGCTGATGAAATATCAGG 
                 1546 
                 X60 
               
               
                   
               
               
                 X 
                 6286346 
                 ATGATAGTGAATAAGCCTCAGG 
                 1547 
                 RNA31 
               
               
                   
               
               
                 X 
                 15276107 
                 AAGATTCATGTACAAACCTCATG 
                 1548 
                 X60 
               
               
                   
               
               
                 X 
                 18754961 
                 AAGACACATGAAGAAAACTGAGG 
                 1549 
                 X60 
               
               
                   
               
               
                 X 
                 19389428 
                 AAGTTGACTGAAACCCTCTGG 
                 1550 
                 RNA32 
               
               
                   
               
               
                 X 
                 25123524 
                 GAGATACCCTAGATAACCCTCAGA 
                 1551 
                 DNA41 
               
               
                   
               
               
                 X 
                 33500389 
                 GTGATAACTGAATAACCCTGCTC 
                 1552 
                 X60 
               
               
                   
               
               
                 X 
                 57574816 
                 GAGATAACTGCATAGCCCTAGGT 
                 1553 
                 X60 
               
               
                   
               
               
                 X 
                 58085053 
                 GAGACACCTGAACGACCTCAGG 
                 1554 
                 RNA41 
               
               
                   
               
               
                 X 
                 58098997 
                 AAGACACCTGACTAACCCCAGGC 
                 1555 
                 X50 
               
               
                   
               
               
                 X 
                 58100889 
                 AAGACACCTGACTAACCCCAGGC 
                 1555 
                 X50 
               
               
                   
               
               
                 X 
                 58123415 
                 TAGACACCTGAACAACCCCAGGC 
                 1556 
                 X60 
               
               
                   
               
               
                 X 
                 58198267 
                 AAGACACCTGGCTGACCCCAGG 
                 1557 
                 RNA41 
               
               
                   
               
               
                 X 
                 58199277 
                 AAGACACCTGAGCAACCTCAGG 
                 1558 
                 RNA31 
               
               
                   
               
               
                 X 
                 58401983 
                 AAGATATCTGAGCAACACTCTGT 
                 1559 
                 X50 
               
               
                   
               
               
                 X 
                 63325513 
                 GAGATATGTGAATAAAACTCTGA 
                 1560 
                 X60 
               
               
                   
               
               
                 X 
                 63786600 
                 CAGAAGCTGAATGACCCTTTGG 
                 1561 
                 RNA41 
               
               
                   
               
               
                 X 
                 63945361 
                 AAAATAACTGATAAATCCTCTGA 
                 1562 
                 X60 
               
               
                   
               
               
                 X 
                 65679966 
                 AAGCTCCTGCATAGCCCACAGG 
                 1563 
                 RNA41 
               
               
                   
               
               
                 X 
                 70140322 
                 AACATACCTAAATATACCCTGG 
                 1564 
                 RNA41 
               
               
                   
               
               
                 X 
                 70754250 
                 AAGCTGACTGAAGAGCCCTTGGG 
                 894 
                 X60 
               
               
                   
               
               
                 X 
                 72198434 
                 TAGATACCTGACTTTCCCACTTG 
                 1565 
                 X60 
               
               
                   
               
               
                 X 
                 73418101 
                 AAGATTCCTGAAGCCTCCTCAGA 
                 1566 
                 X60 
               
               
                   
               
               
                 X 
                 73819519 
                 CATATACTGAATAACCATCTGG 
                 1567 
                 RNA31 
               
               
                   
               
               
                 X 
                 84823869 
                 AAGATATCTGAAATGCCCTAGAG 
                 1568 
                 X60 
               
               
                   
               
               
                 X 
                 89194879 
                 AAATAGCCTGAATAACCCTAGTG 
                 1569 
                 X60 
               
               
                   
               
               
                 X 
                 89316606 
                 AACAAACATGAATAACACTAAGC 
                 1570 
                 X60 
               
               
                   
               
               
                 X 
                 91030898 
                 AAGAAATCTGACAAACCTTTAGG 
                 1571 
                 X60 
               
               
                   
               
               
                 X 
                 91177495 
                 GTGATACTGAATAAGTCTCAGG 
                 1459 
                 RNA41 
               
               
                   
               
               
                 X 
                 99969578 
                 AAGATCCAGAAGAACTCTCTGA 
                 1572 
                 RNA41 
               
               
                   
               
               
                 X 
                 107292941 
                 TAGATACCTAAATAGCCCATGGG 
                 1573 
                 X50 
               
               
                   
               
               
                 X 
                 108681302 
                 AAGAAACCTCTATAAGCCTCTTA 
                 1574 
                 X60 
               
               
                   
               
               
                 X 
                 109943949 
                 AATCTACTGAATAACTCTCAGG 
                 1575 
                 RNA31 
               
               
                   
               
               
                 X 
                 111810669 
                 ATGAATACTGAATAACATTCAGG 
                 1576 
                 X60 
               
               
                   
               
               
                 X 
                 113640910 
                 AAGATACCAATAACCCTCAAA 
                 1577 
                 RNA22 
               
               
                   
               
               
                 X 
                 117399450 
                 AATGTACCCGAACAACCCTCAGG 
                 1578 
                 X40 
               
               
                   
               
               
                 X 
                 120228774 
                 CAGATAGCTGAAAGAACACTCAGG 
                 1579 
                 DNA41 
               
               
                   
               
               
                 X 
                 125113165 
                 AACATAACTGAATAACCATAAGG 
                 1580 
                 X40 
               
               
                   
               
               
                 X 
                 126715614 
                 AATCAACCTGAATATCCATCAGT 
                 1581 
                 X60 
               
               
                   
               
               
                 X 
                 130340267 
                 AAGATACCCATTCACCCTCTGG 
                 1582 
                 RNA31 
               
               
                   
               
               
                 X 
                 130463899 
                 AAAGAACCTAAAAAACCCTCTTG 
                 1583 
                 X60 
               
               
                   
               
               
                 X 
                 132229997 
                 AAGATAGGGGAAAGCCCTCTGG 
                 1584 
                 RNA41 
               
               
                   
               
               
                 X 
                 138021160 
                 AAGGCAAATGAATAACCCACAGC 
                 1585 
                 X60 
               
               
                   
               
               
                 X 
                 139830410 
                 AAAATACAAATAACCCTCAGT 
                 1586 
                 RNA32 
               
               
                   
               
               
                 X 
                 144953782 
                 AAGATCCAGAAGAATTCTCTGG 
                 1587 
                 RNA41 
               
               
                   
               
               
                 X 
                 150328565 
                 AGAATACCTGAATACCCCCAGA 
                 1588 
                 RNA41 
               
               
                   
               
               
                 X 
                 154034869 
                 CAAATACCTTAATCACCATGAGG 
                 1589 
                 X60 
               
               
                   
               
               
                 Y 
                 4605941 
                 AAGAAATCTGACAAACCTTTAGG 
                 1571 
                 X60 
               
               
                   
               
               
                 Y 
                 4641256 
                 GTGATACTGAATAAGTCTCAGG 
                 1459 
                 RNA41 
               
               
                   
               
               
                 Y 
                 6194908 
                 AAGATGCCAACATAAAGCTCAGG 
                 1590 
                 X60 
               
               
                   
               
               
                 Y 
                 13359292 
                 TAAATACCTATAACCCTGAGG 
                 1591 
                 RNA32 
               
               
                   
               
               
                 Y 
                 16565563 
                 AGCACACATGAATAACCCTAAGG 
                 1592 
                 X50 
               
               
                   
               
               
                 Y 
                 22230617 
                 AAGATCCCAGAATAACTACTGG 
                 1593 
                 RNA41 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 ANGPTL3 gRNA off-target site validation 
               
               
                 in human primary hepatocytes 
               
            
           
           
               
               
            
               
                   
                 Editing % 
               
            
           
           
               
               
               
               
               
            
               
                 Chromosome 
                 Location 
                 treated 
                 control 
                 Net Editing % 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 62604219 
                 61.93 
                 0.39 
                 61.54 
               
               
                 1 
                 2.28E+08 
                 0.75 
                 0.81 
                 −0.06 
               
               
                 1 
                   2E+08 
                 0.1 
                 0.03 
                 0.07 
               
               
                 1 
                 59422147 
                 0.32 
                 0.4 
                 −0.08 
               
               
                 1 
                 76436411 
                 0.3 
                 0.26 
                 0.04 
               
               
                 1 
                 19936662 
                 0.26 
                 0.2 
                 0.06 
               
               
                 1 
                 1.8E+08 
                 0.47 
                 0.55 
                 −0.08 
               
               
                 2 
                 1.7E+08 
                 0.18 
                 0.1 
                 0.08 
               
               
                 2 
                 17555591 
                 0.09 
                 0.12 
                 −0.03 
               
               
                 2 
                 87443936 
                 0.27 
                 0.26 
                 0.01 
               
               
                 2 
                 2.26E+08 
                 0.13 
                 0.19 
                 −0.06 
               
               
                 2 
                 1.22E+08 
                 0.48 
                 0.62 
                 −0.14 
               
               
                 2 
                 1.84E+08 
                 0.26 
                 0.36 
                 −0.1 
               
               
                 2 
                 1.88E+08 
                 0.26 
                 0.22 
                 0.04 
               
               
                 3 
                  8738135 
                 0.28 
                 0.23 
                 0.05 
               
               
                 3 
                  539550 
                 1.24 
                 0.44 
                 0.8 
               
               
                 3 
                 1.21E+08 
                 0.26 
                 0.27 
                 −0.01 
               
               
                 3 
                 99319950 
                 0.21 
                 0.22 
                 −0.01 
               
               
                 4 
                 1.73E+08 
                 0.22 
                 0.22 
                 0 
               
               
                 4 
                 1.44E+08 
                 0.48 
                 0.54 
                 −0.06 
               
               
                 4 
                 51965380 
                 0.09 
                 0.08 
                 0.01 
               
               
                 4 
                 1.01E+08 
                 0.09 
                 0.08 
                 0.01 
               
               
                 4 
                 26200297 
                 0.14 
                 0.11 
                 0.03 
               
               
                 4 
                 74170265 
                 0.54 
                 0.3 
                 0.24 
               
               
                 5 
                 1.14E+08 
                 0.16 
                 0.13 
                 0.03 
               
               
                 5 
                 1.56E+08 
                 0.47 
                 0.59 
                 −0.12 
               
               
                 5 
                  8704833 
                 0 
                 0 
                 0 
               
               
                 5 
                 16404802 
                 0.15 
                 0.19 
                 −0.04 
               
               
                 6 
                 1.13E+08 
                 0.09 
                 0.11 
                 −0.02 
               
               
                 6 
                 45506487 
                 0.3 
                 0.36 
                 −0.06 
               
               
                 6 
                 89636030 
                 0.28 
                 0.3 
                 −0.02 
               
               
                 6 
                 18241159 
                 0.08 
                 0.1 
                 −0.02 
               
               
                 6 
                 1.05E+08 
                 0.45 
                 0.39 
                 0.06 
               
               
                 6 
                 84651006 
                 0.15 
                 0.11 
                 0.04 
               
               
                 6 
                 1.26E+08 
                 0.17 
                 0.14 
                 0.03 
               
               
                 6 
                 1.34E+08 
                 0.23 
                 0.29 
                 −0.06 
               
               
                 6 
                 22811364 
                 0.3 
                 0.31 
                 −0.01 
               
               
                 6 
                  6543792 
                 0.08 
                 0.15 
                 −0.07 
               
               
                 6 
                  3993855 
                 0.39 
                 0.45 
                 −0.06 
               
               
                 6 
                 51857715 
                 0.19 
                 0.29 
                 −0.1 
               
               
                 6 
                 90026983 
                 0.28 
                 0.31 
                 −0.03 
               
               
                 6 
                 1.48E+08 
                 0.15 
                 0.17 
                 −0.02 
               
               
                 6 
                 1.46E+08 
                 0.32 
                 0.35 
                 −0.03 
               
               
                 7 
                 1.58E+08 
                 0.5 
                 0.45 
                 0.05 
               
               
                 7 
                 1.05E+08 
                 0.4 
                 0.61 
                 −0.21 
               
               
                 7 
                 18326887 
                 0.03 
                 0 
                 0.03 
               
               
                 7 
                 81886792 
                 0.23 
                 0.47 
                 −0.24 
               
               
                 7 
                 1.24E+08 
                 0.62 
                 0.44 
                 0.18 
               
               
                 7 
                 1.14E+08 
                 0.41 
                 0 
                 0.41 
               
               
                 8 
                 66947451 
                 17.71 
                 0.67 
                 17.04 
               
               
                 8 
                 19687879 
                 0.19 
                 0.26 
                 −0.07 
               
               
                 8 
                 76479079 
                 0.44 
                 0.44 
                 0 
               
               
                 8 
                 1.09E+08 
                 0.14 
                 0.23 
                 −0.09 
               
               
                 8 
                 88225407 
                 0.44 
                 0.57 
                 −0.13 
               
               
                 8 
                  1.4E+08 
                 0.29 
                 0.34 
                 −0.05 
               
               
                 8 
                 1.22E+08 
                 0.42 
                 0.57 
                 −0.15 
               
               
                 9 
                 89929285 
                 0.15 
                 0.21 
                 −0.06 
               
               
                 9 
                 1.01E+08 
                 0.41 
                 0.49 
                 −0.08 
               
               
                 10 
                 32570562 
                 0.17 
                 0.23 
                 −0.06 
               
               
                 11 
                  2376222 
                 0.05 
                 0.11 
                 −0.06 
               
               
                 11 
                 58705310 
                 0.36 
                 0.39 
                 −0.03 
               
               
                 11 
                 80186841 
                 0.24 
                 0.24 
                 0 
               
               
                 11 
                 12052022 
                 0.35 
                 0.32 
                 0.03 
               
               
                 12 
                 26607410 
                 0.41 
                 0.46 
                 −0.05 
               
               
                 12 
                 64120945 
                 0.26 
                 0.27 
                 −0.01 
               
               
                 12 
                 98856708 
                 0.34 
                 0.42 
                 −0.08 
               
               
                 12 
                 71164415 
                 0.42 
                 0.37 
                 0.05 
               
               
                 13 
                 60126783 
                 0.23 
                 0.22 
                 0.01 
               
               
                 13 
                 1.12E+08 
                 0.59 
                 0.76 
                 −0.17 
               
               
                 14 
                 20357994 
                 2.53 
                 0.43 
                 2.1 
               
               
                 14 
                 66348379 
                 0.21 
                 0.22 
                 −0.01 
               
               
                 14 
                 81206628 
                 0.28 
                 0.28 
                 0 
               
               
                 14 
                 22348176 
                 0.74 
                 0.68 
                 0.06 
               
               
                 14 
                 76759328 
                 0.07 
                 0.04 
                 0.03 
               
               
                 15 
                 94867496 
                 0.32 
                 0.42 
                 −0.1 
               
               
                 15 
                 60876750 
                 0.3 
                 0.27 
                 0.03 
               
               
                 15 
                 43079813 
                 100 
                 99.93 
                 0.07 
               
               
                 16 
                 17094952 
                 7.87 
                 1.57 
                 6.3 
               
               
                 16 
                 52525199 
                 0.83 
                 0.69 
                 0.14 
               
               
                 17 
                 39300993 
                 0.08 
                 0.11 
                 −0.03 
               
               
                 18 
                 21942917 
                 0.48 
                 0.65 
                 −0.17 
               
               
                 18 
                 47612935 
                 0.24 
                 0.3 
                 −0.06 
               
               
                 19 
                 37641018 
                 0.4 
                 0.34 
                 0.06 
               
               
                 20 
                 60198378 
                 0.4 
                 0.35 
                 0.05 
               
               
                 21 
                 45566319 
                 0.26 
                 0.35 
                 −0.09 
               
               
                 21 
                 17566032 
                 0.15 
                 0.18 
                 −0.03 
               
               
                 21 
                 17021098 
                 0.19 
                 0.17 
                 0.02 
               
               
                 22 
                 18016369 
                 0.08 
                 0.21 
                 −0.13 
               
               
                 X 
                 1.07E+08 
                 0.25 
                 0.29 
                 −0.04 
               
               
                 X 
                 1.17E+08 
                 0.24 
                 0.2 
                 0.04 
               
               
                 X 
                 21471705 
                 0.42 
                 0.4 
                 0.02 
               
               
                 X 
                 73819519 
                 0.25 
                 0.3 
                 −0.05 
               
               
                 X 
                  6286346 
                 0.58 
                 0.83 
                 −0.25 
               
               
                 X 
                 1.14E+08 
                 0.38 
                 0.38 
                 0 
               
               
                   
               
            
           
         
       
     
     Modifications and/or truncations to either the spacer or tracr portion of the gRNA was assessed by alteration of the gRNA GA100 spacer (5′-AAGATACCTGAATAACCCTC-3′ (SEQ TD NO: 15)) (Table 15). The modifications to the guide can serve to improve on-target editing efficiency and/or improve off-target editing efficiency. Additionally, four different ABE8.8 mRNAs were assessed (MA004, MA040, MA041, MA045; Table 23), in two different experiments (separated in the table below). Each of the gRNAs with an equivalent amount of in vitro transcribed ABE8.8 mRNA (1:1 ratio by weight) were co-transfected into primary human hepatocytes and primary cynomolgus hepatocytes at 5000, 2500, and 1250 ng/RNA/mL and processed as described. 
     
       
         
           
               
             
               
                 TABLE 15 
               
               
                   
               
               
                 gRNA and/or mRNA modifications improve gRNA specificity 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Human Primary Hepatocytes 
               
               
                   
                   
                   
                   
                 Editing % at Position 6 
               
               
                   
                   
                   
                   
                 (Dose, Replicate #) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Protospacer  
                 SEQ ID 
                   
                 5000, 
                 5000, 
                 2500, 
                 2500, 
                 1250, 
                 1250, 
               
               
                 gRNA 
                 (5′-3′) 
                 NO: 
                 mRNA 
                 rep 1 
                 rep 2 
                 rep 1 
                 rep 2 
                 rep 1 
                 rep 2 
               
               
                   
               
               
                 GA441 
                 AAGATACCTG 
                 15 
                 MA004 
                 ND 
                 47.29 
                 52.04 
                 49.69 
                 41.45 
                 42.72 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA442 
                 AAGATACCTG 
                 15 
                 MA004 
                 35.34 
                 32.48 
                 35.2 
                 35.78 
                 31.54 
                 27.9 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA472 
                 AAGATACCTG 
                 15 
                 MA004 
                 37.65 
                 36.08 
                 37.98 
                 32.46 
                 28.85 
                 32.1 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA473 
                 AAGATACCTG 
                 15 
                 MA004 
                 37.9 
                 31.23 
                 37.55 
                 35.45 
                 28.07 
                 26.92 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA474 
                 AAGATACCTG 
                 15 
                 MA004 
                 30.59 
                 39.55 
                 37.46 
                 37.26 
                 28.79 
                 25.37 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA475 
                 AGATACCTGA 
                 248 
                 MA004 
                 36.82 
                 37.79 
                 37.96 
                 40.49 
                 28.11 
                 28.81 
               
               
                   
                 ATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA476 
                 GATACCTGAAT 
                 249 
                 MA004 
                 37.2 
                 37.7 
                 42.27 
                 40.86 
                 31.97 
                 31.48 
               
               
                   
                 AACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA477 
                 ATACCTGAATA 
                 250 
                 MA004 
                 25.59 
                 24.9 
                 27.3 
                 25.25 
                 19.23 
                 19.19 
               
               
                   
                 ACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA441 
                 AAGATACCTG 
                 15 
                 MA040 
                 43.09 
                 45.18 
                 29.68 
                 47.56 
                 41.53 
                 35.98 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA442 
                 AAGATACCTG 
                 15 
                 MA040 
                 41.76 
                 43.74 
                 41.43 
                 41.19 
                 31.04 
                 32.85 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA472 
                 AAGATACCTG 
                 15 
                 MA040 
                 41.15 
                 42.31 
                 40.66 
                 40.5 
                 31.38 
                 28.23 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA473 
                 AAGATACCTG 
                 15 
                 MA040 
                 39.8 
                 39 
                 40.85 
                 34.58 
                 29.19 
                 28.19 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA474 
                 AAGATACCTG 
                 15 
                 MA040 
                 38.99 
                 33.48 
                 30.43 
                 34.63 
                 24.83 
                 22.76 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA475 
                 AGATACCTGA 
                 248 
                 MA040 
                 37.64 
                 39.98 
                 37.03 
                 39.31 
                 28.67 
                 26.83 
               
               
                   
                 ATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA476 
                 GATACCTGAAT 
                 249 
                 MA040 
                 41.08 
                 39.14 
                 37.69 
                 38.01 
                 28.8 
                 27.26 
               
               
                   
                 AACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA477 
                 ATACCTGAATA 
                 250 
                 MA040 
                 20.51 
                 19.43 
                 22.64 
                 22.01 
                 15.69 
                 16.61 
               
               
                   
                 ACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA441 
                 AAGATACCTG 
                 15 
                 MA041 
                 28.97 
                 20.87 
                 31.35 
                 37.98 
                 27.9 
                 27.48 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA442 
                 AAGATACCTG 
                 15 
                 MA041 
                 26.39 
                 25.73 
                 29.01 
                 27.38 
                 20.18 
                 20.53 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA472 
                 AAGATACCTG 
                 15 
                 MA041 
                 27.32 
                 29.91 
                 28.54 
                 28.29 
                 23.4 
                 23.15 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA473 
                 AAGATACCTG 
                 15 
                 MA041 
                 19.75 
                 19.89 
                 19.6 
                 17.62 
                 14.97 
                 14.04 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA474 
                 AAGATACCTG 
                 15 
                 MA041 
                 22.56 
                 20.87 
                 18.86 
                 17.38 
                 15.4 
                 14.14 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA475 
                 AGATACCTGA 
                 248 
                 MA041 
                 31.47 
                 29.18 
                 27.34 
                 23.98 
                 17.67 
                 18.89 
               
               
                   
                 ATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA476 
                 GATACCTGAAT 
                 249 
                 MA041 
                 31.01 
                 33.84 
                 29.37 
                 28.62 
                 19.77 
                 21.75 
               
               
                   
                 AACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA477 
                 ATACCTGAATA 
                 250 
                 MA041 
                 3.7 
                 2.53 
                 3.92 
                 3.47 
                 2.96 
                 3.24 
               
               
                   
                 ACCCTC 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Human Primary Hepatocytes  Editing % 
               
               
                 Verve 
                   
                   
                 Verve 
                 at Position 6 (Dose, Replicate #) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 gRNA 
                 Protospacer 
                 SEQ ID 
                 mRNA 
                 5000, 
                 5000, 
                 2500, 
                 2500, 
                 1250, 
                 1250, 
               
               
                 ID 
                 (5′-3′) 
                 NO: 
                 ID 
                 rep 1 
                 rep 2 
                 rep 1 
                 rep 2 
                 rep 1 
                 rep 2 
               
               
                   
               
               
                 GA441 
                 AAGATACCTG 
                 15 
                 MA004 
                 33.26 
                 34.23 
                 42 
                 36.89 
                 34.57 
                 31.5 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA442 
                 AAGATACCTG 
                 15 
                 MA004 
                 27.1 
                 35.52 
                 36.03 
                 32.59 
                 28.21 
                 32.14 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA472 
                 AAGATACCTG 
                 15 
                 MA004 
                 32.58 
                 22.71 
                 27.15 
                 38.51 
                 29.44 
                 32.3 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA473 
                 AAGATACCTG 
                 15 
                 MA004 
                 28.63 
                 27.84 
                 18.2 
                 36.76 
                 31.84 
                 29.58 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA474 
                 AAGATACCTG 
                 15 
                 MA004 
                 25.26 
                 22.74 
                 26.59 
                 35.61 
                 28.96 
                 26.24 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA475 
                 AGATACCTGA 
                 248 
                 MA004 
                 28.61 
                 34.79 
                 30.52 
                 35.29 
                 29.14 
                 30.03 
               
               
                   
                 ATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA476 
                 GATACCTGAAT 
                 249 
                 MA004 
                 31.45 
                 27.75 
                 31.88 
                 32.11 
                 26.99 
                 30.15 
               
               
                   
                 AACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA477 
                 ATACCTGAATA 
                 250 
                 MA004 
                 15.07 
                 13.4 
                 15.01 
                 18.73 
                 16.78 
                 16.96 
               
               
                   
                 ACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA441 
                 AAGATACCTG 
                 15 
                 MA045 
                 20.49 
                 21.6 
                 26.32 
                 26.13 
                 24.37 
                 25.48 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA442 
                 AAGATACCTG 
                 15 
                 MA045 
                 25.65 
                 17.51 
                 25.36 
                 18.73 
                 18.54 
                 20.34 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA472 
                 AAGATACCTG 
                 15 
                 MA045 
                 20.87 
                 15.13 
                 19.67 
                 19.28 
                 19.07 
                 ND 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA473 
                 AAGATACCTG 
                 15 
                 MA045 
                 14.84 
                 14.05 
                 20.14 
                 15.95 
                 15.95 
                 16.71 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA474 
                 AAGATACCTG 
                 15 
                 MA045 
                 15.81 
                 16.04 
                 17.08 
                 20.15 
                 15.02 
                 14.33 
               
               
                   
                 AATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA475 
                 AGATACCTGA 
                 248 
                 MA045 
                 24.33 
                 31.08 
                 25.52 
                 28.55 
                 20.61 
                 21.1 
               
               
                   
                 ATAACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA476 
                 GATACCTGAAT 
                 249 
                 MA045 
                 25.43 
                 24.26 
                 23.45 
                 26.05 
                 21.47 
                 23.35 
               
               
                   
                 AACCCTC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 GA477 
                 ATACCTGAATA 
                 250 
                 MA045 
                 5.06 
                 2.84 
                 3.07 
                 4.65 
                 3.75 
                 3.01 
               
               
                   
                 ACCCTC 
               
               
                   
               
            
           
         
       
     
     Off-target analysis for altered gRNA/mRNA combinations at the highest dose of 5,000 ng/RNA/mL was performed for two sites previously identified that showed off-target editing (Table 16). The calculated editing percentage across the protospacer was totaled, and the negative control editing % was subtracted. This data shows that modifications to the guide and/or to the ABE mRNA improve off-target editing efficiency. 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Modifications to the guide and/or to the ABE 
               
               
                 mRNA improve off-target editing efficiency. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 gRNA 
                 mRNA 
                 OT Site A 
                 OT Site B 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 Human Primary Hepatocytes-SUM 
                   
               
               
                   
                 total editing % (all positions) 
               
               
                   
                 minus control, AVG of ~2 replicates 
               
            
           
           
               
               
               
               
               
            
               
                   
                 GA441 
                 MA004 
                 9.785 
                 1.605 
               
               
                   
                   
                 MA040 
                 7.17 
                 0.85 
               
               
                   
                   
                 MA041 
                 −0.1 
                 −0.27 
               
               
                   
                 GA442 
                 MA004 
                 7.65 
                 0.77 
               
               
                   
                   
                 MA040 
                 6.97 
                 0.64 
               
               
                   
                   
                 MA041 
                 −0.005 
                 −0.17 
               
               
                   
                 GA472 
                 MA004 
                 0.36 
                 −0.13 
               
               
                   
                   
                 MA040 
                 0.78 
                 −0.185 
               
               
                   
                   
                 MA041 
                 −0.035 
                 −0.205 
               
               
                   
                 GA473 
                 MA004 
                 −0.145 
                 −0.135 
               
               
                   
                   
                 MA040 
                 −0.1 
                 −0.215 
               
               
                   
                   
                 MA041 
                 −0.085 
                 −0.19 
               
               
                   
                 GA474 
                 MA004 
                 1.075 
                 0.03 
               
               
                   
                   
                 MA040 
                 1.545 
                 0.39 
               
               
                   
                   
                 MA041 
                 −0.095 
                 −0.14 
               
               
                   
                 GA475 
                 MA004 
                 2.79 
                 0.39 
               
               
                   
                   
                 MA040 
                 1.68 
                 ND 
               
               
                   
                   
                 MA041 
                 −0.13 
                 −0.205 
               
               
                   
                 GA476 
                 MA004 
                 0.48 
                 0.385 
               
               
                   
                   
                 MA040 
                 0.785 
                 0.295 
               
               
                   
                   
                 MA041 
                 −0.17 
                 ND 
               
               
                   
                 GA477 
                 MA004 
                 −0.13 
                 −0.215 
               
               
                   
                   
                 MA040 
                 −0.48 
                 −0.23 
               
               
                   
                   
                 MA041 
                 −0.225 
                 −0.14 
               
            
           
           
               
               
               
            
               
                   
                 Human Primary Hepatocytes-SUM 
                   
               
               
                   
                 total editing % (all positions) 
               
               
                   
                 AVG of ~2 replicates 
               
            
           
           
               
               
               
               
               
            
               
                   
                 GA441 
                 MA004 
                 6.575 
                 2.345 
               
               
                   
                   
                 MA045 
                 1.36 
                 0.865 
               
               
                   
                 GA442 
                 MA004 
                 7.315 
                 1.75 
               
               
                   
                   
                 MA045 
                 1.355 
                 0.895 
               
               
                   
                 GA472 
                 MA004 
                 2.195 
                 1.005 
               
               
                   
                   
                 MA045 
                 1.19 
                 0.87 
               
               
                   
                 GA473 
                 MA004 
                 1.47 
                 1.025 
               
               
                   
                   
                 MA045 
                 1.495 
                 0.98 
               
               
                   
                 GA474 
                 MA004 
                 2.145 
                 1.055 
               
               
                   
                   
                 MA045 
                 1.405 
                 0.9 
               
               
                   
                 GA475 
                 MA004 
                 3.67 
                 1.825 
               
               
                   
                   
                 MA045 
                 1.39 
                 0.955 
               
               
                   
                 GA476 
                 MA004 
                 1.775 
                 1.675 
               
               
                   
                   
                 MA045 
                 1.505 
                 0.84 
               
               
                   
                 GA477 
                 MA004 
                 1.31 
                 0.975 
               
               
                   
                   
                 MA045 
                 1.135 
                 0.83 
               
               
                   
                   
               
            
           
         
       
     
     Lipid nanoparticles containing ABE8.8 mRNA and a gRNA matching the human/cynomolgus 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13) sequence (GA066) that targets the splice donor at the 5′ end of PCSK9 intron 1 were formulated at a 1:1 ratio by weight. The LNPs were administered to primary human hepatocytes and primary cynomolgus hepatocytes, using various dilutions to assess for editing activity at different concentrations of test article. LNP containing Cas9 mRNA and a gRNA matching a protospacer sequence in another gene, ANGPTL3, as a positive control was used for these experiments. This Cas9 mRNA/gRNA combination were chosen because it had been previously observed to produce high levels of genome-editing activity in primary human hepatocytes, primary cynomolgus hepatocytes, and cynomolgus liver in vivo. It was observed the ABE8.8/GA066 LNP substantially outperformed the control LNP with respect to editing, displaying much higher potency in both human and cynomolgus hepatocytes ( FIG.  15   ). 
     Example 5. Pcsk9 Gene Editing in Mice 
     LNPs containing SpCas9 mRNA and mouse Pcsk9-targeting gRNA with different tracr designs (GA052, GA053, GA054, and GA055) at a 1:1 weight ratio were formulated. Wild-type C57BL/6 mice were dosed with 2 mg/kg of the LNP test article. Seven days after dosing, the mice were euthanized and genomic DNA was harvested from mouse liver, and then assessed for editing of the target site with next-generation sequencing. GA052, GA054, and GA055 all outperformed GA053 that had a previously disclosed tracr design ( FIG.  16   ). 
     Example 6. Pcsk9 Base Editing in Mice 
     LNPs containing ABE8.8 mRNA and a mouse Pcsk9-targeting gRNA at a 1:1 weight ratio were formulated. This gRNA matches the 5′-CCCATACCTTGGAGCAACGG-3′ (SEQ ID NO: 69) protospacer sequence, which is the mouse ortholog of the human 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13) sequence (matched by GA066, GA095, GA096, GA097 and GA346) that targets the splice donor at the 5′ end of PCSK9 intron 1. Wild-type C57BL/6 mice were dosed with 2 mg/kg of the LNP test article via the lateral tail vein or retro-orbital in a total volume of 10 ml/kg. Seven days after dosing, the mice were euthanized and genomic DNA was harvested from mouse liver, and then assessed for base editing of the target splice site with next-generation sequencing. Approximately 60% editing of the target splice site was observed (mouse Pcsk9 intron 1 splice donor) ( FIG.  17   ), providing a preclinical proof of concept of a base-editing therapy knocking down PCSK9 in the liver in vivo. 
     In a subsequent study, LNPs containing ABE8.8 mRNA and a mouse Pcsk9-targeting gRNA at a 1:1 weight ratio were formulated, and dosed in wild-type C57BL/6 mice at doses ranging from 0-2.0 mg total RNA/kg. The low dose of 0.05 mg RNA/kg showed high editing (&gt;45%), while saturation of base editing occurred around 0.25 mg RNA/kg dose ( FIG.  18   ). 
     In an additional study, LNPs containing ABE8.8 mRNA and a mouse Pcsk9-targeting gRNA were formulated at different weight ratios of mRNA and gRNA ranging from 1:1-1:6 and 2:1-6:1. This gRNA matches the 5′-CCCATACCTTGGAGCAACGG-3′ (SEQ ID NO: 69) protospacer sequence, which is the mouse ortholog of the human 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13) sequence (matched by GA066, GA095, GA096, GA097 and GA346) that targets the splice donor at the 5′ end of PCSK9 intron 1. Wild-type mice were dosed with the LNP at 0.05 mg/kg total RNA dose. Additionally, two individual LNPs constituted with the same mRNA and two guides, GA255 and GA257, which matches the 5′-CCCATACCTTGGAGCAACGG-3′ (SEQ ID NO: 69) protospacer sequence but has modifications to the tracr to improve stability, were also injected into mice. Seven days after dosing, the mice were euthanized and genomic DNA was harvested from mouse liver, and then assessed for base editing of the target splice site with next-generation sequencing ( FIG.  19   ). Base editing was comparable between many of the ratios, with 1:6 and 6:1 (gRNA:mRNA) performing the poorest. The guides with modifications to the tracr had increased editing efficiency. 
     Example 7. Angptl3 Base Editing in Mice 
     LNPs containing ABE8.8 mRNA and mouse Angptl3-targeting gRNAs (GA258, GA259, GA260, GA349, GA353) at a 1:1 weight ratio were formulated. Wild-type C57BL/6 mice were dosed with LNP test article at 0.05 mg/kg total RNA dose. The mice were later euthanized and genomic DNA was harvested from mouse liver, and then assessed for base editing of the target splice site with next-generation sequencing ( FIG.  20   ). 
     In Vivo Evaluation in Non-Human Primates (NHP) 
     Example 8. Evaluation of PCSK9 ABE 
     First, a 2-week study was performed to evaluate editing of the PCSK9 gene in cynomolgus monkeys using specific gRNA and base editor nuclease ABE 8.8 through administration of LNPs ( FIG.  21   ). The ABE8.8/GA066 LNP was administered to cynomolgus monkeys via intravenous infusion at two doses, 3 mg/kg (n=3 animals) and 1 mg/kg (n=2 animals) with the intent of producing high-level base editing of PCSK9 gene in the liver. Two weeks after administration of test article, blood samples were collected for clinical chemistry assays and PCSK9 ELISA assay, and the animals underwent necropsy for collection of liver samples, 2 samples each from each of the 4 lobes of the liver (8 in total from each animal). 
     Editing analysis of the liver specimens was performed by next generation sequencing. At the 3 mg/kg dose, a mean of 55% editing of the adenine base at the target splice site in the liver samples was observed; at the 1 mg/kg dose, a mean of 24% editing of the adenine base at the target splice site in the liver samples was observed ( FIG.  22   ). For PCSK9 ELISA analysis, blood samples were collected from the animals on days D-10, D-7 and D-5 pre-dose (average shown) as well as on D8 and D15 post-dose. At 2 weeks after dosing there was a mean 76% reduction in the blood PCSK9 protein level compared to pre-dosing levels in the 3 mg/kg group ( FIG.  23   ), and a mean 32% reduction in the blood PCSK9 protein level in the 1 mg/kg group (Table 17). Low density lipoprotein cholesterol (LDL-C) was determined in serum samples taken pre-dose and on D8 and D15 using a standard clinical analyzer (Table 18). There was a mean 57% reduction in the blood low-density lipoprotein cholesterol (LDL-C) level in the 3 mg/kg group ( FIG.  24   ), and a mean 25% reduction in the blood LDL-C level in the 1 mg/kg group. 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Circulating PCSK9 protein levels decrease after PCSK9  
               
               
                 gRNA/ABE editing 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Dose 
                   
                 Concentration 
                 Reduction from  
               
               
                 PCSK9 
                 (total RNA, 
                 Animal 
                 (ng/ml) 
                 basal on D15 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Group 
                 mg/kg) 
                 ID 
                 Basal 
                 D8 
                 D15 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1 
                 0 
                 1001 
                 432 
                 387 
                 366 
                 15 
               
               
                 2 
                 1 
                 2001 
                 324 
                 285 
                 227 
                 30 
               
               
                 2 
                 1 
                 2002 
                 144 
                 116 
                 95 
                 34 
               
               
                 3 
                 3 
                 3001 
                 254 
                 16 
                 28 
                 89 
               
               
                 3 
                 3 
                 3002 
                 170 
                 34 
                 31 
                 82 
               
               
                 3 
                 3 
                 3003 
                 253 
                 65 
                 109 
                 57 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 LDL-C reduction after editing of the PCSK9 gene 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Dose  
                   
                 LDL-C concentration 
                 Reduction from  
               
               
                   
                 (total RNA,  
                 Animal 
                 (mg/dl) 
                 basal on D15 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Group 
                 mg/kg) 
                 ID 
                 Basal 
                 D8 
                 D15 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1 
                 0 
                 1001 
                 76.7 
                 70 
                 57 
                 26 
               
               
                 2 
                 1 
                 2001 
                 72.3 
                 55 
                 54 
                 25 
               
               
                 2 
                 1 
                 2002 
                 69.0 
                 53 
                 52 
                 25 
               
               
                 3 
                 3 
                 3001 
                 57.0 
                 23 
                 17 
                 70 
               
               
                 3 
                 3 
                 3002 
                 60.0 
                 25 
                 22 
                 63 
               
               
                 3 
                 3 
                 3003 
                 107.7 
                 88 
                 66 
                 39 
               
               
                   
               
            
           
         
       
     
     In a subsequent experiment, LNPs were delivered in monkeys via intravenous infusion at a 1.0 mg/kg dose. For three monkeys that underwent necropsy at 2 weeks after LNP infusion, there was a mean 63% base editing frequency of the PCSK9 splice site adenine in the liver, with no bystander base editing observed elsewhere in the protospacer ( FIG.  25   ); there was a mean indel frequency of 0.5%. The editing was accompanied by a mean 81% reduction in blood PCSK9 levels and a mean 65% reduction in blood LDL-C levels. For two monkeys that underwent necropsy at 24 hours after LNP infusion, there was a mean 48% editing frequency. 
     In a subsequent short-term dose-response study (0.5, 1.0, and 1.5 mg/kg doses, three monkeys each, with necropsy at 2 weeks), all doses achieved &gt;50% mean base editing rates; PCSK9 editing and reductions in PCSK9 protein and LDL-C appeared to saturate at doses ≥1.0 mg/kg ( FIG.  26   ). 
     In these studies we assessed liver function tests and in some groups noted moderate rises in AST and ALT that largely resolved by the end of the first week and entirely resolved by 2 weeks after LNP infusion, with no adverse health events observed in any of the animals. In assaying base editing in a wide variety of tissues, we found that the liver was the predominant site of editing, with much lower editing observed in the spleen and adrenal glands and minimal editing observed elsewhere ( FIG.  27   ). 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 Numeric formats of tissue distribution of editing of the PCSK9 exon 
               
               
                 1 splice donor adenine base in three animals, as shown in FIG. 27. 
               
            
           
           
               
               
               
               
               
            
               
                 Tissue 
                 control NHP 
                 NHP #1 
                 NHP #2 
                 NHP #3 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Liver 
                 0.12 
                 59.49 
                 73.83 
                 55.10 
               
               
                 Spleen 
                 0.02 
                 5.25 
                 7.99 
                 5.67 
               
               
                 Adrenal gland (left) 
                 0.02 
                 1.81 
                 7.49 
                 1.79 
               
               
                 Adrenal gland (right) 
                 0.10 
                 1.54 
                 0.22 
                 2.18 
               
               
                 Kidney (left) 
                 0.10 
                 0.63 
                 0.89 
                 0.24 
               
               
                 Kidney (right) 
                 0.06 
                 0.64 
                 0.27 
                 0.32 
               
               
                 Skin (injection site) 
                 0.13 
                 0.08 
                 1.47 
                 1.56 
               
               
                 Mandibular lymph nodes 
                 0.06 
                 0.15 
                 0.48 
                 0.43 
               
               
                 (left) 
               
               
                 Mandibular lymph nodes 
                 0.06 
                 0.18 
                 0.17 
                 0.96 
               
               
                 (right) 
               
               
                 Mesenteric lymph nodes 
                 0.04 
                 0.12 
                 0.15 
                 0.12 
               
               
                 Testis (left) 
                 0.13 
                 0.11 
                 0.06 
                 0.38 
               
               
                 Testis (right) 
                 0.07 
                 0.16 
                 0.09 
                 0.38 
               
               
                 Epididymis (left) 
                 0.09 
                 0.12 
                 0.58 
                 0.83 
               
               
                 Epididymis (right) 
                 0.80 
                 0.25 
                 0.79 
                 0.52 
               
               
                 Skeletal muscle 
                 0.16 
                 0.40 
                 0.33 
                 0.07 
               
               
                 Duodenum 
                 0.07 
                 0.30 
                 0.83 
                 0.26 
               
               
                 Jejunum 
                 0.06 
                 0.19 
                 0.28 
                 0.58 
               
               
                 Colon 
                 0.08 
                 0.07 
                 0.08 
                 0.13 
               
               
                 Lung (left) 
                 0.01 
                 0.16 
                 0.22 
                 0.18 
               
               
                 Lung (right) 
                 0.10 
                 0.54 
                 0.24 
                 0.16 
               
               
                 Brain 
                 0.12 
                 0.17 
                 0.05 
                 0.15 
               
               
                   
               
            
           
         
       
     
     In an additional study, different tracr designs were assessed. LNPs were delivered in NHPs via intravenous infusion at a 1.0 mg/kg total RNA dose, unless otherwise specified. Base editing of the liver was assessed, and several guides outperformed GA066 with the literature tracr ( FIG.  28   ). 
     In the same NHP study, LNPs containing either: 1) Cas9 mRNA and a gRNA matching 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ TD NO: 13) (GA097) protospacer sequence; or 2) ABE8.8 mRNA and gRNA matching 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13) (GA097) protospacer sequence, were delivered intravenously into NHPs. Importantly, the method by which classical CRISPR/Cas9 disrupts a gene by ultimately introducing an indel, is different than base editing where a base mutation has occurred. Further, a target region that is highly amenable to CRISPR/Cas9 editing does not necessarily mean base editing at that location will occur, and vice versa. This is highlighted in the NHP study, where the LNP containing SpCas9/gRNA resulted in low liver editing in the NHPs (&lt;5%), while the LNP containing ABE8.8/gRNA had significantly higher editing close to 40% ( FIG.  29   ) with a dose (1 mg/kg) lower than the SpCas9/gRNA dose (1.5 mg/kg). 
     In another study, two LNPs were compared for base editing activity after delivery in NHPs via intravenous infusion at doses ranging from 0.5 mg/kg-3.0 mg/kg. Both LNPs showed high efficacy, with the first LNP (LNP #1) outperforming the second LNP (LNP #2), at doses as low as 0.5 mg/kg having greater than 50% average editing ( FIG.  30   ). Similar to past experiments, the PCSK9 protein levels decreased significantly upon high level of base editing at the splice site, decreasing circulating PCSK9 protein by 80-90% ( FIG.  31   ). 
     In a long-term study (four animals, with liver biopsy at 2 weeks) LNPs were introduced via intravenous infusion at a higher dose of 3.0 mg/kg to assess drug tolerability and the durability of PCSK9 protein and LDL-C reductions resulting from PCSK9 editing. The liver biopsy samples showed a mean 66% base editing frequency ( FIG.  32   ). Blood PCSK9 protein levels reached a trough by 1 week and have remained stable thereafter out to at least 6 months, settling at approximately 90% reductions ( FIG.  33   ). Blood LDL-C and lipoprotein(a) [Lp(a)] levels have similarly achieved stable troughs persisting to 8 months, settling at ≈60% and ≈35% reductions, respectively ( FIG.  34   ,  FIG.  35   ). 
     There were transient, moderate rises in AST and ALT that entirely resolved by 2 weeks following LNP infusion ( FIG.  36   ), with no changes in any other liver function tests and no adverse health events observed to date ( FIG.  37   ). Importantly, the persistence of PCSK9 and LDL-C reductions for 8 months with no late AST and ALT elevations demonstrates that such a response, whatever its scale, does not adversely impact the treatment&#39;s efficacy. 
     To evaluate ABE8.8/gRNA (protospacer matching GA346) LNP-mediated off-target editing in primary cyno hepatocytes and monkey liver samples, ONE-seq was performed with a synthetic cynomolgus genomic library selected by homology to the gRNA spacer sequence. This library was treated with ABE8.8 protein and PCSK9 gRNA, and the top 48 ONE-seq-nominated sites ( FIG.  38   )—of which the PCSK9 target site was the very top site—was assessed with next-generation sequencing of targeted PCR amplicons from LNP-treated versus untreated NHP samples ( FIG.  39   ). 
     In LNP-treated primary cynomolgus hepatocytes, besides editing at the PCSK9 target site, there was off-target editing (mean &lt;1%) evident at only one site, designated C5, which has poor homology to the human genome ( FIG.  39   ). Assessing the same 48 sites in liver samples from monkeys that were treated with a 1.0 mg/kg LNP dose (from the aforementioned dose-response study), low-level off-target editing (mean &lt;1%) only at the C5 site ( FIG.  40   ) was observed. No off-target editing with a 0.5 mg/kg LNP dose and only low-level off-target editing with a 1.5 mg/kg LNP dose (mean &lt;1%) were detected. 
     Example 9. Evaluation of ANGPTL3 ABE 
     Similar to the NHP studies performed with the ABE/PCSK9 guide(s), LNPs formulated with ABE8.8 mRNA and a gRNA with the 5′-AAGATACCTGAATAACTCTC-3′ (SEQ ID NO: 14) (GA067) cynomolgus spacer was administered to cynomolgus monkeys via intravenous infusion at two doses, 3 mg/kg (n=3 animals) and 1 mg/kg (n=2 animals) with the intent of producing high-level base editing of ANGPTL3 in the liver. Two weeks after administration of test article, blood samples were collected for clinical chemistry assays and ANGPTL3 ELISA assay, and the animals underwent necropsy for collection of liver samples. Small segments from the center and periphery of each lobe of the liver were excised and flash frozen in liquid nitrogen and stored at −86 to −60° C. Splice-site editing was analyzed by next generation sequencing, confirming that base change occurred. At the highest dose (3 mg/kg), a mean of 61% editing of the adenine base was observed at the target splice site in the liver; at the lowest dose (1 mg/kg), the editing observed was 28% at the target splice site in the liver (Table 19). 
     In addition, blood samples were collected pre-dose, on D7 and D15 post-dose to determine serum ANGPTL3, and triglycerides. At two weeks after dosing there was a mean 90% and 31% reduction in the blood ANGPTL3 protein level compared to pre-dosing levels in the higher (3 mg/kg) and lower (1 mg/kg) dosing groups (Table 20). 
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Liver Editing in cynomolgus monkeys 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Dose 
                 Animal 
                 Mean ± standard 
               
               
                   
                 Group 
                 (total RNA, mg/kg) 
                 ID 
                 deviation 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 1 
                 0 
                 1001 
                  0.25 ± 0.06 
               
               
                   
                 4 
                 1 
                 4001 
                 39.6 ± 2.7 
               
               
                   
                 4 
                 1 
                 4002 
                 15.1 ± 3.9 
               
               
                   
                 5 
                 3 
                 5001 
                 61.2 ± 3.2 
               
               
                   
                 5 
                 3 
                 5002 
                 56.4 ± 5.2 
               
               
                   
                 5 
                 3 
                 5003 
                 64.3 ± 2.6 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Knockdown of circulating cynomolgus monkey ANGPTL3  
               
               
                 following gene editing using ABE 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Dose 
                   
                 Concentration  
                 Reduction from 
               
               
                   
                 (total RNA, 
                 Animal  
                 (ng/ml) 
                 basal on D15  
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Group 
                 mg/kg) 
                 ID 
                 Basal 
                 D8 
                 D15 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1 
                 0 
                 1001 
                 39 
                 47 
                 41 
                 −7 
               
               
                 4 
                 1 
                 4001 
                 56 
                 15 
                 31 
                 45 
               
               
                 4 
                 1 
                 4002 
                 38 
                 22 
                 31 
                 17 
               
               
                 5 
                 3 
                 5001 
                 31 
                 1 
                 1 
                 98 
               
               
                 5 
                 3 
                 5002 
                 33 
                 11 
                 9 
                 72 
               
               
                 5 
                 3 
                 5003 
                 70 
                 1 
                 1 
                 99 
               
               
                   
               
            
           
         
       
     
     Triglycerides were determined in serum samples taken pre-dose and on D8 and D15 using a standard clinical analyzer. Serum triglyceride reduction in response to LNP-mediated ANGPTL3 base editing with ABE8.8 and GA067 are summarized in Table 21. The levels of triglycerides were reduced by 24% and 59% in response to doses of 1 mg/kg and 3 mg/kg base editor ABE8.8 and GA067 targeting ANGPTL3. 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Triglyceride reduction in response to ANGPTL3 gene in liver 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Triglyceride 
                   
               
               
                   
                 Dose  
                   
                 concentration  
                 Reduction from  
               
               
                   
                 (total RNA,  
                 Animal  
                 (mg/dl) 
                 basal on D15  
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Group 
                 mg/kg) 
                 ID 
                 Basal 
                 D8 
                 D15 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1 
                 0 
                 1001 
                 31.3 
                 43 
                 41 
                 −31 
               
               
                 4 
                 1 
                 4001 
                 71.0 
                 46 
                 49 
                 31 
               
               
                 4 
                 1 
                 4002 
                 67.7 
                 52 
                 56 
                 17 
               
               
                 5 
                 3 
                 5001 
                 59.3 
                 23 
                 21 
                 65 
               
               
                 5 
                 3 
                 5002 
                 41.3 
                 33 
                 25 
                 40 
               
               
                 5 
                 3 
                 5003 
                 55.0 
                 27 
                 15 
                 73 
               
               
                   
               
            
           
         
       
     
     In another independent study, the same ABE8.8/GA067 LNP was administered to cynomolgus monkeys via intravenous infusion at a dose of 3 mg/kg (n=3 animals) with the intent of producing high-level base editing of ANGPTL3 in the liver. Two weeks after administration of test article, blood samples were collected for clinical chemistry assays, and the animals underwent a liver biopsy for collection of liver samples. At the 3 mg/kg dose, a mean of 60% editing of the adenine base at the target splice site was achieved in the liver biopsy samples. Concordantly, at 2 weeks after dosing there was a mean 95% reduction in the blood ANGPTL3 protein level compared to pre-dosing levels and there was a mean 64% reduction in the blood triglyceride level ( FIG.  41   ). These results provide an additional preclinical proof of concept of a base-editing therapy knocking down ANGPTL3 in the liver in vivo and effecting reductions in blood ANGPTL3 protein and triglyceride levels. 
     Example 10. Evaluation of Dual PCSK9 and ANGPTL3 Base Editing 
     To assess whether it was possible to affect simultaneous base editing of PCSK9 and ANGPTL3 with a single test article, LNPs were formulated containing a mix of three components: ABE8.8 mRNA, PCSK9-targeting gRNA (GA095), and ANGPTL3-targeting gRNA (GA098) at a 2:1:1 weight ratio. Primary human hepatocytes were incubated with various dilutions of the LNPs. Three days after incubation, genomic DNA was harvested from the hepatocytes, and then assessed for base editing of the target splice site with next-generation sequencing. At the highest LNP concentrations, ≈40% editing of the PCSK9 splice site (intron 1 splice donor) and ≈40% editing of the ANGPTL3 splice site (intron 6 splice donor) was observed ( FIG.  42   ), demonstrating the feasibility of dual gene disruption in human hepatocytes with a single test article and providing a preclinical proof of concept of a single base-editing therapy simultaneously knocking down PCSK9 and ANGPTL3, which would be predicted to substantially reduce both blood LDL cholesterol levels and blood triglyceride levels in human recipients. An advantage of this approach is that because the base editors do not require double strand breaks (DSBs) for editing, in contrast to standard CRISPR-Cas9, there is substantially lower risk of chromosomal rearrangements or other structural changes inherent in the simultaneous targeting of two different sites in the genome. 
     A follow-up NHP study was performed to address: 1) if a second dose of administered LNPs administered can cause editing of the target gene; 2) if a different target can be base edited. LNPs formulated with ABE8.8 mRNA and either a gRNA targeting PCSK9 (GA346) or a gRNA targeting ANGPTL3 (GA347) was administered to cynomolgus monkeys via intravenous infusion at doses ranging from 0.5-2 mg/kg. Two weeks after administration of test article, biopsies were performed to assess for base editing. After 30 days from the initiation of the study, the opposite LNP was administered. Following a second biopsy after an additional 2 weeks, the gDNA was extracted, and base editing was assessed using next generation sequencing. The findings from this study show that high level editing of both the PCSK9 and ANGPTL3 targets are achieved after the first and second doses of LNPs ( FIG.  43   ). Blood samples were collected on both biopsy timepoints and show significant decrease of around 90% in circulating PCSK9 and ANGPTL3 after subsequent dosing with 1 mg/kg LNPs ( FIG.  44   ). In the same study an LNP encpasualting ABE8.8 mRNA, PCSK9 gRNA GA346 and ANGPTL3 gRNA GA347 at 1:0.5:0.5 weight ratio was admnistred at 2 mg/kg total RNA dose, which resulted in rubust synchronized PCSK9 and ANGPTL3 gene editing ( FIG.  44   , top and bottom data labelled GA346+GA347 (D1) - top and bottom panels show PSCK9 and ANGPTL3 editing). The data demonstrate that robust multi-gene editing is possible with single 1-dose administration of ABE base editor mRNA and two or more gRNAs targeting two or more genes of interest in mammals (and/or mammalian cell).  FIG.  44    mirrors corresponding knowckdown of ANGPTL3 (bottom) and PCSK9 (top) proteins. 
     In another study, NHPs were repeat dosed with LNPs ( FIG.  45   ). NHPs were dosed with LNP #1 and LNP #2 formulated with ABE8.8 mRNA MA004 and a gRNA targeting PCSK9 (GA097) via intravenous infusion at a total RNA doses of 0.5 mg/kg. NHPs received an additional LNP dose on day 30 and on day 60. Liver biopsies from Day 14, Day 46, and Day75 were extracted for adenosine base editing analysis. All gDNA was extracted, and base editing was assessed using next generation sequencing. These results demonstrate that repeat dosing of LNP containing ABE8.8 mRNA and PCSK9 gRNA causes additive base editing in the liver, as editing efficiency was near 30% after the first dose of LNP, while over 50% after the third dose LNP. It was also observed that the magnitude of additive editing was LNP-dependent. 
     Further, repeat dosing of LNPs reduced PCSK9 protein levels in NHPs ( FIG.  46   ). The PCSK9 protein levels were monitored over 90 days in NHPs that were repeat dosed with LNPs formulated with ABE8.8 mRNA MA004 and a gRNA targeting PCSK9 (GA097). NHPs were dosed via intravenous infusion at a total RNA dose of 0.5 mg/kg at days 0, 30, and 60 (arrow is illustrated on graph to depict dosing). For description of analysis of PCSK9 protein levels, see detailed methods section. Compared to basal levels, PCSK9 protein dropped by nearly 40% after the initial dose of LNP, but upon additional doses, decreased even further. 
     Liver markers were assessed following repeat dosing of LNPs ( FIG.  47   ). ALT, AST, total bilirubin, and creatine kinase levels were assessed up to 71 days in NHPs that were repeat dosed with LNPs formulated with ABE8.8 mRNA MA004 and a gRNA targeting PCSK9 (GA097). NHPs were dosed via intravenous infusion at a total RNA dose of 0.5 mg/kg at days 0, 30, and 60, which is shown as D1, D2 and D3 on graph, respectively. Liver biopsy was performed at days 14 and 46 which is shown as B1 and B2 on graph, respectively. Blood was collected at multiple timepoints, as described on the graph. Although ALT and AST minimally rose upon dosing, this was a transient response that returned to normal after a period of several days. Similarly, Creatine Kinase levels increased upon dosing, with the largest effect seen after the first dose, and returned to basal levels after a period of several days. 
     The liver enzymes, LDH, GLDH, GGT, and ALP, were assessed in NHPs that were repeat dosed with LNPs formulated with ABE8.8 mRNA MA004 and a gRNA targeting PCSK9 (GA097) ( FIG.  48   ). NHPs were dosed via intravenous infusion at a total RNA doses of 0.5 mg/kg at days 0, 30, and 60. Blood was collected at multiple timepoints, as described on the graph. Although LDH and GLDH rose upon dosing, this was a transient response that returned to normal after a period of several days. 
     Further, long-term adenine base editing of ANGPTL3 was assessed in non-human primates ( FIG.  49   ). Cynomolgus monkeys received an intravenous infusion of a 3 mg/kg dose of an LNP formulation with ABE8.8 mRNA MA004 and ANGPTL3 gRNA GA067. Blood was collected at timepoints specified and the graph, and ANGPTL3 protein levels ( FIG.  49 A ) and triglyceride levels ( FIG.  49 B ) were analyzed. As compared to controls, both ANGPTL3 protein levels (96% reduction) and triglyceride levels substantially decreased upon base editing of the gene, and remain stably reduced for more than 170 days. 
     Cytokine activation and immune response were assessed in NHPs receiving LNPs. In one set of studies, cynomolgus monkeys received intravenous infusions of 0.5 mg/kg doses at three specified time points ( FIG.  50 A  and  FIG.  50 B ) of an LNP formulation with ABE8.8 mRNA MA004 and PCSK9 gRNA GA346. Blood was collected at timepoints specified and the graph, and IP-10 and MCP-1 were analyzed. These results demonstrated: 1) that NHPs receiving LNPs had no evidence of cytokine activation nor immune response, as compared to control; and 2) that repeat dosing of the same LNP does not elicit cytokine activation nor an immune response. 
     In additional studies, IL-6, MCP-1, and SC5b-9 ( FIG.  50 C ,  FIG.  50 D , and  FIG.  50 E , respectively) were analyzed at different time points from blood collected from NHPs that received an intravenous infusion of 1.0 mg/kg total RNA dose of LNP formulated with MA004 and PCSK9 gRNA GA346. This caused minimal cytokine/complement activation that returned to baseline by/before 336 hours, as compared to control. 
     Example 11. Evaluation of SpCa9-Mediated On-Target Editing Efficiency 
     Gene editing of ANGPTL3 or PCSK9 in non-human primates was assessed. Cynomolgus monkeys received an intravenous infusion of a 1.5 mg/kg dose of an LNP formulation with SpCas9 mRNA MS004 and one gRNA targeting either ANGPTL3 (GA261-GA263) or PCSK9 (GA266-GA271). Upon necropsy after 2 weeks, two pieces from each liver lobe (8 pieces total) were isolated and gDNA was extracted. Samples were processed as described in the detailed methods section. Indel % was analyzed for each separate piece and are graphed as individual points ( FIG.  51   ). High editing efficiency was observed in most NHP livers. LDL-C levels were measured ( FIG.  52   ). All NHPs that received LNPs with SpCas9 mRNA/PCSK9 gRNA had at least 35% reduction in circulating LDL-C levels. Although more modest, LNPs with SpCas9 mRNA/ANGPTL3 gRNA had 10-25% reduction in circulating LDL-C levels. Additionally, NHPs that received LNPs with SpCas9 mRNA/ANGPTL3 gRNA had around 10-50% reduction in triglyceride levels ( FIG.  53   ). NHPs that received LNPs with SpCas9 mRNA/PCSK9 gRNA did not show a significant reduction in triglyceride levels. 
     Cynomolgus primary hepatocytes were transfected at 2500, 1250, and 625 ng/test article/mL with SpCas9 mRNA MS002 and a gRNA targeting PCSK9 with modifications to the tracr. Genomic DNA was processed, sequenced, and analyzed as described in the detailed methods section. GA266 was transfected independently twice to serve as two positive controls. Almost all transfections resulted in high editing efficiency compared to positive control, while GA405 had slightly lower editing efficiency. 
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Modifications to guides retain on-target editing efficiency in  
               
               
                 cynomolgus primary hepatocytes. 
               
            
           
           
               
               
            
               
                   
                 Cynomolgus Primary Hepatocytes-% Editing (dose, replicate #) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 2500, 
                 2500, 
                 1250, 
                 1250,  
                 625,  
                 625,  
               
               
                 gRNA 
                 rep1 
                 rep2 
                 rep1 
                 rep2 
                 rep1 
                 rep2 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 GA266, #1 
                 56.51 
                 49.95 
                 51.81 
                 55.2 
                 44.53 
                 42.53 
               
               
                 GA266, #2 
                 50.34 
                 48.3 
                 43.56 
                 46.2 
                 39.75 
                 36.59 
               
               
                 GA395 
                 43.91 
                 49.53 
                 41.48 
                 44.35 
                 33.87 
                 34.57 
               
               
                 GA396 
                 53.98 
                 56.84 
                 56.02 
                 52.02 
                 44.44 
                 41.53 
               
               
                 GA397 
                 56.65 
                 58.59 
                 56.47 
                 58.68 
                 40.72 
                 46.28 
               
               
                 GA398 
                 59.85 
                 63.97 
                 56.45 
                 55.21 
                 39.09 
                 44 
               
               
                 GA399 
                 58.97 
                 62.75 
                 55.55 
                 53.02 
                 37.75 
                 41.8 
               
               
                 GA401 
                 53.74 
                 52.79 
                 49.12 
                 48.83 
                 38.33 
                 38.77 
               
               
                 GA402 
                 45.58 
                 55.34 
                 44.46 
                 47.93 
                 35.05 
                 40.31 
               
               
                 GA403 
                 52.62 
                 55.84 
                 50.5 
                 47.13 
                 38.28 
                 35.58 
               
               
                 GA404 
                 48.3 
                 50.59 
                 41.68 
                 35.04 
                 31.63 
                 30.16 
               
               
                 GA405 
                 41.85 
                 47.33 
                 35.84 
                 35.88 
                 26.2 
                 26.36 
               
               
                 GA406 
                 57.17 
                 51.45 
                 45.11 
                 43.68 
                 37.58 
                 40.31 
               
               
                 GA408 
                 50.4 
                 46.92 
                 47.21 
                 49.8 
                 36.7 
                 37.14 
               
               
                   
               
            
           
         
       
     
     PACE-modifications to the gRNA have been previously demonstrated to reduce off-target editing efficiency. Human primary hepatocytes were transfected at 2500, 1250, 500, and 250 ng/test article/mL with SpCas9 mRNA MS002 and a gRNA targeting PCSK9 with modifications to the tracr. Genomic DNA was processed, sequenced, and analyzed as described in the detailed methods section. GA156 was transfected to serve as a positive control. GA248 and GA249 contain PACE-modifications to the gRNA that have previously been demonstrated to decrease off-target editing efficiency. Indeed, although GA248 and GA249 had lower on-target editing compared to the unmodified gRNA, GA156 ( FIG.  54 A ), GA248 and GA249 showed decreased off-target editing at an identified off-target site ( FIG.  54 B ). 
     ABE mRNA sequences—MA004, MA019, MA020, and MA021—were evaluated for their GC content ( FIG.  55 A ). In general, MA004 sequence has a higher GC content than MA019, MA020, MA021, and ABE8.8 m sequences. GC levels in sequence MA004 are also elevated (above 60%) in certain regions throughout the ABE sequence such as the TadA domain, N-terminal linker, and C-terminal linker, as well as various sub-regions. For all sequences, GC levels below 60% are shaded in gray to provide contrast to the elevated GC regions. GC content is calculated by determining the relative amount of G and C within a given 25 nucleotide stretch across the entire mRNA sequence, i.e. for every 25 nucleotides, the sum of G&#39;s and C&#39;s is divided by the total number of nucleotides. This comparison shows that mRNA MA004 sequence has a distinct distribution and enrichment of G and C nucleotides relative to other mRNA sequences. 
     The region-specific GC characterization of sequence MA004 is further illustrated ( FIG.  55   ). The graph in each row shows the GC content in every 1,000-nucleotide stretch comprising the full ABE coding sequence (4,767 nt). The GC content is particularly high in the 5′ end of the sequence, which includes the TadA region and the N-terminal linker. The GC content is also high various parts of the Cas9 nickase and in the 3′ end of the ABE sequence; there the high GC % is centered around the linker region instead of the NLS. Sub-regions with high GC content are delineated by where GC reaches a threshold level of 60%. GC content in each sub-region is calculated by determining the number of G and C nucleotides divided by the total number of nucleotides for each sub-region. 
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 GC comparison of ABE-encoding nucleotides, MA004, MA019, MA020, 
               
               
                 MA021, and ABE8.8m 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 ABE sequence 
                 MA004 
                 MA019 
                 MA020 
                 MA021 
                 ABE8.8m 
                 AGC* 
               
               
                   
               
            
           
           
               
            
               
                 Region-specific GC % 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Total sequence 
                 63 
                 54 
                 47 
                 54 
                 55 
                 11 
               
               
                 TadA (nt: 1-501) 
                 70 
                 58 
                 54 
                 60 
                 54 
                 14 
               
               
                 N-terminal linker (nt: 502-597) 
                 79 
                 64 
                 65 
                 54 
                 59 
                 19 
               
               
                 Cas9 nickase (nt: 598-4698) 
                 62 
                 54 
                 45 
                 53 
                 56 
                 10 
               
               
                 C-terminal linker (nt: 4699-4710) 
                 83 
                 58 
                 67 
                 58 
                 60 
                 22 
               
               
                 NLS (nt: 4711-4767) 
                 63 
                 53 
                 49 
                 60 
                 48 
                 11 
               
            
           
           
               
            
               
                 Sub-region GC % 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 nt: 27-109 
                 75 
                 59 
                 55 
                 63 
                 59 
                 16 
               
               
                 nt: 111-213 
                 73 
                 56 
                 55 
                 66 
                 57 
                 15 
               
               
                 nt: 250-385 
                 71 
                 60 
                 57 
                 62 
                 57 
                 12 
               
               
                 nt: 389-475 
                 70 
                 59 
                 56 
                 54 
                 53 
                 15 
               
               
                 nt: 479-601 
                 76 
                 62 
                 61 
                 65 
                 55 
                 15 
               
               
                 nt: 615-661 
                 72 
                 64 
                 51 
                 66 
                 68 
                 10 
               
               
                 nt: 699-714 
                 75 
                 63 
                 63 
                 56 
                 69 
                 12 
               
               
                 nt: 735-829 
                 74 
                 62 
                 54 
                 61 
                 61 
                 15 
               
               
                 nt: 1192-1345 
                 67 
                 58 
                 51 
                 55 
                 61 
                 11 
               
               
                 nt: 1363-1627 
                 66 
                 58 
                 52 
                 55 
                 61 
                 10 
               
               
                 nt: 1661-1706 
                 70 
                 57 
                 57 
                 65 
                 63 
                 10 
               
               
                 nt: 1735-1880 
                 66 
                 54 
                 52 
                 57 
                 60 
                 10 
               
               
                 nt: 1938-1999 
                 71 
                 60 
                 55 
                 63 
                 60 
                 12 
               
               
                 nt: 2023-2073 
                 63 
                 55 
                 45 
                 53 
                 63 
                 9 
               
               
                 nt: 2184-2231 
                 67 
                 60 
                 54 
                 63 
                 60 
                 8 
               
               
                 nt: 2542-2600 
                 71 
                 63 
                 51 
                 53 
                 64 
                 13 
               
               
                 nt: 2719-2798 
                 69 
                 56 
                 54 
                 65 
                 64 
                 9 
               
               
                 nt: 3294-3334 
                 73 
                 61 
                 54 
                 61 
                 66 
                 13 
               
               
                 nt: 3542-3584 
                 72 
                 67 
                 58 
                 72 
                 67 
                 6 
               
               
                 nt: 3745-3864 
                 67 
                 54 
                 53 
                 57 
                 62 
                 11 
               
               
                 nt: 3977-4045 
                 65 
                 57 
                 48 
                 59 
                 59 
                 9 
               
               
                 nt: 4207-4286 
                 70 
                 56 
                 54 
                 66 
                 59 
                 11 
               
               
                 nt: 4427-4517 
                 65 
                 60 
                 51 
                 53 
                 55 
                 10 
               
               
                 nt: 4537-4569 
                 73 
                 58 
                 48 
                 64 
                 61 
                 15 
               
               
                 nt: 4583-4741 
                 67 
                 58 
                 49 
                 57 
                 61 
                 11 
               
               
                   
               
               
                 *AGC is the difference between the GC content of sequence MA004 CDS (cDNA sequence) and the average of sequences MA019 CDS, MA020 CDS, MA021 CDS, and ABE8.8m. 
               
            
           
         
       
     
     The editing efficiency of these mRNA constructs were assessed in mice ( FIG.  55 B ). These mRNAs, MA004, MAO 19, MA020, and MA021 code for the same ABE protein sequence but have different nucleotide sequence optimizations. The only difference between these groups is the mRNA sequence; the mRNAs have the same base modifications, and each group used the same LNP formulation and gRNA. Each group was administered intravenously to mouse, and a low dose of 0.05 mg/kg was used in this study to achieve sub-saturating levels of liver editing to resolve efficacy differences. At day 5 post-dose, gDNA was isolated and base editing was assessed by next generation sequencing. These results demonstrate that mRNA sequence changes can affect adenosine base editing performance in vivo. 
     Additional Details of Methods Described 
     Plating, culturing, and transfection of primary hepatocytes. Primary human liver hepatocytes (PHH) and primary cynomolgus liver hepatocytes (PCH) from BioIVT were cultured per the manufacturer&#39;s protocol. Briefly, primary human hepatocytes and primary cyno hepatocytes were obtained as frozen aliquots from BioIVT. Four lots of primary human hepatocytes, each derived from a de-identified individual donor, were used for the experiments: STL (main donor) was used for all experiments, including screening experiments and off-target experiments; HLY, JLP, and TLY were used for off-target experiments. The HFG lot of primary cyno hepatocytes were used for experiments. Following the manufacturer&#39;s instructions, cells were thawed and rinsed prior to plating in 24-well plates that had been coated with bovine collagen overnight, with a density of approximate 350,000 cells/well in INVITROGRO hepatocyte medium supplemented with TORPEDO antibiotic mix (BioIVT). The cells were thawed and resuspended in hepatocyte thawing medium followed by centrifugation at 100 g for 10 min at 4° C. The supernatant was discarded, and the pelleted cells resuspended in hepatocyte plating medium. Each vial contains approximately 5 million cells that were used for plating one 24-well plate. Plated cells were allowed to settle and adhere for 4-6 h in a tissue culture incubator at 37° C. under 5% CO 2  atmosphere. After incubation, cells were checked for monolayer formation. The incubating media were then replaced with fresh hepatocyte maintenance media (complete INVITROGRO medium obtained from BioIVT, the cell line provider). The cells thus became ready for transfection. Each of the gRNAs were co-transfected with an equivalent amount of in vitro transcribed ABE8.8 mRNA (1:1 ratio by molecular weight) into primary human hepatocytes via MessengerMax reagent (Lipofectamine), using various dilutions to assess for editing activity at different concentrations of test article. MessengerMAX from Thermo Fisher is used for transfection. Solution A: desired amount of guide RNA is mixed with 1:1 wt ratio of mRNA in OptiMEM. Solution B: MessengerMAX in OptiMEM. After mixing solutions A and B, the mixture was incubated at room temperature for 20 min. 60 μL of the incubated solution was added dropwise to each cell wells. For protospacer sequences that were a perfect match to the corresponding cynomolgous monkey PCSK9 or ANGPTL3 gene sequence, each gRNA was also co-transfected with an equivalent amount of ABE8.8 mRNA (1:1 ratio by molecular weight) into primary cynomolgous hepatocytes, and followed the same transfection protocol. The cells were then allowed to remain at 37° C. for 3 days. Cells were harvested and prepared for genomic DNA extraction using either a Thermo Kingfisher, or Qiagen DNEasy blood and Tissue Kit, per manufacturer&#39;s instructions. 
     Bioinformatic analysis. Targeted amplicon sequencing data were analyzed with CRISPResso2 v2.0.31 in batch mode (CRISPRessoBatch). For Cas9 experiments, the following parameters were set: “—quantification_window_center-3—quantification_window_size 5—min_frequency_alleles_around_cut_to_plot 0.1—max_rows_alleles_around_cut_to_plot 100”. For ABE experiments, the following parameters were set: “—default_min_aln_score 95—quantification_window_center-10—quantification_window_size 10—base_editor_output-conversion_nuc_from A—conversion_nuc_to G—min_frequency_alleles_around_cut_to_plot 0.1—max_rows_alleles_around_cut_to_plot 100”. For NHP experiments, an additional parameter was set to exclude low quality reads: “—min_single_bp_quality 30”. Moreover, in all cases, the parameter “—max_paired_end_reads_overlap” was set to 2R-F+0.25*F, following FLASH recommendations (http://ccb.jhu.edu/software/FLASH-1/), where R was the read length and F was the amplicon length. 
     Editing was quantified from the “Quantification_window_nucleotide_percentage_table.txt” output table as the percentage of reads that supported any A-to-G/C/T substitution in the main edited position (position 6 of the protospacer DNA sequence). Indels were quantified from the “Alleles_frequency_table_around_sgRNA_*.txt” output table as the percentage of reads that supported insertions or deletions over a 5-bp window on either side of the nick site (at position −3 upstream of the PAM sequence), having excluded reads that supported deletions larger than 30 bp. For candidate off-target sites, editing was quantified from the “Alleles_frequency_table_around_sgRNA_*.txt” output table as the percentage of reads from alleles with an A—&gt;G substitution in the editing window (positions 1-10 in the PAM distal side of the protospacer), having excluded reads from alleles with deletions larger than 30 bp. 
     Reverse transcription. The collected cells were processed with the miRNeasy Mini Kit (QIAGEN) according to the manufacturer&#39;s instructions to isolate both large and small RNA species, with the other part harvested for genomic DNA to establish PCSK9 editing and thereby confirm base editor activity in the cells. Reverse transcription was performed using the iScript Reverse Transcription Supermix reagent according to the manufacturer&#39;s instructions, with four different primer pairs used for PCR amplification of transcripts spanning exon 1 and exon 2, with or without any portions of intron 1. Paired-end reads of 250-bp length generated using an Illumina MiSeq System, as described above, were trimmed for adapters using trimmomatic v0.39 with parameters “ILLUMINACLIP:NexteraPE-PE.fa:2:30:10:1:true LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36”. Reads were then merged with FLASH v1.2.1134 and aligned to the PCSK9 gene body with Bowtie2 v2.4.1 with parameters “—local-very-sensitive-local-k 1-np 0”. Gene annotations were obtained from Ensembl v98 (ftp://ftp.ensembl.org/pub/release-98/gtf/homo_sapiens/Homo_sapiens.GRCh38.98.gtf.gz). Alignments were filtered with samtools v1.10 and converted to BED format with the bedtools v2.25.0 bamtobed function. A minimum of 1000 mapped reads per sample were required and the end positions of mapped reads were tallied. Positions throughout PCSK9 intron 1 supported by a minimum of 10 reads in at least one treated sample are report. 
     ONE-seq analysis to predict candidate off-target sites. The design of a ONE-seq library starts with the computational identification of sites in a reference genome that have sequence homology to the on-target. For human ONE-seq libraries, the reference human genome (GRCh38, Ensembl v98, chromosomes ftp://ftp.ensembl.org/pub/release-98/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.chromosome.{1-22,X,Y,MT}.fa and ftp://ftp.ensembl.org/pub/release-98/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.nonchromosomal.fa), was searched for potential off-target sites with up to 6 mismatches to the protospacer sequence above, and sites with up to 4 mismatches plus up to 2 DNA or RNA bulges, using Cas-Designer v1.2 (http://www.rgenome.net/cas-designer/). 
     For the ONE-seq library pertaining to the cynomolgus monkey, the reference cynomolgus monkey genome (macFas5, Ensembl 98, chromosomes ftp://ftp.ensembl.org/pub/release-98/fasta/ Macaca_fascicularis /dna/ Macaca_fascicularis.Macaca_fascicularis  5.0.dna.chromos ome.{1-20,X,MT}.fa.gz and ftp://ftp.ensembl.org/pub/release-98/fasta/ Macaca_fascicularis /dna/ Macaca_fascicularis.Macaca_fascicularis  5.0.dna.nonchro mosomal.fa.gz) was searched using similar parameters. 
     Sites with up to 6 mismatches and no bulges are referred to using a X&lt;number of mismatches&gt; &lt;number of bulges&gt; code. As such, the on-target site is labelled as X00; a site with 1 mismatch to the on-target and no bulges is labelled as X10, and so on. Sites with DNA bulges are referred to with a similar nomenclature, DNA&lt;number of mismatches&gt; &lt;number of bulges&gt;. As such, a site with 4 mismatches to the on-target and 2 DNA bulges is labelled as DNA42. The same nomenclature is used for RNA bulges, but these are coded as RNA&lt;number of mismatches&lt; &gt;number of bulges&gt;. 
     The protospacer sequences identified were extended by 10 nucleotides (nt) on both sides with adjacent sequence from the respective reference genome (these regions are herein referred to as the genomic context). These extended sequences were then padded by additional sequences up to a final length of approximately 200 nt, including 6 predefined constant regions of different nucleotide composition and sequence length; 2 copies of a 14-nt site-specific barcode, one on each side of the central protospacer sequence; and 2 distinct 11-nt unique molecular identifiers (UMIs), one on each side of the central protospacer sequence. The UMIs are used to correct for bias from PCR amplification, and the barcodes allow for the unambiguous identification of each site during analysis. The barcodes are selected from an initial list of 668,420 barcodes, which contain neither a CC nor a GG in their sequences, and each barcode has a Hamming distance of 2 from any other barcode. A custom Python script was used for designing the final library. 
     The final oligonucleotide libraries are synthesized by a commercial vendor (Agilent Technologies). Each library is PCR-amplified and subjected to 1.25×AMPure XP bead purification (Beckman Coulter). After incubation at 25° C. for 10 minutes in CutSmart buffer (New England Biolabs), RNP comprising 769 nM recombinant ABE8.8-m protein and 1.54 μM gRNA is mixed with 100 ng of the purified library and incubated at 37° C. for 8 hours. The RNP dose is derived from an analysis documenting that it is a super-saturating dose, ie, above the dose that achieves the maximum amount of on-target editing in the biochemical assay. 
     Proteinase K (New England Biolabs) is added to quench the reaction at 37° C. for 45 minutes, followed by 2×AMPure XP bead purification. The reaction is then serially incubated with EndoV (New England Biolabs) at 37° C. for 30 minutes, Klenow Fragment (New England Biolabs) at 37° C. for 30 minutes, and NEBNext Ultra II End Prep Enzyme Mix (New England Biolabs) at 20° C. for 30 minutes followed by 65° C. for 30 minutes, with 2×AMPure XP bead purification after each incubation. The reaction is ligated with an annealed adaptor oligonucleotide duplex at 20° C. for 1 hour to facilitate PCR amplification of the cleaved library products, followed by 2×AMPure XP bead purification. Size selection of the ligated reaction is performed on a PippinHT system (Sage Sciences) to isolate DNA of 150 to 200 bp on a 3% agarose gel cassette, followed by 2 rounds of PCR amplification to generate a barcoded library, which undergoes paired-end sequencing on an Illumina MiSeq System as described above. 
     Two cleavage products are obtained in a ONE-seq experiment. The PROTO side includes the part of the oligonucleotide upstream of the cleavage position, whereas the PAM side includes part of the oligonucleotide downstream of the cleavage position. In an ABE experiment, only the PROTO side is informative of editing activity (an A→G substitution); therefore, only this side is sequenced. 
     Paired-end reads were trimmed for sequencing adapters using trimmomatic v0.39 (Bolger et al., 2014) with custom Nextera adapters (PrefixPE/1: ACACTCTTTCCCTACACGACGCTCTTCCGATCT; PrefixPE/2: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT; as specified in file) and parameters “ILLUMINACLIP:NEB_custom.fa:2:30:10:1:true LEADING:0 TRAILING:0 SLIDINGWINDOW:4:30 MINLEN:36”. For experiments with lower sequencing quality (VOL014), these parameters were set to “ILLUMINACLIP NEB_custom.fa:2:30:10:1:true LEADING:2 TRAILING:0 SLIDINGWINDOW:30:30 MINLEN:36”. Reads were then merged using FLASH v1.2.11 (Magoc and Salzberg, 2011) with parameters “—max-mismatch-density=0.25—max-overlap=160”. Merged reads were scanned for the constant sequences, barcodes and protospacer sequences unique to each site, and filtered to those with evidence of an A→G substitution in the editing window (defined as the 1-10 most PAM-distal positions of the protospacer). Duplicated reads were discarded. 
     For each site, the total number of edited reads was normalized to the total number of edited reads assigned to the on-target site, and this ratio defines the ONE-seq score for the site. Sites were ranked by ONE-seq score, and those with a score equal to or larger than 0.001, were selected for validation. This implies the sponsor follows up on sites that have down to 1000-fold less editing activity in the biochemical assay compared to editing of the on-target site. This threshold is based on the premise that in cells, if there is 100% on-target editing, 1/1000-fold less editing activity would translate to &lt;0.1% off-target editing, which falls below the lower limit of detection of editing by NGS. 
     SureSelect. SureSelect panels were designed and purchased from Agilent Technologies. Bases in raw FASTQ files with quality lower than 30 were masked to Ns using seqtk v1.3-r106 (https://github.com/1h3/seqtk). Adapters were trimmed using the “Trimmer” script in Agilent&#39;s AGeNT v2.0.5 tool. Reads were aligned to the GRCh38 reference human genome (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.15_GRCh38/seq s_for_alignment_pipelines.ucsc_ids/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna) using BWA MEM v 0.7.17-r1188 with parameter “-C”. Aligned reads were processed and duplicates were removed using the “LocatIt” script in Agilent&#39;s AGeNT v2.0.5 tool, with parameters “−1-R -IB -OB -U -1&lt;Covered.bed&gt;”. Nucleotide distributions at each position in the editing window (positions 1-10 in the PAM distal side of the protospacer) were determined using perbase v0.6.3 (https://github.com/sstadick/perbase), with parameters “base-depth-F 3848”. Editing was quantified by summing the percentage of reads supporting an A→G substitution in the editing window. 
     RNA-seq for guide-independent off-target analysis. The RNA samples were processed and sequenced by GENEWIZ; following rRNA depletion, libraries were prepared and underwent 2×150-bp paired-end sequencing on an Illumina HiSeq System, with approximate 50 million reads per sample. RNA-seq variant calling for all samples was executed using GATK Best Practices. In short, reads were aligned using STAR to the GRCh38 reference genome (ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.15_GRCh38/seqs_fo r_alignment_pipelines.ucsc_ids/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.gz) with gencode v34 (ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_34/gencode.v34.primary_assembly.annotation.gtf.gz). PCR duplicates were removed using GATK MarkDuplicates, followed by variant identification using GATK HaplotypeCaller. Variants were then filtered by excluding those with QD (Quality of Depth)&lt;2.0 and FS (Fisher strand—evidence of strand bias)&gt;30. All GATK analyses were performed with gatk4 v4.1.8.1. 
     Variants obtained as above were further filtered by comparison with untreated control samples as follows. (1) Nucleotide distributions at each identified variant in treated cells each untreated control sample and each treated sample using perbase v0.5.1 (https://github.com/sstadick/perbase). (2) For all variants covered by at least 20 reads in both treated and untreated conditions, RNA edits were identified as those that had the reference allele (A or T) in at least 95% of reads in all untreated control samples and the alternate allele (G or C) in at least one read in the treated sample. The above steps were executed with each of the ABE8.8-treated and SpCas9-treated samples. 
     Guide RNA synthesis. The guide RNAs shown in Table 1 were/are synthesized under solid phase oligonucleotide synthesis and deprotection conditions using controlled pore glass support and commercially available phosphoramidite monomers and oligonucleotide synthesis reagents (Methods in Molecular Biology, 1993, 20, 81-114; ACS Chem. Biol. 2015, 10, 1181-1187, incorporated herein by reference in its entirety). The spacer section of the guide RNAs were converted to the corresponding ribonucleotides except the first 1-3 nucleotides from the 5′-end. The first 1-3 nucleotides from the 5′-end was/were converted to the corresponding 2′-O-methylribonucleotide as outlined in Table 1. The deprotected guide RNAs were purified by HPLC and the integrity of each guide RNA was confirmed by mass spectrometric analysis. The observed mass of each guide RNA was conformed to calculated mass. 
     mRNA production by in vitro transcription (IVT) The mRNA described herein are produced by different methods well known in the art. One of such methods is in vitro transcription (IVT) using T7 polymerase or additional RNA polymerase variants. Typically, IVT of mRNA uses a linearized DNA template that comprises a T7 polymerase promoter and associated regulatory sequences, mRNA coding sequence (CDS), 3′ and 5′ untranslated regions (UTRs), poly A tail, and additional sequence elements to enhance mRNA stability and in vivo performance. Prior to IVT, the DNA template is in the form of a plasmid, PCR product, synthetic DNA product, or any other double-stranded DNA construct; linearization of the DNA template, typically with a restriction digestion enzyme, is performed to promote run-off transcription. A typical IVT reaction includes T7 polymerase, DNA template, RNase inhibitor, cap analog, inorganic pyrophosphatase, and naturally occurring ribonucleotide triphosphates (NTPs) such as GTP, ATP, CTP, UTP, or substitutions of natural NTPs with modified NTPs such as pseudouridine, N1-methylpseudouridine, 5′methylcytidine, 5-methoxyuridine, N6-methyladenosine, and N4-acetylcytidine. The cap analog can be added during transcription or supplemented after the IVT reaction using a capping enzyme; in both instances a 2′-O-methyl group, or additional 2′ chemical modification, is added to first initiating nucleotide to produce a cap-1 form of mRNA. In some instances, poly A tail is added to the mRNA after the IVT reaction using an RNA ligase enzyme. After IVT, in some cases DNase is added to the transcription mixture to remove DNA template; alternatively, residual DNA is removed with chromatography, precipitation, or tangential flow filtration. Purification and concentration of mRNA is performed with methods such as ion exchange chromatography, affinity chromatography, precipitation, ion-pairing reversed-phase chromatography, hydrogen bond chromatography, cellulose chromatography, reversed-phase chromatography, enzymatic reactions, size exclusion chromatography, and tangential flow filtration. Similar IVT and purification process are used to produce mRNA encoding luciferase, eGFP, SpCas9, Cas12b, CBE, and ABE; in all cases the DNA template, reaction conditions, and purification parameters are optimized for the specific gene of interest. 
     Lipid nanoparticle formulation and analysis. LNPs used were formulated as previously described (Conway, A. et al. 2019  Mol. Ther.  27, 866-877; Villiger, L. et al 2021  Nat. Biomed. Eng.  5, 179-189) and generated either by (1) microfluidic mixing using the Precision Nanosystems NanoAssemblr system according to the manufacturer&#39;s protocol, with some optimization for individual payloads or by (2) rapid inline mixing of a solution of lipid excipients in an organic solvent and an aqueous solution of gRNA and mRNA. The lipid solution generally comprises of a mixture of four formulation excipients namely: an amino lipid, a monomethoxypolyethylene glycol (or methoxypoluyethyle glycol) of average molecular weight 2000 Da conjugated to a lipid called PEG-Lipid, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), mixed in a predetermined molar ratio in ethanol. LNP composition comprised 40-65% of amino lipid, 2-20% DSPC, 1-5% PEG-Lipid, with the balance being cholesterol (all in mol %). The RNA aqueous solution contains a 1:1 by weight mixture of desired mRNA and guide RNA (gRNA) unless otherwise specified. For evaluating impact of mRNA to gRNA ratio, aqueous solution containing desired weight ratio of mRNA and gRNA were prepared prior to the preparation of corresponding LNPs, for evaluation; for example, the preparation of LNP test article to evaluate 6:1, 3:1, 2:1, 1:1, 1:2, 1:3 and 1:6 mRNA to gRNA ratio in mice (Example 6,  FIG.  19   ). In some other instances, the mRNA and two gRNAs were mixed in 1:0.5:0.5 (mRNA:gRNA1:gRNA2) weight ratio to prepare LNPs containing desired mRNA and two gRNAs in a single test article (Example 10,  FIG.  43   ). The aqueous solution of desired mRNA to gRNA was then mixed with the lipid excipients in ethanol by microfluidic or by rapid inline mixing. All LNPs for reported NHP studies were prepared by following the inline mixing protocols as described WO 2019/036028 A1, WO 2015/199952 A1, WO 2017/004143, WO 2020/081938 A1 and WO 2020/061426 A2. The PEG-Lipid used for the preparation of the NHP LNP test articles was selected from WO 2019/036028 A1, WO 2015/199952 A1. The LNP compositions and ionizable amino lipids were selected from the from Patent Publications WO/2017/004143A1, WO/2017/075531A1 and WO/2018/191719 A1. The ionizable amino lipids used for the preparation of LNP #1 and LNP #2 (Example 8,  FIGS.  30    &amp;  FIG.  45   ) were selected from the publication WO 2018/191719 A1. The resulting LNP formulations were subsequently dialyzed against test article buffer and filtered using a 0.2 μm sterile filter. 
     As an example, the LNPs used for cellular and NHP studies had an average hydrodynamic diameter range of 55 to 65 nm, with a polydispersity index of &lt;0.2 as determined by dynamic light scattering and 85-98% total RNA encapsulation as measured by the Quant-iT Ribogreen Assay. The LNP particle size (Z-Ave, hydrodynamic diameter), polydispersity index and total RNA encapsulation were measured as described in the literature prior to administration. 
     As an example, the LNPs used for cellular and mouse studies had a particle size of 55-120 nm (Z-Ave, hydrodynamic diameter), with a polydispersity index of &lt;0.2 as determined by dynamic light scattering (Malvern NanoZS Zetasizer) and 85-100% total RNA encapsulation as measured by the Quant-iT Ribogreen Assay (Thermo Fisher). 
     Genomic DNA extraction. The whole mouse liver or 100-200 mg of monkey liver was loaded into 2 mL lysing matrix tubes (MP Bio). Livers were lysed with 0.5 ml PBS for the mouse liver or 0.25 mL PBS for the monkey liver, using the FastPrep-24 system (MP Bio) according to the manufacturer&#39;s protocol. Genomic DNA was isolated from approximately 20 L of mouse or monkey liver lysate using a bead-based extraction kit, MagMAX-96 DNA Multi-Sample Kit (Thermo-Fisher Scientific) on the KingFisher Flex automated extraction instrument (Thermo-Fisher Scientific) according to the manufacturer&#39;s protocols. For monkey liver biopsy samples, Qiagen DNEasy Blood &amp; Tissue kit extraction was used to extract genomic DNA according to the manufacturer&#39;s instructions. Extracted genomic DNA was stored at 4° C. until further use or at −80° C. for long term storage. 
     PCSK9 protein levels quantified by ELISA. Blood samples were collected and processed to plasma following blood draw. The plasma cynomolgus PCSK9 levels were determined by ELISA using the described ELISA; briefly, test samples or standards of purified cynomolgus monkey PCSK9 diluted in assay diluent D (Biolegend, part #76384) were incubated with assay buffer A (Biolegend, part #78232) in a 96-well microplate coated with a monoclonal antibody specific for human PCSK9 (Biolegend, part #76157). After four washes with wash buffer (Biolegend, part #78233), a polyclonal antibody specific for human PCSK9 (Biolegend, part #76158) was incubated in individual wells. After four washes, avidin-HRP (Biolegend, part #77897) were next incubated in individual wells. After six washes, substrate solution F (Biolegend, part #79132), which contains TMB, was used to develop the plate. Optical density was determined using on a microplate reader set to 450 nm. Readings at 570 nm were subtracted from the readings at 450 nm to correct for optical imperfections in the plate. Blood samples were collected and processed to plasma following blood draw and stored at −86 to −60° C. until analysis. 
     ANGPTL3 protein levels quantified by ELISA. The plasma cynomolgus ANGPTL3 levels were determined by ELISA; briefly, test samples or standards of purified cynomolgus monkey ANGPTL3 diluted in calibrator diluent RD6Q (R&amp;D, part #895128) were incubated with assay diluent RD1-76 (R&amp;D, part #895812) in a 96-well microplate coated with a monoclonal antibody specific for human ANGPTL3 (R&amp;D, part #893734). After four washes with wash buffer (R&amp;D, part #895003), human ANGPTL3 conjugate (R&amp;D, part #893735) which contains a polyclonal antibody specific for human ANGPTL3 conjugated to horseradish peroxidase (HRP) were next incubated in individual wells. After four washes, TMB substrate solution (R&amp;D, part #895000 and 895001) was used to develop the plate. Optical density was determined using on a microplate reader set to 450 nm. Readings at 540 nm were subtracted from the readings at 450 nm to correct for optical imperfections in the plate. 
     Quantifying lipid levels. Reagent kits each analyte contain reagent, cholesterol, triglycerides and HDL-C are quantified using absorbance measurements of specific enzymatic reaction products. LDL-C is determined indirectly. Most of the circulating cholesterol is found in three major lipoprotein fractions: very low-density lipoproteins (VLDL), LDL and HDL. [Total C]=[VLDL-C]+[LDL-C]+[HDL-C]. Thus the LDL-C can be calculated from measured values of total cholesterol, triglycerides and HDL-C according to the relationship: [LDL-C]=[total C]− [HDL-C]− [TG]/5, where [TG]/5 is an estimate of VLDL-cholesterol expressed. These results provide a preclinical proof of concept of a base-editing therapy knocking down PCSK9 in the human liver in vivo and effecting reductions in blood PCSK9 protein and LDL-C levels. 
     Direct measurement of triglyceride levels. A clinical analyzer instrument is used to measure a ‘lipid panel’ in serum samples. This entails the direct measurement of cholesterol (total C), triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C). A reagent kit specific for triglycerides contains buffers, calibrators, blanks and controls. Using the provided reagents, serum samples from the study are analyzed. Triglycerides are measured using a series of coupled enzymatic reactions. H 2 O 2  is the end product of the last one and its absorbance at 500 nm is used to quantify the analyte. The color intensity is proportional to triglyceride concentrations. All values are reported in mg/dL. 
     LNP treatment of mice. The mouse studies were approved by the Institutional Animal Care and Use Committee of the Charles River Accelerator and Development Lab (CRADL), where the studies were performed. Female C57BL/6J mice were obtained from The Jackson Laboratory and used for experiments at 8-10 weeks of age, with random assignment of animals to various experimental groups. LNP were administered to the mice via injection into the lateral tail vein and/or retro-orbital injection of the of the venous sinus ( Lab Anim  2011; 40(5): 155-160) at mg/kg doses that correspond to mg of total RNA per animal weight (kilogram). The dose is calculated based on total RNA that constitute the amount of mRNA and gRNA, after formulating the LNP. One week following treatment, the mice were euthanized unless otherwise stated, and liver samples were obtained on necropsy and processed with the KingFisher Flex Purification System according to the manufacturer&#39;s instructions to isolate genomic DNA. 
     LNP treatment of NHPs. The NHP studies were approved by the Institutional Animal Care and Use Committees of Envol Biomedical and Altasciences, respectively. NHPs studies were performed at Envol Biomedical (study #VTP2001) and Altasciences (study #1388.02, 04, 05, 09, and 11), with both studies using  Macaca fascicularis ) of Cambodian origin. The animals were 2-3 years of age and 2-3 kilograms in weight at the time of study initiation. All the animals were genotyped at the PCSK9 and/or ANGPTL3 editing site(s) to ensure that any animals receiving LNPs were homozygous for the protospacer DNA sequences perfectly matching the gRNA sequence, and animals were randomly assigned to various experimental groups. The animals were premedicated with 1 mg/kg dexamethasone, 0.5 mg/kg famotidine, and 5 mg/kg diphenhydramine prior to LNP administration unless otherwise stated. The LNP were administered using a temporary catheter inserted into a peripheral vein connected to a primed infusion line, over the course of 1 hour (+/−5 minutes). Dose formulations were administered at a volume of 6 ml/kg, unless otherwise specified, and dosed at mg/kg corresponding to mg of total RNA per animal weight (kilogram). The dose is calculated based on total RNA that constitute the amount of mRNA and gRNA, after formulating the LNP. The appropriate volume based on the weight of the animal was delivered using an infusion pump. Control animals received phosphate-buffered saline instead of LNP under the same infusion conditions. When there are two LNPs constituted from amino lipid 1 and amino lipid 2 used in the same NHP study, the test articles are identified as LNP #1 and LNP #2 where mRNA and gRNA used for preparing these LNPs are the same. If there is only one LNP composition used in a study, the test article is identified as LNP 
     For blood chemistry samples, animals were fasted for at least 4 hours before collection via peripheral venipuncture. NHP studies generally followed collection on the following schedule: day −10, day −7, day −5, day 1 (6 hours after LNP infusion), day 2, day 3, day 5, day 8, and day 15. In the long-term study, samples were also collected at day 21 and day 28 and have generally been collected every 2 weeks thereafter and analyzed by the study site for LDL cholesterol, HDL cholesterol, total cholesterol, triglycerides, AST, and ALT. For each analyte, the mean of the values at day −10, day −7, and day −5 were regarded as the baseline value. A portion of each blood sample was sent to the investigators for PCSK9 or ANGPTL3 protein measurement. 
     Analysis of cytokine levels. The serum cynomolgus IL,-6, MCP-1 and IP-10 levels were determined by U-PLEX Biomarker Group 1 (NP) Assays (Meso Scale Discovery, #K15068L-2) according to manufacturer&#39;s instruction. Briefly, U-PLEX (Meso Scale Discovery, #N05230) plate was incubated overnight at 4° C. with linker-coupled capture antibodies (MCP-1 antibody; Meso Scale Discovery, #C26UG-3, TL-6 antibody; Meso Scale Discovery, #C21TX-3, IP-10 antibody; Meso Scale Discovery, #C21UF-3). After 3 washes with PBS-T (PBS containing 0.050 Tween 20), test samples or Calibrator standards (Calibrator 1; Meso Scale Discovery, #C0060-2, Calibrator 2: Meso Scale Discovery, #C0061-2) were incubated with assay diluent 43 (Meso Scale Discovery, #R50AG-2), which contains serum, blockers and preservatives, at room temperature for an hour. After 3 washes, SULFO-TAG conjugated detection antibodies for TL-6 (Meso Scale Discovery, #D26TX-3), MCP-1 (Meso Scale Discovery, #D26UG-3) and IP-10 (Meso Scale Discovery, #D21UF-3) were incubated in individual wells at at room temperature for an hour. After 3 washes and adding Gold™ read buffer B (Meso Scale Discovery, #R60AM-2) to each well, the plate was analyzed by MSD instrument (Meso Scale Discovery, #R31QQ-3. 
       
     
       
         
           
               
             
               
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     All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. 
     Other Embodiments 
     From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. 
     The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment, any portion of the embodiment, or in combination with any other embodiments or any portion thereof. 
     As is set forth herein, it will be appreciated that the disclosure comprises specific embodiments and examples of base editing systems to effect a nucleobase alteration in a gene and methods of using same for treatment of disease including compositions that comprise such base editing systems, designs and modifications thereto; and specific examples and embodiments describing the synthesis, manufacture, use, and efficacy of the foregoing individually and in combination including as pharmaceutical compositions for treating disease and for in vivo and in vitro delivery of active agents to mammalian cells under described conditions. 
     While specific examples and numerous embodiments have been provided to illustrate aspects and combinations of aspects of the foregoing, it should be appreciated and understood that any aspect, or combination thereof, of an exemplary or disclosed embodiment may be excluded therefrom to constitute another embodiment without limitation and that it is contemplated that any such embodiment can constitute a separate and independent claim. Similarly, it should be appreciated and understood that any aspect or combination of aspects of one or more embodiments may also be included or combined with any aspect or combination of aspects of one or more embodiments and that it is contemplated herein that all such combinations thereof fall within the scope of this disclosure and can be presented as separate and independent claims without limitation. Accordingly, it should be appreciated that any feature presented in one claim may be included in another claim; any feature presented in one claim may be removed from the claim to constitute a claim without that feature; and any feature presented in one claim may be combined with any feature in another claim, each of which is contemplated herein. The following enumerated clauses are further illustrative examples of aspects and combination of aspects of the foregoing embodiments and examples: 
     Following is the first example of enumerated clauses:
     1. A composition for editing a gene target comprising:   (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same,   (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on a PCSK9 gene,   wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the PCSK9 gene in vivo when administered to a mammalian subject,   wherein when the guide RNA and the mRNA is administered at a total amount of at least 0.05 mg/kg, the base alteration occurs in at least 35% of whole liver cells in the mammalian subject as measured by next generation sequencing or Sanger sequencing.   2. The composition of clause 1, wherein the mammalian subject is a cynomolgus monkey,   wherein when the guide RNA and the mRNA is administered at a total amount of at least 0.5 mg/kg, the base alteration occurs in at least 40% of whole liver cells in the cynomolgus monkey as measured by next generation sequencing or Sanger sequencing.   3. The composition of clause 1, wherein the mammalian subject is a cynomolgus monkey,   wherein when the guide RNA and the mRNA is administered at a total amount of at least 1 mg/kg, the base alteration occurs in at least 45% of whole liver cells in the cynomolgus monkey as measured by next generation sequencing or Sanger sequencing.   4. The composition of clause 1, wherein the mammalian subject is a cynomolgus monkey,   wherein when the guide RNA and the mRNA is administered at a total amount of at least 1.5 mg/kg, the base alteration occurs in at least 50% of whole liver cells in the cynomolgus monkey as measured by next generation sequencing or Sanger sequencing.   5. The composition of clause 1, wherein the mammalian subject is a cynomolgus monkey,   wherein when the guide RNA and the mRNA is administered at a total amount of at least 3 mg/kg, the base alteration occurs in at least 55% of whole liver cells in the cynomolgus monkey as measured by next generation sequencing or Sanger sequencing.   6. The composition of clause 1, wherein the mammalian subject is a mouse,   wherein when the guide RNA and the mRNA is administered at a total amount of at least 0.125 mg/kg, the base alteration occurs in at least 40% of whole liver cells in the mouse as measured by next generation sequencing or Sanger sequencing.   7. The composition of clause 1, wherein the mammalian subject is a mouse,   wherein when the guide RNA and the mRNA is administered at a total amount of at least 0.5 mg/kg, the base alteration occurs in at least 45% of whole liver cells in the mouse as measured by next generation sequencing or Sanger sequencing.   8. The composition of clause 1, wherein the mammalian subject is a mouse,   wherein when the guide RNA and the mRNA is administered at a total amount of at least 2 mg/kg, the base alteration occurs in at least 50% of whole liver cells in the mouse as measured by next generation sequencing or Sanger sequencing.   9. The composition of clause 2, wherein the nucleobase alteration results in a reduction of at least 50% in blood low-density lipoprotein cholesterol (LDL-C) level in the subject as compared to prior to the administration.   10. The composition of any one of clauses 1-9, wherein the protospacer is located in a splice site.   11. The composition of any one of clauses 1-9, wherein the protospacer complementary sequence is in the antisense strand of the PCSK9 gene.   12. The composition of any one of clauses 1-9, wherein the protospacer complementary sequence is in the sense strand of the PCSK9 gene.   13. The composition of any one of clauses 1-12, wherein the base alteration happens outside of the protospacer on the PCSK9 gene (off-target sites),   wherein the editing percentages of off-target sites set forth in Table 11 are below or equal to the editing percentages set forth in Table 11, respectively.   14. The composition of any one of clauses 1-13, wherein the deaminase is an adenine deaminase and wherein the nucleobase alteration is a A•T to G•C alteration.   15. The composition of any one of clauses 1-14, wherein the programmable DNA binding domain comprises a nuclease inactive Cas9 or a Cas9 nickase.   16. The composition of any one of clauses 1-15, wherein the nucleobase alteration is at a splice site of the PCSK9 gene.   17. The composition of clause 16, wherein the nucleobase alteration is at a splice donor site of the PCSK9 gene.   18. The composition of clause 17, wherein the splice donor site is at 5′ end of PCSK9 intron 1 as referenced in SEQ ID NO: 5.   19. The composition of clause 16, wherein the nucleobase alteration is at a splice acceptor site of the PCSK9 gene.   20. The composition of any one of clauses 1-19, wherein the nucleobase alteration results in a frame shift, a premature stop codon, an insertion or deletion in a transcript encoded by the PCSK9 gene.   21. The composition of any one of clauses 1-20, wherein the nucleobase alteration results in an aberrant transcript encoded by the PCSK9 gene.   22. The composition of any one of clauses 1-21, wherein the guide RNA is chemically modified.   23. The composition of clause 16, wherein the tracr sequence of the guide RNA is chemically modified following the scheme depicted in  FIG.  7   .   24. The composition of any one of clauses 1-22, wherein the spacer sequence comprises a PCSK9 ABE guide RNA spacer sequence set forth in Table 1.   25. The composition of clause 24, wherein the guide RNA comprises the PCSK9 ABE guide RNA sequence of GA096, GA097, GA343, GA346, GA375-377, GA380-389, GA391, GA439 or GA440 as set forth in Table 1.   26. The composition of any one of clauses 1-23, wherein the protospacer sequence comprises a PCSK9 ABE protospacer sequence set forth in Table 1.   27. The composition of clause 26, wherein the protospacer comprises the sequence 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID No: 13) or 5′-CCGCACCTTGGCGCAGCGG-3′ (SEQ ID No: 247).   28. The composition of any one of clauses 1-27, wherein the base editor fusion protein comprises an amino acid sequence of SEQ ID No: 2137.   29. The composition of any one of clauses 1-28, wherein the GC % content of the mRNA sequence is greater than 50%.   30. The composition of clause 29, wherein the GC % content of the mRNA sequence is greater than 56%.   31. The composition of clause 30, wherein the GC % content of the mRNA sequence is greater than or equal to 63%.   32. The composition of clause 29, wherein the mRNA comprises an adenine tTNA deaminase (TadA) region, a Cas9 region and a nuclear localization sequence (NLS) region.   33. The composition of clause 32, wherein the mRNA further comprises a first linker region which connects the TadA region and the Cas9 region, and a second linker region which connects the Cas9 region and the NLS region.   34. The composition of clause 32 or 33, wherein the GC % content of the TadA region is greater than 60%.   35. The composition of clause 32 or 33, wherein the GC % content of the TadA region is greater than or equal to 70%.   36. The composition of clause 32 or 33, wherein the GC % content of the Cas9 region is greater than 56%.   37. The composition of clause 32 or 33, wherein the GC % content of the Cas9 region is greater than or equal to 62%.   38. The composition of clause 32 or 33, wherein the GC % content of the NLS region is greater than 54%.   39. The composition of clause 32 or 33, wherein the GC % content of the NLS region is greater than or equal to 63%.   40. The composition of clause 33, wherein the GC % content of the first linker region is greater than 65%.   41. The composition of clause 33, wherein the GC % content of the first linker region is greater than or equal to 79%.   42. The composition of clause 33, wherein the GC % content of the second linker region is greater than 67%.   43. The composition of clause 33, wherein the GC % content of the second linker region is greater than or equal to 83%.   44. The composition of clause 33, wherein the GC % content of the TadA region is greater than 60%, the GC % content of the Cas9 region is greater than 56%, the GC % content of the NLS region is greater than 54%, the GC % content of the first linker region is greater than 65%, and the GC % content of the second linker region is greater than 67%.   45. The composition of clause 29, wherein the mRNA comprises a mRNA sequence selected from Table 23.   46. The composition of clause 45, wherein the mRNA comprises a mRNA sequence of SEQ ID No: 2136.   47. The composition of any one of clauses 29-46, wherein the mRNA comprises a poly A tail.   48. The composition of any one of clauses 1-47, further comprising a lipid nanoparticle (LNP) enclosing (i).   49. The composition of clause 48, wherein the LNP further encloses (ii).   50. The composition of clause 48, further comprising a second LNP enclosing (ii).   51. The composition of any one of clauses 1-44, wherein the ratio of the guide RNA and the mRNA encoding the base editor fusion protein is about 1:10 to about 10:1 by weight.   52. The composition of clause 51, wherein the ratio of the guide RNA and the mRNA encoding the base editor fusion protein is about 1:1, 1.5:1, 2:1, 3:1, 4:1, 1:1.5, 1:2, 1:3, or 1:4 by weight.   53. The composition of clause 51, wherein the ratio of the guide RNA and the mRNA encoding the base editor fusion protein is about 1:1 by weight.   54. A pharmaceutical composition comprising the composition of any one of the preceding clauses and a pharmaceutically acceptable carrier or excipient.   55. A method for treating or preventing a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of clause 1.   56. The method of clause 55, wherein the administration is via intravenous infusion.   57. The method of clause 55 or 56, comprising sequential administration of a LNP enclosing (i) and a LNP enclosing (ii).   58. The method of clause 55 or 56, comprising concurrent administration of the t LNP enclosing (i) and the LNP enclosing (ii).   59. The method of clause 57, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 1 day.   60. The method of clause 57, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 2 days.   61. The method of clause 57, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 3 days.   62. The method of clause 57, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 4 days.   63. The method of clause 57, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 5 days.   64. The method of clause 57, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 6 days.   65. The method of clause 57, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 7 days.   66. The method of clause 55 or 56, comprising administering a single dose of the LNP enclosing (i) and (ii).   67. The method of clause 66, wherein the single dose of the LNP is at about 0.3 to about 3 mg/kg.   68. The method of clause 66 or 67, comprising administering a treatment course of one or more treatments to the subject, wherein each one of the one or more treatment comprises one or more of the single doses of the LNP.   69. The method of clause 68, comprising administering a treatment course of two to ten treatments.   70. The method of clause 68, comprising administering a treatment course of two to five treatments.   71. The method of clause 68, comprising administering a treatment course of two treatments.   72. The method of clause 68, comprising administering a treatment course of three treatments.   73. The method of clause 68, comprising administering a treatment course of four treatments.   74. The method of clause 68, comprising administering a treatment course of five treatments.   75. The method of any one of clauses 55-74, wherein the condition is an atherosclerotic cardiovascular disease.   76. The method of any one of clauses 55-74, wherein the condition is an atherosclerotic vascular disease.   77. The method of any one of clauses 55-74, wherein the subject is a human.   78. A composition for editing a gene target comprising:   (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same,   (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on a PCSK9 gene,   wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the PCSK9 gene in vivo when administered to a mammalian subject, and   wherein the guide RNA comprises the PCSK9 ABE guide RNA sequences as set forth in Table 1.   79. A composition for editing a gene target comprising:   (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same,   (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on a PCSK9 gene,   wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the PCSK9 gene in vivo when administered to a mammalian subject, and   wherein the mRNA comprises a sequence selected from Table 23.   80. A method for treating or preventing an atherosclerotic cardiovascular disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a first composition, comprising   (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same,   (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on a PCSK9 gene,   wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the PCSK9 gene in vivo when administered to a mammalian subject,   wherein when the guide RNA and the mRNA is administered at a total amount of at least 0.05 mg/kg, the base alteration occurs in at least 35% of whole liver cells in the mammalian subject as measured by next generation sequencing or Sanger sequencing; and   a second composition, comprising   (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same,   (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on a ANGPTL3 gene,   wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the ANGPTL3 gene in vivo when administered to a mammalian subject,   wherein when the guide RNA and the mRNA is administered at a total amount of at least 0.5 mg/kg, the base alteration occurs in at least 35% of whole liver cells in the mammalian subject as measured by next generation sequencing or Sanger sequencing.   81. The method of clause 80, comprising sequential administration of the first composition and the second composition.   82. The method of clause 81, comprising administering one or more doses of the first composition followed by one or more dose of the second composition.   83. The method of clause 82, comprising administering one or more doses of the second composition followed by one or more dose of the first composition.   84. The method of clause 80, comprising concurrent administration of the first composition and the second composition.   85. The method of clause 84, comprising one or more doses of the first composition and the second composition.   86. A composition for editing a gene target comprising:   (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same,   (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on a APOC3 gene,   wherein the guide RNA comprises the APOC3 ABE guide RNA sequence of GA300-303 as set forth in Table 24.   87. A composition for editing a gene target comprising:   (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same,   (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on a PCSK9 gene,   wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the PCSK9 gene in vitro,   wherein when the guide RNA and the mRNA is administered at a total amount of at least 2.5 mg/kg, the base alteration occurs in at least 35% of whole liver cells in the mammalian subject as measured by next generation sequencing or Sanger sequencing.   88. A composition for editing a PCSK9 gene comprising:   (a) a mRNA encoding an adenine base editor protein having an editing window, and   (b) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor protein and a spacer sequence that serves to guide the base editor protein to a protospacer sequence on the PCSK9 gene,   wherein the spacer sequence is complimentary, at least in part, to a splice site or an exon region of the PCSK9 gene.   89. The composition for editing the PCSK9 gene of clause 88, wherein when the base editor protein is operatively bound to the guide RNA and the guide RNA is hybridized with the complementary strand to the protospacer sequence on the PCSK9 gene, the editing window encompasses the splice site of the PCSK9 gene.   90. The composition for editing the PCSK9 gene of clause 88, wherein when the base editor protein is operatively bound to the guide RNA and the guide RNA is hybridized with the complementary strand to the protospacer sequence on the PCSK9 gene, the editing window encompasses a region of an intron of the PCSK9 gene.   91. The composition for editing the PCSK9 gene of clause 88, wherein when the base editor protein is operatively bound to the guide RNA and the guide RNA is hybridized with the complementary strand to the protospacer sequence on the PCSK9 gene, the editing window encompasses a region of intron 1, intron 3 or intron 4 of the PCSK9 gene.   92. The composition for editing the PCSK9 gene of clause 88, wherein when the base editor protein is operatively bound to the guide RNA and the guide RNA is hybridized with the complementary strand to the protospacer sequence on the PCSK9 gene, the editing window encompasses a region of intron 1 of the PCSK9 gene.   93. The composition for editing the PCSK9 gene of clause 88, wherein the spacer sequence has a 80-100% nucleotide sequence identity to a spacer sequence selected from the group of guide RNA sequences identified as GA066, GA073 and GA074.   94. The composition for editing the PCSK9 gene of clause 88, wherein the tracr sequence has a 80-100% nucleotide sequence identity to a tracr sequence selected from the group of guide RNA sequences identified as GA066, GA095, GA096, GA097, GA343, GA346, GA375, GA376, GA377, GA380, GA381, GA382, GA383, GA384, GA385, GA386, GA387, GA388, GA389, GA439, and GA440.   95. The composition for editing the PCSK9 gene of clause 88, wherein the mRNA has an 80-100%0 sequence identity to the mRNA sequences identified as MA002, MA004, MA040, MA0041, or MA045.   96. The composition for editing the PCSK9 gene of clause 88, wherein the mRNA has one or more of the GC nucleotide region percentages set forth in the following table:   

     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Nucleotide region 
                 Average GC Nucleotide Content 
               
               
                   
                   
               
             
            
               
                   
                  27-213 
                 67-73% 
               
               
                   
                 389-661 
                 67-71% 
               
               
                   
                 735-829 
                 63-74% 
               
               
                   
                 4207-4286 
                 67-70% 
               
               
                   
                 4537-4569 
                 65-73% 
               
               
                   
                 4683-4741 
                 62-67% 
               
               
                   
                   
               
            
           
         
       
         
         97. The composition for editing the PCSK9 gene of clause 88, wherein the mRNA has one or more of the GC nucleotide region percentages set forth in the following table: 
       
    
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Nucleotide region 
                 Average GC Nucleotide Content 
               
               
                   
                   
               
             
            
               
                   
                  27-213 
                 At least 73% 
               
               
                   
                 389-661 
                 At least 71% 
               
               
                   
                 735-829 
                 At least 74% 
               
               
                   
                 4207-4286 
                 At least 70% 
               
               
                   
                 4537-4569 
                 At least 73% 
               
               
                   
                 4683-4741 
                 At least 67% 
               
               
                   
                   
               
            
           
         
       
         
         98. The composition for editing the PCSK9 gene of clause 88, wherein the mRNA and gRNA are encapsulated within a lipid nanoparticle. 
         99. The composition for editing the PCSK9 gene of clause 88, wherein the mRNA and gRNA are encapsulated within a lipid nanoparticle having the following:
       LNP composition (mol %):   40-65% iLipid   2-20% DSPC   1-5%0 PEG   Remaining mol % balance is cholesterol;   LNP Particle size: 55-120 nm Z average hydrodynamic diameter; and   Polydispersity index of &lt;0.2 as determined by dynamic light scattering.   
     
         100. The composition for editing the PCSK9 gene of clause 88, wherein the mRNA and gRNA are encapsulated within the lipid nanoparticle having an LNP particle size between 50-70 nm Z average hydrodynamic diameter. 
         101. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 40 percent. 
         102. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 50 percent. 
         103. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 30 percent. 
         104. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 40 percent. 
         105. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 50 percent. 
         106. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 60 percent. 
         107. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 70 percent. 
         108. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 40 percent. 
         109. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 50 percent. 
         110. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 60 percent. 
         111. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 70 percent. 
         112. The composition for editing the PCSK9 gene of clause 88, wherein the composition when at administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 80 percent. 
         113. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 40 percent. 
         114. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 50 percent. 
         115. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 60 percent. 
         116. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 70 percent. 
         117. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the PCSK9 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 80 percent. 
         118. The compositions for editing the PCSK9 gene of clauses 87-117, wherein the percent editing is determined at 15 days after dosing through analysis of dosed cynomolgus monkey liver either via liver biopsy or necropsy of the monkey. 
         119. The compositions for editing of the PCSK9 gene of clauses 87-117, wherein the percent editing is determined to be durably maintained by periodic liver biopsy testing of the dosed cynomolgus monkeys over a span of at least 168 days after dosing. 
         120. The compositions for editing of the PCSK9 gene of clauses 87-117, wherein the percent editing is determined to be durably maintained by periodic liver biopsy testing of the dosed cynomolgus monkeys over a span of at least 300 days after dosing. 
         121. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         122. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         123. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         124. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed monkeys on average of at least 60 percent as compared to baseline. 
         125. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 70 percent as compared to baseline. 
         126. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 80 percent as compared to baseline. 
         127. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         128. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         129. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         130. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 60 percent as compared to baseline. 
         131. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 70 percent as compared to baseline. 
         132. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 80 percent as compared to baseline. 
         133. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         134. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         135. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         136. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 60 percent as compared to baseline. 
         137. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 70 percent as compared to baseline. 
         138. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 80 percent as compared to baseline. 
         139. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         140. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         141. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         142. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 60 percent as compared to baseline. 
         143. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 70 percent as compared to baseline. 
         144. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing PCSK9 protein in the plasma of the dosed cynomolgus monkeys on average of at least 80 percent as compared to baseline. 
         145. The compositions for editing of the PCSK9 gene of clauses 121-144, wherein the reduction in plasma protein is determined at 15 days after dosing via blood sampling and analysis of the dosed cynomolgus monkey. 
         146. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of monkey weight is capable of reducing LDL-C in the plasma of the dosed monkeys on average of at least 20 percent as compared to baseline. 
         147. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 25 percent as compared to baseline. 
         148. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 30 percent as compared to baseline. 
         149. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         150. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         151. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 45 percent as compared to baseline. 
         152. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 20 percent as compared to baseline. 
         153. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 25 percent as compared to baseline. 
         154. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 30 percent as compared to baseline. 
         155. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         156. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         157. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 45 percent as compared to baseline. 
         158. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         159. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 55 percent as compared to baseline. 
         160. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 60 percent as compared to baseline. 
         161. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 20 percent as compared to baseline. 
         162. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 25 percent as compared to baseline. 
         163. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 30 percent as compared to baseline. 
         164. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         165. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         166. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 45 percent as compared to baseline. 
         167. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         168. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 55 percent as compared to baseline. 
         169. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 60 percent as compared to baseline. 
         170. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 65 percent as compared to baseline. 
         171. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 20 percent as compared to baseline. 
         172. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 25 percent as compared to baseline. 
         173. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 30 percent as compared to baseline. 
         174. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         175. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         176. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 45 percent as compared to baseline. 
         177. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         178. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 55 percent as compared to baseline. 
         179. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 60 percent as compared to baseline. 
         180. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing LDL-C in the plasma of the dosed cynomolgus monkeys on average of at least 65 percent as compared to baseline. 
         181. The compositions for editing the PCSK9 gene of clauses 148-182, wherein the reduction in LDL-C is determined at 15 days after dosing via blood sampling and analysis of the dosed cynomolgus monkey. 
         182. The compositions for editing the PCSK9 gene of clauses 148-182, wherein the reduction in LDL-C is determined to be durably maintained over a span of at least 168 days by periodic blood sampling and analysis of the dosed cynomolgus monkey. 
         183. The compositions for editing the PCSK9 gene of clauses 148-182, wherein the reduction in LDL-C is determined to be durably maintained over a span of at least 300 days by periodic blood sampling and analysis of the dosed cynomolgus monkey. 
         184. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing lipoprotein(a) in the plasma of the dosed cynomolgus monkeys on average of at least 10 percent as compared to baseline. 
         185. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing lipoprotein(a) level in the plasma of the dosed cynomolgus monkeys on average of at least 15 percent as compared to baseline. 
         186. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing lipoprotein(a) level in the plasma of the dosed cynomolgus monkeys on average of at least 20 percent as compared to baseline. 
         187. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing lipoprotein(a) level in the plasma of the dosed cynomolgus monkeys on average of at least 25 percent as compared to baseline. 
         188. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing lipoprotein(a) level in the plasma of the dosed cynomolgus monkeys on average of at least 30 percent as compared to baseline. 
         189. The composition for editing the PCSK9 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing lipoprotein(a) level in the plasma of the dosed cynomolgus monkeys on average of approximately 35 percent as compared to baseline. 
         190. The compositions for editing the PCSK9 gene of clauses 186-191, wherein the reduction in lipoprotein(a) is determined at 15 days after dosing via blood sampling and analysis of the dosed cynomolgus monkey. 
         191. The compositions for editing the PCSK9 gene of clauses 186-191, wherein the reduction in lipoprotein(a) is determined to be durably maintained over a span of at least 224 days by periodic blood sampling and analysis of the dosed cynomolgus monkey. 
         192. The compositions for editing the PCSK9 gene of clauses 186-191, wherein the reduction in lipoprotein(a) is determined to be durably maintained over a span of at least 300 days by periodic blood sampling and analysis of the dosed cynomolgus monkey. 
         193. The compositions for editing the PCSK9 gene of clauses 101-194, wherein to the extent that the dosing of the cynomolgus monkeys results in elevation of AST, ALT, or Cytokines, the elevations resulting from the dosing of the composition are transient and resolved back to approximately baseline levels within 3-15 days after dosing. 
         194. The composition for editing the PCSK9 gene of clauses 101-103, 121-126, 148-153, wherein the percent editing of PCSK9 is negligible outside of the liver, spleen and adrenal glands tissues as illustrated in  FIG.  27   . 
         195. The compositions for editing the PCSK9 gene of clauses 101-107, 121-132, 148-162, wherein repeat dosing results is additive with respect to the editing percentage of PCSK9 editing percentage. 
         196. The compositions for editing the PCSK9 gene of clause 195, wherein the repeat dosing does not elicit cytokine activation nor an immune response. 
         197. The composition for editing the PCSK9 gene of clause 88, wherein the spacer sequence has at least 80% nucleotide correlation with the nucleotide sequence of a targeted protospacer on the PCSK9 gene, wherein an RNA nucleotide on the spacer sequence is in correlation with a DNA nucleotide of the protospacer if it has the same nucleotide as the DNA nucleotide in the same order and wherein uracil and thymine bases are considered the same nucleotide for purposes of determining correlation. 
         198. The composition for editing the PCSK9 gene of clause 197, wherein the spacer sequence has at least 85% nucleotide correlation with the nucleotide sequence of a targeted protospacer on the PCSK9 gene. 
         199. The composition for editing the PCSK9 gene of clause 197, wherein the spacer sequence has at least 90% nucleotide correlation with the nucleotide sequence of a targeted protospacer on the PCSK9 gene. 
         200. The composition for editing the PCSK9 gene of clause 197, wherein the spacer sequence has at least 95% nucleotide correlation with the nucleotide sequence of a targeted protospacer on the PCSK9 gene. 
         201. The composition for editing the PCSK9 gene of clause 197, wherein the spacer sequence has at least 99% nucleotide correlation with the nucleotide sequence of a targeted protospacer on the PCSK9 gene. 
         202. The composition for editing the PCSK9 gene of clause 197, wherein the spacer sequence has at least 100% nucleotide correlation with the nucleotide sequence of a targeted protospacer on the PCSK9 gene. 
         203. A method for treating or preventing an atherosclerotic cardiovascular disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of 
         (a) a mRNA encoding an adenine base editor protein having an editing window, 
         (b) a first guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor protein and a spacer sequence that serves to guide the base editor protein to a protospacer sequence on a PCSK9 gene, wherein the spacer sequence is complimentary, at least in part, to a splice site or an exon region of the PCSK9 gene; and 
         (c) a second guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor protein and a spacer sequence that serves to guide the base editor protein to a protospacer sequence on a ANGPTL3 gene, wherein the spacer sequence is complimentary, at least in part, to a splice site or an exon region of the ANGPTL3 gene. 
         204. The method of clause 203, further comprising a first LNP enclosing (a). 
         205. The method of clause 204, wherein the first LNP encloses (b) and (c). 
         206. The method of clause 204, wherein the first LNP was administered repeatedly. 
         207. The method of clause 204, wherein the first LNP was administered repeatedly at an interval of one to sixty days. 
         208. The method of clause 204, wherein the first LNP was administered repeatedly at an interval of seven days. 
         209. The method of clause 204, wherein the first LNP further encloses (b). 
         210. The method of clause 209, further comprising a second LNP enclosing (a) and (c). 
         211. The method of clause 210, wherein the first LNP and the second LNP are administered sequentially. 
         212. The method of clause 211, wherein the first LNP and the second LNP are administered sequentially at an interval of one day to 12 months. 
         213. The method of clause 212, wherein the interval is one day. 
         214. The method of clause 212, wherein the interval is five days. 
         215. The method of clause 212, wherein the interval is ten days. 
         216. The method of clause 212, wherein the interval is fifteen days. 
         217. The method of clause 212, wherein the interval is twenty days. 
         218. The method of clause 212, wherein the interval is twenty-five days. 
         219. The method of clause 212, wherein the interval is one month. 
         220. The method of clause 212, wherein the interval is two months. 
         221. The method of clause 212, wherein the interval is three months. 
         222. The method of clause 212, wherein the interval is five months. 
         223. The method of clause 212, wherein the interval is eight months. 
         224. The method of clause 212, wherein the interval is ten months. 
         225. The method of clause 212, wherein the interval is twelve months. 
         Following is the second example of enumerated clauses: 
         1. A single guide RNA that comprises: (a) a spacer sequence, wherein the spacer sequence comprises (i) one or more chemical modification(s) and (ii) one or more unmodified nucleotide(s) at select position(s), and (b) a tracr sequence having at least 70% identity to SEQ ID NO: 61, wherein the tracr sequence serve as a binding scaffold for a Type II Cas protein, and wherein the tracr sequence comprises (i) one or more chemical modification(s) and (ii) one or more unmodified nucleotide(s) at select position(s). 
         2. The single guide RNA of clause 1, wherein the spacer sequence hybridizes with a target polynucleotide sequence in a gene of interest when contacted with the target polynucleotide sequence, wherein the single guide RNA directs the Cas protein to effect alteration in the gene when administered to a mammalian subject. 
         3. The single guide RNA of clause 1 or 2, wherein the tracr sequence comprises unmodified nucleotides at positions 2 to 7, 23 to 25, 27, 29, 31, 38, 39, 42 to 45 48, 49 and 62 as numbered in SEQ ID NO: 61 or a corresponding position thereof. 
         4. The single guide RNA of clause 1 or 2, wherein the tracr sequence comprises unmodified nucleotides at positions 2 to 7, 13, 23 to 25, 27, 29, 31, 38, 39, 42 to 45, 48, 49, 53 and 62 as numbered in SEQ ID NO: 61 or a corresponding position thereof. 
         5. The single guide RNA of clause 1 or 2, wherein the tracr sequence comprises unmodified nucleotides at positions 2 to 7, 13, 23 to 25, 27, 29, 31, 38, 39, 42 to 45, 48, 49, 53, 61, 68, 70 and 11 as numbered in SEQ ID NO: 61 or a corresponding position thereof. 
         6. The single guide RNA of any one of clauses 1 to 3, wherein the tracr sequence comprises modified nucleotides at positions 1, 8 to 22, 26, 28, 30, 32 to 37, 40, 41, 46, 47, 50 to 61, and 63 to 80 as numbered in SEQ ID NO: 61 or a corresponding position thereof. 
         7. The single guide RNA of any one of clauses 1 to 3, wherein the tracr sequence comprises modified nucleotides at positions 1, 8 to 12, 14 to 22, 26, 28, 30, 32 to 37, 40, 41, 46, 47, 50 to 52, 54 to 61, and 63 to 80 as numbered in SEQ ID NO: 61 or a corresponding position thereof. 
         8. The single guide RNA of any one of clauses 1 to 7, wherein the tracr sequence comprises modified nucleotides at positions 1, 8 to 12, 14 to 22, 26, 28, 30, 32 to 34, 37, 41, 46, 47, 50 to 52, 54 to 60, 62 to 67, 69 and 72 to 80 as numbered in SEQ ID NO: 61 or a corresponding position thereof. 
         9. The single guide RNA of any one of clauses 3 to 8, wherein more than 60% of the nucleotides in the tracr sequence with SEQ ID NO: 61 are modified. 
         10. The single guide RNA of clauses 9, wherein more than 70% of the nucleotides in the tracr sequence with SEQ ID NO: 61 are modified. 
         11. The single guide RNA of any one of clauses 1 to 10, wherein the tracr sequence comprises unmodified nucleotides at positions 13, 49, 53 and 62 as numbered in SEQ ID NO: 61 or a corresponding position thereof. 
         12. The single guide RNA of any one of clauses 11, wherein the tracr sequence comprises unmodified nucleotides at positions 49 and 62 as numbered in SEQ ID NO: 61 or a corresponding position thereof. 
         13. The single guide RNA of any one of clauses 1 to 10, wherein the tracr sequence comprises unmodified nucleotides at positions 13, 40, 49, 53, 61, 68, 70 and 71 as numbered in SEQ ID NO: 61 or a corresponding position thereof. 
         14. The single guide RNA of any one of clause 13, wherein the tracr sequence comprises unmodified nucleotides at positions 13, 49 and 53 as numbered in SEQ ID NO: 61 or a corresponding position thereof. 
         15. The single guide RNA of any one of clauses 1 to 10, wherein the tracr sequence comprises modified nucleotides at positions 1, 8, 21, 22, 26, 28, 30, 32 to 37, 40, 41, 46 and 47 as numbered in SEQ ID NO: 61 or a corresponding position thereof. 
         16. The single guide RNA of any one of clauses 1-15, wherein the tracr sequence comprises at least 80% identity to SEQ ID NO: 61. 
         17. The single guide RNA of any one of clauses 1-15, wherein the tracr sequence comprises at least 90% identity to SEQ ID NO: 61. 
         18. The single guide RNA of any one of clauses 1-17, wherein the chemical modification comprises a 2′-OMe modification. 
         19. The single guide RNA of any one of clauses 1-17, wherein the chemical modification comprises a nebularin or a deoxynebularin. 
         20. The single guide RNA of any one of clauses 1-17, wherein the chemical modification comprises a phosphorothioate linkage. 
         21. The single guide RNA of any one of clauses 1-20, wherein the tracr sequence comprises SEQ ID No. 61, and wherein the single guide RNA further comprises one or more phosphorothioate linkage at a 5′ end, at a 3′ end, at select internal positions or any combinations thereof. 
         22. The single guide RNA of clause 21, wherein the single guide RNA further comprises two and no more than two contiguous phosphorothioate linkages at the 5′ end, at the 3′ end or both. 
         23. The single guide RNA of clause 21, wherein the single guide RNA further comprises three contiguous phosphorothioate linkages at the 5′ end, at the 3′ end or both. 
         24. The single guide RNA of any one of clauses 1-21, wherein the single guide RNA comprises the sequence 5′- ususuNNN-3′ at the 3′end, wherein N independently indicates a unmodified ribonucleotide, and wherein each u indicates 2′-O-methyluridine and each s indicates phosphorothioate linkage. 
         25. The single guide RNA of clauses 24, wherein each N is uridine. 
         26. The single guide RNA of any one of clauses 1-21, wherein the single guide RNA comprises the sequence 5′- ususuNNn-3′ at the 3′end, wherein each N independently indicates a unmodified ribonucleotide, wherein the n indicates a modified nucleotide, wherein each u indicates 2′-O-methyluridine and wherein each s indicates phosphorothioate linkage. 
         27. The single guide RNA of clauses 26, wherein each N is uridine and the n is 2′-O-methyluridine. 
         28. A single guide RNA that comprises (i) a spacer sequence and (ii) a tracr sequence, wherein the spacer sequence hybridizes with a target polynucleotide sequence in a PCSK9 gene or an ANGPTL3 gene when contacted with the target polynucleotide sequence, wherein the tracr sequence binds a Type II Cas protein when contacted with the Type II Cas protein, and wherein the single guide RNA comprises a nebularine, a deoxynebularine, or a 2′-O-methylnebularine. 
         29. A single guide RNA that comprises (i) a spacer sequence and (ii) a tracr sequence, wherein the spacer sequence hybridizes with a target polynucleotide sequence in a PCSK9 gene or an ANGPTL3 gene when contacted with the target polynucleotide sequence, wherein the tracr sequence binds a Type II Cas protein when contacted with the Type II Cas protein, and wherein the single guide RNA comprises two and no more than two phosphorothioate linkages at a 5′ end or at a 3′ end. 
         30. The single guide RNA of clause 29, wherein the single guide RNA comprises two and no more than two phosphorothioate linkages at the 5′ end and at the 3′ end. 
         31. The single guide RNA of clause 29, wherein the single guide RNA comprises three phosphorothioate linkages at the 5′ end. 
         32. The single guide RNA of clause 29, wherein the single guide RNA comprises three phosphorothioate linkages at the 3′ end. 
         33. The single guide RNA of clause 29, wherein the two phosphorothioate linkages at the 5′ end are two contiguous phosphorothioate linkages at the first two nucleotide positions of the 5′ end. 
         34. The single guide RNA of clause 29, wherein the two phosphorothioate linkages at the 5′ end are within the first 3-10 nucleotides of the 5′ end. 
         35. The single guide RNA of clause 29, wherein the two phosphorothioate linkages at the 3′ end are two contiguous phosphorothioate linkages at the last two nucleotide positions of the 3′ end. 
         36. The single guide RNA of clause 29, wherein the two phosphorothioate linkages at the 3′ end are within the last 3-10 nucleotides of the 3′ end. 
         37. The single guide RNA of clause 29 comprising the sequence 5′-UsUsUs-3′ at the 3′ end, wherein U indicates a uridine and s indicates a phosphorothioate linkage. 
         38. The single guide RNA of clause 29 comprising the sequence 5′-UUU-3′ at the 3′end. 
         39. The single guide RNA of any one of clauses 1-27, wherein the tracr sequence binds the Type II Cas protein with increased binding affinity compared to a tracr sequence in an unmodified single guide RNA. 
         40. The single guide RNA of any one of clauses 28-38, wherein the Type II Cas protein is a Cas9 protein. 
         41. The single guide RNA of clause 40, wherein the Cas9 protein is a  Streptococcus pyogenes  Cas9. 
         42. A single guide RNA that comprises a guide RNA sequence selected from Table 1, wherein a, u, g, and c indicate 2′-OMe modified adenine, uridine, guanine, and cytidine, wherein s indicates a phosphorothioate linkage, wherein X indicates a nebularine, wherein x indicates a 2′-O-methylnebularine and wherein dX indicates a 2′-deoxynebularine. 
         43. A pharmaceutical composition for gene modification comprising the single guide RNA of any one of clauses 1-42 and a Type II Cas protein or a nucleic acid sequence encoding the Type II Cas protein. 
         44. The pharmaceutical composition of clause 43 further comprising a vector that comprises the nucleic acid sequence encoding the Type II Cas protein. 
         45. The pharmaceutical composition of clause 43 or 44, wherein the Type II Cas protein is a Cas9. 
         46. The pharmaceutical composition of any one of clauses 43-45 further comprising a pharmaceutically acceptable carrier. 
         47. A lipid nanoparticle comprising the pharmaceutical composition of any one of clauses 43-46. 
         48. A method for modifying a target polynucleotide sequence in a cell comprising introducing into the cell the pharmaceutical composition of any one of clauses 43-46, wherein the single guide RNA directs the Type II Cas protein to effect a modification in the target polynucleotide sequence in the cell. 
         49. The method of clause 48, wherein the target polynucleotide sequence is in a PCSK9 gene. 
         50. The method of clause 49, wherein the modification reduces expression of functional PCSK9 protein encoded by the PCSK9 gene in the cell. 
         51. The method of clause 50, wherein the target polynucleotide sequence is in an ANGPTL3 gene. 
         52. The method of clause 51 wherein the modification reduces expression of functional ANGPTL3 protein encoded by the ANGPTL3 gene in the cell. 
         53. The method of any one of clauses 48-52, wherein the introduction is performed via a lipid nanoparticle that comprises the composition. 
         54. A method for treating or preventing a condition in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of any one of clauses 43-46 or the lipid nanoparticle of clause 47, wherein the single guide RNA directs the Type II Cas protein to effect a modification in a target polynucleotide sequence in a cell of the subject, thereby treating or preventing the condition. 
         55. The method of clause 54, wherein the target polynucleotide sequence is in a PCSK9 gene. 
         56. The method of clause 54, wherein the target polynucleotide sequence is in an ANGPTL3 gene. 
         57. The method of clause 55, wherein the modification reduces expression of functional PCSK9 protein encoded by the PCSK9 gene in the subject. 
         58. The method of clause 56, wherein the modification reduces expression of functional ANGPTL3 protein encoded by the ANGPTL3 gene in the subject. 
         59. The method of any one of clauses 48-58 wherein the condition is an atherosclerotic vascular disease. 
         60. The method of any one of clauses 48-58, wherein the condition is an atherosclerotic vascular disease, hypertriglyceridemia, or diabetes. 
         61. The method of any one of clauses 48-60, wherein the subject exhibits a reduced blood LDL cholesterol level, and/or a reduced blood triglycerides level as compared to before the administration. 
         Following is the third example of enumerated clauses: 
         1. A composition for editing a gene target comprising: 
         (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same, 
         (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on a ANGPTL3 gene, 
         wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the ANGPTL3 gene in vivo when administered to a mammalian subject, 
         wherein when the guide RNA and the mRNA is administered at a total amount of at least 0.5 mg/kg, the base alteration occurs in at least 35% of whole liver cells in the mammalian subject as measured by next generation sequencing or Sanger sequencing. 
         2. The composition of clause 1, wherein the mammalian subject is a cynomolgus monkey, 
         wherein when the guide RNA and the mRNA is administered at a total amount of about 1 mg/kg, the base alteration occurs in at least 35% of whole liver cells in the cynomolgus monkey as measured by next generation sequencing or Sanger sequencing. 
         3. The composition of clause 1, wherein the mammalian subject is a cynomolgus monkey, 
         wherein when the guide RNA and the mRNA is administered at a total amount of about 3 mg/kg, the base alteration occurs in at least 50% of whole liver cells in the cynomolgus monkey as measured by next generation sequencing or Sanger sequencing. 
         4. The composition of clause 2, wherein the nucleobase alteration results in a reduction of at least 20% in blood triglyceride level in the cynomolgus monkey as compared to prior to the administration. 
         5. The composition of clause 3, wherein the nucleobase alteration results in a reduction of at least 50% in blood triglyceride level in the cynomolgus monkey as compared to prior to the administration. 
         6. The composition of any one of clauses 1-5, wherein the protospacer is located in a splice site. 
         7. The composition of any one of clauses 1-5, wherein the protospacer complementary sequence is in the antisense strand of the ANGPTL3 gene. 
         8. The composition of any one of clauses 1-5, wherein the protospacer complementary sequence is in the sense strand of the ANGPTL3 gene. 
         9. The composition of any one of clauses 1-8, wherein the base alteration happens outside of the protospacer on the ANGPTL3 gene (off-target sites), 
         wherein the editing percentages of off-target sites set forth in Table 14 are below or equal to the editing percentages set forth in Table 14, respectively. 
         10. The composition of any one of clauses 1-9, wherein the deaminase is an adenine deaminase and wherein the nucleobase alteration is a A•T to G•C alteration. 
         11. The composition of any one of clauses 1-10, wherein the programmable DNA binding domain comprises a nuclease inactive Cas9 or a Cas9 nickase. 
         12. The composition of any one of clauses 1-11, wherein the nucleobase alteration is at a splice site of the ANGPTL3 gene. 
         13. The composition of clause 12, wherein the nucleobase alteration is at a splice donor site of the ANGPTL3 gene. 
         14. The composition of clause 13, wherein the splice donor site is at 5′ end of ANGPTL3 intron 6 as referenced in SEQ ID NO: 7. 
         15. The composition of clause 12, wherein the nucleobase alteration is at a splice acceptor site of the ANGPTL3 gene. 
         16. The composition of any one of clauses 1-15, wherein the nucleobase alteration results in a frame shift, a premature stop codon, an insertion or deletion in a transcript encoded by the ANGPTL3 gene. 
         17. The composition of any one of clauses 1-16, wherein the nucleobase alteration results in an aberrant transcript encoded by the ANGPTL3 gene. 
         18. The composition of any one of clauses 1-17, wherein the guide RNA is chemically modified. 
         19. The composition of clause 16, wherein the tracr sequence of the guide RNA is chemically modified following the scheme depicted in  FIG.  7   . 
         20. The composition of any one of clauses 1-19, wherein the spacer sequence comprises an ANGPTL3 ABE guide RNA spacer sequence set forth in Table 1. 
         21. The composition of clause 20, wherein the guide RNA comprises the ANGPTL3 ABE guide RNA sequence of GA067, GA091, GA098, GA099, GA100, GA101, GA102, GA103, GA347, GA441, GA442, GA472, GA473, GA474, GA475, GA476, GA517 or GA547 as set forth in Table 1. 
         22. The composition of any one of clauses 1-19, wherein the protospacer sequence comprises an ANGPTL3 ABE protospacer sequence set forth in Table 1. 
         23. The composition of clause 22, wherein the protospacer comprises the sequence 5′-AAGATACCTGAATAACTCTC-3′ (SEQ ID No: 14), 5′-AAGATACCTGAATAACCCTC-3′ (SEQ ID No: 15), 5′-GATACCTGAATAACTCTC-3′ (SEQ ID No: 1606), 5′-AGATACCTGAATAACCCTC-3′ (SEQ ID No: 248), or 5′-GATACCTGAATAACCCTC-3′ (SEQ ID No: 249). 
         24. The composition of any one of clauses 1-23, wherein the base editor fusion protein comprises an amino acid sequence of SEQ ID No: 2137. 
         25. The composition of any one of clauses 1-24, wherein the GC % content of the mRNA sequence is greater than 50%. 
         26. The composition of clause 25, wherein the GC % content of the mRNA sequence is greater than 56%. 
         27. The composition of clause 26, wherein the GC % content of the mRNA sequence is greater than or equal to 63%. 
         28. The composition of clause 25, wherein the mRNA comprises an adenine tTNA deaminase (TadA) region, a Cas9 region and a nuclear localization sequence (NLS) region. 
         29. The composition of clause 28, wherein the mRNA further comprises a first linker region which connects the TadA region and the Cas9 region, and a second linker region which connects the Cas9 region and the NLS region. 
         30. The composition of clause 28 or 29, wherein the GC % content of the TadA region is greater than 60%. 
         31. The composition of clause 28 or 29, wherein the GC % content of the TadA region is greater than or equal to 70%. 
         32. The composition of clause 28 or 29, wherein the GC % content of the Cas9 region is greater than 56%. 
         33. The composition of clause 28 or 29, wherein the GC % content of the Cas9 region is greater than or equal to 62%. 
         34. The composition of clause 28 or 29, wherein the GC % content of the NLS region is greater than 54%. 
         35. The composition of clause 28 or 29, wherein the GC % content of the NLS region is greater than or equal to 63%. 
         36. The composition of clause 29, wherein the GC % content of the first linker region is greater than 65%. 
         37. The composition of clause 29, wherein the GC % content of the first linker region is greater than or equal to 79%. 
         38. The composition of clause 29, wherein the GC % content of the second linker region is greater than 67%. 
         39. The composition of clause 29, wherein the GC % content of the second linker region is greater than or equal to 83%. 
         40. The composition of clause 29, wherein the GC % content of the TadA region is greater than 60%, the GC % content of the Cas9 region is greater than 56%, the GC % content of the NLS region is greater than 54%, the GC % content of the first linker region is greater than 65%, and the GC % content of the second linker region is greater than 67%. 
         41. The composition of clause 25, wherein the mRNA comprises a mRNA sequence selected from Table 23. 
         42. The composition of clause 41, wherein the mRNA comprises a mRNA sequence of SEQ ID No: 2136. 
         43. The composition of any one of clauses 25-42, wherein the mRNA comprises a poly A tail. 
         44. The composition of any one of clauses 1-43, further comprising a lipid nanoparticle (LNP) enclosing (i). 
         45. The composition of clause 44, wherein the LNP further encloses (ii). 
         46. The composition of clause 44, further comprising a second LNP enclosing (ii). 
         47. The composition of any one of clauses 1-44, wherein the ratio of the guide RNA and the mRNA encoding the base editor fusion protein is about 1:10 to about 10:1 by weight. 
         48. The composition of clause 47, wherein the ratio of the guide RNA and the mRNA encoding the base editor fusion protein is about 1:1, 1.5:1, 2:1, 3:1, 4:1, 1:1.5, 1:2, 1:3, or 1:4 by weight. 
         49. The composition of clause 48, wherein the ratio of the guide RNA and the mRNA encoding the base editor fusion protein is about 1:1 by weight. 
         50. A pharmaceutical composition comprising the composition of any one of the preceding clauses and a pharmaceutically acceptable carrier or excipient. 
         51. A method for treating or preventing a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of clause 1. 
         52. The method of clause 51, wherein the administration is via intravenous infusion. 
         53. The method of clause 51 or 52, comprising sequential administration of a LNP enclosing (i) and a LNP enclosing (ii). 
         54. The method of clause 51 or 52, comprising concurrent administration of the LNP enclosing (i) and the LNP enclosing (ii). 
         55. The method of clause 53, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 1 day. 
         56. The method of clause 53, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 2 days. 
         57. The method of clause 53, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 3 days. 
         58. The method of clause 53, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 4 days. 
         59. The method of clause 53, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 5 days. 
         60. The method of clause 53, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 6 days. 
         61. The method of clause 53, comprising administering a single dose of the LNP enclosing (ii) followed by staggered doses of the LNP enclosing (i) over an interval of 7 days. 
         62. The method of clause 51 or 52, comprising administering a single dose of the LNP enclosing (i) and (ii). 
         63. The method of clause 62, wherein the single dose of the LNP is at about 0.3 to about 3 mg/kg. 
         64. The method of clause 62 or 63, comprising administering a treatment course of one or more treatments to the subject, wherein each one of the one or more treatment comprises one or more of the single doses of the LNP. 
         65. The method of clause 64, comprising administering a treatment course of two to ten treatments. 
         66. The method of clause 64, comprising administering a treatment course of two to five treatments. 
         67. The method of clause 64, comprising administering a treatment course of two treatments. 
         68. The method of clause 64, comprising administering a treatment course of three treatments. 
         69. The method of clause 64, comprising administering a treatment course of four treatments. 
         70. The method of clause 62, comprising administering a treatment course of five treatments. 
         71. The method of any one of clauses 51-70, wherein the condition is an atherosclerotic cardiovascular disease. 
         72. The method of any one of clauses 51-70, wherein the condition is an atherosclerotic vascular disease. 
         73. The method of any one of clauses 51-72, wherein the subject is a human. 
         74. A composition for editing a gene target comprising: 
         (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same, 
         (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on a ANGPTL3 gene, 
         wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the ANGPTL3 gene in vivo when administered to a mammalian subject, and 
         wherein the guide RNA comprises the ANGPTL3 ABE guide RNA sequences as set forth in Table 1. 
         75. A composition for editing a gene target comprising: 
         (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same, 
         (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on a ANGPTL3 gene, 
         wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the ANGPTL3 gene in vivo when administered to a mammalian subject, and 
         wherein the mRNA comprises a sequence selected from Table 23. 
         76. A method for treating or preventing an atherosclerotic cardiovascular disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a first composition, comprising 
         (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same, 
         (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on a PCSK9 gene, 
         wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the PCSK9 gene in vivo when administered to a mammalian subject, 
         wherein when the guide RNA and the mRNA is administered at a total amount of at least 0.5 mg/kg, the base alteration occurs in at least 35% of whole liver cells in the mammalian subject as measured by next generation sequencing or Sanger sequencing; and 
         a second composition, comprising 
         (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same, 
         (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on a ANGPTL3 gene, 
         wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the ANGPTL3 gene in vivo when administered to a mammalian subject, 
         wherein when the guide RNA and the mRNA is administered at a total amount of at least 1 mg/kg, the base alteration occurs in at least 35% of whole liver cells in the mammalian subject as measured by next generation sequencing or Sanger sequencing. 
         77. The method of clause 76, comprising sequential administration of the first composition and the second composition. 
         78. The method of clause 77, comprising administering one or more doses of the first composition followed by one or more dose of the second composition. 
         79. The method of clause 78, comprising administering one or more doses of the second composition followed by one or more dose of the first composition. 
         80. The method of clause 76, comprising concurrent administration of the first composition and the second composition. 
         81. The method of clause 80, comprising one or more doses of the first composition and the second composition. 
         82. A composition for editing a gene target comprising: 
         (i) a base editor fusion protein comprising a programmable DNA binding domain and a deaminase, or a mRNA encoding the same, 
         (ii) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor fusion protein, and a spacer sequence that corresponds to a protospacer on a ANGPTL3 gene, 
         wherein the guide RNA directs the base editor fusion protein to effect a nucleobase alteration in the ANGPTL3 gene in vitro, 
         wherein when the guide RNA and the mRNA is administered at a total amount of at least 0.5 mg/kg, the base alteration occurs in at least 35% of whole liver cells in the mammalian subject as measured by next generation sequencing or Sanger sequencing. 
         83. The composition of clause 82, wherein when the guide RNA and the mRNA is administered at a total amount of at least 1 mg/kg, the base alteration occurs in at least 40% of whole liver cells in the mammalian subject as measured by next generation sequencing or Sanger sequencing. 
         84. The composition of clause 82, wherein when the guide RNA and the mRNA is administered at a total amount of at least 1.5 mg/kg, the base alteration occurs in at least 45% of whole liver cells in the mammalian subject as measured by next generation sequencing or Sanger sequencing. 
         85. The composition of clause 82, wherein when the guide RNA and the mRNA is administered at a total amount of at least 2 mg/kg, the base alteration occurs in at least 
         50% of whole liver cells in the mammalian subject as measured by next generation sequencing or Sanger sequencing. 
         86. The composition of clause 82, wherein when the guide RNA and the mRNA is administered at a total amount of at least 2.5 mg/kg, the base alteration occurs in at least 55% of whole liver cells in the mammalian subject as measured by next generation sequencing or Sanger sequencing. 
         87. The composition of clause 82, wherein when the guide RNA and the mRNA is administered at a total amount of at least 3 mg/kg, the base alteration occurs in at least 60% of whole liver cells in the mammalian subject as measured by next generation sequencing or Sanger sequencing. 
         88. A composition for editing an ANGPTL3 gene comprising:
       (a) a mRNA encoding an adenine base editor protein having an editing window, and   (b) a guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor protein and a spacer sequence that serves to guide the base editor protein to a protospacer sequence on the ANGPTL3 gene,   
     
         wherein the spacer sequence is complimentary, at least in part, to a splice site or an exon region of the ANGPTL3 gene. 
         89. The composition for editing the ANGPTL3 gene of clause 83, wherein when the base editor protein is operatively bound to the guide RNA and the guide RNA is hybridized with the complementary strand to the protospacer sequence on the ANGPTL3 gene, the editing window encompasses the splice site of the ANGPTL3 gene. 
         90. The composition for editing the ANGPTL3 gene of clause 88, wherein when the base editor protein is operatively bound to the guide RNA and the guide RNA is hybridized with the complementary strand to the protospacer sequence on the ANGPTL3 gene, the editing window encompasses a region of an intron of the ANGPTL3 gene. 
         91. The composition for editing the ANGPTL3 gene of clause 88, wherein when the base editor protein is operatively bound to the guide RNA and the guide RNA is hybridized with the complementary strand to the protospacer sequence on the ANGPTL3 gene, the editing window encompasses a region of intron 1, intron 3 or intron 4 of the ANGPTL3 gene. 
         92. The composition for editing the ANGPTL3 gene of clause 88, wherein when the base editor protein is operatively bound to the guide RNA and the guide RNA is hybridized with the complementary strand to the protospacer sequence on the ANGPTL3 gene, the editing window encompasses a region of intron 1 of the ANGPTL3 gene. 
         93. The composition for editing the ANGPTL3 gene of clause 88, wherein the spacer sequence has a 80-100% nucleotide sequence identity to a spacer sequence selected from the group of guide RNA sequences identified as GA067, GA100 and GA574. 
         94. The composition for editing the ANGPTL3 gene of clause 88, wherein the tracr sequence has a 80-100% nucleotide sequence identity to a tracr sequence selected from the group of guide RNA sequences identified as GA067, GA091, GA098, GA099, GA100, GA101, GA102, GA103, GA347, GA441, GA442, GA472, GA473, GA474, GA475, GA476, GA517 and GA547. 
         95. The composition for editing the ANGPTL3 gene of clause 88, wherein the mRNA has an 80-100% sequence identity to the mRNA sequences identified as MA002, MA004, MA040, MA0041, or MA045. 
         96. The composition for editing the ANGPTL3 gene of clause 88, wherein the mRNA has one or more of the GC nucleotide region percentages set forth in the following table: 
       
    
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Nucleotide region 
                 Average GC Nucleotide Content 
               
               
                   
                   
               
             
            
               
                   
                  27-213 
                 67-73% 
               
               
                   
                 389-661 
                 67-71% 
               
               
                   
                 735-829 
                 63-74% 
               
               
                   
                 4207-4286 
                 67-70% 
               
               
                   
                 4537-4569 
                 65-73% 
               
               
                   
                 4683-4741 
                 62-67% 
               
               
                   
                   
               
            
           
         
       
         
         97. The composition for editing the ANGPTL3 gene of clause 88, wherein the mRNA has one or more of the GC nucleotide region percentages set forth in the following table: 
       
    
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Nucleotide region 
                 Average GC Nucleotide Content 
               
               
                   
                   
               
             
            
               
                   
                  27-213 
                 At least 73% 
               
               
                   
                 389-661 
                 At least 71% 
               
               
                   
                 735-829 
                 At least 74% 
               
               
                   
                 4207-4286 
                 At least 70% 
               
               
                   
                 4537-4569 
                 At least 73% 
               
               
                   
                 4683-4741 
                 At least 67% 
               
               
                   
                   
               
            
           
         
       
         
         98. The composition for editing the ANGPTL3 gene of clause 88, wherein the mRNA and gRNA are encapsulated within a lipid nanoparticle. 
         99. The composition for editing the ANGPTL3 gene of clause 88, wherein the mRNA and gRNA are encapsulated within a lipid nanoparticle having the following: 
         LNP composition (mol %): 
         40-65% iLipid 
         2-20% DSPC 
         1-5% PEG 
         Remaining mol % balance is cholesterol; 
         LNP Particle size: 55-120 nm Z average hydrodynamic diameter; and 
         Polydispersity index of &lt;0.2 as determined by dynamic light scattering. 
         100. The composition for editing the ANGPTL3 gene of clause 88, wherein the mRNA and gRNA are encapsulated within the lipid nanoparticle having an LNP particle size between 50-70 nm Z average hydrodynamic diameter. 
         101. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 40 percent. 
         102. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 50 percent. 
         103. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 30 percent. 
         104. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 40 percent. 
         105. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 50 percent. 
         106. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 60 percent. 
         107. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 70 percent. 
         108. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 40 percent. 
         109. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 50 percent. 
         110. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 60 percent. 
         111. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 70 percent. 
         112. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when at administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 80 percent. 
         113. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 40 percent. 
         114. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 50 percent. 
         115. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 60 percent. 
         116. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 70 percent. 
         117. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of the cynomolgus monkey weight is capable of inducing adenine base editing at the ANGPTL3 target splice site in the liver of the cynomolgus monkeys with an average editing percentage of greater than 80 percent. 
         118. The compositions for editing the ANGPTL3 gene of clauses 87-117, wherein the percent editing is determined at 15 days after dosing through analysis of dosed cynomolgus monkey liver either via liver biopsy or necropsy of the monkey. 
         119. The compositions for editing of the ANGPTL3 gene of clauses 87-117, wherein the percent editing is determined to be durably maintained by periodic liver biopsy testing of the dosed cynomolgus monkeys over a span of at least 168 days after dosing. 
         120. The compositions for editing of the ANGPTL3 gene of clauses 87-117, wherein the percent editing is determined to be durably maintained by periodic liver biopsy testing of the dosed cynomolgus monkeys over a span of at least 300 days after dosing. 
         121. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         122. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         123. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         124. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed monkeys on average of at least 60 percent as compared to baseline. 
         125. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 70 percent as compared to baseline. 
         126. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 80 percent as compared to baseline. 
         127. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         128. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         129. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         130. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 60 percent as compared to baseline. 
         131. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 70 percent as compared to baseline. 
         132. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 80 percent as compared to baseline. 
         133. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         134. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         135. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         136. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 60 percent as compared to baseline. 
         137. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 70 percent as compared to baseline. 
         138. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 80 percent as compared to baseline. 
         139. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         140. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         141. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         142. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 60 percent as compared to baseline. 
         143. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 70 percent as compared to baseline. 
         144. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing ANGPTL3 protein in the plasma of the dosed cynomolgus monkeys on average of at least 80 percent as compared to baseline. 
         145. The compositions for editing of the ANGPTL3 gene of clauses 121-144, wherein the reduction in plasma protein is determined at 15 days after dosing via blood sampling and analysis of the dosed cynomolgus monkey. 
         146. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of monkey weight is capable of reducing triglyceride level in the plasma of the dosed monkeys on average of at least 20 percent as compared to baseline. 
         147. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 25 percent as compared to baseline. 
         148. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 30 percent as compared to baseline. 
         149. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         150. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         151. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 0.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 45 percent as compared to baseline. 
         152. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 20 percent as compared to baseline. 
         153. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 25 percent as compared to baseline. 
         154. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 30 percent as compared to baseline. 
         155. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         156. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         157. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 45 percent as compared to baseline. 
         158. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         159. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 55 percent as compared to baseline. 
         160. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 60 percent as compared to baseline. 
         161. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 20 percent as compared to baseline. 
         162. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 25 percent as compared to baseline. 
         163. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 30 percent as compared to baseline. 
         164. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         165. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         166. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 45 percent as compared to baseline. 
         167. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         168. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 55 percent as compared to baseline. 
         169. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 60 percent as compared to baseline. 
         170. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 1.5 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 65 percent as compared to baseline. 
         171. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 20 percent as compared to baseline. 
         172. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 25 percent as compared to baseline. 
         173. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 30 percent as compared to baseline. 
         174. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 35 percent as compared to baseline. 
         175. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 40 percent as compared to baseline. 
         176. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 45 percent as compared to baseline. 
         177. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 50 percent as compared to baseline. 
         178. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 55 percent as compared to baseline. 
         179. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 60 percent as compared to baseline. 
         180. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing triglyceride level in the plasma of the dosed cynomolgus monkeys on average of at least 65 percent as compared to baseline. 
         181. The compositions for editing the ANGPTL3 gene of clauses 148-182, wherein the reduction in triglyceride level is determined at 15 days after dosing via blood sampling and analysis of the dosed cynomolgus monkey. 
         182. The compositions for editing the ANGPTL3 gene of clauses 148-182, wherein the reduction in triglyceride level is determined to be durably maintained over a span of at least 168 days by periodic blood sampling and analysis of the dosed cynomolgus monkey. 
         183. The compositions for editing the ANGPTL3 gene of clauses 148-182, wherein the reduction in triglyceride level is determined to be durably maintained over a span of at least 300 days by periodic blood sampling and analysis of the dosed cynomolgus monkey. 
         184. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing lipoprotein(a) in the plasma of the dosed cynomolgus monkeys on average of at least 10 percent as compared to baseline. 
         185. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing lipoprotein(a) level in the plasma of the dosed cynomolgus monkeys on average of at least 15 percent as compared to baseline. 
         186. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing lipoprotein(a) level in the plasma of the dosed cynomolgus monkeys on average of at least 20 percent as compared to baseline. 
         187. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing lipoprotein(a) level in the plasma of the dosed cynomolgus monkeys on average of at least 25 percent as compared to baseline. 
         188. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing lipoprotein(a) level in the plasma of the dosed cynomolgus monkeys on average of at least 30 percent as compared to baseline. 
         189. The composition for editing the ANGPTL3 gene of clause 88, wherein the composition when administered to a group of cynomolgus monkeys via intravenous infusion at a dose of approximately 3 mg of the guide RNA and mRNA combined total weight per kg of cynomolgus monkey weight is capable of reducing lipoprotein(a) level in the plasma of the dosed cynomolgus monkeys on average of approximately 35 percent as compared to baseline. 
         190. The compositions for editing the ANGPTL3 gene of clauses 186-191, wherein the reduction in lipoprotein(a) is determined at 15 days after dosing via blood sampling and analysis of the dosed cynomolgus monkey. 
         191. The compositions for editing the ANGPTL3 gene of clauses 186-191, wherein the reduction in lipoprotein(a) is determined to be durably maintained over a span of at least 224 days by periodic blood sampling and analysis of the dosed cynomolgus monkey. 
         192. The compositions for editing the ANGPTL3 gene of clauses 186-191, wherein the reduction in lipoprotein(a) is determined to be durably maintained over a span of at least 300 days by periodic blood sampling and analysis of the dosed cynomolgus monkey. 
         193. The compositions for editing the ANGPTL3 gene of clauses 101-194, wherein to the extent that the dosing of the cynomolgus monkeys results in elevation of AST, ALT, or Cytokines, the elevations resulting from the dosing of the composition are transient and resolved back to approximately baseline levels within 3-15 days after dosing. 
         194. The composition for editing the ANGPTL3 gene of clauses 101-103, 121-126, 148-153, wherein the percent editing of ANGPTL3 is negligible outside of the liver, spleen and adrenal glands tissues as illustrated in  FIG.  27   . 
         195. The compositions for editing the ANGPTL3 gene of clauses 101-107, 121-132, 148-162, wherein repeat dosing results is additive with respect to the editing percentage of ANGPTL3 editing percentage. 
         196. The compositions for editing the ANGPTL3 gene of clause 195, wherein the repeat dosing does not elicit cytokine activation nor an immune response. 
         197. The composition for editing the ANGPTL3 gene of clause 88, wherein the spacer sequence has at least 80% nucleotide correlation with the nucleotide sequence of a targeted protospacer on the ANGPTL3 gene, wherein an RNA nucleotide on the spacer sequence is in correlation with a DNA nucleotide of the protospacer if it has the same nucleotide as the DNA nucleotide in the same order and wherein uracil and thymine bases are considered the same nucleotide for purposes of determining correlation. 
         198. The composition for editing the ANGPTL3 gene of clause 197, wherein the spacer sequence has at least 85% nucleotide correlation with the nucleotide sequence of a targeted protospacer on the ANGPTL3 gene. 
         199. The composition for editing the ANGPTL3 gene of clause 197, wherein the spacer sequence has at least 90% nucleotide correlation with the nucleotide sequence of a targeted protospacer on the ANGPTL3 gene. 
         200. The composition for editing the ANGPTL3 gene of clause 197, wherein the spacer sequence has at least 95% nucleotide correlation with the nucleotide sequence of a targeted protospacer on the ANGPTL3 gene. 
         201. The composition for editing the ANGPTL3 gene of clause 197, wherein the spacer sequence has at least 99% nucleotide correlation with the nucleotide sequence of a targeted protospacer on the ANGPTL3 gene. 
         202. The composition for editing the ANGPTL3 gene of clause 197, wherein the spacer sequence has at least 100% nucleotide correlation with the nucleotide sequence of a targeted protospacer on the ANGPTL3 gene. 
         203. A method for treating or preventing an atherosclerotic cardiovascular disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of
       (a) a mRNA encoding an adenine base editor protein having an editing window,   (b) a first guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor protein and a spacer sequence that serves to guide the base editor protein to a protospacer sequence on a PCSK9 gene, wherein the spacer sequence is complimentary, at least in part, to a splice site or an exon region of the PCSK9 gene; and   (c) a second guide RNA comprising a tracr sequence that serves as a binding scaffold for the base editor protein and a spacer sequence that serves to guide the base editor protein to a protospacer sequence on a ANGPTL3 gene, wherein the spacer sequence is complimentary, at least in part, to a splice site or an exon region of the ANGPTL3 gene.   
     
         204. The method of clause 203, further comprising a first LNP enclosing (a). 
         205. The method of clause 204, wherein the first LNP encloses (b) and (c). 
         206. The method of clause 204, wherein the first LNP was administered repeatedly. 
         207. The method of clause 204, wherein the first LNP was administered repeatedly at an interval of one to sixty days. 
         208. The method of clause 204, wherein the first LNP was administered repeatedly at an interval of seven days. 
         209. The method of clause 204, wherein the first LNP further encloses (b). 
         210. The method of clause 209, further comprising a second LNP enclosing (a) and (c). 
         211. The method of clause 210, wherein the first LNP and the second LNP are administered sequentially. 
         212. The method of clause 211, wherein the first LNP and the second LNP are administered sequentially at an interval of one day to 12 months. 
         213. The method of clause 212, wherein the interval is one day. 
         214. The method of clause 212, wherein the interval is five days. 
         215. The method of clause 212, wherein the interval is ten days. 
         216. The method of clause 212, wherein the interval is fifteen days. 
         217. The method of clause 212, wherein the interval is twenty days. 
         218. The method of clause 212, wherein the interval is twenty-five days. 
         219. The method of clause 212, wherein the interval is one month. 
         220. The method of clause 212, wherein the interval is two months. 
         221. The method of clause 212, wherein the interval is three months. 
         222. The method of clause 212, wherein the interval is five months. 
         223. The method of clause 212, wherein the interval is eight months. 
         224. The method of clause 212, wherein the interval is ten months. 
         225. The method of clause 212, wherein the interval is twelve months. 
       
    
     It will also be appreciated from reviewing the present disclosure, that it is contemplated that the one or more aspects or features presented in one of or a group of related clauses may also be included in other clauses or in combination with the one or more aspects or features in other clauses. 
       
     
       
         
           
               
             
               
                 LENGTHY TABLES 
               
             
            
               
                 The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (https://seqdata.uspto.gov/?pageRequest=docDetail&amp;DocID=US20230158174A1). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).