Patent Publication Number: US-2020289628-A1

Title: Compositions and methods for expressing factor ix

Description:
This application claims the benefit of priority from U.S. Provisional Application No. 62/747,509, filed on Oct. 18, 2018, U.S. Provisional Application No. 62/829,009, filed on Apr. 3, 2019, U.S. Provisional Application No. 62/829,621, filed on Apr. 4, 2019 and U.S. Provisional Application No. 62/840,352, filed on Apr. 29, 2019. The specifications of each of the foregoing applications are incorporated herein by reference in their entirety. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 21, 2019, is named 1861884-0002-004-101_SL.txt and is 194,154 bytes in size. 
     Bleeding disorders are caused by inadequate blood clotting. This deficiency may be caused by congenital coagulation disorders, acquired coagulation disorders, or hemorrhagic conditions induced by trauma. Bleeding is one of the most serious and significant manifestations of disease, and may occur from a local site or be generalized. Localized bleeding may be associated with lesions and may be further complicated by a defective haemostatic mechanism. Congenital or acquired deficiencies of any of the coagulation factors may be associated with a hemorrhagic tendency. Classic examples of bleeding disorders include hemophilia, such as hemophilia A, which results from a deficiency in factor VIII, or hemophilia B (Christmas Disease), which results from a deficiency in factor IX. Hemophilia occurs in all racial and ethnic groups, and affects many people in the United States and worldwide. 
     Traditional therapy for bleeding disorders includes parenteral replacement of deficient clotting factors, such as factor VII, factor VIII or factor IX. For example, current treatments for Hemophilia B rely on chronic, repeated intravenous infusions of purified recombinant Factor IX. However, those treatments suffer from a number of drawbacks including the need for repeated intravenous infusions, being associated with inhibitor formation, and generally being more prophylactic rather than curative. See, e.g., Petrini 2001 , Hemophilia  7:99; Fischer et al. 2002 , Blood  99 (7):2337. 
     Gene therapy, which involves introducing a copy of a missing or defective gene into a patient, provide one possible method of introducing Factor IX to patients for a longer duration. However, there exists a need for additional compositions and methods that offer improved, long term expression of Factor IX. 
     The present disclosure provides compositions and methods useful for expressing Factor IX in a host cell or a population of host cells (in vitro or in vivo), and for treating hemophilia (e.g., hemophilia B). Provided herein are guide RNAs for use in targeted insertion of a sequence encoding Factor IX into a human genomic locus, e.g., a safe harbor site, such as an albumin safe harbor site. Also provided are donor constructs (e.g., bidirectional constructs), comprising a sequence encoding Factor IX, for use in targeted insertion into a safe harbor site, such as intron 1 of the albumin safe harbor site. In some embodiments, the guide RNA disclosed herein can be used in combination with an RNA-guided DNA binding agent (e.g., Cas nuclease) and a donor construct (e.g., bidirectional construct) comprising a Factor IX transgene. In some embodiments, the donor construct (e.g., bidirectional construct) can be used with a gene editing system (e.g., CRISPR/Cas system; zinc finger nuclease (ZFN) system; transcription activator-like effector nuclease (TALEN) system). In some embodiments, the guide RNA disclosed herein can be used in combination with an RNA-guided DNA binding agent (e.g., Cas nuclease) and a donor construct (e.g., bidirectional construct) that comprises a Factor IX transgene. The following embodiments are provided. 
     In some aspects, provided herein is a method of introducing a Factor IX nucleic acid to a cell or a population of cells, comprising administering: i) a nucleic acid construct comprising a Factor IX protein coding sequence; ii) an RNA-guided DNA binding agent; and iii) a guide RNA (gRNA) comprising a sequence. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID Nos: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNA comprises a sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNA comprises a sequence that is a sequence selected from the group consisting of SEQ ID NOs: 34, 40, 45, 51, 60, 61, 63, 64, 65, 66, 72, 77, 83, 92, 93, 95, 96, and 97. In some embodiments, the guide RNA comprises a sequence that is a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNA comprises a sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNA comprises a sequence that is selected from the group consisting of SEQ ID NOs: 34-97. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 98-119. In some embodiments, the guide RNA comprises a sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 98-119. In some embodiments, the guide RNA comprises a sequence that is a sequence selected from the group consisting of SEQ ID NOs: 120-163. 
     In some aspects, provided herein is a method of expressing Factor IX in a cell or population of cells, comprising administering: i) a nucleic acid construct comprising a Factor IX protein coding sequence; ii) an RNA-guided DNA binding agent; and iii) a guide RNA (gRNA) comprising a sequence. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID Nos: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNA comprises a sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNA comprises a sequence that is selected from the group consisting of SEQ ID NOs: 34, 40, 45, 51, 60, 61, 63, 64, 65, 66, 72, 77, 83, 92, 93, 95, 96, and 97. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNA comprises a sequence that is least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNA comprises a sequence selected from the group consisting of SEQ ID NOs: 34-97. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 98-119. In some embodiments, the guide RNA comprises a sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 98-119. In some embodiments, the guide RNA comprises a sequence that is selected from the group consisting of SEQ ID NOs: 120-163. 
     In some aspects, provided herein is a method of introducing or expressing Factor IX in a cell or population of cells, comprising administering: i) a nucleic acid construct comprising a Factor IX protein coding sequence; ii) an RNA-guided DNA binding agent; and iii) a guide RNA (gRNA) comprising a sequence wherein the administration is in vitro. 
     In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID Nos: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNA comprises at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNA comprises a sequence selected from the group consisting of SEQ ID NOs: 34, 40, 45, 51, 60, 61, 63, 64, 65, 66, 72, 77, 83, 92, 93, 95, 96, and 97. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNA comprises at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNA comprises a sequence selected from the group consisting of SEQ ID NOs: 34-97. 
     In some embodiments, the nucleic acid construct is administered in a nucleic acid vector and/or a lipid nanoparticle. In some embodiments, the RNA-guided DNA binding agent and/or gRNA is administered in a nucleic acid vector and/or lipid nanoparticle. In some embodiments, the nucleic acid vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of an adeno associate viral (AAV) vector, adenovirus vector, retrovirus vector, and lentivirus vector. In some embodiments, the AAV vector is selected from the group consisting of AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV-DJ, and AAV2/8. 
     In some embodiments, the nucleic acid construct, RNA-guided DNA binding agent, and gRNA are administered sequentially, in any order and/or in any combination. In some embodiments, wherein the nucleic acid construct, RNA-guided DNA binding agent, and gRNA, individually or in any combination, are administered simultaneously. In some embodiments, the RNA-guided DNA binding agent, or RNA-guided DNA binding agent and gRNA in combination, is administered prior to administering the nucleic acid construct. In some embodiments, the nucleic acid construct is administered prior to administering the gRNA and/or RNA-guided DNA binding agent. 
     In some embodiments, the RNA-guided DNA binding agent is a Cas nuclease. In some embodiments, the Cas nuclease is a class 2 Cas nuclease. In some embodiments the Cas nuclease is Cas9. In some embodiments, the Cas nuclease is an  S. pyogenes  Cas9 nuclease. In some embodiments, the Cas nuclease is a nickase. 
     In some embodiments, the nucleic acid construct is a bidirectional nucleic acid construct. In some embodiments, the nucleic acid construct is single-stranded or double-stranded. In some embodiments, the nucleic acid construct is a single-stranded DNA or a double-stranded DNA. In some embodiments, the bidirectional construct does not comprise a promoter that drives the expression of the Factor IX protein. In some embodiments, the cell or population of cells expresses Factor IX with a heterologous peptide, such as an albumin signal peptide. 
     In some embodiments, the cell or population of cells includes a liver cell. In some embodiments, the liver cell is a hepatocyte. 
     In some embodiments, the nucleic acid encodes a wild-type Factor IX protein. In some embodiments, the nucleic acid encodes a mutant Factor IX protein. In some embodiments, the nucleic acid encodes a Factor IX protein having a mutation R338L. 
     In some aspects, provided herein is a method of introducing a Factor IX nucleic acid to a cell or population of cells, comprising administering to the cell or population of cells a bidirectional nucleic acid construct comprising a Factor IX protein coding sequence, thereby expressing Factor IX in the cell or population of cells. Provided herein is a method of expressing Factor IX in a cell or population of cells, comprising administering to the cell or population of cells a bidirectional nucleic acid construct comprising a Factor IX protein coding sequence, thereby expressing Factor IX expression in the cell or population of cells. 
     In some embodiments, the bidirectional nucleic acid construct comprises: a) a first segment comprising a coding sequence for Factor IX; and b) a second segment comprising a reverse complement of a coding sequence of Factor IX, wherein the construct does not comprise a promoter that drives the expression of Factor IX. In some embodiments, the bidirectional nucleic acid construct comprises: a) a first segment comprising a coding sequence for Factor IX; and b) a second segment comprising a reverse complement of a coding sequence of a second polypeptide, wherein the construct does not comprise a promoter that drives the expression of the polypeptide. 
     In some embodiments, the method of introducing a Factor IX nucleic acid to a cell or population of cells further comprises administering an RNA-guided DNA binding agent. In some embodiments, the method further comprises administering a gRNA. In some embodiments, the bidirectional nucleic acid construct is administered in a nucleic acid vector and/or a lipid nanoparticle. In some embodiments, the RNA-guided DNA binding agent is administered in a nucleic acid vector and/or lipid nanoparticle. In some embodiments, the gRNA is administered in a nucleic acid vector and/or lipid nanoparticle. In some embodiments, the nucleic acid vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of an adeno associate viral (AAV) vector, adenovirus vector, retrovirus vector, and lentivirus vector. In some embodiments, the AAV vector is selected from the group consisting of AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV-DJ, and AAV2/8. 
     In some embodiments, the bidirectional nucleic acid construct, RNA-guided DNA binding agent, and gRNA are administered sequentially, in any order and/or in any combination. In some embodiments, the bidirectional nucleic acid construct, RNA-guided DNA binding agent, and gRNA, in any combination, are administered simultaneously. In some embodiments, the RNA-guided DNA binding agent, or RNA-guided DNA binding agent and gRNA in combination, is administered prior to administering the bidirectional nucleic acid construct. In some embodiments, the bidirectional nucleic acid construct is administered prior to administering the gRNA and/or RNA-guided DNA binding agent. 
     In some embodiments, the RNA-guided DNA binding agent is a Cas nuclease. In some embodiments, the Cas nuclease is a class 2 Cas nuclease. In some embodiments, the Cas nuclease is selected from the group consisting of  S. pyogenes  nuclease,  S. aureus  nuclease,  C. jejuni  nuclease,  S. thermophilus  nuclease,  N. meningitidis  nuclease, and variants thereof. In some embodiments, the Cas nuclease is Cas9. In some embodiments, the Cas nuclease is a nickase. 
     In some embodiments, the bidirectional construct does not comprise a promoter that drives the expression of the Factor IX protein. In some embodiments, the bidirectional construct is single-stranded or double-stranded. In some embodiments, the nucleic acid construct is a single-stranded DNA or a double-stranded DNA. In some embodiments, the gRNA comprises at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-33 or a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-33. 
     In some aspects, provided herein is a composition for use in expressing Factor IX in a cell, wherein the composition comprises: i) a nucleic acid construct comprising a Factor IX protein coding sequence; ii) an RNA-guided DNA binding agent; and iii) a guide RNA (gRNA) comprising a guide sequence selected from the group consisting of SEQ ID NOs: 2-33 or a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-33. Provided herein is a composition for use in expressing Factor IX in a cell or population of cells, wherein the composition comprises a bidirectional nucleic acid construct comprising a Factor IX protein coding sequence. In some embodiments, a host cell is made by the method of any preceding embodiment. 
     In some embodiments, the host cell is a liver cell. In some embodiments, the host cell is a non-dividing cell type. In some embodiments, the host cell expresses the Factor IX polypeptide encoded by the bidirectional construct. In some embodiments, the host cell is a hepatocyte. 
     In some embodiments of the method, construct, or host cell of any above method, the gRNA comprises SEQ ID NO: 401. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows construct formats as represented in AAV genomes. SA=splice acceptor; pA=polyA signal sequence; HA=homology arm; LHA=left homology arm; RHA=right homology arm 
         FIG. 2  shows vectors without homology arms are not effective in an immortalized liver cell line (Hepa1-6). An scAAV derived from plasmid P00204 comprising 200 bp homology arms resulted in expression of hFIX in the dividing cells. Use of the AAV vectors derived from P00123 (scAAV lacking homology arms) and P00147 (ssAAV bidirectional construct lacking homology arms) did not result in detectable expression of hFIX. 
         FIGS. 3A and 3B  show results from in vivo testing of insertion templates with and without homology arms using vectors derived from P00123, P00147, or P00204.  FIG. 3A  shows liver editing levels as measured by indel formation of ˜60% were detected in each group of animals treated with LNPs comprising CRISPR/Cas9 components.  FIG. 3B  shows animals receiving the ssAAV vectors without homology arms (derived from P00147) in combination with LNP treatment resulted in the highest level of hFIX expression in serum. 
         FIGS. 4A and 4B  show results from in vivo testing of ssAAV insertion templates with and without homology arms.  FIG. 4A  compares targeted insertion with vectors derived from plasmids P00350, P00356, P00362 (having asymmetrical homology arms as shown), and P00147 (bidirectional construct as shown in  FIG. 4B ).  FIG. 4B  compares insertion into a second site targeted with vectors derived from plasmids P00353, P00354 (having symmetrical homology arms as shown), and P00147. 
         FIGS. 5A-5D  show results of targeted insertion of bidirectional constructs across 20 target sites in primary mouse hepatocytes.  FIG. 5A  shows the schematics of each of the vectors tested.  FIG. 5B  shows editing as measured by indel formation for each of the treatment groups across each combination tested.  FIG. 5C  and  FIG. 5D  show that significant levels of editing (as indel formation at a specific target site) did not necessarily result in more efficient insertion or expression of the transgenes. hSA=human F9 splice acceptor; mSA=mouse albumin splice acceptor; HiBit=tag for luciferase based detection; pA=polyA signal sequence; Nluc=nanoluciferase reporter; GFP=green fluorescent reporter. 
         FIG. 6  shows results from in vivo screening of targeted insertion with bidirectional constructs across 10 target sites using with ssAAV derived from P00147. As shown, significant levels of indel formation do not necessarily result in high levels of transgene expression. 
         FIGS. 7A-7D  show results from in vivo screening of bidirectional constructs across 20 target sites using ssAAV derived from P00147.  FIG. 7A  shows varied levels of editing as measured by indel formation were detected for each of the treatment groups across each LNP/vector combination tested.  FIG. 7B  provides corresponding targeted insertion data. The results show poor correlation between indel formation and insertion or expression of the bidirectional constructs ( FIG. 7B  and  FIG. 7D ), and a positive correlation between in vitro and in vivo results ( FIG. 7C ). 
         FIGS. 8A and 8B  show insertion of the bidirectional construct at the cellular level using in situ hybridization method using probes that can detect the junctions between the hFIX transgene and the mouse albumin exon 1 sequence ( FIG. 8A ). Circulating hFIX levels correlated with the number of cells that were positive for the hybrid transcript ( FIG. 8B ). 
         FIG. 9  shows the effect on targeted insertion of varying the timing between delivery of the ssAAV comprising the bidirectional hFIX construct and LNP. 
         FIG. 10  shows the effect on targeted insertion of varying the number of LNP doses (e.g., 1, 2, or 3) following delivery of the bidirectional hFIX construct. 
         FIG. 11A  shows the durability of hFIX expression in vivo.  FIG. 11B  demonstrates expression from intron 1 of albumin was sustained. 
         FIG. 12A  and  FIG. 12B  show that varying AAV or LNP dose can modulate the amount of expression of hFIX from intron 1 of the albumin gene in vivo. 
         FIGS. 13A-13C  show results from screening bidirectional constructs across target sites in primary cynomolgus hepatocytes.  FIG. 13A  shows varied levels of editing as measured by indel formation detected for each of the samples.  FIG. 13B  and  FIG. 13C  show that significant levels of indel formation was not predictive for insertion or expression of the bidirectional constructs into intron 1 of albumin. 
         FIGS. 14A-14C  show results from screening bidirectional constructs across target sites in primary human hepatocytes.  FIG. 14A  shows editing as measured by indel formation detected for each of the samples.  FIG. 14B ,  FIGS. 14C, and 14D  show that significant levels of indel formation was not predictive for insertion or expression of the bidirectional constructs into intron 1 of the albumin gene. 
         FIG. 15  shows the results of in vivo studies where non-human primates were dosed with LNPs along with a bi-directional hFIX insertion template (derived from P00147). Systemic hFIX levels were achieved only in animals treated with both LNPs and AAV, with no hFIX detectable using AAV or LNPs alone. 
         FIG. 16A  and  FIG. 16B  show human Factor IX expression levels in the plasma samples at week 6 post-injection. 
         FIG. 17  shows week 7 serum levels and % positive cells across the multiple lobes for each animal. 
         FIG. 18  shows human Factor IX expression levels in the plasma samples in each group at weeks 1, 2, and 4 post-injection. 
         FIG. 19  shows insertion of the hF9 transgene and clotting function in the aPTT assay. 
         FIG. 20A  and  FIG. 20B  show insertion of the hF9 transgene and thrombin generation in TGA-EA analysis. 
         FIG. 21  shows insertion of the hF9 transgene and thrombin generation. 
     
    
    
     DETAILED DESCRIPTION 
     Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention is described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended embodiments. 
     Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended embodiments, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a conjugate” includes a plurality of conjugates and reference to “a cell” includes a plurality or population of cells and the like. As used herein, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. 
     Numeric ranges are inclusive of the numbers defining the range. Measured and measureable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings. 
     Unless specifically noted in the specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of” or “consisting essentially of” the recited components; embodiments in the specification that recite “consisting of” various components are also contemplated as “comprising” or “consisting essentially of” the recited components; and embodiments in the specification that recite “consisting essentially of” various components are also contemplated as “consisting of” or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims). The term “or” is used in an inclusive sense, i.e., equivalent to “and/or,” unless the context clearly indicates otherwise. The term “about”, when used before a list, modifies each member of the list. The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. 
     The term “about”, when used before a list, modifies each member of the list. The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. 
     The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any material incorporated by reference contradicts any term defined in this specification or any other express content of this specification, this specification controls. 
     I. Definitions 
     Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings: 
     “Polynucleotide” and “nucleic acid” are used herein to refer to a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. A nucleic acid “backbone” can be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2′ methoxy or 2′ halide substitutions. Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N 4 -methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, and O 4 -alkyl-pyrimidines; U.S. Pat. No. 5,378,825 and PCT No. WO 93/13121). For general discussion see  The Biochemistry of the Nucleic Acids  5-36, Adams et al., ed., 11 th  ed., 1992). Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (U.S. Pat. No. 5,585,481). A nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional bases with 2′ methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acid includes “locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004 , Biochemistry  43(42):13233-41). RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA. 
     “Guide RNA”, “gRNA”, and simply “guide” are used herein interchangeably to refer to either a guide that comprises a guide sequence, e.g., crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or, for example, in two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or “gRNA” refers to each type. The trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations compared to naturally-occurring sequences. Guide RNAs, such as sgRNAs or dgRNAs, can include modified RNAs as described herein. 
     As used herein, a “guide sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binding agent. A “guide sequence” may also be referred to as a “targeting sequence,” or a “spacer sequence.” A guide sequence can be 20 base pairs in length, e.g., in the case of  Streptococcus pyogenes  (i.e., Spy Cas9) and related Cas9 homologs/orthologs. Shorter or longer sequences can also be used as guides, e.g., 15-, 16-, 17-, 18-, 19-, 21-, 22-, 23-, 24-, or 25-nucleotides in length. For example, in some embodiments, the guide sequence comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:2-33. In some embodiments, the target sequence is in a gene or on a chromosome, for example, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. For example, in some embodiments, the guide sequence comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 2-33. In some embodiments, the guide sequence and the target region may be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, where the total length of the target sequence is at least 15, 16, 17, 18, 19, 20 or more base pairs. In some embodiments, the guide sequence and the target region may contain 1-4 mismatches where the guide sequence comprises at least 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides. 
     Target sequences for RNA-guided DNA binding agents include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence&#39;s reverse complement), as a nucleic acid substrate for an RNA-guided DNA binding agent is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be “complementary to a target sequence”, it is to be understood that the guide sequence may direct a guide RNA to bind to the reverse complement of a target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence. 
     As used herein, an “RNA-guided DNA-binding agent” means a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. The term RNA-guided DNA binding-agent also includes nucleic acids encoding such polypeptides. Exemplary RNA-guided DNA-binding agents include Cas cleavases/nickases. Exemplary RNA-guided DNA-binding agents may include inactivated forms thereof (“dCas DNA-binding agents”), e.g. if those agents are modified to permit DNA cleavage, e.g. via fusion with a FokI cleavase domain. “Cas nuclease”, as used herein, encompasses Cas cleavases and Cas nickases. Cas cleavases and Cas nickases include a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases. As used herein, a “Class 2 Cas nuclease” is a single-chain polypeptide with RNA-guided DNA binding activity. Class 2 Cas nucleases include Class 2 Cas cleavases/nickases (e.g., H840A, D10A, or N863A variants), which further have RNA-guided DNA cleavases or nickase activity, and Class 2 dCas DNA-binding agents, in which cleavase/nickase activity is inactivated”), if those agents are modified to permit DNA cleavage. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g, K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpf1 protein, Zetsche et al.,  Cell,  163: 1-13 (2015), also contains a RuvC-like nuclease domain. Cpf1 sequences of Zetsche are incorporated by reference in their entirety. See, e.g., Zetsche, Tables S1 and S3. See, e.g., Makarova et al.,  Nat Rev Microbiol,  13(11): 722-36 (2015); Shmakov et al.,  Molecular Cell,  60:385-397 (2015). As used herein, delivery of an RNA-guided DNA-binding agent (e.g. a Cas nuclease, a Cas9 nuclease, or an  S. pyogenes  Cas9 nuclease) includes delivery of the polypeptide or mRNA. 
     As used herein, “ribonucleoprotein” (RNP) or “RNP complex” refers to a guide RNA together with an RNA-guided DNA binding agent, such as a Cas nuclease, e.g., a Cas cleavase, Cas nickase, or dCas DNA binding agent (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binding agent such as Cas9 to a target sequence, and the guide RNA hybridizes with and the agent binds to the target sequence; in cases where the agent is a cleavase or nickase, binding can be followed by cleaving or nicking. 
     As used herein, a first sequence is considered to “comprise a sequence with at least X % identity to” a second sequence if an alignment of the first sequence to the second sequence shows that X % or more of the positions of the second sequence in its entirety are matched by the first sequence. For example, the sequence AAGA comprises a sequence with 100% identity to the sequence AAG because an alignment would give 100% identity in that there are matches to all three positions of the second sequence. The differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs such as modified uridines do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5′-AXG where X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5′-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well-known in the art. One skilled in the art will understand what choice of algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and expected identity &gt;50% for amino acids or &gt;75% for nucleotides, the Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server is generally appropriate. 
     As used herein, a first sequence is considered to be “X % complementary to” a second sequence if X % of the bases of the first sequence base pairs with the second sequence. For example, a first sequence 5′AAGA3′ is 100% complementary to a second sequence 3′TTCT5′, and the second sequence is 100% complementary to the first sequence. In some embodiments, a first sequence 5′AAGA3′ is 100% complementary to a second sequence 3′TTCTGTGA5′, whereas the second sequence is 50% complementary to the first sequence. 
     As used herein, “mRNA” is used herein to refer to a polynucleotide that is entirely or predominantly RNA or modified RNA and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2′-methoxy ribose residues. In some embodiments, the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2′-methoxy ribose residues, or a combination thereof. 
     Guide sequences useful in the guide RNA compositions and methods described herein are shown in Table 1 throughout the application. 
     As used herein, “indels” refer to insertion/deletion mutations consisting of a number of nucleotides that are either inserted or deleted at the site of double-stranded breaks (DSBs) in a target nucleic acid. 
     As used herein, “Factor IX” is used interchangeably with “FIX” or “F9”, and is also known as Christmas Factor. The human wild-type Factor IX protein sequence is available at NCBI NP_000124; gene sequence is available at NCBI NM_000133. Examples of the Factor IX protein sequence are described herein (e.g. SEQ ID NO: 700, SEQ ID NO: 701, and/or SEQ ID NO: 702). As used herein, Factor IX also encompasses a variant of Factor IX, e.g., a variant that possesses increased coagulation activity as compared to wild type Factor IX. A hyperactive variant of Factor IX may comprise a substitution of R338. An example of such a variant Factor IX comprises the mutation R338L relative to SEQ ID NO: 701. The terms hyperactive and hyperfunctional are being used interchangeably herein. Further examples of variant Factor IX comprise an amino acid at residue 338 chosen from alanine, leucine, valine, isoleucine, phenylalanine, tryptophan, methionine, serine, and threonine. Further Factor IX variants comprise an amino acid at residue 338 chosen from leucine, cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, or tyrosine. As used herein, Factor IX also encompasses a variant that is 80%, 85%, 90%, 93%, 95%, 97%, 99% identical to SEQ ID NO: 700, having at least 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 100%, or more, activity as compared to wild-type Factor IX. As used herein, Factor IX also encompasses a variant that is 80%, 85%, 90%, 93%, 95%, 97%, 99% identical to SEQ ID NO: 700, having at least 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 100%, or more, activity as compared to SEQ ID NO: 701 or SEQ ID NO: 702. As used herein, Factor IX also encompasses a fragment that possesses at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 100%, or more, activity as compared to wild type Factor IX. In some embodiments, a Factor IX variant may be a hyperactive Factor IX variant. In certain instances, the Factor IX variant possesses between about 80% and about 100%, 120%, 140%, 160%, 180%, or 200% of the activity as compared to the wild-type Factor IX. The specific activity of the Factor IX variant can be used to calculate its functionally normalized activity, for example as described in Example 13. The specific activities of Factor IX variants, e.g. R338L, are known in the literature and can be calculated using known methods. A hyperfunctional Factor IX variant may have about 1.2, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, or 15 times the specific activity of a corresponding wild type Factor IX protein. In one embodiment, the hyperfunctional Factor IX may have about 8-12 times the specific activity of a corresponding wild type Factor IX protein. In another embodiment, the hyperfunctional Factor IX may have 1.2-5 times the specific activity of a corresponding wild type Factor IX protein. Exemplary sequences are known in the art, and include sequences in U.S. Pat. Nos. 4,770,999, 4,994,371, 5,521,070, 6,046,380, 6,531,298, and 8,383,388, for example. 
     As used herein, a “target sequence” refers to a sequence of nucleic acid in a target gene that has complementarity to the guide sequence of the gRNA. The interaction of the target sequence and the guide sequence directs an RNA-guided DNA binding agent to bind, and potentially nick or cleave (depending on the activity of the agent), within the target sequence. 
     As used herein, “hemophilia” refers to a disorder caused by a missing or defective Factor IX gene or polypeptide. The disorder includes conditions that are inherited and/or acquired (e.g., caused by a spontaneous mutation in the gene), and includes hemophilia B. In some embodiments, the defective Factor IX gene or polypeptide results in reduced Factor IX level in the plasma and/or a reduced coagulation activity of Factor IX. As used herein, hemophilia includes mild, moderate, and severe hemophilia. For example, individuals with less than about 1% active factor are classified as having severe haemophilia, those with about 1-5% active factor have moderate haemophilia, and those with mild haemophilia have between about 5-40% of normal levels of active clotting factor. 
     As used herein, “normal” or “healthy” individuals include those having between 50 and 160% of normal pooled plasma level of Factor IX activity and antigen levels. Based on its purification from human plasma, the concentration of Factor IX in the normal adult (normal pooled plasma level of Factor IX) is about 300-400 μg/ml of plasma. In some embodiments, the level of Factor IX, e.g., circulating Factor IX, can be measured by a coagulation and/or an immunologic assay, e.g., an sandwich immunoassay, ELISA (see, e.g., Example 13), MSD (see, e.g., Example 14). Factor IX procoagulant activity is determined by the ability of the patient&#39;s plasma to correct the clotting time of Factor IX-deficient plasma. 
     As used herein, “treatment” refers to any administration or application of a therapeutic for disease or disorder in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of one or more symptoms of the disease. For example, treatment of hemophilia may comprise alleviating symptoms of hemophilia. 
     As used herein, a “bidirectional nucleic acid construct” (interchangeably referred to herein as a “bidirectional construct”) comprises at least two nucleic acid segments, wherein one segment (the first segment) comprises a coding sequence that encodes a polypeptide of interest (the coding sequence may be referred to herein as “transgene” or a first transgene), while the other segment (the second segment) comprises a sequence wherein the complement of the sequence encodes a polypeptide of interest, or a second transgene. That is, the at least two segments can encode identical or different polypeptides. When the two segments encode the identical polypeptide, the coding sequence of the first segment need not be identical to the complement of the sequence of the second segment. In some embodiments, the sequence of the second segment is a reverse complement of the coding sequence of the first segment. A bidirectional construct can be single-stranded or double-stranded. The bidirectional construct disclosed herein encompasses a construct that is capable of expressing any polypeptide of interest. 
     In some embodiments, a bidirectional nucleic acid construct comprises a first segment that comprises a coding sequence that encodes a first polypeptide (a first transgene), and a second segment that comprises a sequence wherein the complement of the sequence encodes a second polypeptide (a second transgene). In some embodiments, the first and the second polypeptides are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some embodiments, the first and the second polypeptides comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, e.g. across 50, 100, 200, 500, 1000 or more amino acid residues. 
     As used herein, a “reverse complement” refers to a sequence that is a complement sequence of a reference sequence, wherein the complement sequence is written in the reverse orientation. For example, for a hypothetical sequence 5′CTGGACCGA3′ (SEQ ID NO: 500), the “perfect” complement sequence is 3′GACCTGGCT5′ (SEQ ID NO: 501), and the “perfect” reverse complement is written 5′TCGGTCCAG3′ (SEQ ID NO: 502). A reverse complement sequence need not be “perfect” and may still encode the same polypeptide or a similar polypeptide as the reference sequence. Due to codon usage redundancy, a reverse complement can diverge from a reference sequence that encodes the same polypeptide. As used herein, “reverse complement” also includes sequences that are, e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the reverse complement sequence of a reference sequence. 
     As used herein, “polypeptide” refers to a wild-type or variant protein (e.g., mutant, fragment, fusion, or combinations thereof). A variant polypeptide may possess at least or about 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% functional activity of the wild-type polypeptide. In some embodiments, the variant is at least 70%, 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of the wild-type polypeptide. In some embodiments, a variant polypeptide may be a hyperactive variant. In certain instances, the variant possesses between about 80% and about 120%, 140%, 160%, 180%, 200%, 300%, 400%, 500%, or more of a functional activity of the wild-type polypeptide. 
     As used herein, a “heterologous gene” refers to a gene that has been introduced as an exogenous source to a site within a host cell genome (e.g., at a genomic locus such as a safe harbor locus including an albumin intron 1 site). That is, the introduced gene is heterologous with respect to its insertion site. A polypeptide expressed from such heterologous gene is referred to as a “heterologous polypeptide.” The heterologous gene can be naturally-occuring or engineered, and can be wild type or a variant. The heterologous gene may include nucleotide sequences other than the sequence that encodes the heterologous polypeptide (e.g., an internal ribosomal entry site). The heterologous gene can be a gene that occurs naturally in the host genome, as a wild type or a variant (e.g., mutant). For example, although the host cell contains the gene of interest (as a wild type or as a variant), the same gene or variant thereof can be introduced as an exogenous source for, e.g., expression at a locus that is highly expressed. The heterologous gene can also be a gene that is not naturally occurring in the host genome, or that expresses a heterologous polypeptide that does not naturally occur in the host genome. “Heterologous gene”, “exogenous gene”, and “transgene” are used interchangeably. In some embodiments, the heterologous gene or transgene includes an exogenous nucleic acid sequence, e.g. a nucleic acid sequence is not endogenous to the recipient cell. In some embodiments, the heterologous gene or transgene includes an exogenous nucleic acid sequence, e.g. a nucleic acid sequence that does not naturally occur in the recipient cell. For example, a heterologous gene may be heterologous with respect to its insertion site and with respect to its recipient cell. 
     A “safe harbor” locus is a locus within the genome wherein a gene may be inserted without significant deleterious effects on the host cell, e.g. hepatocyte, e.g., without causing apoptosis, necrosis, and/or senescence, or without causing more than 5%, 10%, 15%, 20%, 25%, 30%, or 40% apoptosis, necrosis, and/or senescence as compared to a control cell. See, e.g., Hsin et al., “Hepatocyte death in liver inflammation, fibrosis, and tumorigenesis,” 2017. In some embodiments, a safe harbor locus allows overexpression of an exogenous gene without significant deleterious effects on the host cell, e.g. hepatocyte, e.g., without causing apoptosis, necrosis, and/or senescence, or without causing more than 5%, 10%, 15%, 20%, 25%, 30%, or 40% apoptosis, necrosis, and/or senescence as compared to a control cell. In some embodiments, a desirable safe harbor locus may be one in which expression of the inserted gene sequence is not perturbed by read-through expression from neighboring genes. The safe harbor may be within an albumin gene, such as a human albumin gene. The safe harbor may be within an albumin intron 1 region, e.g., human albumin intron 1. The safe harbor may be a human safe harbor, e.g., for a liver tissue or hepatocyte host cell. In some embodiments, a safe harbor allows overexpression of an exogenous gene without significant deleterious effects on the host cell or cell population, such as hepatocytes or liver cells, e.g. without causing apoptosis, necrosis, and/or senescence, or without causing more than 5%, 10%, 15%, 20%, 25%, 30%, or 40% apoptosis, necrosis, and/or senescence as compared to a control cell or cell population. 
     II. Compositions 
     A. Compositions Comprising Guide RNA (gRNAs) 
     Provided herein are guide RNA compositions and methods useful for inserting and expressing a Factor IX gene within a genomic locus, e.g., a safe harbor site of a host cell or a population of host cells. In particular, as exemplified herein, targeting and inserting an exogenous gene at the albumin locus (e.g., at intron 1) allows the use of albumin&#39;s endogenous promoter to drive robust expression of the exogenous gene. The present disclosure is based, in part, on the identification of guide RNAs that specifically target sites within intron 1 of the albumin gene, and which provide efficient insertion and expression of the Factor IX gene. As shown in the Examples and further described herein, the ability of identified gRNAs to mediate high levels of editing as measured through indel forming activity, unexpectedly does not necessarily correlate with use of the same gRNAs to mediate efficient insertion of transgenes as measured through, e.g., expression of the transgene. That is, certain gRNAs that are able to achieve a high level of indel formation are not necessarily able to mediate efficient insertion, and conversely, some gRNAs shown to achieve low levels of indel formation may mediate efficient insertion and expression of a transgene. 
     In some embodiments, provided herein are compositions and methods useful for inserting and expressing a Factor IX gene within a region of an albumin locus (e.g., intron 1) of a host cell. In some embodiments, disclosed herein are compositions useful for introducing or inserting a heterologous Factor IX nucleic acid within an albumin locus of a host cell, e.g., using a guide RNA disclosed herein with an RNA-guided DNA binding agent, and a construct (e.g., donor construct or template) comprising a heterologous Factor IX nucleic acid (“Factor IX transgene”). In some embodiments, disclosed herein are compositions useful for expressing a heterologous Factor IX from an albumin locus of a host cell, e.g., using a guide RNA disclosed herein with an RNA-guided DNA binding agent and a construct (e.g., donor) comprising a heterologous Factor IX nucleic acid. In some embodiments, disclosed herein are compositions useful for expressing a heterologous Factor IX from an albumin locus of a host cell, e.g., using a guide RNA disclosed herein with an RNA-guided DNA binding agent and a bidirectional construct comprising a heterologous Factor IX nucleic acid. In some embodiments, disclosed herein are compositions useful for inducing a break (e.g., double-stranded break (DSB) or single-stranded break (nick)) within the serum albumin gene of a host cell, e.g., using a guide RNA disclosed herein with an RNA-guided DNA binding agent (e.g., a CRISPR/Cas system). The compositions may be used in vitro or in vivo for, e.g., treating hemophilia. 
     In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that binds, or is capable of binding, within an intron of an albumin locus. In some embodiments, the guide RNAs disclosed herein bind within a region of intron 1 of the human albumin gene (SEQ ID NO: 1). It will be appreciated that not every base of the guide sequence must bind within the recited regions. For example, in some embodiments, 15, 16, 17, 18, 19, 20, or more bases of the guide RNA sequence bind with the recited regions. For example, in some embodiments, 15, 16, 17, 18, 19, 20, or more contiguous bases of the guide RNA sequence bind with the recited regions. 
     In some embodiments, the guide RNAs disclosed herein mediate a target-specific cutting by an RNA-guided DNA binding agent (e.g., Cas nuclease) at a site within human albumin intron 1 (SEQ ID NO: 1). It will be appreciated that, in some embodiments, the guide RNAs comprise guide sequences that bind to, or are capable of binding to, said regions. 
     In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. 
     In some embodiments, the guide RNAs disclosed herein comprise a guide sequence having at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID Nos: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NO: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 34, 40, 45, 51, 60, 61, 63, 64, 65, 66, 72, 77, 83, 92, 93, 95, 96, and 97. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is selected from the group consisting of SEQ ID NOs: 34-97. 
     In some embodiments, the guide RNAs disclosed herein comprise a guide sequence having at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 34-37, 42-49, 53-59, 61-69, 74-81, 85-91, and 93-97. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is selected from the group consisting of SEQ ID NOs: 34-37, 42-49, 53-59, 61-69, 74-81, 85-91, and 93-97. 
     In some embodiments, the guide RNAs disclosed herein mediate a target-specific cutting resulting in a double-stranded break (DSB). In some embodiments, the guide RNAs disclosed herein mediate a target-specific cutting resulting in a single-stranded break (nick). 
     In some embodiments, the guide RNAs disclosed herein bind to a region upstream of a propospacer adjacent motif (PAM). As would be understood by those of skill in the art, the PAM sequence occurs on the strand opposite to the strand that contains the target sequence. That is, the PAM sequence is on the complement strand of the target strand (the strand that contains the target sequence to which the guide RNA binds). In some embodiments, the PAM is selected from the group consisting of NGG, NNGRRT, NNGRR(N), NNAGAAW, NNNNG(A/C)TT, and NNNNRYAC. 
     In some embodiments, the guide RNA sequences provided herein are complementary to a sequence adjacent to a PAM sequence. 
     In some embodiments, the guide RNA sequence comprises a sequence that is complementary to a sequence within a genomic region selected from the tables herein according to coordinates in human reference genome hg38. In some embodiments, the guide RNA sequence comprises a sequence that is complementary to a sequence that comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides from within a genomic region selected from the tables herein. In some embodiments, the guide RNA sequence comprises a sequence that is complementary to a sequence that comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides spanning a genomic region selected from the tables herein. 
     The guide RNAs disclosed herein mediate a target-specific cutting resulting in a double-stranded break (DSB). The guide RNAs disclosed herein mediate a target-specific cutting resulting in a single-stranded break (SSB or nick). 
     In some embodiments, the guide RNAs disclosed herein mediates target-specific cutting by an RNA-guided DNA binding agent (e.g., a Cas nuclease, as disclosed herein), resulting in insertion of a heterologous Factor IX nucleic acid within intron 1 of an albumin gene. In some embodiments, the guide RNA and/or cutting results in a rate of between 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95 and 99% insertion of a heterologous Factor IX gene. In some embodiments, the guide RNA and/or cutting results in a rate of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% insertion of a heterologous Factor IX nucleic acid. Insertion rates can be measured in vitro or in vivo. For example, in some embodiments, rate of insertion can be determined by detecting and measuring the inserted Factor IX nucleic acid within a population of cells, and calculating a percentage of the population that contains the inserted Factor IX nucleic acid. Methods of measuring insertion rates are known and available in the art. In some embodiments, the guide RNA allows between 5 and 10%, 10 and 15%, 15 and 20%, 20 and 25%, 25 and 30%, 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, 95 and 99% or more increased expression of a heterologous Factor IX gene. Increased expression of a heterologous Factor IX gene can be measured in vitro or in vivo. For example, in some embodiments, increased expression can be determined by detecting and measuring the Factor IX polypeptide level and comparing the level against the Factor IX polypeptide level before, e.g., treating the cells or administration to a subject. In some embodiments, the guide RNA allows between 5 and 10%, 10 and 15%, 15 and 20%, 20 and 25%, 25 and 30%, 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, 95 and 99% or more increased activity that results from expression of a heterologous Factor IX gene. For example, increased activity can be determined by detecting and measuring the coagulation activity and comparing the activity against the the coagulation activity before, e.g., treating the cells or administration to a subject. In some embodiments, increased activity can be determined using by assessing clotting function in an aPTT assay and/or thrombin generation in an TGA-EA assay. Such methods are available and known in the art (e.g. Simioni et al, NEJM 2009). 
     Each of the guide sequences shown in Table 1 at SEQ ID NOs:2-33 may further comprise additional nucleotides to form a crRNA and/or guide RNA, e.g., with the following exemplary nucleotide sequence following the guide sequence at its 3′ end: GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 400) in 5′ to 3′ orientation. Genomic coordinates are according to human reference genome hg38. In the case of a sgRNA, the above guide sequences may further comprise additional nucleotides to form a sgRNA, e.g., with the following exemplary nucleotide sequence following the 3′ end of the guide sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 401) 
               
               
                 GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC 
               
               
                   
               
               
                 AACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU 
               
               
                 or 
               
               
                   
               
               
                 in 5′ to 3′ orientation 
               
               
                 (SEQ ID NO: 402) 
               
               
                 GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC 
               
               
                   
               
               
                 AACUUGAAAAAGUGGCACCGAGUCGGUGC. 
               
            
           
         
       
     
     Each of the guide sequences in Table 1 at SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33 may further comprise additional nucleotides to form a crRNA, e.g., with the following exemplary nucleotide sequence following the guide sequence at its 3′ end: GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 400) in 5′ to 3′ orientation. In the case of a sgRNA, the above guide sequences may further comprise additional nucleotides to form a sgRNA, e.g., with the following exemplary nucleotide sequence following the 3′ end of the guide sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 401) 
               
               
                 GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC 
               
               
                   
               
               
                 AACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU 
               
               
                 or 
               
               
                   
               
               
                 in 5′ to 3′ orientation 
               
               
                 (SEQ ID NO: 402) 
               
               
                 GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC 
               
               
                   
               
               
                 AACUUGAAAAAGUGGCACCGAGUCGGUGC. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Human guide RNA sequences and 
               
               
                 chromosomal coordinates 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Human Genomic 
                 SEQ 
               
               
                   
                 Guide 
                   
                 Coordinates 
                 ID 
               
               
                   
                 ID 
                 Guide Sequence 
                 (hg38) 
                 NO: 
               
               
                   
                   
               
               
                   
                 G009844 
                 GAGCAACCUC 
                 chr4: 73405113- 
                  2 
               
               
                   
                   
                 ACUCUUGUCU 
                 73405133 
                   
               
               
                   
                   
               
               
                   
                 G009851 
                 AUGCAUUUGU 
                 chr4: 73405000- 
                  3 
               
               
                   
                   
                 UUCAAAAUAU 
                 73405020 
                   
               
               
                   
                   
               
               
                   
                 G009852 
                 UGCAUUUGUU 
                 chr4: 73404999- 
                  4 
               
               
                   
                   
                 UCAAAAUAUU 
                 73405019 
                   
               
               
                   
                   
               
               
                   
                 G009857 
                 AUUUAUGAGA 
                 chr4: 73404761- 
                  5 
               
               
                   
                   
                 UCAACAGCAC 
                 73404781 
                   
               
               
                   
                   
               
               
                   
                 G009858 
                 GAUCAACAGC 
                 chr4: 73404753- 
                  6 
               
               
                   
                   
                 ACAGGUUUUG 
                 73404773 
                   
               
               
                   
                   
               
               
                   
                 G009859 
                 UUAAAUAAAG 
                 chr4: 73404727- 
                  7 
               
               
                   
                   
                 CAUAGUGCAA 
                 73404747 
                   
               
               
                   
                   
               
               
                   
                 G009860 
                 UAAAGCAUAG 
                 chr4: 73404722- 
                  8 
               
               
                   
                   
                 UGCAAUGGAU 
                 73404742 
                   
               
               
                   
                   
               
               
                   
                 G009861 
                 UAGUGCAAUG 
                 chr4: 73404715- 
                  9 
               
               
                   
                   
                 GAUAGGUCUU 
                 73404735 
                   
               
               
                   
                   
               
               
                   
                 G009866 
                 UACUAAAACU 
                 chr4: 73404452- 
                 10 
               
               
                   
                   
                 UUAUUUUACU 
                 73404472 
                   
               
               
                   
                   
               
               
                   
                 G009867 
                 AAAGUUGAAC 
                 chr4: 73404418- 
                 11 
               
               
                   
                   
                 AAUAGAAAAA 
                 73404438 
                   
               
               
                   
                   
               
               
                   
                 G009868 
                 AAUGCAUAAU 
                 chr4: 73405013- 
                 12 
               
               
                   
                   
                 CUAAGUCAAA 
                 73405033 
                   
               
               
                   
                   
               
               
                   
                 G009874 
                 UAAUAAAAUU 
                 chr4: 73404561- 
                 13 
               
               
                   
                   
                 CAAACAUCCU 
                 73404581 
                   
               
               
                   
                   
               
               
                   
                 G012747 
                 GCAUCUUUAA 
                 chr4: 73404478- 
                 14 
               
               
                   
                   
                 AGAAUUAUUU 
                 73404498 
                   
               
               
                   
                   
               
               
                   
                 G012748 
                 UUUGGCAUUU 
                 chr4: 73404496- 
                 15 
               
               
                   
                   
                 AUUUCUAAAA 
                 73404516 
                   
               
               
                   
                   
               
               
                   
                 G012749 
                 UGUAUUUGUG 
                 chr4: 73404529- 
                 16 
               
               
                   
                   
                 AAGUCUUACA 
                 73404549 
                   
               
               
                   
                   
               
               
                   
                 G012750 
                 UCCUAGGUAA 
                 chr4: 73404577- 
                 17 
               
               
                   
                   
                 AAAAAAAAAA 
                 73404597 
                   
               
               
                   
                   
               
               
                   
                 G012751 
                 UAAUUUUCUU 
                 chr4: 73404620- 
                 18 
               
               
                   
                   
                 UUGCGCACUA 
                 73404640 
                   
               
               
                   
                   
               
               
                   
                 G012752 
                 UGACUGAAAC 
                 chr4: 73404664- 
                 19 
               
               
                   
                   
                 UUCACAGAAU 
                 73404684 
                   
               
               
                   
                   
               
               
                   
                 G012753 
                 GACUGAAACU 
                 chr4: 73404665- 
                 20 
               
               
                   
                   
                 UCACAGAAUA 
                 73404685 
                   
               
               
                   
                   
               
               
                   
                 G012754 
                 UUCAUUUUAG 
                 chr4: 73404803- 
                 21 
               
               
                   
                   
                 UCUGUCUUCU 
                 73404823 
                   
               
               
                   
                   
               
               
                   
                 G012755 
                 AUUAUCUAAG 
                 chr4: 73404859- 
                 22 
               
               
                   
                   
                 UUUGAAUAUA 
                 73404879 
                   
               
               
                   
                   
               
               
                   
                 G012756 
                 AAUUUUUAAA 
                 chr4: 73404897- 
                 23 
               
               
                   
                   
                 AUAGUAUUCU 
                 73404917 
                   
               
               
                   
                   
               
               
                   
                 G012757 
                 UGAAUUAUUC 
                 chr4: 73404924- 
                 24 
               
               
                   
                   
                 UUCUGUUUAA 
                 73404944 
                   
               
               
                   
                   
               
               
                   
                 G012758 
                 AUCAUCCUGA 
                 chr4: 73404965- 
                 25 
               
               
                   
                   
                 GUUUUUCUGU 
                 73404985 
                   
               
               
                   
                   
               
               
                   
                 G012759 
                 UUACUAAAAC 
                 chr4: 73404453- 
                 26 
               
               
                   
                   
                 UUUAUUUUAC 
                 73404473 
                   
               
               
                   
                   
               
               
                   
                 G012760 
                 ACCUUUUUUU 
                 chr4: 73404581- 
                 27 
               
               
                   
                   
                 UUUUUUACCU 
                 73404601 
                   
               
               
                   
                   
               
               
                   
                 G012761 
                 AGUGCAAUGG 
                 chr4: 73404714- 
                 28 
               
               
                   
                   
                 AUAGGUCUUU 
                 73404734 
                   
               
               
                   
                   
               
               
                   
                 G012762 
                 UGAUUCCUAC 
                 chr4: 73404973- 
                 29 
               
               
                   
                   
                 AGAAAAACUC 
                 73404993 
                   
               
               
                   
                   
               
               
                   
                 G012763 
                 UGGGCAAGGG 
                 chr4: 73405094- 
                 30 
               
               
                   
                   
                 AAGAAAAAAA 
                 73405114 
                   
               
               
                   
                   
               
               
                   
                 G012764 
                 CCUCACUCUU 
                 chr4: 73405107- 
                 31 
               
               
                   
                   
                 GUCUGGGCAA 
                 73405127 
                   
               
               
                   
                   
               
               
                   
                 G012765 
                 ACCUCACUCU 
                 chr4: 73405108- 
                 32 
               
               
                   
                   
                 UGUCUGGGCA 
                 73405128 
                   
               
               
                   
                   
               
               
                   
                 G012766 
                 UGAGCAACCU 
                 chr4: 73405114- 
                 33 
               
               
                   
                   
                 CACUCUUGUC 
                 73405134 
               
               
                   
                   
               
            
           
         
       
     
     The guide RNA may further comprise a trRNA. In each composition and method embodiment described herein, the crRNA and trRNA may be associated as a single RNA (sgRNA) or may be on separate RNAs (dgRNA). In the context of sgRNAs, the crRNA and trRNA components may be covalently linked, e.g., via a phosphodiester bond or other covalent bond. In some embodiments, the sgRNA comprises one or more linkages between nucleotides that is not a phosphodiester linkage. 
     In each of the composition, use, and method embodiments described herein, the guide RNA may comprise two RNA molecules as a “dual guide RNA” or “dgRNA”. The dgRNA comprises a first RNA molecule comprising a crRNA comprising, e.g., a guide sequence shown in Table 1, and a second RNA molecule comprising a trRNA. The first and second RNA molecules may not be covalently linked, but may form a RNA duplex via the base pairing between portions of the crRNA and the trRNA. 
     In each of the composition, use, and method embodiments described herein, the guide RNA may comprise a single RNA molecule as a “single guide RNA” or “sgRNA”. The sgRNA may comprise a crRNA (or a portion thereof) comprising a guide sequence shown in Table 1 covalently linked to a trRNA. The sgRNA may comprise 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a guide sequence shown in Table 1. In some embodiments, the crRNA and the trRNA are covalently linked via a linker. In some embodiments, the sgRNA forms a stem-loop structure via the base pairing between portions of the crRNA and the trRNA. In some embodiments, the crRNA and the trRNA are covalently linked via one or more bonds that are not a phosphodiester bond. 
     In some embodiments, the trRNA may comprise all or a portion of a trRNA sequence derived from a naturally-occurring CRISPR/Cas system. In some embodiments, the trRNA comprises a truncated or modified wild type trRNA. The length of the trRNA depends on the CRISPR/Cas system used. In some embodiments, the trRNA comprises or consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides. In some embodiments, the trRNA may comprise certain secondary structures, such as, for example, one or more hairpin or stem-loop structures, or one or more bulge structures. 
     In some embodiments, the target sequence or region within intron 1 of a human albumin locus (SEQ ID NO: 1) may be complementary to the guide sequence of the guide RNA. In some embodiments, the degree of complementarity or identity between a guide sequence of a guide RNA and its corresponding target sequence may be at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the target sequence and the guide sequence of the gRNA may be 100% complementary or identical. In other embodiments, the target sequence and the guide sequence of the gRNA may contain at least one mismatch. For example, the target sequence and the guide sequence of the gRNA may contain 1, 2, 3, 4, or 5 mismatches, where the total length of the guide sequence is about 20, or 20. In some embodiments, the target sequence and the guide sequence of the gRNA may contain 1-4 mismatches where the guide sequence is about 20, or 20 nucleotides. 
     In some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease, is provided, used, or administered. 
     B. Modified gRNAs and mRNAs 
     In some embodiments, the gRNA is chemically modified. A gRNA comprising one or more modified nucleosides or nucleotides is called a “modified” gRNA or “chemically modified” gRNA, 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 gRNA is synthesized with a non-canonical nucleoside or nucleotide, is here called “modified.” 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, 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). 
     Chemical modifications such as those listed above can be combined to provide modified gRNAs and/or mRNAs comprising nucleosides and nucleotides (collectively “residues”) that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase. In some embodiments, every base of a gRNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group. In certain embodiments, all, or substantially all, of the phosphate groups of an gRNA molecule are replaced with phosphorothioate groups. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 5′ end of the RNA. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 3′ end of the RNA. Certain gRNAs comprise at least one modified residue at or near the 5′ end and 3′ end of the RNA. 
     In some embodiments, the gRNA comprises one, two, three or more modified residues. In some embodiments, at least 5% (e.g., 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%, or 100%) of the positions in a modified gRNA are modified nucleosides or nucleotides. 
     Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the gRNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases. In some embodiments, the modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. 
     In some embodiments of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified residue, e.g., modified residue present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution. 
     Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp). The backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens. 
     The phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. 
     Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. 
     The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e. at sugar modification. For example, the 2′ hydroxyl group (OH) can be modified, e.g. replaced with a number of different “oxy” or “deoxy” substituents. In some embodiments, modifications to the 2′ hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2′-alkoxide ion. 
     Examples of 2′ hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH 2 CH 2 O) n CH 2 CH 2 OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the 2′ hydroxyl group modification can be 2′-O-Me. In some embodiments, the 2′ hydroxyl group modification can be a 2′-fluoro modification, which replaces the 2′ hydroxyl group with a fluoride. In some embodiments, the 2′ hydroxyl group modification can be a 2′-H, which replaces the 2′ hydroxyl group with a hydrogen. In some embodiments, the 2′ hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2′ hydroxyl can be connected, e.g., by a C 1-6  alkylene or C 1-6  heteroalkylene bridge, to the 4′ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH 2 ) n -amino, (wherein amino can be, e.g., NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the 2′ hydroxyl group modification can include “unlocked” nucleic acids (UNA) in which the ribose ring lacks the C2′-C3′ bond. In some embodiments, the 2′ hydroxyl group modification can include the methoxyethyl group (MOE), (OCH 2 CH 2 OCH 3 , e.g., a PEG derivative). 
     “Deoxy” 2′ modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH 2 CH 2 NH) n CH2CH 2 — amino (wherein amino can be, e.g., as described herein), —NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein. 
     The sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L-nucleosides. 
     The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base. 
     In embodiments employing a dual guide RNA, each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA and/or tracr RNA. In embodiments comprising an sgRNA, one or more residues at one or both ends of the sgRNA may be chemically modified, and/or internal nucleosides may be modified, and/or the entire sgRNA may be chemically modified. Certain embodiments comprise a 5′ end modification. Certain embodiments comprise a 3′ end modification. 
     In some embodiments, the guide RNAs disclosed herein comprise one of the modification patterns disclosed in WO2018/107028 A1, filed Dec. 8, 2017, titled “Chemically Modified Guide RNAs,” the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in US20170114334, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures/modification patterns disclosed in WO2017/136794, WO2017004279, US2018187186, US2019048338, the contents of which are hereby incorporated by reference in their entirety. 
     In some embodiments, the sgRNA of the present disclosure comprises the modification patterns shown below in Table 2. “Full Sequence” in Table 2 refers to an sgRNA sequence for each of the guides listed in Table 1. “Full Sequence Modified” shows a modification pattern for each sgRNA. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 sgRNA and modification patterns 
               
               
                 to sgRNA of human albumin guide sequences 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                   
                 SEQ 
               
               
                 Guide 
                   
                 ID 
                   
                 ID 
               
               
                 ID 
                 Full Sequence 
                 NO: 
                 Full Sequence Modified 
                 NO: 
               
               
                   
               
               
                 G009844 
                 GAGCAACCUCACUCUUGUCUGUUUU 
                 34 
                 mG*mA*mG*CAACCUCACUCUUGUCUGU 
                 66 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UUUAGAmGmCmUmAmGmAmAmAmUm 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AmGmCAAGUUAAAAUAAGGCUAGUCC 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 GUUAUCAmAmCmUmUmGmAmAmAmAm 
                   
               
               
                   
                   
                   
                 AmGmUmGmGmCmAmCmCmGmAmGmUm 
                   
               
               
                   
                   
                   
                 CmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009851 
                 AUGCAUUUGUUUCAAAAUAUGUUUU 
                 35 
                 mA*mU*mG*CAUUUGUUUCAAAAUAUG 
                 67 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UUUUAGAmGmCmUmAmGmAmAmAmUm 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AmGmCAAGUUAAAAUAAGGCUAGUCCG 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 UUAUCAmAmCmUmUmGmAmAmAmAmAm 
                   
               
               
                   
                   
                   
                 GmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                   
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009852 
                 UGCAUUUGUUUCAAAAUAUUGUUUU 
                 36 
                 mU*mG*mC*AUUUGUUUCAAAAUAUUGU 
                 68 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UUUAGAmGmCmUmAmGmAmAmAmUmAm 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 GmCAAGUUAAAAUAAGGCUAGUCCGUUA 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 UCAmAmCmUmUmGmAmAmAmAmAmGmUm 
                   
               
               
                   
                   
                   
                 GmGmCmAmCmCmGmAmGmUmCmGmGmUm 
                   
               
               
                   
                   
                   
                 GmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009857 
                 AUUUAUGAGAUCAACAGCACGUUUU 
                 37 
                 mA*mU*mU*UAUGAGAUCAACAGCACGU 
                 69 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UUUAGAmGmCmUmAmGmAmAmAmUmAm 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 GmCAAGUUAAAAUAAGGCUAGUCCGUUA 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 UCAmAmCmUmUmGmAmAmAmAmAmGm 
                   
               
               
                   
                   
                   
                 UmGmGmCmAmCmCmGmAmGmUmCmGmGm 
                   
               
               
                   
                   
                   
                 UmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009858 
                 GAUCAACAGCACAGGUUUUGGUUUU 
                 38 
                 mG*mA*mU*CAACAGCACAGGUUUUGGU 
                 70 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UUUAGAmGmCmUmAmGmAmAmAmUmAm 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 GmCAAGUUAAAAUAAGGCUAGUCCGUUA 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 UCAmAmCmUmUmGmAmAmAmAmAmGm 
                   
               
               
                   
                   
                   
                 UmGmGmCmAmCmCmGmAmGmUmCmGm 
                   
               
               
                   
                   
                   
                 GmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009859 
                 UUAAAUAAAGCAUAGUGCAAGUUUU 
                 39 
                 mU*mU*mA*AAUAAAGCAUAGUGCAAGUUU 
                 71 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G009860 
                 UAAAGCAUAGUGCAAUGGAUGUUUU 
                 40 
                 mU*mA*mA*AGCAUAGUGCAAUGGAUGUUU 
                 72 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G009861 
                 UAGUGCAAUGGAUAGGUCUUGUUUU 
                 41 
                 mU*mA*mG*UGCAAUGGAUAGGUCUUGUUU 
                 73 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G009866 
                 UACUAAAACUUUAUUUUACUGUUUU 
                 42 
                 mU*mA*mC*UAAAACUUUAUUUUACUGUUU 
                 74 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G009867 
                 AAAGUUGAACAAUAGAAAAAGUUUU 
                 43 
                 mA*mA*mA*GUUGAACAAUAGAAAAAGUUU 
                 75 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G009868 
                 AAUGCAUAAUCUAAGUCAAAGUUUU 
                 44 
                 mA*mA*mU*GCAUAAUCUAAGUCAAAGUUU 
                 76 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G009874 
                 UAAUAAAAUUCAAACAUCCUGUUUU 
                 45 
                 mU*mA*mA*UAAAAUUCAAACAUCCUGUUU 
                 77 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012747 
                 GCAUCUUUAAAGAAUUAUUUGUUUU 
                 46 
                 mG*mC*mA*UCUUUAAAGAAUUAUUUGUUU 
                 78 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012748 
                 UUUGGCAUUUAUUUCUAAAAGUUUU 
                 47 
                 mU*mU*mU*GGCAUUUAUUUCUAAAAGUUU 
                 79 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012749 
                 UGUAUUUGUGAAGUCUUACAGUUUU 
                 48 
                 mU*mG*mU*AUUUGUGAAGUCUUACAGUUU 
                 80 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012750 
                 UCCUAGGUAAAAAAAAAAAAGUUUU 
                 49 
                 mU*mC*mC*UAGGUAAAAAAAAAAAAGUUU 
                 81 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012751 
                 UAAUUUUCUUUUGCGCACUAGUUUU 
                 50 
                 mU*mA*mA*UUUUCUUUUGCGCACUAGUUU 
                 82 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012752 
                 UGACUGAAACUUCACAGAAUGUUUU 
                 51 
                 mU*mG*mA*CUGAAACUUCACAGAAUGUUU 
                 83 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012753 
                 GACUGAAACUUCACAGAAUAGUUUU 
                 52 
                 mG*mA*mC*UGAAACUUCACAGAAUAGUUU 
                 84 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012754 
                 UUCAUUUUAGUCUGUCUUCUGUUUU 
                 53 
                 mU*mU*mC*AUUUUAGUCUGUCUUCUGUUU 
                 85 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012755 
                 AUUAUCUAAGUUUGAAUAUAGUUUU 
                 54 
                 mA*mU*mU*AUCUAAGUUUGAAUAUAGUUU 
                 86 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012756 
                 AAUUUUUAAAAUAGUAUUCUGUUUU 
                 55 
                 mA*mA*mU*UUUUAAAAUAGUAUUCUGUUU 
                 87 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012757 
                 UGAAUUAUUCUUCUGUUUAAGUUUU 
                 56 
                 mU*mG*mA*AUUAUUCUUCUGUUUAAGUUU 
                 88 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012758 
                 AUCAUCCUGAGUUUUUCUGUGUUUU 
                 57 
                 mA*mU*mC*AUCCUGAGUUUUUCUGUGUUU 
                 89 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012759 
                 UUACUAAAACUUUAUUUUACGUUUU 
                 58 
                 mU*mU*mA*CUAAAACUUUAUUUUACGUUU 
                 90 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012760 
                 ACCUUUUUUUUUUUUUACCUGUUUU 
                 59 
                 mA*mC*mC*UUUUUUUUUUUUUACCUGUUU 
                 91 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012761 
                 AGUGCAAUGGAUAGGUCUUUGUUUU 
                 60 
                 mA*mG*mU*GCAAUGGAUAGGUCUUUGUUU 
                 92 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012762 
                 UGAUUCCUACAGAAAAACUCGUUUU 
                 61 
                 mU*mG*mA*UUCCUACAGAAAAACUCGUUU 
                 93 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012763 
                 UGGGCAAGGGAAGAAAAAAAGUUUU 
                 62 
                 mU*mG*mG*GCAAGGGAAGAAAAAAAGUUU 
                 94 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012764 
                 CCUCACUCUUGUCUGGGCAAGUUUU 
                 63 
                 mC*mC*mU*CACUCUUGUCUGGGCAAGUUU 
                 95 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012765 
                 ACCUCACUCUUGUCUGGGCAGUUUU 
                 64 
                 mA*mC*mC*UCACUCUUGUCUGGGCAGUUU 
                 96 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
                   
               
               
                   
               
               
                 G012766 
                 UGAGCAACCUCACUCUUGUCGUUUU 
                 65 
                 mU*mG*mA*GCAACCUCACUCUUGUCGUUU 
                 97 
               
               
                   
                 AGAGCUAGAAAUAGCAAGUUAAAAU 
                   
                 UAGAmGmCmUmAmGmAmAmAmUmAmGmC 
                   
               
               
                   
                 AAGGCUAGUCCGUUAUCAACUUGAA 
                   
                 AAGUUAAAAUAAGGCUAGUCCGUUAUCAm 
                   
               
               
                   
                 AAAGUGGCACCGAGUCGGUGCUUUU 
                   
                 AmCmUmUmGmAmAmAmAmAmGmUmGmGm 
                   
               
               
                   
                   
                   
                 CmAmCmCmGmAmGmUmCmGmGmUmGmCm 
                   
               
               
                   
                   
                   
                 U*mU*mU*mU 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the modified sgRNA comprises the following sequence: mN*mN*mN* GUUUUAGAmGmCmUmAmGmAmAmAmUmA mGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 300), where “N” may be any natural or non-natural nucleotide, and wherein the totality of N&#39;s comprise an albumin intron 1 guide sequence as described in Table 1. For example, encompassed herein is SEQ ID NO: 300, where the N&#39;s are replaced with any of the guide sequences disclosed herein in Table 1 (SEQ ID Nos: 2-33). 
     For example, encompassed herein is SEQ ID NO: 300, where the N&#39;s are replaced with any of the guide sequences disclosed herein in Table 1 (SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33). 
     Any of the modifications described below may be present in the gRNAs and mRNAs described herein. 
     The terms “mA,” “mC,” “mU,” or “mG” may be used to denote a nucleotide that has been modified with 2′-O-Me. 
     Modification of 2′-O-methyl can be depicted as follows: 
     
       
         
         
             
             
         
       
     
     Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution. For example, 2′-fluoro (2′-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability. 
     In this application, the terms “fA,” “fC,” “fU,” or “fG” may be used to denote a nucleotide that has been substituted with 2′-F. 
     Substitution of 2′-F can be depicted as follows: 
     
       
         
         
             
             
         
       
     
     Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one nonbridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S-oligos. 
     A “*” may be used to depict a PS modification. In this application, the terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3′) nucleotide with a PS bond. 
     In this application, the terms “mA*,” “mC*,” “mU*,” or “mG*” may be used to denote a nucleotide that has been substituted with 2′-O-Me and that is linked to the next (e.g., 3′) nucleotide with a PS bond. 
     The diagram below shows the substitution of S— into a nonbridging phosphate oxygen, generating a PS bond in lieu of a phosphodiester bond: 
     
       
         
         
             
             
         
       
     
     Abasic nucleotides refer to those which lack nitrogenous bases. The figure below depicts an oligonucleotide with an abasic (also known as apurinic) site that lacks a base: 
     
       
         
         
             
             
         
       
     
     Inverted bases refer to those with linkages that are inverted from the normal 5′ to 3′ linkage (i.e., either a 5′ to 5′ linkage or a 3′ to 3′ linkage). For example: 
     
       
         
         
             
             
         
       
     
     An abasic nucleotide can be attached with an inverted linkage. For example, an abasic nucleotide may be attached to the terminal 5′ nucleotide via a 5′ to 5′ linkage, or an abasic nucleotide may be attached to the terminal 3′ nucleotide via a 3′ to 3′ linkage. An inverted abasic nucleotide at either the terminal 5′ or 3′ nucleotide may also be called an inverted abasic end cap. 
     In some embodiments, one or more of the first three, four, or five nucleotides at the 5′ terminus, and one or more of the last three, four, or five nucleotides at the 3′ terminus are modified. In some embodiments, the modification is a 2′-O-Me, 2′-F, inverted abasic nucleotide, PS bond, or other nucleotide modification well known in the art to increase stability and/or performance. 
     In some embodiments, the first four nucleotides at the 5′ terminus, and the last four nucleotides at the 3′ terminus are linked with phosphorothioate (PS) bonds. 
     In some embodiments, the first three nucleotides at the 5′ terminus, and the last three nucleotides at the 3′ terminus comprise a 2′-O-methyl (2′-O-Me) modified nucleotide. In some embodiments, the first three nucleotides at the 5′ terminus, and the last three nucleotides at the 3′ terminus comprise a 2′-fluoro (2′-F) modified nucleotide. In some embodiments, the first three nucleotides at the 5′ terminus, and the last three nucleotides at the 3′ terminus comprise an inverted abasic nucleotide. 
     In some embodiments, the guide RNA comprises a modified sgRNA. In some embodiments, the sgRNA comprises the modification pattern shown in SEQ ID No: 300, where N is any natural or non-natural nucleotide, and where the totality of the N&#39;s comprise a guide sequence that directs a nuclease to a target sequence in human albumin intron 1, e.g., as shown in Table 1. 
     In some embodiments, the guide RNA comprises a sgRNA shown in any one of SEQ ID No: 34-97. In some embodiments, the guide RNA comprises a sgRNA comprising any one of the guide sequences of SEQ ID No: 2-33 and the nucleotides of SEQ ID No: 300 wherein the nucleotides of SEQ ID No: 300 are on the 3′ end of the guide sequence, and wherein the sgRNA may be modified, e.g., as shown in SEQ ID NO: 300. 
     In some embodiments, the guide RNA comprises a sgRNA shown in any one of SEQ ID NOs: 34-37, 42-49, 53-59, 61-69, 74-81, 85-91, and 93-97. In some embodiments, the guide RNA comprises a sgRNA comprising any one of the guide sequences of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33 and the nucleotides of SEQ ID No: 300 wherein the nucleotides of SEQ ID NO: 300 are on the 3′ end of the guide sequence, and wherein the sgRNA may be modified, e.g., as shown in SEQ ID NO: 300. 
     As noted above, in some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease, is provided, used, or administered. As described below, the mRNA comprising a Cas nuclease may comprise a Cas9 nuclease, such as an  S. pyogenes  Cas9 nuclease having cleavase, nickase, and/or site-specific DNA binding activity. In some embodiments, the ORF encoding an RNA-guided DNA nuclease is a “modified RNA-guided DNA binding agent ORF” or simply a “modified ORF,” which is used as shorthand to indicate that the ORF is modified. 
     Cas9 ORFs, including modified Cas9 ORFs, are provided herein and are known in the art. As one example, the Cas9 ORF can be codon optimized, such that coding sequence includes one or more alternative codons for one or more amino acids. An “alternative codon” as used herein refers to variations in codon usage for a given amino acid, and may or may not be a preferred or optimized codon (codon optimized) for a given expression system. Preferred codon usage, or codons that are well-tolerated in a given system of expression, is known in the art. The Cas9 coding sequences, Cas9 mRNAs, and Cas9 protein sequences of WO2013/176772, WO2014/065596, WO2016/106121, and WO2019/067910 are hereby incorporated by reference. In particular, the ORFs and Cas9 amino acid sequences of the table at paragraph [0449] WO2019/067910, and the Cas9 mRNAs and ORFs of paragraphs [0214]-[0234] of WO2019/067910 are hereby incorporated by reference. 
     In some embodiments, the modified ORF may comprise a modified uridine at least at one, a plurality of, or all uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5 position, e.g., with a halogen, methyl, or ethyl. In some embodiments, the modified uridine is a pseudouridine modified at the 1 position, e.g., with a halogen, methyl, or ethyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine. 
     In some embodiments, an mRNA disclosed herein comprises a 5′ cap, such as a Cap0, Cap1, or Cap2. A 5′ cap is generally a 7-methylguanine ribonucleotide (which may be further modified, as discussed below e.g. with respect to ARCA) linked through a 5′-triphosphate to the 5′ position of the first nucleotide of the 5′-to-3′ chain of the mRNA, i.e., the first cap-proximal nucleotide. In Cap0, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2′-hydroxyl. In Cap1, the riboses of the first and second transcribed nucleotides of the mRNA comprise a 2′-methoxy and a 2′-hydroxyl, respectively. In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2′-methoxy. See, e.g., Katibah et al. (2014)  Proc Natl Acad Sci USA  111(33):12025-30; Abbas et al. (2017)  Proc Natl Acad Sci USA  114(11):E2106-E2115. Most endogenous higher eukaryotic mRNAs, including mammalian mRNAs such as human mRNAs, comprise Cap1 or Cap2. Cap0 and other cap structures differing from Cap1 and Cap2 may be immunogenic in mammals, such as humans, due to recognition as “non-self” by components of the innate immune system such as IFIT-1 and IFIT-5, which can result in elevated cytokine levels including type I interferon. Components of the innate immune system such as IFIT-1 and IFIT-5 may also compete with eIF4E for binding of an mRNA with a cap other than Cap1 or Cap2, potentially inhibiting translation of the mRNA. 
     A cap can be included co-transcriptionally. For example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific Cat. No. AM8045) is a cap analog comprising a 7-methylguanine 3′-methoxy-5′-triphosphate linked to the 5′ position of a guanine ribonucleotide which can be incorporated in vitro into a transcript at initiation. ARCA results in a Cap0 cap in which the 2′ position of the first cap-proximal nucleotide is hydroxyl. See, e.g., Stepinski et al., (2001) “Synthesis and properties of mRNAs containing the novel ‘anti-reverse’ cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl(3′deoxy)GpppG,” RNA 7: 1486-1495. The ARCA structure is shown below. 
     
       
         
         
             
             
         
       
     
     CleanCap™ AG (m7G(5′)ppp(5′)(2′OMeA)pG; TriLink Biotechnologies Cat. No. N-7113) or CleanCap™ GG (m7G(5′)ppp(5′)(2′OMeG)pG; TriLink Biotechnologies Cat. No. N-7133) can be used to provide a Cap1 structure co-transcriptionally. 3′-O-methylated versions of CleanCap™ AG and CleanCap™ GG are also available from TriLink Biotechnologies as Cat. Nos. N-7413 and N-7433, respectively. The CleanCap™ AG structure is shown below. 
     
       
         
         
             
             
         
       
     
     Alternatively, a cap can be added to an RNA post-transcriptionally. For example, Vaccinia capping enzyme is commercially available (New England Biolabs Cat. No. M2080S) and has RNA triphosphatase and guanylyltransferase activities, provided by its D1 subunit, and guanine methyltransferase, provided by its D12 subunit. As such, it can add a 7-methylguanine to an RNA, so as to give Cap0, in the presence of S-adenosyl methionine and GTP. See, e.g., Guo, P. and Moss, B. (1990)  Proc. Natl. Acad. Sci. USA  87, 4023-4027; Mao, X. and Shuman, S. (1994)  J. Biol. Chem.  269, 24472-24479. 
     In some embodiments, the mRNA further comprises a poly-adenylated (poly-A) tail. In some embodiments, the poly-A tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines. In some embodiments, the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. 
     C. Donor Constructs 
     The compositions and methods described herein include the use of a nucleic acid construct that comprises a sequence encoding a heterologous Factor IX gene to be inserted into a cut site created by a guide RNA of the present disclosure and an RNA-guided DNA binding agent. As used herein, such a construct is sometimes referred to as a “donor construct/template”. In some embodiments, the construct is a DNA construct. Methods of designing and making various functional/structural modifications to donor constructs are known in the art. In some embodiments, the construct may comprise any one or more of a polyadenylation tail sequence, a polyadenylation signal sequence, splice acceptor site, or selectable marker. In some embodiments, the polyadenylation tail sequence is encoded, e.g., as a “poly-A” stretch, at the 3′ end of the coding sequence. Methods of designing a suitable polyadenylation tail sequence and/or polyadenylation signal sequence are well known in the art. For example, the polyadenylation signal sequence AAUAAA (SEQ ID NO: 800) is commonly used in mammalian systems, although variants such as UAUAAA (SEQ ID NO: 801) or AU/GUAAA (SEQ ID NO: 802) have been identified. See, e.g., NJ Proudfoot, Genes &amp; Dev. 25(17):1770-82, 2011. 
     In some embodiments, the donor construct comprises a sequence encoding Factor IX, wherein the Factor IX sequence is wild type Factor IX, e.g., SEQ ID NO: 700. In some embodiments, the donor construct comprises a sequence encoding Factor IX, wherein the Factor IX sequence is wild type Factor IX, e.g., SEQ ID NO: 701. In some embodiments, the sequence encodes a variant of Factor IX. For example, the variant can possess increased coagulation activity than wild type Factor IX. For example, the variant Factor IX can comprise one or mutations, such as an amino acid substitution in position R338 (e.g., R338L), relative to SEQ ID NO: 701. In some embodiments, the sequence encodes a Factor IX variant that is 80%, 85%, 90%, 93%, 95%, 97%, 99% identical to SEQ ID NO: 700, SEQ ID NO: 701, or SEQ ID NO: 702, having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 100%, or more, activity as compared to wild type Factor IX. In some embodiments, the sequence encodes a fragment of Factor IX, wherein the fragment possesses at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 100%, or more, activity as compared to wild type Factor IX. 
     In some embodiments, the donor construct comprises a sequence encoding a Factor IX variant, wherein the Factor IX variant activates coagulation in the absence of its cofactor, Factor VIII. Such Factor IX variants can further maintain the activity of wild type Factor IX. Such Factor IX variants can be used to treat hemophilia, such as hemophilia B. For example, such a Factor IX variant can comprise an amino acid substation at position L6, V181, K265, I383, E185, or a combination thereof relative to wild type Factor IX (e.g., relative to SEQ ID NO: 701). For example, such a Factor IX variant can comprise an L6F mutation, a V181I mutation, a K265A mutation, an I383V mutation, an E185D mutation, or a combination thereof relative to wild type Factor IX (e.g., relative to SEQ ID NO: 701). 
     In one example, the Factor IX protein can comprise amino acid substitutions at positions L6 and V181. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6 and K265. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6 and I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6 and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181 and K265. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181 and an I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181 and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions K265 and I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions K265 and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions I383 and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, V181, and K265. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, V181, and I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, V181, and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, K265, and I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, K265, and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, I383, and E186. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181, K265, and I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181, K265, and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181, I383, and E186. In another example, the Factor IX protein can comprise amino acid substitutions at positions K265, I383, and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, V181, K265, and I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, V181, I383, and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, K265, I383, and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181, K265, I383, and E185. 
     In a specific example, the Factor IX protein can comprise amino acid substitutions at positions V181, K265, and I383. In another specific example, the Factor IX protein can comprise amino acid substitutions at positions V181, K265, I383, and E185. In another specific example, the Factor IX protein can comprise amino acid substitutions at positions L6, V181, K265, and I383. 
     In one example, the Factor IX protein can comprise an L6F mutation and a V181I mutation. In another example, the Factor IX protein can comprise an L6F mutation and a K265A mutation. In another example, the Factor IX protein can comprise an L6F mutation and an I383V mutation. In another example, the Factor IX protein can comprise an L6F mutation and an E185D mutation. In another example, the Factor IX protein can comprise a V181I mutation and a K265A mutation. In another example, the Factor IX protein can comprise a V181I mutation and an I383V mutation. In another example, the Factor IX protein can comprise a V181I mutation and an E185D mutation. In another example, the Factor IX protein can comprise a K265A mutation and an I383V mutation. In another example, the Factor IX protein can comprise a K265A mutation and an E185D mutation. In another example, the Factor IX protein can comprise an I383V mutation and an E185D mutation. In another example, the Factor IX protein can comprise an L6F mutation, a V181I mutation, and a K265A mutation. In another example, the Factor IX protein can comprise an L6F mutation, a V181I mutation, and an I383V mutation. In another example, the Factor IX protein can comprise an L6F mutation, a V181I mutation and an E185D mutation. In another example, the Factor IX protein can comprise an L6F mutation, a K265A mutation, and an I383V mutation. In another example, the Factor IX protein can comprise an L6F mutation, a K265A mutation, and an E185D mutation. In another example, the Factor IX protein can comprise an L6F mutation, an I383V mutation, and an E186D mutation. In another example, the Factor IX protein can comprise a V181I mutation, a K265A mutation, and an I383V mutation. In another example, the Factor IX protein can comprise a V181I mutation, a K265A mutation, and an E185D mutation. In another example, the Factor IX protein can comprise a V181I mutation, an I383V mutation, and an E186D mutation. In another example, the Factor IX protein can comprise a K265A mutation, an I383V mutation, and an E185D mutation. In another example, the Factor IX protein can comprise an L6F mutation, a V181I mutation, a K265A mutation, and an I383V mutation. In another example, the Factor IX protein can comprise an L6F mutation, a V181I mutation, an I383V mutation, and an E185D mutation. In another example, the Factor IX protein can comprise an L6F mutation, a K265A mutation, an I383V mutation, and an E185D mutation. In another example, the Factor IX protein can comprise a V181I mutation, a K265A mutation, an I383V mutation, and an E185D mutation. 
     In a specific example, the Factor IX protein can comprise a V181I mutation, an K265A mutation, and an I383V mutation. In another specific example, the Factor IX protein can comprise a V181I mutation, a K265A mutation, an I383V mutation, and an E185D mutation. In some embodiments, the Factor IX variant is at least 80%, 85%, 90%, 93%, 95%, 97%, 99% identical to SEQ ID NO: 700, having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 100%, or more, activity as compared to wild type Factor IX. In certain embodiments, the Factor IX variant is at least 80%, 85%, 90%, 93%, 95%, 97%, 99% identical to SEQ ID NO: 700, having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 100%, or more, activity as compared to wild type Factor IX and comprises a V181I mutation, a K265A mutation, an I383V mutation, and/or an E185D mutation. In another specific example, the Factor IX protein can comprise an L6F mutation, a V181I mutation, a K265A mutation, and an I383V mutation. In some embodiments, the Factor IX variant is at least 80%, 85%, 90%, 93%, 95%, 97%, 99% identical to SEQ ID NO: 700, having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 100%, or more, activity as compared to wild type Factor IX and comprises an L6F mutation, a V181I mutation, a K265A mutation, and/or an I383V mutation. 
     The length of the construct can vary, depending on the size of the gene to be inserted, and can be, for example, from 200 base pairs (bp) to about 5000 bp, such as about 200 bp to about 2000 bp, such as about 500 bp to about 1500 bp. In some embodiments, the length of the DNA donor template is about 200 bp, or is about 500 bp, or is about 800 bp, or is about 1000 base pairs, or is about 1500 base pairs. In other embodiments, the length of the donor template is at least 200 bp, or is at least 500 bp, or is at least 800 bp, or is at least 1000 bp, or is at least 1500 bp. In other embodiments, the length of the donor template is at least 200 bp, or is at least 500 bp, or is at least 800 bp, or is at least 1000 bp, or is at least 1500 bp, or at least 2000, or at least 2500, or at least 3000, or at least 3500, or at least 4000, or at least 4500, or at least 5000. 
     The construct can be DNA or RNA, single-stranded, double-stranded or partially single- and partially double-stranded and can be introduced into a host cell in linear or circular (e.g., minicircle) form. See, e.g., U.S. Patent Publication Nos. 2010/0047805, 2011/0281361, 2011/0207221. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3′ terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987)  Proc. Natl. Acad. Sci. USA  84:4959-4963; Nehls et al. (1996)  Science  272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. A construct can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. A construct may omit viral elements. Moreover, donor constructs can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus). 
     In some embodiments, the construct may be inserted so that its expression is driven by the endogenous promoter at the insertion site (e.g., the endogenous albumin promoter when the donor is integrated into the host cell&#39;s albumin locus). In such cases, the transgene may lack control elements (e.g., promoter and/or enhancer) that drive its expression (e.g., a promoterless construct). Nonetheless, it will be apparent that in other cases the construct may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue specific (e.g., liver- or platelet-specific) promoter that drives expression of the functional protein upon integration. The construct may comprise a sequence encoding a heterologous Factor IX protein downstream of and operably linked to a signal sequence encoding a signal peptide. In some embodiments, the nucleic acid construct works in homology-independent insertion of a nucleic acid that encodes a Factor IX protein. In some embodiments, the nucleic acid construct works in non-dividing cells, e.g., cells in which NHEJ, not HR, is the primary mechanism by which double-stranded DNA breaks are repaired. The nucleic acid may be a homology-independent donor construct. 
     Some donor constructs comprising a heterologous Factor IX nucleic acid (Factor IX transgene) are capable of insertion into a cut site in a target DNA sequence for a gene editing system (e.g., capable of insertion into a safe harbor gene, such as an albumin locus) by non-homologous end joining. In some cases, such constructs do not comprise homology arms. For example, such constructs can be inserted into a blunt end double-strand break following cleavage with a gene editing system (e.g., CRISPR/Cas system) as disclosed herein. In a specific example, the construct can be delivered via AAV and can be capable of insertion by non-homologous end joining (e.g., the construct can be one that does not comprise homology arms). 
     In a specific example, the construct can be inserted via homology-independent targeted integration. For example, the heterologous Factor IX nucleic acid in the construct can be flanked on each side by a target site for a gene editing system (e.g., the same target site as in the target DNA sequence for targeted insertion (e.g., in a safe harbor gene), and the same gene editing system being used to cleave the target DNA sequence for targeted insertion). The gene editing system can then cleave the target sites flanking the heterologous Factor IX nucleic acid. In a specific example, the construct is delivered AAV-mediated delivery, and cleavage of the target sites flanking the heterologous Factor IX nucleic acid can remove the inverted terminal repeats (ITRs) of the AAV. In some methods, the target DNA sequence for targeted insertion (e.g., target DNA sequence in a safe harbor locus, e.g., a gRNA target sequence including the flanking protospacer adjacent motif) is no longer present if the heterologous Factor IX nucleic acid is inserted into the cut site or target DNA sequence in the correct orientation but it is reformed if the heterologous Factor IX nucleic acid is inserted into the cut site or target DNA sequence in the opposite orientation. This can help ensure that the heterologous Factor IX nucleic acid is inserted in the correct orientation for expression. 
     Also described herein are bidirectional nucleic acid constructs that allow enhanced insertion and expression of a Factor IX gene. Briefly, various bidirectional constructs disclosed herein comprise at least two nucleic acid segments, wherein one segment (the first segment) comprises a coding sequence that encodes Factor IX (sometimes interchangeably referred to herein as “transgene”), while the other segment (the second segment) comprises a sequence wherein the complement of the sequence encodes Factor IX. 
     In one embodiment, a bidirectional construct comprise at least two nucleic acid segments in cis, wherein one segment (the first segment) comprises a coding sequence (sometimes interchangeably referred to herein as “transgene”), while the other segment (the second segment) comprises a sequence wherein the complement of the sequence encodes a transgene. The first transgene and the second transgene may be the same or different. The bidirectional constructs may comprise at least two nucleic acid segments in cis, wherein one segment (the first segment) comprises a coding sequence that encodes a heterologous gene in one orientation, while the other segment (the second segment) comprises a sequence wherein its complement encodes the heterologous gene in the other orientation. That is, the first segment is a complement of the second segment (not necessarily a perfect complement); the complement of the second segment is the reverse complement of the first segment (not necessarily a perfect reverse complement though both encode the same heterologous protein). A bidirectional construct may comprise a first coding sequence that encodes a heterologous gene linked to a splice acceptor and a second coding sequence wherein the complement encodes a heterologous gene in the other orientation, also linked to a splice acceptor. 
     When used in combination with a gene editing system (e.g., CRISPR/Cas system; zinc finger nuclease (ZFN) system; transcription activator-like effector nuclease (TALEN) system) as described herein, the bidirectionality of the nucleic acid constructs allows the construct to be inserted in either direction (is not limited to insertion in one direction) within a target insertion site, allowing the expression of Factor IX from either a) a coding sequence of one segment (e.g., the left segment encoding “Human F9” of  FIG. 1  upper left ssAAV construct), orb) a complement of the other segment (e.g., the complement of the right segment encoding “Human F9” indicated upside down in the upper left ssAAV construct  FIG. 1 ), thereby enhancing insertion and expression efficiency, as exemplified herein. Various known gene editing systems can be used in the practice of the present disclosure, including, e.g., CRISPR/Cas system; zinc finger nuclease (ZFN) system; transcription activator-like effector nuclease (TALEN) system. 
     The bidirectional constructs disclosed herein can be modified to include any suitable structural feature as needed for any particular use and/or that confers one or more desired function. In some embodiments, the bidirectional nucleic acid construct disclosed herein does not comprise a homology arm. In some embodiments, the bidirectional nucleic acid construct disclosed herein is a homology-independent donor construct. In some embodiments, owing in part to the bidirectional function of the nucleic acid construct, the bidirectional construct can be inserted into a genomic locus in either direction as described herein to allow for efficient insertion and/or expression of a polypeptide of interest (e.g., Factor IX). 
     In some embodiments, the bidirectional nucleic acid construct does not comprise a promoter that drives the expression of Factor IX. For example, the expression of Factor IX is driven by a promoter of the host cell (e.g., the endogenous albumin promoter when the transgene is integrated into a host cell&#39;s albumin locus). 
     In some embodiments, the bidirectional nucleic acid construct comprises a first segment comprising a coding sequence for Factor IX and a second segment comprising a reverse complement of a coding sequence of Factor IX. Thus, the coding sequence in the first segment is capable of expressing Factor IX, while the complement of the reverse complement in the second segment is also capable of expressing Factor IX. As used herein, “coding sequence” when referring to the second segment comprising a reverse complement sequence refers to the complementary (coding) strand of the second segment (i.e., the complement coding sequence of the reverse complement sequence in the second segment). 
     In some embodiments, the coding sequence that encodes Factor IX in the first segment is less than 100% complementary to the reverse complement of a coding sequence that also encodes Factor IX. That is, in some embodiments, the first segment comprises a coding sequence (1) for Factor IX, and the second segment is a reverse complement of a coding sequence (2) for Factor IX, wherein the coding sequence (1) is not identical to the coding sequence (2). For example, coding sequence (1) and/or coding sequence (2) that encodes for Factor IX can be codon optimized, such that coding sequence (1) and the reverse complement of coding sequence (2) possess less than 100% complementarity. In some embodiments, the coding sequence of the second segment encodes Factor IX using one or more alternative codons for one or more amino acids of the same (i.e., same amino acid sequence) Factor IX encoded by the coding sequence in the first segment. An “alternative codon” as used herein refers to variations in codon usage for a given amino acid, and may or may not be a preferred or optimized codon (codon optimized) for a given expression system. Preferred codon usage, or codons that are well-tolerated in a given system of expression is known in the art. 
     In some embodiments, the second segment comprises a reverse complement sequence that adopts different codon usage from that of the coding sequence of the first segment in order to reduce hairpin formation. Such a reverse complement forms base pairs with fewer than all nucleotides of the coding sequence in the first segment, yet it optionally encodes the same polypeptide. In such cases, the coding sequence, e.g. for Polypeptide A, of the first segment many be homologous to, but not identical to, the coding sequence, e.g. for Polypeptide A of the second half of the bidirectional construct. In some embodiments, the second segment comprises a reverse complement sequence that is not substantially complementary (e.g., not more than 70% complementary) to the coding sequence in the first segment. In some embodiments, the second segment comprises a reverse complement sequence that is highly complementary (e.g., at least 90% complementary) to the coding sequence in the first segment. In some embodiments, the second segment comprises a reverse complement sequence having at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, or about 99% complementarity to the coding sequence in the first segment. 
     In some embodiments, the second segment comprises a reverse complement sequence having 100% complementarity to the coding sequence in the first segment. That is, the sequence in the second segment is a perfect reverse complement of the coding sequence in the first segment. By way of example, the first segment comprises a hypothetical sequence 5′ CTGGACCGA 3′ (SEQ ID NO: 500) and the second segment comprises the reverse complement of SEQ ID NO: 1—i.e., 5′ TCGGTCCAG 3′ (SEQ ID NO: 502). 
     In some embodiments, the bidirectional nucleic acid construct comprises a first segment comprising a coding sequence for Factor IX (a first polypeptide) and a second segment comprising a reverse complement of a coding sequence of a (second) polypeptide. In some embodiments, the first and second segments each comprise a coding sequence that encodes the same polypeptide (e.g., Factor IX), as described above. In some embodiments, the first and second segments each comprise a coding sequence that encodes different polypeptides. For example, the first polypeptide is Factor IX and the second polypeptide is Polypeptide B. As a further example, the first polypeptide is Factor IX and the second polypeptide is a variant (e.g., a fragment, mutant, fusion) of Factor IX (e.g., having R338L mutation described herein). A coding sequence that encodes a polypeptide may optionally comprise one or more additional sequences, such as sequences encoding amino- or carboxy-terminal amino acid sequences such as a signal sequence, label sequence (e.g. HiBit), or heterologous functional sequence (e.g. nuclear localization sequence (NLS) or self-cleaving peptide) linked to the polypeptide. A coding sequence that encodes a polypeptide may optionally comprise sequences encoding one or more amino-terminal signal peptide sequences. Each of these additional sequences can be the same or different in the first segment and second segment of the construct. 
     In some embodiments, the bidirectional nucleic acid construct is linear. For example, the first and second segments are joined in a linear manner through a linker sequence. In some embodiments, the 5′ end of the second segment that comprises a reverse complement sequence is linked to the 3′ end of the first segment. In some embodiments, the 5′ end of the first segment is linked to the 3′ end of the second segment that comprises a reverse complement sequence. In some embodiments, the linker sequence is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 500, 1000, 1500, 2000 or more nucleotides in length. As would be appreciate by those of skill in the art, other structural elements in addition to, or instead of a linker sequence, can be inserted between the first and second segments. 
     The bidirectional constructs disclosed herein can be modified to include any suitable structural feature as needed for any particular use and/or that confers one or more desired function. In some embodiments, the bidirectional nucleic acid construct disclosed herein does not comprise a homology arm. In some embodiments, owing in part to the bidirectional function of the nucleic acid construct, the bidirectional construct can be inserted into a genomic locus in either direction (orientation) as described herein to allow for efficient insertion and/or expression of a polypeptide of interest (e.g., a heterologous Factor IX). 
     In some embodiments, one or both of the first and second segment comprises a polyadenylation tail sequence. Methods of designing a suitable polyadenylation tail sequence are well known in the art. 
     In some embodiments, one or both of the first and second segment comprises a polyadenylation tail sequence and/or a polyadenylation signal sequence downstream of an open reading frame. In some embodiments, the polyadenylation tail sequence is encoded, e.g., as a “poly-A” stretch, at the 3′ end of the first and/or second segment. In some embodiments, a polyadenylation tail sequence is provided co-transcriptionally as a result of a polyadenylation signal sequence that is encoded at or near the 3′ end of the first and/or second segment. In some embodiments, a poly-A tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines. In some embodiments, the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. Methods of designing a suitable polyadenylation tail sequence and/or polyadenylation signal sequence are well known in the art. Suitable splice acceptor sequences are disclosed and exemplified herein, including mouse albumin and human FIX splice acceptor sites. In some embodiments, the polyadenylation signal sequence AAUAAA (SEQ ID NO: 800) is commonly used in mammalian systems, although variants such as UAUAAA (SEQ ID NO: 801) or AU/GUAAA (SEQ ID NO: 802) have been identified. See, e.g., NJ Proudfoot, Genes &amp; Dev. 25(17):1770-82, 2011. In some embodiments, a polyA tail sequence is included. 
     In some embodiments, the constructs disclosed herein can be DNA or RNA, single-stranded, double-stranded, or partially single- and partially double-stranded. For example, the constructs can be single- or double-stranded DNA. In some embodiments, the nucleic acid can be modified (e.g., using nucleoside analogs), as described herein. 
     In some embodiments, the constructs disclosed herein comprise a splice acceptor site on either or both ends of the construct, e.g., 5′ of an open reading frame in the first and/or second segments, or 5′ of one or both transgene sequences. In some embodiments, the splice acceptor site comprises NAG. In further embodiments, the splice acceptor site consists of NAG. In some embodiments, the splice acceptor is an albumin splice acceptor, e.g., an albumin splice acceptor used in the splicing together of exons 1 and 2 of albumin. In some embodiments, the splice acceptor is derived from the human albumin gene. In some embodiments, the splice acceptor is derived from the mouse albumin gene. In some embodiments, the splice acceptor is a F9 (or “FIX”) splice acceptor, e.g., the F9 splice acceptor used in the splicing together of exons 1 and 2 of F9. In some embodiments, the splice acceptor is derived from the human F9 gene. In some embodiments, the splice acceptor is derived from the mouse F9 gene. Additional suitable splice acceptor sites useful in eukaryotes, including artificial splice acceptors are known and can be derived from the art. See, e.g., Shapiro, et al., 1987, Nucleic Acids Res., 15, 7155-7174, Burset, et al., 2001, Nucleic Acids Res., 29, 255-259. 
     In some embodiments, the bidirectional constructs disclosed herein can be modified on either or both ends to include one or more suitable structural features as needed, and/or to confer one or more functional benefit. For example, structural modifications can vary depending on the method(s) used to deliver the constructs disclosed herein to a host cell—e.g., use of viral vector delivery or packaging into lipid nanoparticles for delivery. Such modifications include, without limitation, e.g., terminal structures such as inverted terminal repeats (ITR), hairpin, loops, and other structures such as toroid. In some embodiments, the constructs disclosed herein comprise one, two, or three ITRs. In some embodiments, the constructs disclosed herein comprise no more than two ITRs. Various methods of structural modifications are known in the art. 
     In some embodiments, one or both ends of the construct can be protected (e.g., from exonucleolytic degradation) by methods known in the art. For example, one or more dideoxynucleotide residues are added to the 3′ terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987)  Proc. Natl. Acad. Sci. USA  84:4959-4963; Nehls et al. (1996)  Science  272:886-889. Additional methods for protecting the constructs from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. 
     In some embodiments, the constructs disclosed herein can be introduced into a cell as part of a vector having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. In some embodiments, the constructs can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome, polymer, or poloxamer, or can be delivered by viral vectors (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus). 
     In some embodiments, although not required for expression, the constructs disclosed herein may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding peptides, and/or polyadenylation signals. 
     In some embodiments, the constructs comprising a coding sequence for Factor IX may include one or more of the following modifications: codon optimization (e.g., to human codons) and/or addition of one or more glycosylation sites. See, e.g., McIntosh et al. (2013)  Blood  (17):3335-44. 
     D. Gene Editing System 
     Various known gene editing systems can be used for targeted insertion of the Factor IX gene in the practice of the present disclosure, including, e.g., CRISPR/Cas system; zinc finger nuclease (ZFN) system; transcription activator-like effector nuclease (TALEN) system. Generally, the gene editing systems involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick (e.g., a single strand break, or SSB) in a target DNA sequence. Cleavage or nicking can occur through the use of specific nucleases such as engineered ZFN, TALENs, or using the CRISPR/Cas system with an engineered guide RNA to guide specific cleavage or nicking of a target DNA sequence. Further, targeted nucleases are being developed based on the Argonaute system (e.g., from  T. thermophilus , known as ‘TtAgo’, see Swarts et al (2014)  Nature  507(7491): 258-261), which also may have the potential for uses in genome editing and gene therapy. 
     It will be appreciated that for methods that use the guide RNAs disclosed herein, the methods include the use of the CRISPR/Cas system (and any of the donor construct disclosed herein that comprises a sequence encoding Factor IX). It will also be appreciated that the present disclosure contemplates methods of targeted insertion and expression of Factor IX using the bidirectional constructs disclosed herein, which can be performed with or without the guide RNAs disclosed herein (e.g., using a ZFN system to cause a break in a target DNA sequence, creating a site for insertion of the bidirectional construct). 
     In some embodiments, a CRISPR/Cas system (e.g., a guide RNA and RNA-guided DNA binding agent) can be used to create a site of insertion at a desired locus within a host genome, at which site a donor construct (e.g., bidirectional construct) comprising a sequence encoding Factor IX disclosed herein can be inserted to express Factor IX. The Factor IX may be heterologous with respect to its insertion site or locus, for example a safe harbor locus from which Factor IX is not normally expressed, as described herein. Alternatively, in some embodiments, Factor IX may be non-heterologous with respect to its insertion site, for example, insertion of a wild type Factor IX into the endogenous locus to correct a defective Factor IX gene. The safe harbor may be within an albumin gene, such as a human albumin gene. The safe harbor may be within an albumin intron 1 region, e.g., human albumin intron 1. The safe harbor may be a human safe harbor, e.g., for a liver tissue or hepatocyte host cell. In some embodiments, a guide RNA described herein can be used according to the present methods with an RNA-guided DNA binding agent (e.g., Cas nuclease) to create a site of insertion, at which site a donor construct (e.g., bidirectional construct) comprising a sequence encoding Factor IX can be inserted to express Factor IX. The guide RNAs useful for targeted insertion of Factor IX into intron 1 of the human albumin locus are exemplified and described herein (see, e.g., Table 1). 
     Methods of using various RNA-guided DNA-binding agents, e.g., a nuclease, such as a Cas nuclease, e.g., Cas9, are also well known in the art. While the use of a bidirectional nucleic acid with a CRISPR/Cas system is exemplified herein, it will be appreciated that suitable variations to the system can also be used. It will be appreciated that, depending on the context, the RNA-guided DNA-binding agent can be provided as a nucleic acid (e.g., DNA or mRNA) or as a protein. In some embodiments, the present method can be practiced in a host cell that already comprises and/or expresses an RNA-guided DNA-binding agent. 
     In some embodiments, the RNA-guided DNA-binding agent, such as a Cas9 nuclease, has cleavase activity, which can also be referred to as double-strand endonuclease activity. In some embodiments, the RNA-guided DNA-binding agent, such as a Cas9 nuclease, has nickase activity, which can also be referred to as single-strand endonuclease activity. In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nuclease. Examples of Cas nucleases include those of the type II CRISPR systems of  S. pyogenes, S. aureus , and other prokaryotes (see, e.g., the list in the next paragraph), and variant or mutant (e.g., engineered, non-naturally occurring, naturally occurring, or or other variant) versions thereof. See, e.g., US2016/0312198 A1; US 2016/0312199 A1. 
     Non-limiting exemplary species that the Cas nuclease can be derived from include  Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus  sp.,  Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas  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 thermopropionicum, 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, Oscillatoria  sp.,  Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus  sp.,  Lachnospiraceae  bacterium ND2006, and  Acaryochloris marina.    
     In some embodiments, the Cas nuclease is the Cas9 nuclease from  Streptococcus pyogenes . In some embodiments, the Cas nuclease is the Cas9 nuclease from  Streptococcus thermophilus . In some embodiments, the Cas nuclease is the Cas9 nuclease from  Neisseria meningitidis . In some embodiments, the Cas nuclease is the Cas9 nuclease is from  Staphylococcus aureus . In some embodiments, the Cas nuclease is the Cpf1 nuclease from  Francisella novicida . In some embodiments, the Cas nuclease is the Cpf1 nuclease from Acidaminococcus sp. In some embodiments, the Cas nuclease is the Cpf1 nuclease from  Lachnospiraceae bacterium  ND2006. In further embodiments, the Cas nuclease is the Cpf1 nuclease from  Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens , or  Porphyromonas macacae . In certain embodiments, the Cas nuclease is a Cpf1 nuclease from an Acidaminococcus or Lachnospiraceae. 
     In some embodiments, the gRNA together with an RNA-guided DNA-binding agent is called a ribonucleoprotein complex (RNP). In some embodiments, the RNA-guided DNA-binding agent is a Cas nuclease. In some embodiments, the gRNA together with a Cas nuclease is called a Cas RNP. In some embodiments, the RNP comprises Type-I, Type-II, or Type-III components. In some embodiments, the Cas nuclease is the Cas9 protein from the Type-II CRISPR/Cas system. In some embodiment, the gRNA together with Cas9 is called a Cas9 RNP. 
     Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 protein comprises more than one RuvC domain and/or more than one HNH domain. In some embodiments, the Cas9 protein is a wild type Cas9. In each of the composition, use, and method embodiments, the Cas induces a double strand break in target DNA. 
     In some embodiments, chimeric Cas nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease such as FokI. In some embodiments, a Cas nuclease may be a modified nuclease. 
     In other embodiments, the Cas nuclease may be from a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a Type-III CRISPR/Cas system. In some embodiments, the Cas nuclease may have an RNA cleavage activity. 
     In some embodiments, the RNA-guided DNA-binding agent has single-strand nickase activity, i.e., can cut one DNA strand to produce a single-strand break, also known as a “nick.” In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nickase. A nickase is an enzyme that creates a nick in dsDNA, i.e., cuts one strand but not the other of the DNA double helix. In some embodiments, a Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which an endonucleolytic active site is inactivated, e.g., by one or more alterations (e.g., point mutations) in a catalytic domain. See, e.g., U.S. Pat. No. 8,889,356 for discussion of Cas nickases and exemplary catalytic domain alterations. In some embodiments, a Cas nickase such as a Cas9 nickase has an inactivated RuvC or HNH domain. 
     In some embodiments, the RNA-guided DNA-binding agent is modified to contain only one functional nuclease domain. For example, the agent protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase is used having a RuvC domain with reduced activity. In some embodiments, a nickase is used having an inactive RuvC domain. In some embodiments, a nickase is used having an HNH domain with reduced activity. In some embodiments, a nickase is used having an inactive HNH domain. 
     In some embodiments, a conserved amino acid within a Cas protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, a Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the  S. pyogenes  Cas9 protein). See, e.g., Zetsche et al. (2015)  Cell  October 22:163(3): 759-771. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the  S. pyogenes  Cas9 protein). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the  Francisella novicida  U112 Cpf1 (FnCpf1) sequence (UniProtKB-A0Q7Q2 (CPF1_FRATN)). 
     In some embodiments, a nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNAs direct the nickase to a target sequence and introduce a DSB by generating a nick on opposite strands of the target sequence (i.e., double nicking). In some embodiments, a nickase is used together with two separate guide RNAs targeting opposite strands of DNA to produce a double nick in the target DNA. In some embodiments, a nickase is used together with two separate guide RNAs that are selected to be in close proximity to produce a double nick in the target DNA. 
     In some embodiments, the RNA-guided DNA-binding agent comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide). 
     In some embodiments, the heterologous functional domain may facilitate transport of the RNA-guided DNA-binding agent into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA-binding agent may be fused with 1-10 NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with 1-5 NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with one NLS. Where one NLS is used, the NLS may be linked at the N-terminus or the C-terminus of the RNA-guided DNA-binding agent sequence. It may also be inserted within the RNA-guided DNA-binding agent sequence. In other embodiments, the RNA-guided DNA-binding agent may be fused with more than one NLS. In some embodiments, the RNA-guided DNA-binding agent may be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLSs. In certain circumstances, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the RNA-guided DNA-binding agent is fused to two SV40 NLS sequences linked at the carboxy terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLSs, one linked at the N-terminus and one at the C-terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with 3 NLSs. In some embodiments, the RNA-guided DNA-binding agent may be fused with no NLS. In some embodiments, the NLS may be a monopartite sequence, such as, e.g., the SV40 NLS, PKKKRKV (SEQ ID NO: 600) or PKKKRRV (SEQ ID NO: 601). In some embodiments, the NLS may be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 602). In a specific embodiment, a single PKKKRKV (SEQ ID NO: 600) NLS may be linked at the C-terminus of the RNA-guided DNA-binding agent. One or more linkers are optionally included at the fusion site. 
     III. Delivery Methods 
     The guide RNA, RNA-guided DNA binding agents (e.g., Cas nuclease), and nucleic acid constructs (e.g., bidirectional construct) disclosed herein can be delivered to a host cell or population of host cells or a subject, in vivo or ex vivo, using various known and suitable methods available in the art. The guide RNA, RNA-guided DNA binding agents, and nucleic acid constructs can be delivered individually or together in any combination, using the same or different delivery methods as appropriate. 
     Conventional viral and non-viral based gene delivery methods can be used to introduce the guide RNA disclosed herein as well as the RNA-guided DNA binding agent and donor construct in cells (e.g., mammalian cells) and target tissues. As further provided herein, non-viral vector delivery systems nucleic acids such as non-viral vectors, plasmid vectors, and, e.g naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome, lipid nanoparticle (LNP), or poloxamer. Viral vector delivery systems include DNA and RNA viruses. 
     Methods and compositions for non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, LNPs, polycation or lipid:nucleic acid conjugates, naked nucleic acid (e.g., naked DNA/RNA), artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. 
     Additional exemplary nucleic acid delivery systems include those provided by AmaxaBiosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Ma.) and Copernicus Therapeutics Inc., (see for example U.S. Pat. No. 6,008,336). Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™) The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known in the art, and as described herein. 
     Various delivery systems (e.g., vectors, liposomes, LNPs) containing the guide RNAs, RNA-guided DNA binding agent, and donor construct, singly or in combination, can also be administered to an organism for delivery to cells in vivo or administered to a cell or cell culture ex vivo. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood, fluid, or cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art. 
     In certain embodiments, the present disclosure provides DNA or RNA vectors encoding any of the compositions disclosed herein—e.g., a guide RNA comprising any one or more of the guide sequences described herein; or a construct (e.g., bidirectional construct) comprising a sequence encoding Factor IX. In some embodiments, the vector also comprises a sequence encoding an RNA-guided DNA binding agent. In certain embodiments, the invention comprises DNA or RNA vectors encoding any one or more of the compositions described herein, or in any combination. In some embodiments, the vectors further comprise, e.g., promoters, enhancers, and regulatory sequences. In some embodiments, the vector that comprises a bidirectional construct comprising a sequence that encodes Factor IX does not comprise a promoter that drives Factor IX expression. For example, the expression of the Factor IX polypeptide is driven by a promoter of the host cell (e.g., the endogenous albumin promoter when the transgene is integrated into a host cell&#39;s albumin locus). In some embodiments, the bidirectional nucleic acid construct includes a first segment and a second segment, each having a splice acceptor upstream of a transgene. In certain embodiments, the splice acceptor is compatible with the splice donor sequence of the host cell&#39;s safe harbor site, e.g. the splice donor of intron 1 of a human albumin gene. In some embodiments, the vector that comprises a guide RNA comprising any one or more of the guide sequences described herein also comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA and trRNA, as disclosed herein. 
     In some embodiments, the vector comprises a nucleotide sequence encoding a guide RNA described herein. In some embodiments, the vector comprises one copy of the guide RNA. In other embodiments, the vector comprises more than one copy of the guide RNA. In embodiments with more than one guide RNA, the guide RNAs may be non-identical such that they target different target sequences, or may be identical in that they target the same target sequence. In some embodiments where the vectors comprise more than one guide RNA, each guide RNA may have other different properties, such as activity or stability within a complex with an RNA-guided DNA nuclease, such as a Cas RNP complex. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or translational control sequence, such as a promoter, a 3′ UTR, or a 5′ UTR. In one embodiment, the promoter may be a tRNA promoter, e.g., tRNA Lys3 , or a tRNA chimera. See Mefferd et al.,  RNA.  2015 21:1683-9; Scherer et al.,  Nucleic Acids Res.  2007 35: 2620-2628. In some embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters include U6 and H1 promoters. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In other embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human H1 promoter. In embodiments with more than one guide RNA, the promoters used to drive expression may be the same or different. In some embodiments, the nucleotide encoding the crRNA of the guide RNA and the nucleotide encoding the trRNA of the guide RNA may be provided on the same vector. In some embodiments, the nucleotide encoding the crRNA and the nucleotide encoding the trRNA may be driven by the same promoter. In some embodiments, the crRNA and trRNA may be transcribed into a single transcript. For example, the crRNA and trRNA may be processed from the single transcript to form a double-molecule guide RNA. Alternatively, the crRNA and trRNA may be transcribed into a single-molecule guide RNA (sgRNA). In other embodiments, the crRNA and the trRNA may be driven by their corresponding promoters on the same vector. In yet other embodiments, the crRNA and the trRNA may be encoded by different vectors. 
     In some embodiments, the nucleotide sequence encoding the guide RNA may be located on the same vector comprising the nucleotide sequence encoding an RNA-guided DNA binding agent such as a Cas protein. In some embodiments, expression of the guide RNA and of the RNA-guided DNA binding agent such as a Cas protein may be driven by their own corresponding promoters. In some embodiments, expression of the guide RNA may be driven by the same promoter that drives expression of the RNA-guided DNA binding agent such as a Cas protein. In some embodiments, the guide RNA and the RNA-guided DNA binding agent such as a Cas protein transcript may be contained within a single transcript. For example, the guide RNA may be within an untranslated region (UTR) of the RNA-guided DNA binding agent such as a Cas protein transcript. In some embodiments, the guide RNA may be within the 5′ UTR of the transcript. In other embodiments, the guide RNA may be within the 3′ UTR of the transcript. In some embodiments, the intracellular half-life of the transcript may be reduced by containing the guide RNA within its 3′ UTR and thereby shortening the length of its 3′ UTR. In additional embodiments, the guide RNA may be within an intron of the transcript. In some embodiments, suitable splice sites may be added at the intron within which the guide RNA is located such that the guide RNA is properly spliced out of the transcript. In some embodiments, expression of the RNA-guided DNA binding agent such as a Cas protein and the guide RNA from the same vector in close temporal proximity may facilitate more efficient formation of the CRISPR RNP complex. 
     In some embodiments, the nucleotide sequence encoding the guide RNA and/or RNA-guided DNA binding agent may be located on the same vector comprising the construct that comprises a Factor IX gene. In some embodiments, proximity of the construct comprising the Factor IX gene and the guide RNA (and/or the RNA-guided DNA binding agent) on the same vector may facilitate more efficient insertion of the construct into a site of insertion created by the guide RNA/RNA-guided DNA binding agent. 
     In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a sgRNA and an mRNA encoding an RNA-guided DNA binding agent, which can be a Cas protein, such as Cas9 or Cpf1. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, and an mRNA encoding an RNA-guided DNA binding agent, which can be a Cas protein, such as, Cas9 or Cpf1. In one embodiment, the Cas9 is from  Streptococcus pyogenes  (i.e., Spy Cas9). In some embodiments, the nucleotide sequence encoding the crRNA, trRNA, or crRNA and trRNA (which may be a sgRNA) comprises or consists of a guide sequence flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR/Cas system. The nucleic acid comprising or consisting of the crRNA, trRNA, or crRNA and trRNA may further comprise a vector sequence wherein the vector sequence comprises or consists of nucleic acids that are not naturally found together with the crRNA, trRNA, or crRNA and trRNA. 
     In some embodiments, the crRNA and the trRNA are encoded by non-contiguous nucleic acids within one vector. In other embodiments, the crRNA and the trRNA may be encoded by a contiguous nucleic acid. In some embodiments, the crRNA and the trRNA are encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and the trRNA are encoded by the same strand of a single nucleic acid. 
     In some embodiments, the vector comprises a donor construct (e.g., the bidirectional nucleic acid construct) comprising a sequence that encodes Factor IX, as disclosed herein. In some embodiments, in addition to the donor construct (e.g., bidirectional nucleic acid construct) disclosed herein, the vector may further comprise nucleic acids that encode the guide RNAs described herein and/or nucleic acid encoding an RNA-guided DNA-binding agent (e.g., a Cas nuclease such as Cas9). In some embodiments, a nucleic acid encoding an RNA-guided DNA-binding agent are each or both on a separate vector from a vector that comprises the donor construct (e.g., bidirectional construct) disclosed herein. In any of the embodiments, the vector may include other sequences that include, but are not limited to, promoters, enhancers, regulatory sequences, as described herein. In some embodiments, the promoter does not drive the expression of Factor IX of the donor construct (e.g., bidirectional construct). In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA and trRNA. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a sgRNA and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas nuclease (e.g., Cas9). In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas nuclease, such as, Cas9. In some embodiments, the Cas9 is from  Streptococcus pyogenes  (i.e., Spy Cas9). In some embodiments, the nucleotide sequence encoding the crRNA, trRNA, or crRNA and trRNA (which may be a sgRNA) comprises or consists of a guide sequence flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR/Cas system. The nucleic acid comprising or consisting of the crRNA, trRNA, or crRNA and trRNA may further comprise a vector sequence wherein the vector sequence comprises or consists of nucleic acids that are not naturally found together with the crRNA, trRNA, or crRNA and trRNA. 
     In some embodiments, the vector may be circular. In other embodiments, the vector may be linear. In some embodiments, the vector may be enclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors. 
     In some embodiments, the vector may be a viral vector. In some embodiments, the viral vector may be genetically modified from its wild type counterpart. For example, the viral vector may comprise an insertion, deletion, or substitution of one or more nucleotides to facilitate cloning or such that one or more properties of the vector is changed. Such properties may include packaging capacity, transduction efficiency, immunogenicity, genome integration, replication, transcription, and translation. In some embodiments, a portion of the viral genome may be deleted such that the virus is capable of packaging exogenous sequences having a larger size. In some embodiments, the viral vector may have an enhanced transduction efficiency. In some embodiments, the immune response induced by the virus in a host may be reduced. In some embodiments, viral genes (such as, e.g., integrase) that promote integration of the viral sequence into a host genome may be mutated such that the virus becomes non-integrating. In some embodiments, the viral vector may be replication defective. In some embodiments, the viral vector may comprise exogenous transcriptional or translational control sequences to drive expression of coding sequences on the vector. In some embodiments, the virus may be helper-dependent. For example, the virus may need one or more helper virus to supply viral components (such as, e.g., viral proteins) required to amplify and package the vectors into viral particles. In such a case, one or more helper components, including one or more vectors encoding the viral components, may be introduced into a host cell or population of host cells along with the vector system described herein. In other embodiments, the virus may be helper-free. For example, the virus may be capable of amplifying and packaging the vectors without a helper virus. In some embodiments, the vector system described herein may also encode the viral components required for virus amplification and packaging. 
     Non-limiting exemplary viral vectors include adeno-associated virus (AAV) vector, lentivirus vectors, adenovirus vectors, helper dependent adenoviral vectors (HDAd), herpes simplex virus (HSV-1) vectors, bacteriophage T4, baculovirus vectors, and retrovirus vectors. In some embodiments, the viral vector may be an AAV vector. In other embodiments, the viral vector may a lentivirus vector. 
     In some embodiments, “AAV” refers all serotypes, subtypes, and naturally-occuring AAV as well as recombinant AAV. “AAV” may be used to refer to the virus itself or a derivative thereof. The term “AAV” includes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2/8, AAVrh10, AAVLK03, AV10, AAV11, AAV12, rh10, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, nonprimate AAV, and ovine AAV. The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. A “AAV vector” as used herein refers to an AAV vector comprising a heterologous sequence not of AAV origin (i.e., a nucleic acid sequence heterologous to AAV), typically comprising a sequence encoding a heterologous polypeptide of interest. The construct may comprise an AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2/8, AAVrh10, AAVLK03, AV10, AAV11, AAV12, rh10, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, nonprimate AAV, and ovine AAV capsid sequence. In general, the heterologous nucleic acid sequence (the transgene) is flanked by at least one, and generally by two, AAV inverted terminal repeat sequences (ITRs). An AAV vector may either be single-stranded (ssAAV) or self-complementary (scAAV). 
     In some embodiments, the lentivirus may be non-integrating. In some embodiments, the viral vector may be an adenovirus vector. In some embodiments, the adenovirus may be a high-cloning capacity or “gutless” adenovirus, where all coding viral regions apart from the 5′ and 3′ inverted terminal repeats (ITRs) and the packaging signal (‘I’) are deleted from the virus to increase its packaging capacity. In yet other embodiments, the viral vector may be an HSV-1 vector. In some embodiments, the HSV-1-based vector is helper dependent, and in other embodiments it is helper independent. For example, an amplicon vector that retains only the packaging sequence requires a helper virus with structural components for packaging, while a 30 kb-deleted HSV-1 vector that removes non-essential viral functions does not require helper virus. In additional embodiments, the viral vector may be bacteriophage T4. In some embodiments, the bacteriophage T4 may be able to package any linear or circular DNA or RNA molecules when the head of the virus is emptied. In further embodiments, the viral vector may be a baculovirus vector. In yet further embodiments, the viral vector may be a retrovirus vector. In embodiments using AAV or lentiviral vectors, which have smaller cloning capacity, it may be necessary to use more than one vector to deliver all the components of a vector system as disclosed herein. For example, one AAV vector may contain sequences encoding an RNA-guided DNA binding agent such as a Cas protein (e.g., Cas9), while a second AAV vector may contain one or more guide sequences. 
     In some embodiments, the vector system may be capable of driving expression of one or more nuclease components in a cell. In some embodiments, the bidirectional construct, optionally as part of a vector system, may comprise a promoter capable of driving expression of a coding sequence in a cell. In some embodiments, the vector does not comprise a promoter that drives expression of one or more coding sequences once it is integrated in a cell (e.g., uses the host cell&#39;s endogenous promoter such as when inserted at intron 1 of an albumin locus, as exempflied herein). In some embodiments, the cell may be a eukaryotic cell, such as, e.g., a yeast, plant, insect, or mammalian cell. In some embodiments, the eukaryotic cell may be a mammalian cell. In some embodiments, the eukaryotic cell may be a rodent cell. In some embodiments, the eukaryotic cell may be a human cell. Suitable promoters to drive expression in different types of cells are known in the art. In some embodiments, the promoter may be wild type. In other embodiments, the promoter may be modified for more efficient or efficacious expression. In yet other embodiments, the promoter may be truncated yet retain its function. For example, the promoter may have a normal size or a reduced size that is suitable for proper packaging of the vector into a virus. 
     In some embodiments, the vector may comprise a nucleotide sequence encoding an RNA-guided DNA binding agent such as a Cas protein (e.g., Cas9) described herein. In some embodiments, the nuclease encoded by the vector may be a Cas protein. In some embodiments, the vector system may comprise one copy of the nucleotide sequence encoding the nuclease. In other embodiments, the vector system may comprise more than one copy of the nucleotide sequence encoding the nuclease. In some embodiments, the nucleotide sequence encoding the nuclease may be operably linked to at least one transcriptional or translational control sequence. In some embodiments, the nucleotide sequence encoding the nuclease may be operably linked to at least one promoter. 
     In some embodiments, the vector may comprise any one or more of the constructs comprising a heterologous Factor IX gene described herein. In some embodiments, the Factor IX gene may be operably linked to at least one transcriptional or translational control sequence. In some embodiments, the Factor IX gene may be operably linked to at least one promoter. In some embodiments, the Factor IX gene is not linked to a promoter that drives the expression of the heterologous gene. 
     In some embodiments, the promoter may be constitutive, inducible, or tissue-specific. In some embodiments, the promoter may be a constitutive promoter. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-alpha (EF1a) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EF1a promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech). 
     In some embodiments, the promoter may be a tissue-specific promoter, e.g., a promoter specific for expression in the liver. 
     In some embodiments, the compositions comprise a vector system. In some embodiments, the vector system may comprise one single vector. In other embodiments, the vector system may comprise two vectors. In additional embodiments, the vector system may comprise three vectors. When different guide RNAs are used for multiplexing, or when multiple copies of the guide RNA are used, the vector system may comprise more than three vectors. 
     In some embodiments, the vector system may comprise inducible promoters to start expression only after it is delivered to a target cell. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech). 
     In additional embodiments, the vector system may comprise tissue-specific promoters to start expression only after it is delivered into a specific tissue. 
     The vector comprising: a guide RNA, RNA-binding DNA binding agent, or donor construct comprising a sequence encoding Factor IX, individually or in any combination, may be delivered by liposome, a nanoparticle, an exosome, or a microvesicle. The vector may also be delivered by a lipid nanoparticle (LNP). One or more guide RNA, RNA-binding DNA binding agent (e.g. mRNA), or donor construct comprising a sequence encoding a heterologous protein, individually or in any combination, may be delivered by liposome, a nanoparticle, an exosome, or a microvesicle. One or more guide RNA, RNA-binding DNA binding agent (e.g. mRNA), or donor construct comprising a sequence encoding a heterologous protein, individually or in any combination, may be delivered by LNP. 
     Lipid nanoparticles (LNPs) are a well-known means for delivery of nucleotide and protein cargo, and may be used for delivery of any of the guide RNAs, RNA-guided DNA binding agent, and/or donor construct (e.g., bidirectional construct) disclosed herein. In some embodiments, the LNPs deliver the compositions in the form of nucleic acid (e.g., DNA or mRNA), or protein (e.g., Cas nuclease), or nucleic acid together with protein, as appropriate. 
     In some embodiments, provided herein is a method for delivering any of the guide RNAs described herein and/or donor construct (e.g., bidirectional construct) disclosed herein, alone or in combination, to a host cell or a population of host cells or a subject, wherein any one or more of the components is associated with an LNP. In some embodiments, the method further comprises an RNA-guided DNA binding agent (e.g., Cas9 or a sequence encoding Cas9). 
     In some embodiments, provided herein is a composition comprising any of the guide RNAs described herein and/or donor construct (e.g., bidirectional construct) disclosed herein, alone or in combination, with an LNP. In some embodiments, the composition further comprises an RNA-guided DNA binding agent (e.g., Cas9 or a sequence encoding Cas9). 
     In some embodiments, the LNPs comprise cationic lipids. In some embodiments, the LNPs comprise (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. See, e.g., lipids of PCT/US2018/053559 (filed Sep. 28, 2018), WO/2017/173054, WO2015/095340, and WO2014/136086, as well as references provided therein. In some embodiments, the LNPs comprise molar ratios of a cationic lipid amine to RNA phosphate (N:P) of about 4.5, 5.0, 5.5, 6.0, or 6.5. In some embodiments, the term cationic and ionizable in the context of LNP lipids is interchangeable, e.g., wherein ionizable lipids are cationic depending on the pH. 
     In some embodiments, LNPs associated with the bidirectional construct disclosed herein are for use in preparing a medicament for treating a disease or disorder. The disease or disorder may be a Factor IX deficiency such as hemophilia B. 
     In some embodiments, any of the guide RNAs described herein, RNA-guided DNA binding agents, and/or donor construct (e.g., bidirectional construct) disclosed herein, alone or in combination, whether naked or as part of a vector, is formulated in or administered via a lipid nanoparticle; see e.g., WO/2017/173054 the contents of which are hereby incorporated by reference in their entirety. 
     In some embodiments, an LNP composition is encompassed comprising: an RNA component and a lipid component, wherein the lipid component comprises an amine lipid such as a biodegradable, ionizable lipid. In some instances, the lipid component comprises biodegradable, ionizable lipid, cholesterol, DSPC, and PEG-DMG. 
     It will be apparent that a guide RNA disclosed herein, an RNA-guided DNA binding agent (e.g., Cas nuclease or a nucleic acid encoding a Cas nuclease), and a donor construct (e.g., bidirectional construct) comprising a sequence encoding Factor IX can be delivered using the same or different systems. For example, the guide RNA, Cas nuclease, and construct can be carried by the same vector (e.g., AAV). Alternatively, the Cas nuclease (as a protein or mRNA) and/or gRNA can be carried by a plasmid or LNP, while the donor construct can be carried by a vector such as AAV. Furthermore, the different delivery systems can be administered by the same or different routes (e.g. by infusion; by injection, such as intramuscular injection, tail vein injection, or other intravenous injection; by intraperitoneal administration and/or intramuscular injection). 
     The different delivery systems can be delivered in vitro or in vivo simultaneously or in any sequential order. In some embodiments, the donor construct, guide RNA, and Cas nuclease can be delivered in vitro or in vivo simultaneously, e.g., in one vector, two vectors, individual vectors, one LNP, two LNPs, individual LNPs, or a combination thereof. In some embodiments, the donor construct can be delivered in vivo or in vitro, as a vector and/or associated with a LNP, prior to (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days) delivering the guide RNA and/or Cas nuclease, as a vector and/or associated with a LNP singly or together as a ribonucleoprotein (RNP). As a further example, the guide RNA and Cas nuclease, as a vector and/or associated with a LNP singly or together as a ribonucleoprotein (RNP), can be delivered in vivo or in vitro, prior to (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days) delivering the construct, as a vector and/or associated with a LNP. 
     In some embodiments, the present disclosure also provides pharmaceutical formulations for administering any of the guide RNAs disclosed herein. In some embodiments, the pharmaceutical formulation includes an RNA-guided DNA binding agent (e.g., Cas nuclease) and a donor construct comprising a coding sequence of a therapeutic heterologous gene, as disclosed herein. Pharmaceutical formulations suitable for delivery into a subject (e.g., human subject) are well known in the art. 
     IV. Methods of Use 
     The gRNAs, donor construct (e.g., bidirectional construct comprising a sequence encoding Factor IX), and RNA-guided DNA binding agents described herein are useful for introducing a Factor IX nucleic acid to a host cell or population of host cells, in vivo or in vitro. In some embodiments, the gRNAs, donor construct (e.g., bidirectional construct comprising a sequence encoding Factor IX), and RNA-guided DNA binding agents described herein are useful for expressing Factor IX in a host cell or population of host cells, or in a subject in need thereof. In some embodiments, the gRNAs, donor construct (e.g., bidirectional construct comprising a sequence encoding Factor IX), and RNA-guided DNA binding agents described herein are useful for treating hemophilia (e.g., hemophilia B) in a subject in need thereof. Administration of any one or more of the gRNAs, donor construct (e.g., bidirectional construct comprising a sequence encoding Factor IX), and RNA-guided DNA binding agents described herein will increase Factor IX protein levels and/or Factor IX activity levels, e.g. circulating, serum, or plasma levels. In some embodiments, the effectiveness of the treatment can be assessed by measuring serum or plasma Factor IX activity, wherein an increase in the subject&#39;s plasma level and/or activity of Factor IX indicates effectiveness of the treatment. In some embodiments, the effectiveness of the treatment can be assessed by measuring serum or plasma Factor IX protein and/or activity levels, wherein an increase in the subject&#39;s plasma level and/or activity of Factor IX indicates effectiveness of the treatment. In some embodiments, effectiveness of the treatment can be determined by assessing clotting function in an aPTT assay and/or thrombin generation in an TGA-EA assay. In some embodiments, effectiveness of the treatment can be determined by assessing the level of Factor IX, e.g., circulating Factor IX, can be measured by a coagulation and/or an immunologic assay, e.g., an sandwich immunoassay, ELISA (see, e.g., Example 13), MSD (see, e.g., Example 14). 
     In normal or healthy individuals, Factor IX activity and antigen levels vary between about 50 and 160% of normal pooled plasma which is about 3-5 μg/ml, based on its purification from adult human plasma Amiral et al., Clin. Chem. 30(9), 1512-16, 1984 at Table 2; see also Osterud et al., 1978. Individuals having less than 50% of normal plasma level of Factor IX activity and/or antigen levels are classified as having hemophilia. In particular, individuals with less than about 1% active factor are classified as having severe haemophilia, while those with about 1-5% active factor have moderate haemophilia. Individuals with mild haemophilia have between about 6-49% of normal levels of active clotting factor. In some embodiments, the level of circulating factor IX can be measured by a coagulation and/or an immunologic assay, which methods are well known in the art (e.g. Simioni et al, NEJM 2009, Adcock et al., Coagulation Handbook, Esoterix Laboratory Services, 2006). An immunologic method for detecting hFIX protein, and a method of functionally normalizing Factor IX activity of a hyperfunctional hFIX variant is found in Example 13. In some embodiments, Factor IX, e.g., circulating Factor IX, can be measured by a coagulation and/or an immunologic assay, e.g., an sandwich immunoassay, ELISA (see, e.g., Example 13), MSD (see, e.g., Example 14). 
     Accordingly, in some embodiments, the compositions and methods disclosed herein are useful for increasing plasma levels of Factor IX or Factor IX activity levels in a subject having hemophilia to about 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, of normal level. 
     In some embodiments, the compositions and methods disclosed herein are useful for increasing Factor IX activity and/or levels, for example increasing circulating FIX protein levels to about 0.05, 0.1, 0.2, 0.5, 1, 2, 3, or 4 μg/ml. FIX protein levels may reach about 150 μg/ml, or more. In some embodiments, the compositions and methods disclosed herein are useful for increasing Factor IX protein levels to about 4 μg/ml. In some embodiments, the compositions and methods disclosed herein are useful for increasing Factor IX protein levels to about 4 μg/ml to about 5 μg/ml, about 4 μg/ml to 6 μg/ml, about 4 μg/ml to 8 μg/ml, about 4 μg/ml to about 10 μg/ml, or more. In some embodiments, the compositions and methods disclosed herein are useful for increasing Factor IX protein levels to about 0.1 μg/ml to about 10 μg/ml, about 1 μg/ml to about 10 μg/ml, about 0.1 μg/ml to about 6 μg/ml, about 1 μg/ml to about 6 μg/ml, about 2 μg/ml to about 5 μg/ml, or about 3 μg/ml to about 5 μg/ml. For example, the compositions and methods disclosed herein are useful for increasing plasma levels of Factor IX in a subject having hemophilia to about 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 μg/ml, or more. 
     In some embodiments, the compositions and methods disclosed herein are useful for increasing plasma levels of Factor IX activity and/or levels in a subject having hemophilia by about 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%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, as compared to the subject&#39;s plasma level and/or activity of Factor IX before administration. 
     In some embodiments, the compositions and methods disclosed herein are useful for increasing Factor IX protein and/or Factor IX activity in a host cell or population of host cells by about 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%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more as compared to a Factor IX level and/or activity before administration to the host cell or population of host cells, e.g. a normal level. In some embodiments, the cell is a liver cell or a population of liver cells. In some embodiments, the liver cell is hepatocyte or the population of liver cells are hepatocytes. 
     In some embodiments, the method comprises administering a guide RNA and an RNA-guided DNA binding agent (such as an mRNA encoding a Cas9 nuclease) in an LNP. In further embodiments, the method comprises administering an AAV nucleic acid construct encoding a Factor IX protein, such as an bidirectional FIX construct. CRISPR/Cas9 LNP, comprising guide RNA and an mRNA encoding a Cas9, can be administered intravenously. AAV FIX donor construct can be administered intravenously. Exemplary dosing of CRISPR/Cas9 LNP includes about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, or 10 mpk (RNA). The units mg/kg and mpk are being used interchangably herein. Exemplary dosing of AAV comprising a nucleic acid encoding a FIX protein includes an MOI of about 10 11 , 10 12 , 10 13 , and 10 14  vg/kg, optionally the MOI may be about 1×10 13  to 1×10 14  vg/kg. 
     In some embodiments, the method comprises expressing a therapeutically effective amount of the Factor IX protein. In some embodiments, the method comprises achieving a therapeutically effective level of circulating Factor IX coagulation activity in an individual. In particular embodiments, the method comprises achieving Factor IX activity of at least about 5% to about 50% of normal. The method may comprise achieving Factor IX activity of at least about 50% to about 150% of normal. In certain embodiments, the method comprises achieving an increase in Factor IX activity over the patient&#39;s baseline Factor IX activity of at least about 1% to about 50% of normal Factor IX activity, or at least about 5% to about 50% of normal Factor IX activity, or at least about 50% to about 150% of normal Factor IX activity. 
     In some embodiments, the method further comprises achieving a durable effect, e.g. at least 1 month, 2 months, 6 months, 1 year, or 2 year effect. In some embodiments, the method further comprises achieving the therapeutic effect in a durable and sustained manner, e.g. at least 1 month, 2 months, 6 months, 1 year, or 2 year effect. In some embodiments, the level of circulating Factor IX activity and/or level is stable for at least 1 month, 2 months, 6 months, 1 year, or more. In some embodiments a steady-state activity and/or level of FIX protein is achieved by at least 7 days, at least 14 days, or at least 28 days. In additional embodiments, the method comprises maintaining Factor IX activity and/or levels after a single dose for at least 1, 2, 4, or 6 months, or at least 1, 2, 3, 4, or 5 years. 
     In additional embodiments involving insertion into the albumin locus, the individual&#39;s circulating albumin levels are normal. The method may comprise maintaining the individual&#39;s circulating albumin levels within ±5%, ±10%, ±15%, ±20%, or ±50% of normal circulating albumin levels. In certain embodiments, the individual&#39;s albumin levels are unchanged as compared to the albumin levels of untreated individuals by at least week 4, week 8, week 12, or week 20. In certain embodiments, the individual&#39;s albumin levels transiently drop then return to normal levels. In particular, the methods may comprise detecting no significant alterations in levels of plasma albumin. 
     In some embodiments, the invention comprises a method or use of modifying (e.g., creating a double strand break in) an albumin gene, such as a human albumin gene, comprising, administering or delivering to a host cell or population of host cells any one or more of the gRNAs, donor construct (e.g., bidirectional construct comprising a sequence encoding Factor IX), and RNA-guided DNA binding agents (e.g., Cas nuclease) described herein. In some embodiments, the invention comprises a method or use of modifying (e.g., creating a double strand break in) an albumin intron 1 region, such as a human albumin intron 1, comprising, administering or delivering to a host cell or population of host cells any one or more of the gRNAs, donor construct (e.g., bidirectional construct comprising a sequence encoding Factor IX), and RNA-guided DNA binding agents (e.g., Cas nuclease) described herein. In some embodiments, the invention comprises a method or use of modifying (e.g., creating a double strand break in) a human safe harbor, such as liver tissue or hepatocyte host cell, comprising, administering or delivering to a host cell or population of host cells any one or more of the gRNAs, donor construct (e.g., bidirectional construct comprising a sequence encoding Factor IX), and RNA-guided DNA binding agents (e.g., Cas nuclease) described herein. Insertion within a safe harbor locus, such as an albumin locus, allows overexpression of the Factor IX gene without significant deleterious effects on the host cell or cell population, such as hepatocytes or liver cells. In some embodiments, the invention comprises a method or use of modifying (e.g., creating a double strand break in) intron 1 of a human albumin locus comprising, administering or delivering to a host cell or population of host cells any one or more of the gRNAs, donor construct (e.g., bidirectional construct comprising a sequence encoding Factor IX), and RNA-guided DNA binding agents (e.g., Cas nuclease) described herein. In some embodiments, the guide RNA comprises a guide sequence that contains at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides that bind within intron 1 of a human albumin locus (SEQ ID NO: 1). In some embodiments, the guide RNA comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs:2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NO: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 34, 40, 45, 51, 60, 61, 63, 64, 65, 66, 72, 77, 83, 92, 93, 95, 96, and 97. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is selected from the group consisting of SEQ ID NOs: 34-97. In some embodiments, the guide RNA comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 34-37, 42-49, 53-59, 61-69, 74-81, 85-91, and 93-97. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is selected from the group consisting of SEQ ID NOs: 34-37, 42-49, 53-59, 61-69, 74-81, 85-91, and 93-97. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo. In some embodiments, the donor construct is a bidirectional construct that comprises a sequence encoding Factor IX. In some embodiments, the host cell is a liver cell, such as. In additional embodiments, the liver cell is a hepatocyte. 
     In some embodiments, the invention comprises a method or use of introducing a Factor IX nucleic acid to a host cell or population of host cells comprising, administering or delivering any one or more of the gRNAs, donor construct (e.g., bidirectional construct comprising a sequence encoding Factor IX), and RNA-guided DNA binding agents (e.g., Cas nuclease) described herein. In some embodiments, the guide RNA comprises a guide sequence that contains at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides that are capable of binding to a region within intron 1 of human albumin locus (SEQ ID NO: 1). In some embodiments, the guide RNA comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NO: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 34, 40, 45, 51, 60, 61, 63, 64, 65, 66, 72, 77, 83, 92, 93, 95, 96, and 97. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is selected from the group consisting of SEQ ID NOs: 34-97. In some embodiments, the guide RNA comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 34-37, 42-49, 53-59, 61-69, 74-81, 85-91, and 93-97. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is selected from the group consisting of SEQ ID NOs: 34-37, 42-49, 53-59, 61-69, 74-81, 85-91, and 93-97. In some embodiments, the method is in vitro. In some embodiments, the method is in vivo. In some embodiments, the donor construct is a bidirectional construct that comprises a sequence encoding Factor IX. In some embodiments, the host cell is a liver cell, or the population of host cells are liver cells, such as hepatocyte. 
     In some embodiments, the invention comprises a method or use of expressing Factor IX in a host cell or a population of host cells comprising, administering or delivering any one or more of the gRNAs, donor construct (e.g., bidirectional construct comprising a sequence encoding Factor IX), and RNA-guided DNA binding agents (e.g., Cas nuclease) described herein. In some embodiments, the guide RNA comprises a guide sequence that contains at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides that are capable of binding to a region within intron 1 of human albumin locus (SEQ ID NO: 1). In some embodiments, the guide RNA comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs:2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NO: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 34, 40, 45, 51, 60, 61, 63, 64, 65, 66, 72, 77, 83, 92, 93, 95, 96, and 97. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is selected from the group consisting of SEQ ID NOs: 34-97. In some embodiments, the guide RNA comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 34-37, 42-49, 53-59, 61-69, 74-81, 85-91, and 93-97. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is selected from the group consisting of SEQ ID NOs: 34-37, 42-49, 53-59, 61-69, 74-81, 85-91, and 93-97. In some embodiments, the method is in vitro. In some embodiments, the method is in vivo. In some embodiments, the donor construct is a bidirectional construct that comprises a sequence encoding Factor IX. In some embodiments, the host cell is a liver cell, or the population of host cells are liver cells, such as hepatocyte. 
     In some embodiments, the invention comprises a method or use of treating hemophilia (e.g., hemophilia B) comprising, administering or delivering any one or more of the gRNAs, donor construct (e.g., bidirectional construct comprising a sequence encoding Factor IX), and RNA-guided DNA binding agents (e.g., Cas nuclease) described herein to a subject in need thereof. In some embodiments, the guide RNA comprises a guide sequence that contains at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides that are capable of binding to a region within intron 1 of human albumin locus (SEQ ID NO: 1). In some embodiments, the guide RNA comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NO: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 13, 19, 28, 29, 31, 32, 33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 34, 40, 45, 51, 60, 61, 63, 64, 65, 66, 72, 77, 83, 92, 93, 95, 96, and 97. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is selected from the group consisting of SEQ ID NOs: 34-97. In some embodiments, the guide RNA comprises at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNA comprises a sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence selected from the group consisting of SEQ ID NOs: 34-37, 42-49, 53-59, 61-69, 74-81, 85-91, and 93-97. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 95%, 90%, 85%, 80%, or 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 2-5, 10-17, 21-27, and 29-33. In some embodiments, the guide RNAs disclosed herein comprise a guide sequence that is selected from the group consisting of SEQ ID NOs: 34-37, 42-49, 53-59, 61-69, 74-81, 85-91, and 93-97. In some embodiments, the donor construct is a bidirectional construct that comprises a sequence encoding Factor IX. In some embodiments, the host cell is a liver cell, or the population of host cells are liver cells, such as hepatocytes. 
     As described herein, the donor construct (e.g., bidirectional construct) comprising a sequence encoding Factor IX, guide RNA, and RNA-guided DNA binding agent can be delivered using any suitable delivery system and method known in the art. The compositions can be delivered in vitro or in vivo simultaneously or in any sequential order. In some embodiments, the donor construct, guide RNA, and Cas nuclease can be delivered in vitro or in vivo simultaneously, e.g., in one vector, two vectors, individual vectors, one LNP, two LNPs, individual LNPs, or a combination thereof. In some embodiments, the donor construct can be delivered in vivo or in vitro, as a vector and/or associated with a LNP, prior to (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days) delivering the guide RNA and/or Cas nuclease, as a vector and/or associated with a LNP singly or together as a ribonucleoprotein (RNP). In some embodiments, the donor construct can be delivered in multiple administrations, e.g., every day, every two days, every three days, every four days, every week, every two weeks, every three weeks, or every four weeks. In some embodiments, the donor construct can be delivered at one-week intervals, e.g., at week 1, week 2, and week 3, etc. As a further example, the guide RNA and Cas nuclease, as a vector and/or associated with a LNP singly or together as a ribonucleoprotein (RNP), can be delivered in vivo or in vitro, prior to (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days) delivering the construct, as a vector and/or associated with a LNP. In some embodiments, the albumin guide RNA can be delivered in multiple administrations, e.g., every day, every two days, every three days, every four days, every week, every two weeks, every three weeks, or every four weeks. In some embodiments, the the albumin guide RNA can be delivered at one-week intervals, e.g., at week 1, week 2, and week 3, etc. In some embodiments, the Cas nuclease can be delivered in multiple administrations, e.g., can be delivered every day, every two days, every three days, every four days, every week, every two weeks, every three weeks, or every four weeks. In some embodiments, the Cas nuclease can be delivered at one-week intervals, e.g., at week 1, week 2, and week 3, etc. In some embodiments, the guide RNA and Cas nuclease are associated with an LNP and delivered to the host cell or the population of host cells prior to delivering the Factor IX donor construct. 
     In some embodiments, the donor construct comprises a sequence encoding Factor IX, wherein the Factor IX sequence is wild type Factor IX, e.g., SEQ ID NO: 700. In some embodiments, the donor construct comprises a sequence encoding Factor IX, wherein the Factor IX sequence is wild type Factor IX, e.g., SEQ ID NO: 701. In some embodiments, the sequence encodes a variant of Factor IX. For example, the variant possesses increased coagulation activity than wild type Factor IX. For example, the variant Factor IX comprises one or more mutations, such as an amino acid substitution in position R338 (e.g., R338L), relative to SEQ ID NO: 701. In some embodiments, the sequence encodes a Factor IX variant that is 80%, 85%, 90%, 93%, 95%, 97%, 99% identical to SEQ ID NO: 700, SEQ ID NO: 701, or SEQ ID NO: 702, having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 100%, or more, activity as compared to wild type Factor IX. In some embodiments, the sequence encodes a fragment of Factor IX, wherein the fragment possesses at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 100%, or more, activity as compared to wild type Factor IX. 
     In one example, the Factor IX protein can comprise amino acid substitutions at positions L6 and V181. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6 and K265. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6 and I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6 and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181 and K265. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181 and an I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181 and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions K265 and I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions K265 and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions 1383 and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, V181, and K265. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, V181, and I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, V181, and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, K265, and I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, K265, and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, 1383, and E186. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181, K265, and I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181, K265, and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181, I383, and E186. In another example, the Factor IX protein can comprise amino acid substitutions at positions K265, I383, and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, V181, K265, and I383. In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, V181, I383, and E185. 
     In another example, the Factor IX protein can comprise amino acid substitutions at positions L6, K265, I383, and E185. In another example, the Factor IX protein can comprise amino acid substitutions at positions V181, K265, I383, and E185. 
     In some embodiments, the donor construct comprises a sequence encoding a Factor IX variant, wherein the Factor IX variant activates coagulation in the absence of its cofactor, Factor VIII (expression results in therapeutically relevant FVIII mimetic activity). Such Factor IX variants can further maintain the activity of wild type Factor IX. For example, such a Factor IX variant can comprise an amino acid substation at position L6, V181, K265, I383, E185, or a combination thereof relative to wild type Factor IX (e.g., relative to SEQ ID NO: 701). For example, such a Factor IX variant can comprise an L6F mutation, a V181I mutation, a K265A mutation, an I383V mutation, an E185D mutation, or a combination thereof relative to wild type Factor IX (e.g., relative to SEQ ID NO: 701). 
     In a specific example, the Factor IX protein can comprise amino acid substitutions at positions V181, K265, and I383. In another specific example, the Factor IX protein can comprise amino acid substitutions at positions V181, K265, I383, and E185. In another specific example, the Factor IX protein can comprise amino acid substitutions at positions L6, V181, K265, and I383. 
     In one example, the Factor IX protein can comprise an L6F mutation and a V181I mutation. In another example, the Factor IX protein can comprise an L6F mutation and a K265A mutation. In another example, the Factor IX protein can comprise an L6F mutation and an I383V mutation. In another example, the Factor IX protein can comprise an L6F mutation and an E185D mutation. In another example, the Factor IX protein can comprise a V181I mutation and a K265A mutation. In another example, the Factor IX protein can comprise a V181I mutation and an I383V mutation. In another example, the Factor IX protein can comprise a V181I mutation and an E185D mutation. In another example, the Factor IX protein can comprise a K265A mutation and an I383V mutation. In another example, the Factor IX protein can comprise a K265A mutation and an E185D mutation. In another example, the Factor IX protein can comprise an I383V mutation and an E185D mutation. In another example, the Factor IX protein can comprise an L6F mutation, a V181I mutation, and a K265A mutation. In another example, the Factor IX protein can comprise an L6F mutation, a V181I mutation, and an I383V mutation. In another example, the Factor IX protein can comprise an L6F mutation, a V181I mutation and an E185D mutation. In another example, the Factor IX protein can comprise an L6F mutation, a K265A mutation, and an I383V mutation. In another example, the Factor IX protein can comprise an L6F mutation, a K265A mutation, and an E185D mutation. In another example, the Factor IX protein can comprise an L6F mutation, an I383V mutation, and an E186D mutation. In another example, the Factor IX protein can comprise a V181I mutation, a K265A mutation, and an I383V mutation. In another example, the Factor IX protein can comprise a V181I mutation, a K265A mutation, and an E185D mutation. In another example, the Factor IX protein can comprise a V181I mutation, an I383V mutation, and an E186D mutation. In another example, the Factor IX protein can comprise a K265A mutation, an I383V mutation, and an E185D mutation. In another example, the Factor IX protein can comprise an L6F mutation, a V181I mutation, a K265A mutation, and an I383V mutation. In another example, the Factor IX protein can comprise an L6F mutation, a V181I mutation, an I383V mutation, and an E185D mutation. In another example, the Factor IX protein can comprise an L6F mutation, a K265A mutation, an I383V mutation, and an E185D mutation. In another example, the Factor IX protein can comprise a V181I mutation, a K265A mutation, an I383V mutation, and an E185D mutation. 
     In a specific example, the Factor IX protein can comprise a V181I mutation, an K265A mutation, and an I383V mutation. In another specific example, the Factor IX protein can comprise a V181I mutation, a K265A mutation, an I383V mutation, and an E185D mutation. In some embodiments, the Factor IX protein is at least 80%, 85%, 90%, 93%, 95%, 97%, 99% identical to SEQ ID NO: 700, having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 100%, or more, activity as compared to wild type Factor IX. In certain embodiments, the Factor IX variant is at least 80%, 85%, 90%, 93%, 95%, 97%, 99% identical to SEQ ID NO: 700, having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 100%, or more, activity as compared to wild type Factor IX and comprises a V181I mutation, a K265A mutation, an I383V mutation, and/or an E185D mutation. In another specific example, the Factor IX protein can comprise an L6F mutation, a V181I mutation, a K265A mutation, and an I383V mutation. In certain embodiments, the Factor IX variant is at least 80%, 85%, 90%, 93%, 95%, 97%, 99% identical to SEQ ID NO: 700, having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99%, 100%, or more, activity as compared to wild type Factor IX and comprises a V181I mutation, a K265A mutation, and an I383V mutation. 
     In some embodiments, the host cell is a liver cell, or the population of host cells are liver cells. In some embodiments, the host cell is, or the population of host cells are, any suitable non-dividing cell. As used herein, a “non-dividing cell” refers to cells that are terminally differentiated and do not divide, as well as quiescent cells that do not divide but retains the ability to re-enter cell division and proliferation. Liver cells, for example, retain the ability to divide (e.g., when injured or resected), but do not typically divide. During mitotic cell division, homologous recombination is a mechanism by which the genome is protected and double-stranded breaks are repaired. In some embodiments, a “non-dividing” cell refers to a cell in which homologous recombination (HR) is not the primary mechanism by which double-stranded DNA breaks are repaired in the cell, e.g., as compared to a control dividing cell. In some embodiments, a “non-dividing” cell refers to a cell in which non-homologous end joining (NHEJ) is the primary mechanism by which double-stranded DNA breaks are repaired in the cell, e.g., as compared to a control dividing cell. Non-dividing cell types have been described in the literature, e.g. by active NHEJ double-stranded DNA break repair mechanisms. See, e.g. Iyama, DNA Repair (Amst.) 2013, 12(8): 620-636. In some embodiments, the host cell includes, but is not limited to, a liver cell, a muscle cell, or a neuronal cell. In some embodiments, the host cell, or the population of host cells are, is a hepatocyte, such as a mouse, cyno, or human hepatocyte. In some embodiments, the host cell is a myocyte, such as a mouse, cyno, or human myocyte. In some embodiments, provided herein is a host cell composition comprising any one or more guide RNA described herein, alone or in combination with an RNA-guided DNA binding protein. In some embodiments, provided herein is a host cell composition comprising any one or more of the vectors described herein. 
     In some embodiments, the donor construct (e.g., bidirectional construct) is administered in a nucleic acid vector, such as an AAV vector, e.g., AAV8. In some embodiments, the donor construct does not comprise a homology arm. 
     In some embodiments, the subject is a mammal. In some embodiments, the subject is human. In some embodiments, the subject is cow, pig, monkey, sheep, dog, cat, fish, or poultry. 
     In some embodiments, the donor construct (e.g., bidirectional construct) comprising a sequence encoding Factor IX, guide RNA, and RNA-guided DNA binding agent are administered intravenously. In some embodiments, the donor construct (e.g., bidirectional construct) comprising a sequence encoding Factor IX, guide RNA, and RNA-guided DNA binding agent are administered into the hepatic circulation. 
     In some embodiments, a single administration of a donor construct (e.g., bidirectional construct) comprising a sequence encoding Factor IX, guide RNA, and RNA-guided DNA binding agent is sufficient to increase expression of Factor IX to a desirable level. In other embodiments, more than one administration of a composition comprising a donor construct (e.g., bidirectional construct) comprising a sequence encoding Factor IX, guide RNA, and RNA-guided DNA binding agent may be beneficial to maximize therapeutic effects. 
     In some embodiments, the present disclosure includes combination therapies comprising any one or more of the gRNAs, donor construct (e.g., bidirectional construct comprising a sequence encoding Factor IX), and RNA-guided DNA binding agents (e.g., Cas nuclease) described herein together with an additional therapy suitable for treating hemophilia, as described above. For example, the methods of the present disclosure can be combined with the use of other hemostatic agents, blood factors, and medications. For example, the subject may be administered a therapeutically effective amount of one or more factors selected from the group consisting of factor XI, factor XII, prekallikrein, high molecular weight kininogen (HMWK), factor V, factor VII, factor VIII, factor X, factor XIII, factor II, factor VIIa, and von Willebrands factor. 
     In some embodiments, treatment may further comprise administering a procoagulant, such as an activator of the intrinsic coagulation pathway, including factor Xa, factor IXa, factorXIa, factor XIIa, and VIIIa, prekallekrein, and high-molecular weight kininogen; or an activator of the extrinsic coagulation pathway, including tissue factor, factor VIIa, factor Va, and factor Xa. 
     This description and exemplary embodiments should not be taken as limiting. For the purposes of this specification and appended embodiments, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and embodiments, are to be understood as being modified in all instances by the term “about,” to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached embodiments are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the embodiments, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. 
     
       
         
           
               
               
            
               
                 Human Factor IX Protein Sequence (SEQ ID NO: 700) NCBI Ref: NP_000124: 
                   
               
               
                 MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNL 
               
               
                   
               
               
                 ERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCP 
               
               
                   
               
               
                 FGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGR 
               
               
                   
               
               
                 VSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPW 
               
               
                   
               
               
                 QVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRII 
               
               
                   
               
               
                 PHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVF 
               
               
                   
               
               
                 HKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVE 
               
               
                   
               
               
                 GTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT 
               
               
                   
               
               
                 Human Factor IX Nucleotide Sequence (SEQ ID NO: 706) NCBI Ref: NM_000133): 
               
            
           
           
               
               
               
            
               
                    1 
                 accactttca caatctgcta gcaaaggtta tgcagcgcgt gaacatgatc atggcagaat 
                   
               
               
                   
               
               
                   61 
                 caccaggcct catcaccatc tgccttttag gatatctact cagtgctgaa tgtacagttt 
               
               
                   
               
               
                  121 
                 ttcttgatca tgaaaacgcc aacaaaattc tgaatcggcc aaagaggtat aattcaggta 
               
               
                   
               
               
                  181 
                 aattggaaga gtttgttcaa gggaaccttg agagagaatg tatggaagaa aagtgtagtt 
               
               
                   
               
               
                  241 
                 ttgaagaagc acgagaagtt tttgaaaaca ctgaaagaac aactgaattt tggaagcagt 
               
               
                   
               
               
                  301 
                 atgttgatgg agatcagtgt gagtccaatc catgtttaaa tggcggcagt tgcaaggatg 
               
               
                   
               
               
                  361 
                 acattaattc ctatgaatgt tggtgtccct ttggatttga aggaaagaac tgtgaattag 
               
               
                   
               
               
                  421 
                 atgtaacatg taacattaag aatggcagat gcgagcagtt ttgtaaaaat agtgctgata 
               
               
                   
               
               
                  481 
                 acaaggtggt ttgctcctgt actgagggat atcgacttgc agaaaaccag aagtcctgtg 
               
               
                   
               
               
                  541 
                 aaccagcagt gccatttcca tgtggaagag tttctgtttc acaaacttct aagctcaccc 
               
               
                   
               
               
                  601 
                 gtgctgagac tgtttttcct gatgtggact atgtaaattc tactgaagct gaaaccattt 
               
               
                   
               
               
                  661 
                 tggataacat cactcaaagc acccaatcat ttaatgactt cactcgggtt gttggtggag 
               
               
                   
               
               
                  721 
                 aagatgccaa accaggtcaa ttcccttggc aggttgtttt gaatggtaaa gttgatgcat 
               
               
                   
               
               
                  781 
                 tctgtggagg ctctatcgtt aatgaaaaat ggattgtaac tgctgcccac tgtgttgaaa 
               
               
                   
               
               
                  841 
                 ctggtgttaa aattacagtt gtcgcaggtg aacataatat tgaggagaca gaacatacag 
               
               
                   
               
               
                  901 
                 agcaaaagcg aaatgtgatt cgaattattc ctcaccacaa ctacaatgca gctattaata 
               
               
                   
               
               
                  961 
                 agtacaacca tgacattgcc cttctggaac tggacgaacc cttagtgcta aacagctacg 
               
               
                   
               
               
                 1021 
                 ttacacctat ttgcattgct gacaaggaat acacgaacat cttcctcaaa tttggatctg 
               
               
                   
               
               
                 1081 
                 gctatgtaag tggctgggga agagtcttcc acaaagggag atcagcttta gttcttcagt 
               
               
                   
               
               
                 1141 
                 accttagagt tccacttgtt gaccgagcca catgtcttcg atctacaaag ttcaccatct 
               
               
                   
               
               
                 1201 
                 ataacaacat gttctgtgct ggcttccatg aaggaggtag agattcatgt caaggagata 
               
               
                   
               
               
                 1261 
                 gtgggggacc ccatgttact gaagtggaag ggaccagttt cttaactgga attattagct 
               
               
                   
               
               
                 1321 
                 ggggtgaaga gtgtgcaatg aaaggcaaat atggaatata taccaaggta tcccggtatg 
               
               
                   
               
               
                 1381 
                 tcaactggat taaggaaaaa acaaagctca cttaatgaaa gatggatttc caaggttaat 
               
               
                   
               
               
                 1441 
                 tcattggaat tgaaaattaa cagggcctct cactaactaa tcactttccc atcttttgtt 
               
               
                   
               
               
                 1501 
                 agatttgaat atatacattc tatgatcatt gctttttctc tttacagggg agaatttcat 
               
               
                   
               
               
                 1561 
                 attttacctg agcaaattga ttagaaaatg gaaccactag aggaatataa tgtgttagga 
               
               
                   
               
               
                 1621 
                 aattacagtc atttctaagg gcccagccct tgacaaaatt gtgaagttaa attctccact 
               
               
                   
               
               
                 1681 
                 ctgtccatca gatactatgg ttctccacta tggcaactaa ctcactcaat tttccctcct 
               
               
                   
               
               
                 1741 
                 tagcagcatt ccatcttccc gatcttcttt gcttctccaa ccaaaacatc aatgtttatt 
               
               
                   
               
               
                 1801 
                 agttctgtat acagtacagg atctttggtc tactctatca caaggccagt accacactca 
               
               
                   
               
               
                 1861 
                 tgaagaaaga acacaggagt agctgagagg ctaaaactca tcaaaaacac tactcctttt 
               
               
                   
               
               
                 1921 
                 cctctaccct attcctcaat cttttacctt ttccaaatcc caatccccaa atcagttttt 
               
               
                   
               
               
                 1981 
                 ctctttctta ctccctctct cccttttacc ctccatggtc gttaaaggag agatggggag 
               
               
                   
               
               
                 2041 
                 catcattctg ttatacttct gtacacagtt atacatgtct atcaaaccca gacttgcttc 
               
               
                   
               
               
                 2101 
                 cgtagtggag acttgctttt cagaacatag ggatgaagta aggtgcctga aaagtttggg 
               
               
                   
               
               
                 2161 
                 ggaaaagttt ctttcagaga gttaagttat tttatatata taatatatat ataaaatata 
               
               
                   
               
               
                 2221 
                 taatatacaa tataaatata tagtgtgtgt gtatgcgtgt gtgtagacac acacgcatac 
               
               
                   
               
               
                 2281 
                 acacatataa tggaagcaat aagccattct aagagcttgt atggttatgg aggtctgact 
               
               
                   
               
               
                 2341 
                 aggcatgatt tcacgaaggc aagattggca tatcattgta actaaaaaag ctgacattga 
               
               
                   
               
               
                 2401 
                 cccagacata ttgtactctt tctaaaaata ataataataa tgctaacaga aagaagagaa 
               
               
                   
               
               
                 2461 
                 ccgttcgttt gcaatctaca gctagtagag actttgagga agaattcaac agtgtgtctt 
               
               
                   
               
               
                 2521 
                 cagcagtgtt cagagccaag caagaagttg aagttgccta gaccagagga cataagtatc 
               
               
                   
               
               
                 2581 
                 atgtctcctt taactagcat accccgaagt ggagaagggt gcagcaggct caaaggcata 
               
               
                   
               
               
                 2641 
                 agtcattcca atcagccaac taagttgtcc ttttctggtt tcgtgttcac catggaacat 
               
               
                   
               
               
                 2701 
                 tttgattata gttaatcctt ctatcttgaa tcttctagag agttgctgac caactgacgt 
               
               
                   
               
               
                 2761 
                 atgtttccct ttgtgaatta ataaactggt gttctggttc at 
               
               
                   
               
            
           
           
               
               
            
               
                 Human Factor IX polypeptide (SEQ ID No: 701) 
                   
               
               
                 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYE 
               
               
                   
               
               
                 CWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQT 
               
               
                   
               
               
                 SKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSI 
               
               
                   
               
               
                 VNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLV 
               
               
                   
               
               
                 LNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFC 
               
               
                   
               
               
                 AGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT 
               
            
           
         
       
     
     EXAMPLES 
     The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way. 
     Example 1—Materials and Methods 
     Cloning and Plasmid Preparation 
     A bidirectional insertion construct flanked by ITRs was synthesized and cloned into pUC57-Kan by a commercial vendor. The resulting construct (P00147) was used as the parental cloning vector for other vectors. The other insertion constructs (without ITRs) were also commercially synthesized and cloned into pUC57. Purified plasmid was digested with BglII restriction enzyme (New England BioLabs, cat # R0144S), and the insertion constructs were cloned into the parental vector. Plasmid was propagated in Stbl3™ Chemically Competent  E. coli  (Thermo Fisher, Cat # C737303). 
     AAV Production 
     Triple transfection in HEK293 cells was used to package genomes with constructs of interest for AAV8 and AAV-DJ production and resulting vectors were purified from both lysed cells and culture media through iodixanol gradient ultracentrifugation method (See, e.g., Lock et al., Hum Gene Ther. 2010 October; 21(10):1259-71). The plasmids used in the triple transfection that contained the genome with constructs of interest are referenced in the Examples by a “PXXXX” number, see also e.g., Table 9. Isolated AAV was dialyzed in storage buffer (PBS with 0.001% Pluronic F68). AAV titer was determined by qPCR using primers/probe located within the ITR region. 
     In Vitro Transcription (“IVT”) of Nuclease mRNA 
     Capped and polyadenylated  Streptococcus pyogenes  (“Spy”) Cas9 mRNA containing N1-methyl pseudo-U was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. Generally, plasmid DNA containing a T7 promoter and a 100 nt poly (A/T) region was linearized by incubating at 37° C. with XbaI to complete digestion followed by heat inactivation of XbaI at 65° C. The linearized plasmid was purified from enzyme and buffer salts. The IVT reaction to generate Cas9 modified mRNA was incubated at 37° C. for 4 hours in the following conditions: 50 ng/μL linearized plasmid; 2 mM each of GTP, ATP, CTP, and N1-methyl pseudo-UTP (Trilink); 10 mM ARCA (Trilink); 5 U/μL T7 RNA polymerase (NEB); 1 U/μL Murine Rnase inhibitor (NEB); 0.004 U/μL Inorganic  E. coli  pyrophosphatase (NEB); and 1× reaction buffer. TURBO Dnase (ThermoFisher) was added to a final concentration of 0.01 U/μL, and the reaction was incubated for an additional 30 minutes to remove the DNA template. The Cas9 mRNA was purified using a MegaClear Transcription Clean-up kit according to the manufacturer&#39;s protocol (ThermoFisher). Alternatively, the Cas9 mRNA was purified using LiCl precipitation, ammonium acetate precipitation, and sodium acetate precipitation or using a LiCl precipitation method followed by further purification by tangential flow filtration. The transcript concentration was determined by measuring the light absorbance at 260 nm (Nanodrop), and the transcript was analyzed by capillary electrophoresis by Bioanlayzer (Agilent). 
     Cas9 mRNAs below comprise Cas9 ORF SEQ ID NO: 703 or SEQ ID NO: 704 or a sequence of Table 24 of PCT/US2019/053423 (which is hereby incorporated by reference). 
     Lipid Formulations for Delivery of Cas9 mRNA and gRNA 
     Cas9 mRNA and gRNA were delivered to cells and animals utilizing lipid formulations comprising ionizable lipid ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate), cholesterol, DSPC, and PEG2k-DMG. 
     For experiments utilizing pre-mixed lipid formulations (referred to herein as “lipid packets”), the components were reconstituted in 100% ethanol at a molar ratio of ionizable lipid:cholesterol:DSPC:PEG2k-DMG of 50:38:9:3, prior to being mixed with RNA cargos (e.g., Cas9 mRNA and gRNA) at a lipid amine to RNA phosphate (N:P) molar ratio of about 6.0, as further described herein. 
     For experiments utilizing the components formulated as lipid nanoparticles (LNPs), the components were dissolved in 100% ethanol at various molar ratios. The RNA cargos (e.g., Cas9 mRNA and gRNA) were dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL. 
     For the experiments described in Example 2, the LNPs were formed by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems NanoAssemblr™ Benchtop Instrument, according to the manufacturer&#39;s protocol. A 2:1 ratio of aqueous to organic solvent was maintained during mixing using differential flow rates. After mixing, the LNPs were collected, diluted in water (approximately 1:1 v/v), held for 1 hour at room temperature, and further diluted with water (approximately 1:1 v/v) before final buffer exchange. The final buffer exchange into 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS) was completed with PD-10 desalting columns (GE). If required, formulations were concentrated by centrifugation with Amicon 100 kDa centrifugal filters (Millipore). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNP was stored at −80° C. until further use. The LNPs were formulated at a molar ratio of ionizable lipid:cholesterol:DSPC:PEG2k-DMG of 45:44:9:2, with a lipid amine to RNA phosphate (N:P) molar ratio of about 4.5, and a ratio of gRNA to mRNA of 1:1 by weight. 
     For the experiments described in other examples, the LNPs were prepared using a cross-flow technique utilizing impinging jet mixing of the lipid in ethanol with two volumes of RNA solutions and one volume of water. The lipid in ethanol was mixed through a mixing cross with the two volumes of RNA solution. A fourth stream of water was mixed with the outlet stream of the cross through an inline tee (See WO2016010840  FIG. 2 .). The LNPs were held for 1 hour at room temperature, and further diluted with water (approximately 1:1 v/v). Diluted LNPs were concentrated using tangential flow filtration on a flat sheet cartridge (Sartorius, 100 kD MWCO) and then buffer exchanged by diafiltration into 50 mM Tris, 45 mM NaCl, 5% (w/v) sucrose, pH 7.5 (TSS). Alternatively, the final buffer exchange into TSS was completed with PD-10 desalting columns (GE). If required, formulations were concentrated by centrifugation with Amicon 100 kDa centrifugal filters (Millipore). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNP was stored at 4° C. or −80° C. until further use. The LNPs were formulated at a molar ratio of ionizable lipid:cholesterol:DSPC:PEG2k-DMG of 50:38:9:3, with a lipid amine to RNA phosphate (N:P) molar ratio of about 6.0, and a ratio of gRNA to mRNA of 1:1 by weight. 
     Cell Culture and In Vitro Delivery of Cas9 mRNA, gRNA, and Insertion Constructs 
     Hepa1-6 Cells 
     Hepa 1-6 cells were plated at density of 10,000 cells/well in 96-well plates. 24 hours later, cells were treated with LNP and AAV. Before treatment the media was aspirated off from the wells. LNP was diluted to 4 ng/ul in DMEM+10% FBS media and further diluted to 2 ng/ul in 10% FBS (in DMEM) and incubated at 37° C. for 10 min (at a final concentration of 5% FBS). Target MOI of AAV was 1e6, diluted in DMEM+10% FBS media. 50 μl of the above diluted LNP at 2 ng/ul was added to the cells (delivering a total of 100 ng of RNA cargo) followed by 50 μl of AAV. The treatment of LNP and AAV were minutes apart. Total volume of media in cells was 100 μl. After 72 hours post-treatment and 30 days post-treatment, supernatant from these treated cells were collected for human FIX ELISA analysis as described below. 
     Primary Hepatocytes 
     Primary mouse hepatocytes (PMH), primary cyno hepatocytes (PCH) and primary human hepatocytes (PHH) were thawed and resuspended in hepatocyte thawing medium with supplements (ThermoFisher) followed by centrifugation. The supernatant was discarded, and the pelleted cells resuspended in hepatocyte plating medium plus supplement pack (ThermoFisher). Cells were counted and plated on Bio-coat collagen I coated 96-well plates at a density of 33,000 cells/well for PHH and 50,000 cells/well for PCH and 15,000 cells/well for PMH. Plated cells were allowed to settle and adhere for 5 hours in a tissue culture incubator at 37° C. and 5% CO 2  atmosphere. After incubation cells were checked for monolayer formation and were washed thrice with hepatocyte maintenance prior and incubated at 37° C. 
     For experiments utilizing lipid packet delivery, Cas9 mRNA and gRNA were each separately diluted to 2 mg/ml in maintenance media and 2.9 μl of each were added to wells (in a 96-well Eppendorf plate) containing 12.5 μl of 50 mM sodium citrate, 200 mM sodium chloride at pH 5 and 6.9 μl of water. 12.5 μl of lipid packet formulation was then added, followed by 12.5 μl of water and 150 μl of TSS. Each well was diluted to 20 ng/μ1 (with respect to total RNA content) using hepatocyte maintenance media, and then diluted to 10 ng/μ1 (with respect to total RNA content) with 6% fresh mouse serum. Media was aspirated from the cells prior to transfection and 40 μl of the lipid packet/RNA mixtures were added to the cells, followed by addition of AAV (diluted in maintenance media) at an MOI of 1e5. Media was collected 72 hours post-treatment for analysis and cells were harvested for further analysis, as described herein. 
     Luciferase Assays 
     For experiments involving NanoLuc detection in cell media, one volume of Nano-Glo® Luciferase Assay Substrate was combined with 50 volumes of Nano-Glo® Luciferase Assay Buffer. The assay was run on a Promega Glomax runner at an integration time of 0.5 sec using 1:10 dilution of samples (50 μl of reagent+40 μl water+10 μl cell media). 
     For experiments involving detection of the HiBit tag in cell media, LgBiT Protein and Nano-GloR HiBiT Extracellular Substrate were diluted 1:100 and 1:50, respectively, in room temperature Nano-GloR HiBiT Extracellular Buffer. The assay was run on a Promega Glomax runner at an integration time of 1.0 sec using 1:10 dilution of samples (50 μl of reagent+40 μl water+10 μl cell media). 
     In Vivo Delivery of LNP and/or AAV 
     Mice were dosed with AAV, LNP, both AAV and LNP, or vehicle (PBS+0.001% Pluronic for AAV vehicle, TSS for LNP vehicle) via the lateral tail vein. AAV were administered in a volume of 0.1 mL per animal with amounts (vector genomes/mouse, “vg/ms”) as described herein. LNPs were diluted in TSS and administered at amounts as indicated herein, at about 5 μl/gram body weight. Typically, mice were injected first with AAV and then with LNP, if applicable. At various times points post-treatment, serum and/or liver tissue was collected for certain analyses as described further below. 
     Human Factor IX (hFIX) ELISA Analysis 
     For in vitro studies, total human Factor IX levels secreted in cell media were determined using a Human Factor IX ELISA Kit (Abcam, Cat # ab188393) according to manufacturer&#39;s protocol. Secreted hFIX levels were quantitated off a standard curve using 4 parameter logistic fit and expressed as ng/ml of media. 
     For in vivo studies, blood was collected and the serum or plasma was isolated as indicated. The total human Factor IX levels were determined using a Human Factor IX ELISA Kit (Abcam, Cat # ab188393) according to manufacturer&#39;s protocol. Serum or plasma hFIX levels were quantitated off a standard curve using 4 parameter logistic fit and expressed as μg/mL of serum. 
     Next-Generation Sequencing (“NGS”) and Analysis for On-Target Cleavage Efficiency 
     Deep sequencing was utilized to identify the presence of insertions and deletions introduced by gene editing, e.g., within intron 1 of albumin. PCR primers were designed around the target site and the genomic area of interest was amplified. Primer sequence design was done as is standard in the field. 
     Additional PCR was performed according to the manufacturer&#39;s protocols (Illumina) to add chemistry for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument. The reads were aligned to the reference genome after eliminating those having low quality scores. The resulting files containing the reads were mapped to the reference genome (BAM files), where reads that overlapped the target region of interest were selected and the number of wild type reads versus the number of reads which contain an insertion or deletion (“indel”) was calculated. 
     The editing percentage (e.g., the “editing efficiency” or “percent editing”) is defined as the total number of sequence reads with insertions or deletions (“indels”) over the total number of sequence reads, including wild type. 
     In Situ Hybridization Analysis 
     BaseScope (ACDbio, Newark, Calif.) is a specialized RNA in situ hybridization technology that can provide specific detection of exon junctions, e.g., in a hybrid mRNA transcript that contains an insertion transgene (hFIX) and coding sequence from the site of insertion (e.g. exon 1 of albumin). BaseScope was used to measure the percentage of liver cells expressing the hybrid mRNA. 
     To detect the hybrid mRNA, two probes against the hybrid mRNAs that may arise following insertion of a bidirectional construct were designed by ACDbio (Newark, Calif.). One of the probes was designed to detect a hybrid mRNA resulting from insertion of the construct in one orientation, while the other probe was designed to detect a hybrid mRNA resulting from insertion of the construct in the other orientation. Livers from different groups of mice were collected and fresh-frozen sectioned. The BaseScope assay, using a single probe or pooled probes was performed according to the manufacture&#39;s protocol. Slides were scanned and analyzed by the HALO software. The background (saline treated group) of this assay was 0.58%. 
     Example 2—In Vitro Testing of Insertion Templates with and without Homology Arms 
     In this Example, Hepa1-6 cells were cultured and treated with AAV harboring insertion templates of various forms (e.g., having either a single-stranded genome (“ssAAV”) or a self-complementary genome (“scAAV”)), in the presence or absence of LNP delivering Cas9 mRNA and G000551 e.g., as described in Example 1 (n=3). The AAV and LNP were prepared as described in Example 1. Following treatment, the media was collected for human Factor IX levels as described in Example 1. 
     Hepa1-6 cells are an immortalized mouse liver cell line that continues to divide in culture. As shown in  FIG. 2  (72 hour post-treatment time point), only the vector (scAAV derived from plasmid P00204) comprising 200 bp homology arms resulted in detectable expression of hFIX. Use of the AAV vectors derived from P00123 (scAAV lacking homology arms) and P00147 (ssAAV bidirectional construct lacking homology arms) did not result in any detectable expression of hFIX in this experiment. The cells were kept in culture and these results were confirmed when re-assayed at 30 days post-treatment (data not shown). 
     Example 3—In Vivo Testing of Insertion Templates with and without Homology Arms 
     In this Example, mice were treated with AAV derived from the same plasmids (P00123, P00204, and P00147) as tested in vitro in Example 2. The dosing materials were prepared and dosed as described in Example 1. C57Bl/6 mice were dosed (n=5 for each group) with 3e11 vector genomes each (vg/ms) followed by LNP comprising G000551 (“G551”) at a dose of 4 mg/kg (with respect to total RNA cargo content). Four weeks post dose, the animals were euthanized and liver tissue and sera were collected for editing and hFIX expression, respectively. 
     As shown in  FIG. 3A  and Table 12, liver editing levels of ˜60% were detected in each group of animals treated with LNP comprising gRNA targeting intron 1 of murine albumin. However, despite robust and consistent levels of editing in each treatment group, animals receiving the bi-directional vector without homology arms (ssAAV vector derived from P00147) in combination with LNP treatment resulted in the highest level of hFIX expression in serum ( FIG. 3B  and Table 13). 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 % Indel 
               
            
           
           
               
               
               
            
               
                 Template 
                 Average Indel (%) 
                 St. Dev Indel (%) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 scAAV Blunt (P00123) 
                 66.72 
                 4.09 
               
               
                 ssAAV Blunt (P00147) 
                 68.10 
                 2.27 
               
               
                 ssAAV HR (P00204) 
                 70.16 
                 3.68 
               
               
                 LNP only 
                 68.24 
                 6.47 
               
               
                 Vehicle 
                 0.28 
                 0.08 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Factor IX Levels 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Average Factor IX 
                 St.Dev Factor IX 
               
               
                   
                 Template 
                 (ug/mL) 
                 (ug/mL) 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 scAAV Blunt (P00123) 
                 0.75 
                 0.28 
               
               
                   
                 ssAAV Blunt (P00147) 
                 2.92 
                 1.04 
               
               
                   
                 ssAAV HR (P00204) 
                 0.96 
                 0.35 
               
               
                   
                 LNP only 
                 0 
                 0 
               
               
                   
                 Vehicle 
                 0 
                 0 
               
               
                   
                   
               
            
           
         
       
     
     Example 4—In Vivo Testing of ssAAV Insertion Templates with and without Homology Arms 
     The experiment described in this example examined the effect of incorporating homology arms into ssAAV vectors in vivo. 
     The dosing materials used in this experiment were prepared and dosed as described in Example 1. C57Bl/6 mice were dosed (n=5 for each group) with 3e11 vg/ms followed by LNP comprising G000666 (“G666”) or G000551 (“G551”) at a dose of 0.5 mg/kg (with respect to total RNA cargo content). Four weeks post dose, the animals sera was collected for hFIX expression. 
     As shown in  FIG. 4A  and Table 14, use of the ssAAV vectors with asymmetrical homology arms (300/600 bp arms, 300/2000 bp arms, and 300/1500 bp arms for vectors derived from plasmids P00350, P00356, and P00362, respectively) for insertion into the site targeted by G551 resulted in levels of circulating hFIX that were below the lower limit of detection for the assay. However, use of the ssAAV vector (derived from P00147) without homology arms and having two hFIX open reading frames (ORF) in a bidirectional orientation resulted in detectable levels of circulating hFIX in each animal. 
     Similarly, use of the ssAAV vectors with asymmetrical homology arms (500 bp arms and 800 bp arms for vectors derived from plasmids P00353 and P00354, respectively) for insertion into the site targeted by G666 resulted in lower but detectable levels, as compared to use of the bidirectional vector without homology arms (derived from P00147) (see  FIG. 4B  and Table 15). 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Serum hFIX Levels 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Average Serum FIX 
                 St.Dev Serum FIX 
               
               
                   
                 AAV 
                 (ug/mL) 
                 (ug/mL) 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 P00147 
                 5.13 
                 1.31 
               
               
                   
                 P00350 
                 −0.22 
                 0.08 
               
               
                   
                 P00356 
                 −0.23 
                 0.04 
               
               
                   
                 P00362 
                 −0.09 
                 0.16 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Serum hFIX Levels 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Average Serum FIX 
                 St.Dev Serum FIX 
               
               
                   
                 AAV 
                 (ug/mL) 
                 (ug/mL) 
               
               
                   
                   
               
               
                   
                 P00147 
                 7.72 
                 4.67 
               
               
                   
                 P00353 
                 0.20 
                 0.23 
               
               
                   
                 P00354 
                 0.46 
                 0.26 
               
               
                   
                   
               
            
           
         
       
     
     Example 5—In Vitro Screening of Bidirectional Constructs Across Target Sites in Primary Mouse Hepatocytes 
     Having demonstrated that bidirectional constructs lacking homology arms outperformed vectors with other configurations, the experiment described in this Example examined the effects of altering the splice acceptors used to form the hybrid transcript between hFIX and exon 1 of albumin and altering the gRNAs for targeting CRISPR/Cas9-mediated insertion. These varied bidirectional constructs were tested across a panel of target sites utilizing 20 different gRNAs targeting intron 1 of murine albumin in primary mouse hepatocytes (PMH). 
     The ssAAV and lipid packet delivery materials tested in this Example were prepared and delivered to PMH as described in Example 1, with the AAV at an MOI of 1e5. Following treatment, isolated genomic DNA and cell media was collected for editing and transgene expression analysis, respectively. Each of the vectors comprised a reporter that can be measured through luciferase-based fluorescence detection as described in Example 1, plotted in  FIG. 5C  as relative luciferase units (“RLU”). The vectors comprised a HiBit peptide fused at the 3′ ends of the hFIX ORF, which allows for sensitive detection of relative expression. Schematics of each vector tested are provided in  FIG. 5A . The gRNAs tested are shown in  FIGS. 5B and 5C , using a shortened number for those listed in Table 5 (e.g., where the leading zeros are omitted, for example where “G551” corresponds to “G000551” in Table 5). 
     As shown in  FIG. 5B  and Table 16, consistent but varied levels of editing were detected for each of the treatment groups across each combination tested. Tansgene expression using various combinations of template and guide RNA is shown in  FIG. 5C  and Table 17. As shown in  FIG. 5D , a significant level of indel formation did not necessarily result in more efficient expression of the transgenes. Using P00411- and P00418-derived templates, the R 2  values were 0.54 and 0.37, respectively, when guides with less than 10% editing are not included. The mouse albumin splice acceptor and human FIX splice acceptor each resulted in effective transgene expression. 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 % Indel 
               
            
           
           
               
               
               
               
            
               
                   
                 P00411 
                 P00418 
                 P00415 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Average 
                 St. Dev 
                 Average 
                 St. Dev 
                 Average 
                 St. Dev 
               
               
                 Guide ID 
                 Indel (%) 
                 Indel (%) 
                 Indel (%) 
                 Indel (%) 
                 Indel (%) 
                 Indel (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 G000551 
                 67.4 
                 1.42 
                 70.67 
                 2.29 
                 66.73 
                 4.90 
               
               
                 G000552 
                 90.93 
                 0.15 
                 91.10 
                 2.43 
                 90.37 
                 1.01 
               
               
                 G000553 
                 77.80 
                 3.83 
                 77.47 
                 1.87 
                 80.50 
                 0.85 
               
               
                 G000554 
                 72.37 
                 6.49 
                 70.53 
                 3.16 
                 70.60 
                 2.91 
               
               
                 G000555 
                 35.37 
                 2.63 
                 35.77 
                 9.34 
                 40.47 
                 4.75 
               
               
                 G000666 
                 62.47 
                 3.87 
                 50.90 
                 19.41 
                 65.90 
                 3.99 
               
               
                 G000667 
                 30.57 
                 2.73 
                 25.30 
                 3.67 
                 31.67 
                 2.29 
               
               
                 G000668 
                 63.60 
                 2.02 
                 66.65 
                 4.60 
                 68.30 
                 4.90 
               
               
                 G000669 
                 19.10 
                 2.51 
                 19.33 
                 1.53 
                 18.70 
                 1.25 
               
               
                 G000670 
                 47.80 
                 3.27 
                 49.10 
                 4.42 
                 51.97 
                 2.06 
               
               
                 G011722 
                 4.20 
                 0.72 
                 4.27 
                 1.20 
                 4.20 
                 0.26 
               
               
                 G011723 
                 5.63 
                 1.27 
                 6.07 
                 0.15 
                 5.93 
                 0.15 
               
               
                 G011724 
                 6.10 
                 1.28 
                 8.50 
                 2.69 
                 7.13 
                 1.27 
               
               
                 G011725 
                 1.93 
                 0.29 
                 2.60 
                 0.79 
                 2.53 
                 0.65 
               
               
                 G011726 
                 10.73 
                 1.46 
                 11.70 
                 0.50 
                 12.43 
                 1.33 
               
               
                 G011727 
                 14.20 
                 1.56 
                 14.80 
                 2.36 
                 16.20 
                 2.69 
               
               
                 G011728 
                 10.55 
                 1.20 
                 13.65 
                 0.92 
                 15.50 
                 1.56 
               
               
                 G011729 
                 5.00 
                 0.10 
                 5.63 
                 0.25 
                 6.00 
                 1.01 
               
               
                 G011730 
                 7.83 
                 0.97 
                 9.13 
                 0.59 
                 7.33 
                 0.59 
               
               
                 G011731 
                 23.70 
                 0.66 
                 25.27 
                 1.21 
                 24.87 
                 1.01 
               
               
                 AAV Only 
                 0.15 
                 0.07 
                 0.05 
                 0.07 
                 0.10 
                 0.00 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Luciferase Levels 
               
            
           
           
               
               
               
               
            
               
                   
                 P00411 
                 P00418 
                 P00415 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Average 
                 St. Dev 
                 Average 
                 St. Dev 
                 Average 
                 St. Dev 
               
               
                   
                 Luciferase 
                 Luciferase 
                 Luciferase 
                 Luciferase 
                 Luciferase 
                 Luciferase 
               
               
                 Guide ID 
                 (RLU) 
                 (RLU) 
                 (RLU) 
                 (RLU) 
                 (RLU) 
                 (RLU) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 G000551 
                 58000.00 
                 4331.28 
                 41800.00 
                 2165.64 
                 78633.33 
                 20274.70 
               
               
                 G000552 
                 95700.00 
                 10573.08 
                 80866.67 
                 27911.35 
                 205333.33 
                 30664.86 
               
               
                 G000553 
                 205333.33 
                 52993.71 
                 177333.33 
                 32929.22 
                 471666.67 
                 134001.00 
               
               
                 G000554 
                 125333.33 
                 55949.38 
                 91933.33 
                 19194.10 
                 232666.67 
                 67002.49 
               
               
                 G000555 
                 59933.33 
                 11566.04 
                 77733.33 
                 11061.80 
                 155666.67 
                 15947.83 
               
               
                 G000666 
                 88500.00 
                 28735.87 
                 93266.67 
                 30861.19 
                 313000.00 
                 15394.80 
               
               
                 G000667 
                 75333.33 
                 22653.11 
                 68966.67 
                 27222.11 
                 153000.00 
                 30805.84 
               
               
                 G000668 
                 164000.00 
                 56320.51 
                 133400.00 
                 65111.29 
                 429000.00 
                 120751.80 
               
               
                 G000669 
                 28933.33 
                 11636.29 
                 22033.33 
                 2413.16 
                 46466.67 
                 6543.19 
               
               
                 G000670 
                 162666.67 
                 32959.57 
                 200000.00 
                 33867.39 
                 424666.67 
                 36473.73 
               
               
                 G011722 
                 16766.67 
                 3384.28 
                 8583.33 
                 4103.10 
                 24000.00 
                 8915.16 
               
               
                 G011723 
                 22733.33 
                 7252.82 
                 17133.33 
                 4905.44 
                 26100.00 
                 8109.87 
               
               
                 G011724 
                 17300.00 
                 2400.00 
                 28033.33 
                 9091.94 
                 30933.33 
                 3365.02 
               
               
                 G011725 
                 8253.33 
                 1163.20 
                 8890.00 
                 1429.27 
                 20366.67 
                 13955.05 
               
               
                 G011726 
                 12223.33 
                 3742.54 
                 11610.00 
                 2490.44 
                 14950.00 
                 8176.03 
               
               
                 G011727 
                 35600.00 
                 8128.35 
                 36300.00 
                 12301.22 
                 86700.00 
                 5023.94 
               
               
                 G011728 
                 14900.00 
                 5011.99 
                 22466.67 
                 7130.45 
                 38166.67 
                 13829.08 
               
               
                 G011729 
                 10460.00 
                 2543.95 
                 11223.33 
                 2220.28 
                 26966.67 
                 16085.50 
               
               
                 G011730 
                 14833.33 
                 2307.24 
                 21700.00 
                 8681.59 
                 41233.33 
                 25687.03 
               
               
                 G011731 
                 16433.33 
                 3274.65 
                 22566.67 
                 2205.30 
                 20756.67 
                 13096.20 
               
               
                 AAV Only 
                 217.00 
                 15.56 
                 215.00 
                 15.56 
                 207.00 
                 1.41 
               
               
                   
               
            
           
         
       
     
     Example 6—In Vivo Screening of Bidirectional Constructs Across Target Sites 
     The ssAAV and LNPs tested in this Example were prepared and delivered to C57Bl/6 mice as described in Example 1 to assess the performance of the bidirectional constructs across target sites in vivo. Four weeks post dose, the animals were euthanized and liver tissue and sera were collected for editing and hFIX expression, respectively. 
     In an initial experiment, 10 different LNP formulations containing 10 different gRNA targeting intron 1 of albumin were delivered to mice along with ssAAV derived from P00147. The AAV and LNP were delivered at 3e11 vg/ms and 4 mg/kg (with respect to total RNA cargo content), respectively (n=5 for each group). The gRNAs tested in this experiment are shown in  FIG. 6  and tabulated in Table 18. As shown in  FIG. 6  and as observed in vitro, a significant level of indel formation was not predictive for insertion or expression of the transgenes. 
     In a separate experiment, a panel of 20 gRNAs targeting the 20 different target sites tested in vitro in Example 5 were tested in vivo. To this end, LNP formulations containing the 20 gRNAs targeting intron 1 of albumin were delivered to mice along with ssAAV derived from P00147. The AAV and LNP were delivered at 3e11 vg/ms and 1 mg/kg (with respect to total RNA cargo content), respectively. The gRNAs tested in this experiment are shown in  FIGS. 7A and 7B . 
     As shown, in  FIG. 7A  and tabulated in Table 19, varied levels of editing were detected for each of the treatment groups across each LNP/vector combination tested. However, as shown in  FIG. 7B  and Table 20 and consistent with the in vitro data described in Example 5, higher levels of editing did not necessarily result in higher levels of expression of the transgenes in vivo, indicating a lack of correlation between editing and insertion/expression of the bidirectional hFIX constructs. Indeed, very little correlation exists between the amount of editing achieved and the amount of hFIX expression as viewed in the plot provided in  FIG. 7D . In particular, an R 2  value of only 0.34 is calculated between the editing and expression data sets for this experiment, when those gRNAs achieving less than 10% editing are removed from the analysis. Interestingly, as shown in  FIG. 7C , a correlation plot is provided comparing the levels of expression as measured in RLU from the in vitro experiment of Example 5 to the transgene expression levels in vivo detected in this experiment, with an R 2  value of 0.70, demonstrating a positive correlation between the primary cell screening and the in vivo treatments. 
     To assess insertion of the bidirectional construct at the cellular level, liver tissues from treated animals were assayed using an in situ hybridization method (BaseScope), e.g., as described in Example 1. This assay utilized probes that can detect the junctions between the hFIX transgene and the mouse albumin exon 1 sequence, as a hybrid transcript. As shown in  FIG. 8A , cells positive for the hybrid transcript were detected in animals that received both AAV and LNP. Specifically, when AAV alone is administered, less than 1.0% of cells were positive for the hybrid transcript. With administration of LNPs comprising G011723, G000551, or G000666, 4.9%, 19.8%, or 52.3% of cells were positive for the hybrid transcript. Additionally, as shown in  FIG. 8B , circulating hFIX levels correlated with the number of cells that were positive for the hybrid transcript. Lastly, the assay utilized pooled probes that can detect insertion of the bidirectional hFIX construct in either orientation. However, when a single probe was used that only detects a single orientation, the amount of cells that were positive for the hybrid transcript was about half that detected using the pooled probes (in one example, 4.46% vs 9.68%), suggesting that the bidirectional construct indeed is capable of inserting in either orientation giving rise to expressed hybrid transcripts that correlate with the amount of transgene expression at the protein level. These data show that the circulating hFIX levels achieved are dependent on the guide used for insertion. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 hFIX Serum Levels and % Indel 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Average  
                 St. Dev  
                 Average hFIX 
                 St. Dev hFIX 
               
               
                 Guide 
                 Indel (%) 
                 Indel (%) 
                 Serum Levels 
                 Serum Levels 
               
               
                   
               
               
                 G000551 
                 75.02 
                 1.27 
                  3.82 
                 3.38 
               
               
                 G000555 
                 51.18 
                 1.19 
                 32.56 
                 9.05 
               
               
                 G000553 
                 62.78 
                 2.64 
                 25.07 
                 4.04 
               
               
                 G000667 
                 52.96 
                 4.96 
                 32.03 
                 6.74 
               
               
                 G000554 
                 55.24 
                 2.28 
                 29.48 
                 7.34 
               
               
                 G000552 
                 67.56 
                 1.73 
                 14.79 
                 5.34 
               
               
                 G000668 
                 43.14 
                 5.78 
                 26.72 
                 7.97 
               
               
                 G000669 
                 50.68 
                 2.97 
                 10.70 
                 4.43 
               
               
                 G000666 
                 64.62 
                 1.34 
                 26.19 
                 5.56 
               
               
                 G000670 
                 55.90 
                 1.30 
                 30.96 
                 8.44 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 % Liver Editing 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Average Liver 
                 St. Dev Liver 
               
               
                   
                 Guide 
                 Editing (%) 
                 Editing (%) 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 G000551 
                 59.48 
                 4.02 
               
               
                   
                 G000555 
                 58.72 
                 3.65 
               
               
                   
                 G000553 
                 51.26 
                 2.81 
               
               
                   
                 G000554 
                 33.04 
                 8.76 
               
               
                   
                 G000555 
                 12.72 
                 4.46 
               
               
                   
                 G000666 
                 53.60 
                 4.92 
               
               
                   
                 G000667 
                 26.74 
                 4.98 
               
               
                   
                 G000668 
                 39.22 
                 3.04 
               
               
                   
                 G000669 
                 33.34 
                 4.77 
               
               
                   
                 G000670 
                 47.50 
                 5.58 
               
               
                   
                 G011722 
                 10.34 
                 1.68 
               
               
                   
                 G011723 
                 4.02 
                 0.84 
               
               
                   
                 G011724 
                 2.46 
                 0.64 
               
               
                   
                 G011725 
                 8.26 
                 1.24 
               
               
                   
                 G011726 
                 6.90 
                 1.01 
               
               
                   
                 G011727 
                 13.33 
                 6.43 
               
               
                   
                 G011728 
                 35.78 
                 9.34 
               
               
                   
                 G011729 
                 4.62 
                 1.46 
               
               
                   
                 G011730 
                 12.68 
                 3.14 
               
               
                   
                 G011731 
                 26.70 
                 1.86 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Serum hFIX Levels 
               
            
           
           
               
               
               
               
            
               
                   
                 Week 1 
                 Week 2 
                 Week 4 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Average 
                 St.Dev 
                 Average 
                 St.Dev 
                 Average 
                 St.Dev 
               
               
                   
                 FIX 
                 FIX 
                 FIX 
                 FIX 
                 FIX 
                 FIX 
               
               
                 Guide 
                 (ug/mL) 
                 (ug/mL) 
                 (ug/mL) 
                 (ug/mL) 
                 (ug/mL) 
                 (ug/mL) 
               
               
                   
               
               
                 G000551 
                 10.88 
                 2.74 
                 10.25 
                 2.51 
                  9.39 
                 3.48 
               
               
                   
               
               
                 G000555 
                 13.34 
                 2.09 
                 12.00 
                 2.75 
                 12.43 
                 2.57 
               
               
                   
               
               
                 G000553 
                 17.64 
                 4.34 
                 20.27 
                 6.35 
                 15.31 
                 2.43 
               
               
                   
               
               
                 G000554 
                 12.79 
                 4.99 
                 14.29 
                 6.09 
                 12.74 
                 4.93 
               
               
                   
               
               
                 G000555 
                 11.94 
                 5.79 
                 11.99 
                 5.76 
                  8.61 
                 4.02 
               
               
                   
               
               
                 G000666 
                 21.63 
                 1.32 
                 20.65 
                 1.55 
                 17.23 
                 0.62 
               
               
                   
               
               
                 G000667 
                 16.77 
                 2.86 
                 12.35 
                 2.85 
                 12.57 
                 5.60 
               
               
                   
               
               
                 G000668 
                 21.35 
                 1.51 
                 18.20 
                 3.18 
                 17.72 
                 2.25 
               
               
                   
               
               
                 G000669 
                  5.76 
                 2.10 
                  6.72 
                 2.93 
                  3.39 
                 0.78 
               
               
                   
               
               
                 G000670 
                 18.18 
                 2.17 
                 19.16 
                 3.05 
                 15.49 
                 3.61 
               
               
                   
               
               
                 G011722 
                  8.07 
                 1.74 
                  7.74 
                 2.41 
                  8.07 
                 1.74 
               
               
                   
               
               
                 G011723 
                  2.11 
                 0.28 
                  1.65 
                 0.28 
                  2.11 
                 0.28 
               
               
                   
               
               
                 G011724 
                  0.92 
                 0.43 
                  0.60 
                 0.30 
                  0.92 
                 0.43 
               
               
                   
               
               
                 G011725 
                  1.75 
                 0.77 
                  1.14 
                 0.67 
                  1.75 
                 0.77 
               
               
                   
               
               
                 G011726 
                  0.59 
                 0.30 
                  1.01 
                 0.64 
                  0.59 
                 0.30 
               
               
                   
               
               
                 G011727 
                  6.71 
                 2.80 
                  6.90 
                 3.68 
                  6.71 
                 2.80 
               
               
                   
               
               
                 G011728 
                 11.77 
                 3.12 
                 12.29 
                 3.43 
                 11.77 
                 3.12 
               
               
                   
               
               
                 G011729 
                  0.94 
                 0.35 
                  0.89 
                 0.29 
                  0.94 
                 0.35 
               
               
                   
               
               
                 G011730 
                  5.93 
                 1.77 
                  6.33 
                 1.73 
                  5.93 
                 1.77 
               
               
                   
               
               
                 G011731 
                  3.56 
                 0.87 
                  3.78 
                 0.50 
                  3.56 
                 0.87 
               
               
                   
               
               
                 AAV Only 
                  0.00 
                 0.00 
                  0.00 
                 0.00 
                  0.00 
                 0.00 
               
               
                   
               
               
                 Vehicle 
                  0.00 
                 0.00 
                  0.00 
                 0.00 
                  0.00 
                 0.00 
               
               
                   
               
               
                 Human Serum 
                  3.63 
                 0.32 
                  3.61 
                 0.35 
                  3.28 
                 0.03 
               
               
                   
               
            
           
         
       
     
     Example 7—Timing of AAV and LNP Delivery In Vivo 
     In this Example, the timing between delivery of ssAAV comprising the bidirectional hFIX construct and LNP was examined in C57Bl/6 mice. 
     The ssAAV and LNPs tested in this Example were prepared and delivered to mice as described in Example 1. The LNP formulation contained G000551 and the bidirectional template was delivered as ssAAV derived from P00147. The AAV and LNP were delivered at 3e11 vg/ms and 4 mg/kg (with respect to total RNA cargo content), respectively (n=5 for each group). A “Template only” cohort received AAV only, and a “PBS” cohort received no AAV or LNP. One cohort received AAV and LNP sequentially (minutes apart) at day 0 (“Template+LNP day 0”); another cohort received AAV at day 0 and LNP at day 1 (“Template+LNP day 1”); and a final cohort received AAV at day 0 and LNP at day 7(“Template+LNP day 7”). At 1 week, 2 weeks and 6 weeks, plasma was collected for hFIX expression analysis. 
     As shown in  FIG. 9 , hFIX was detected in each cohort at each time assayed, except for the 1 week timepoint for the cohort that received the LNP at day 7 post AAV delivery. 
     Example 8—Multiple Dosing of LNP Following Delivery of AAV 
     In this Example, the effects of repeat dosing of LNP following administration of ssAAV was examined. 
     The ssAAV and LNPs tested in this Example were prepared and delivered to C57Bl/6 mice as described in Example 1. The LNP formulation contained G000551 and the ssAAV was derived from P00147. The AAV and LNP were delivered at 3e11 vg/ms and 0.5 mg/kg (with respect to total RNA cargo content), respectively (n=5 for each group). A “Template only” cohort received AAV only, and a “PBS” cohort received no AAV or LNP. One cohort received AAV and LNP sequentially (minutes apart) at day 0 with no further treatments (“Template+LNP(1×)” in  FIG. 10 ); another cohort received AAV and LNP sequentially (minutes apart) at day 0 and a second dose at day 7 (“Template+LNP(2×)” in  FIG. 10 ); and a final cohort received AAV and LNP sequentially (minutes apart) at day 0, a second dose of LNP at day 7 and a third dose of LNP at day 14 (“Template+LNP(3×)” in  FIG. 10 ). At 1, 2, 4 and 6 weeks post-administration of AAV, plasma was collected for hFIX expression analysis. 
     As shown in  FIG. 10 , hFIX was detected in each cohort at each time assayed, and multiple subsequent doses of LNP did not significantly increase the amount of hFIX expression. 
     Example 9—Durability of hFIX Expression In Vivo 
     The durability of hFIX expression over time in treated animals was assessed in this Example. To this end, hFIX was measured in the serum of treated animals post-dose, as part of a one-year durability study. 
     The ssAAV and LNPs tested in this Example were prepared and delivered to C57Bl/6 mice as described in Example 1. The LNP formulation contained G000551 and the ssAAV was derived from P00147. The AAV was delivered at 3e11 vg/ms and the LNP was delivered at either 0.25 or 1.0 mg/kg (with respect to total RNA cargo content) (n=5 for each group). 
     As shown in  FIG. 11A  and Table 21, hFIX expression was sustained at each time point assessed for both groups out to 41 weeks. A drop in the levels observed at 8 weeks is believed to be due to the variability of the ELISA assay. Serum albumin levels were measured by ELISA at week 2 and week 41, showing that circulating albumin levels are maintained across the study. 
     As shown in  FIG. 11B  and Table 22, hFIX expression was sustained at each time point assessed for both groups out to 52 weeks. 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 FIX Levels 
               
            
           
           
               
               
            
               
                   
                 Dose 
               
            
           
           
               
               
               
            
               
                   
                 0.25 mpk LNP 
                 1 mpk LNP 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Average hFIX 
                 StDev hFIX 
                 Average hFIX 
                 StDev hFIX 
               
               
                 Week 
                 (ug/mL) 
                 (ug/mL) 
                 (ug/mL) 
                 (ug/mL) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 2 
                 0.48 
                 0.21 
                 2.24 
                 1.12 
               
               
                 4 
                 0.55 
                 0.18 
                 2.82 
                 1.67 
               
               
                 8 
                 0.40 
                 0.17 
                 1.72 
                 0.77 
               
               
                 12 
                 0.48 
                 0.20 
                 2.85 
                 1.34 
               
               
                 20 
                 0.48 
                 0.27 
                 2.45 
                 1.26 
               
               
                 41 
                 0.79 
                 0.49 
                 4.63 
                 0.95 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 FIX Levels 
               
            
           
           
               
               
            
               
                   
                 Dose 
               
            
           
           
               
               
               
            
               
                   
                 0.25 mpk LNP 
                 1 mpk LNP 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Average hFIX 
                 StDev hFIX 
                 Average hFIX 
                 StDev hFIX 
               
               
                 Week 
                 (ug/mL) 
                 (ug/mL) 
                 (ug/mL) 
                 (ug/mL) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 2 
                 0.87 
                 0.15 
                 4.02 
                 1.75 
               
               
                 8 
                 0.99 
                 0.15 
                 4.11 
                 1.41 
               
               
                 12 
                 0.93 
                 0.14 
                 4.15 
                 1.35 
               
               
                 20 
                 0.83 
                 0.22 
                 4.27 
                 1.54 
               
               
                 41 
                 0.83 
                 0.37 
                 4.76 
                 1.62 
               
               
                 52 
                 0.82 
                 0.25 
                 4.72 
                 1.54 
               
               
                   
               
            
           
         
       
     
     Example 10—Effects of Varied Doses of AAV and LNP to Modulate hFIX Expression In Vivo 
     In this Example, the effects of varying the dose of both AAV and LNP to modulate expression of hFIX was assessed in C57Bl/6 mice. 
     The ssAAV and LNPs tested in this Example were prepared and delivered to mice as described in Example 1. The LNP formulation contained G000553 and the ssAAV was derived from P00147. The AAV was delivered at 1e11, 3e11, 1e12 or 3e12 vg/ms and the LNP was delivered at 0.1, 0.3, or 1.0 mg/kg (with respect to total RNA cargo content) (n=5 for each group). Two weeks post-dose, the animals were euthanized. Sera were collected at two timepoints for hFIX expression analysis. 
     As shown in  FIG. 12A  (1 week),  FIG. 12B  (2 weeks) and Table 23, varying the dose of either AAV or LNP can modulate the amount of expression of hFIX in vivo. 
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Serum hFIX 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 RNP Dose 
                 AAV Dose 
                 Mean FIX 
                   
                   
               
               
                 Timepoint 
                 (mg/kg) 
                 (MOI) 
                 (ng/ml) 
                 SD 
                 N 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Week 1 
                 0.1 
                 1E+11 
                 0.08 
                 0.02 
                 2 
               
               
                   
                   
                 3E+11 
                 0.11 
                 0.04 
                 5 
               
               
                   
                   
                 1E+12 
                 0.41 
                 0.15 
                 5 
               
               
                   
                   
                 3E+12 
                 0.61 
                 0.17 
                 5 
               
               
                   
                 0.3 
                 1E+11 
                 0.36 
                 0.14 
                 5 
               
               
                   
                   
                 3E+11 
                 0.67 
                 0.26 
                 5 
               
               
                   
                   
                 1E+12 
                 1.76 
                 0.14 
                 5 
               
               
                   
                   
                 3E+12 
                 4.70 
                 2.40 
                 5 
               
               
                   
                 1.0 
                 1E+11 
                 3.71 
                 0.31 
                 4 
               
               
                   
                   
                 3E+11 
                 8.00 
                 0.51 
                 5 
               
               
                   
                   
                 1E+12 
                 14.17 
                 1.38 
                 5 
               
               
                   
                   
                 3E+12 
                 20.70 
                 2.79 
                 5 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Human serum 1:1000 
                 6.62 
                 — 
                 1 
               
            
           
           
               
               
               
               
               
               
            
               
                 Week 2 
                 0.1 
                 1E+11 
                 0.12 
                 0.01 
                 2 
               
               
                   
                   
                 3E+11 
                 0.26 
                 0.07 
                 5 
               
               
                   
                   
                 1E+12 
                 0.83 
                 0.24 
                 5 
               
               
                   
                   
                 3E+12 
                 1.48 
                 0.35 
                 5 
               
               
                   
                 0.3 
                 1E+11 
                 0.70 
                 0.26 
                 4 
               
               
                   
                   
                 3E+11 
                 1.42 
                 0.37 
                 5 
               
               
                   
                   
                 1E+12 
                 3.53 
                 0.49 
                 5 
               
               
                   
                   
                 3E+12 
                 8.94 
                 4.39 
                 5 
               
               
                   
                 1.0 
                 1E+11 
                 5.40 
                 0.47 
                 4 
               
               
                   
                   
                 3E+11 
                 12.31 
                 2.45 
                 5 
               
               
                   
                   
                 1E+12 
                 17.89 
                 1.95 
                 5 
               
               
                   
                   
                 3E+12 
                 25.52 
                 3.62 
                 5 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Human serum 1:1000 
                 4.47 
                 — 
                 1 
               
               
                   
               
            
           
         
       
     
     Example 11—In Vitro Screening of Bidirectional Constructs Across Target Sites in Primary Cynomolgus and Primary Human Hepatocytes 
     In this Example, ssAAV vectors comprising a bidirectional construct were tested across a panel of target sites utilizing gRNAs targeting intron 1 of cynomolgus (“cyno”) and human albumin in primary cyno (PCH) and primary human hepatocytes (PHH), respectively. 
     The ssAAV and lipid packet delivery materials tested in this Example were prepared and delivered to PCH and PHH as described in Example 1. Following treatment, isolated genomic DNA and cell media was collected for editing and transgene expression analysis, respectively. Each of the vectors comprised a reporter that can be measured through luciferase-based fluorescence detection as described in Example 1 (derived from plasmid P00415), plotted in  FIGS. 13B and 14B  as relative luciferase units (“RLU”). For example, the AAV vectors contained the NanoLuc ORF (in addition to GFP). Schematics of the vectors tested are provided in  FIGS. 13B  and  14 B. The gRNAs tested are shown in each of the Figures using a shortened number for those listed in Table 1 and Table 7. 
     As shown in  FIG. 13A  for PCH and  FIG. 14A  for PHH, varied levels of editing were detected for each of the combinations tested (editing data for some combinations tested in the PCH experiment are not reported in  FIG. 13A  and Table 3 due to failure of certain primer pairs used for the amplicon based sequencing). The editing data shown in  FIGS. 13A and 14A  graphically, are reproduced numerically in Table 3 and Table 4 below. However, as shown in  FIGS. 13B, 13C  and  FIGS. 14B and 14C , a significant level of indel formation was not predictive for insertion or expression of the transgenes, indicating little correlation between editing and insertion/expression of the bidirectional constructs in PCH and PHH, respectively. As one measure, the R 2  value calculated in  FIG. 13C  is 0.13, and the R 2  value of  FIG. 14D  is 0.22. 
     Additionally, ssAAV vectors comprising a bidirectional construct were tested across a panel of target sites utilizing single guide RNAs targeting intron 1 of human albumin in primary human hepatocytes (PHH). 
     The ssAAV and LNP materials were prepared and delivered to PHH as described in Example 1. Following treatment, isolated genomic DNA and cell media was collected for editing and transgene expression analysis, respectively. Each of the vectors comprised a reporter that can be measured through luciferase-based fluorescence detection as described in Example 1 (derived from plasmid P00415), plotted in  FIG. 14D  as relative luciferase units (“RLU”) and tabulated in Table 24 below. For example, the AAV vectors contained the NanoLuc ORF (in addition to GFP). Schematics of the vectors tested are provided in  FIGS. 13B and 14B . The gRNAs tested are shown in  FIG. 14D  using a shortened number for those listed in Table 1 and Table 7. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Albumin intron 1 editing data for sgRNAs 
               
               
                 delivered to primary cynomolgus hepatocytes 
               
            
           
           
               
               
               
               
            
               
                   
                 GUIDE ID 
                 Avg % Edit 
                 Std Dev % Edit 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 G009867 
                 25.05 
                 0.21 
               
               
                   
                 G009866 
                 18.7 
                 3.96 
               
               
                   
                 G009876 
                 14.85 
                 4.88 
               
               
                   
                 G009875 
                 12.85 
                 2.33 
               
               
                   
                 G009874 
                 28.25 
                 6.01 
               
               
                   
                 G009873 
                 42.65 
                 5.59 
               
               
                   
                 G009865 
                 59.15 
                 0.21 
               
               
                   
                 G009872 
                 48.15 
                 3.46 
               
               
                   
                 G009871 
                 46.5 
                 5.23 
               
               
                   
                 G009864 
                 33.2 
                 8.34 
               
               
                   
                 G009863 
                 54.8 
                 12.45 
               
               
                   
                 G009862 
                 44.6 
                 7.21 
               
               
                   
                 G009861 
                 28.65 
                 0.21 
               
               
                   
                 G009860 
                 33.2 
                 7.07 
               
               
                   
                 G009859 
                 0.05 
                 0.07 
               
               
                   
                 G009858 
                 14.65 
                 1.77 
               
               
                   
                 G009857 
                 23 
                 0.99 
               
               
                   
                 G009856 
                 14.8 
                 0.99 
               
               
                   
                 G009851 
                 1.5 
                 0.42 
               
               
                   
                 G009868 
                 12.15 
                 2.47 
               
               
                   
                 G009850 
                 63.45 
                 13.93 
               
               
                   
                 G009849 
                 57.55 
                 8.27 
               
               
                   
                 G009848 
                 33 
                 5.37 
               
               
                   
                 G009847 
                 66.75 
                 7 
               
               
                   
                 G009846 
                 61.85 
                 5.02 
               
               
                   
                 G009845 
                 54.4 
                 7.5 
               
               
                   
                 G009844 
                 47.15 
                 2.05 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Albumin intron 1 editing data for sgRNAs 
               
               
                 delivered to primary human hepatocytes 
               
            
           
           
               
               
               
               
            
               
                   
                 GUIDE ID 
                 Avg % Edit 
                 Std Dev % Edit 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 G009844 
                 19.07 
                 2.07 
               
               
                   
                 G009851 
                 0.43 
                 0.35 
               
               
                   
                 G009852 
                 47.20 
                 3.96 
               
               
                   
                 G009857 
                 0.10 
                 0.14 
               
               
                   
                 G009858 
                 8.63 
                 9.16 
               
               
                   
                 G009859 
                 3.07 
                 3.50 
               
               
                   
                 G009860 
                 18.80 
                 4.90 
               
               
                   
                 G009861 
                 10.27 
                 2.51 
               
               
                   
                 G009866 
                 13.60 
                 13.55 
               
               
                   
                 G009867 
                 12.97 
                 3.04 
               
               
                   
                 G009868 
                 0.63 
                 0.32 
               
               
                   
                 G009874 
                 49.13 
                 0.60 
               
               
                   
                 G012747 
                 3.83 
                 0.23 
               
               
                   
                 G012748 
                 1.30 
                 0.35 
               
               
                   
                 G012749 
                 9.77 
                 1.50 
               
               
                   
                 G012750 
                 42.73 
                 4.58 
               
               
                   
                 G012751 
                 7.77 
                 1.16 
               
               
                   
                 G012752 
                 32.93 
                 2.27 
               
               
                   
                 G012753 
                 21.20 
                 2.95 
               
               
                   
                 G012754 
                 0.60 
                 0.10 
               
               
                   
                 G012755 
                 1.10 
                 0.10 
               
               
                   
                 G012756 
                 2.17 
                 0.40 
               
               
                   
                 G012757 
                 1.07 
                 0.25 
               
               
                   
                 G012758 
                 0.90 
                 0.10 
               
               
                   
                 G012759 
                 2.60 
                 0.35 
               
               
                   
                 G012760 
                 39.10 
                 6.58 
               
               
                   
                 G012761 
                 36.17 
                 2.43 
               
               
                   
                 G012762 
                 8.50 
                 0.57 
               
               
                   
                 G012763 
                 47.07 
                 3.07 
               
               
                   
                 G012764 
                 44.57 
                 5.83 
               
               
                   
                 G012765 
                 19.90 
                 1.68 
               
               
                   
                 G012766 
                 8.50 
                 0.28 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 hAlb Guide Screen Luciferase 
               
            
           
           
               
               
               
            
               
                   
                 Average 
                 St. Dev 
               
               
                 Guide 
                 Luciferase (RLU) 
                 Luciferase (RLU) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 G009844 
                 3700000 
                 509116.9 
               
               
                 G009852 
                 281000 
                 69296.46 
               
               
                 G009857 
                 1550000 
                 127279.2 
               
               
                 G009858 
                 551000 
                 108894.4 
               
               
                 G009859 
                 1425000 
                 77781.75 
               
               
                 G009860 
                 2240000 
                 183847.8 
               
               
                 G009861 
                 663500 
                 238295 
               
               
                 G009866 
                 274000 
                 11313.71 
               
               
                 G009867 
                 44700 
                 565.6854 
               
               
                 G009874 
                 2865000 
                 431335.1 
               
               
                 G012747 
                 651000 
                 59396.97 
               
               
                 G012749 
                 867000 
                 93338.1 
               
               
                 G012752 
                 4130000 
                 268700.6 
               
               
                 G012753 
                 1145000 
                 162634.6 
               
               
                 G012757 
                 579000 
                 257386.9 
               
               
                 G012760 
                 129000 
                 36769.55 
               
               
                 G012761 
                 4045000 
                 728320 
               
               
                 G012762 
                 2220000 
                 127279.2 
               
               
                 G012763 
                 1155000 
                 205061 
               
               
                 G012764 
                 11900000 
                 1555635 
               
               
                 G012765 
                 1935000 
                 134350.3 
               
               
                 G012766 
                 2050000 
                 169705.6 
               
               
                 LNP 
                 8430 
                 212.132 
               
               
                   
               
            
           
         
       
     
     Example 12—In Vivo Testing of Factor IX Expression from an Alternative Safe Harbor Locus 
     In this Example, insertion of ssAAV comprising a bidirectional hFIX construct at an alternative safe harbor locus was evaluated. To test the insertion into an alternative safe harbor locus, AAV was prepared as described above. Mice were administered with AAVs at a dose of 3e11 vg/mouse immediately followed by administration of LNPs formulated with Cas9 mRNAs and guide RNAs at a dose of 0.3 mg/kg. Animals were sacrificed 4 weeks post-dose, and liver and blood samples were collected. Editing in the liver samples was determined by NGS. Human hFIX levels in the serum was determined by ELISA. The NGS and ELISA data showed effective insertion and expression of hFIX within the alternative safe harbor locus. 
     Example 13—In Vivo Testing of the Human Factor IX Gene Insertion in Non-Human Primates 
     In this example, an 8 week study was performed to evaluate the human Factor IX gene insertion and hFIX protein expression in cynomolgus monkeys through administration of adeno-associated virus (AAV) and/or lipid nanoparticles (LNP) with various guides. This study was conducted with LNP formulations and AAV formulations prepared as described above. Each LNP formulation contained Cas9 mRNA and guide RNA (gRNA) with an mRNA:gRNA ratio of 2:1 by weight. The ssAAV was derived from P00147. 
     Male cynomologus monkeys were treated in cohorts of n=3. Animals were dosed with AAV by slow bolus injection or infusion in the doses described in Table 10. Following AAV treatment, animals received buffer or LNP as described in Table 10 by slow bolus or infusion. 
     Two weeks post-dose, liver specimens were collected through single ultrasound-guided percutaneous biopsy. Each biopsy specimen was flash frozen in liquid nitrogen and stored at −86 to −60° C. Editing analysis of the liver specimens was performed by NGS Sequencing as previously described. 
     For Factor IX ELISA analysis, blood samples were collected from the animals on days 7, 14, 28, and 56 post-dose. Blood samples were collected and processed to plasma following blood draw and stored at −86 to −60° C. until analysis. 
     The total human Factor IX levels were determined from plasma samples by ELISA. Briefly, Reacti-Bind 96-well microplate (VWR Cat # PI15041) were coated with capture antibody (mouse mAB to human Factor IX antibody (HTI, Cat # AHIX-5041)) at a concentration of 1 μg/ml then blocked using 1×PBS with 5% Bovine Serum Albumin. Test samples or standards of purified human Factor IX protein (ERL, Cat # HFIX 1009, Lot # HFIX4840) diluted in Cynomolgus monkey plasma were next incubated in individual wells. The detection antibody (Sheep anti-human Factor 9 polyclonal antibody, Abcam, Cat # ab128048) was adsorbed at a concentration of 100 ng/ml. The secondary antibody (Donkey anti-Sheep IgG pAbs with HRP, Abcam, Cat # ab97125) was used at 100 ng/mL. TMB Substrate Reagent set (BD OptEIA Cat #555214) was used to develop the plate. Optical density was assessed spectrophotometrically at 450 nm on a microplate reader (Molecular Devices i3 system) and analyzed using SoftMax pro 6.4. 
     Indel formation was detected, confirming that editing occurred. The NGS data showed effective indel formation. Expression of hFIX from the albumin locus in NHPs was measured by ELISA and is depicted in Table 11 and  FIG. 15 . Plasma levels of hFIX reached levels previously described as therapeutically effective (George, et al., NEJM 377(23), 2215-27, 2017). 
     As measured, circulating hFIX protein levels were sustained through the eight week study (see  FIG. 15 , showing day 7, 14, 28, and 56 average levels of ˜135, ˜140, ˜150, and ˜110 ng/mL, respectively), achieving protein levels ranging from ˜75 ng/mL to ˜250 ng/mL. Plasma hFIX levels were calculated using a specific activity of ˜8 fold higher for the R338L hyperfunctional hFIX variant (Simioni et al., NEJM 361(17), 1671-75, 2009) (which reports a protein-specific activity of hFIX-R338L of 390±28 U per milligram, and a protein-specific activity for wild-type factor IX of 45±2.4 U per milligram). Calculating the functionally normalized Factor IX activity for the hyperfunctional Factor IX variant tested in this example, the experiment achieved stable levels of human Factor IX protein in the NHPs over the 8 week study that correspond to about 20-40% of wild type Factor IX activity (range spans 12-67% of wild type Factor IX activity). 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Editing in liver 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 F9-AAV 
                   
                 LNP 
               
               
                 Animal 
                   
                 F9-AAV 
                 Volume 
                 LNP 
                 Volume 
               
               
                 ID 
                 Guide ID 
                 (vg/kg) 
                 (mL/kg) 
                 (mg/kg) 
                 (mL/kg) 
               
               
                   
               
               
                 4001 
                 G009860 
                 3E+13 
                 1 
                 3 
                 2 
               
               
                 4002 
                 G009860 
                 3E+13 
                 1 
                 3 
                 2 
               
               
                 4003 
                 G009860 
                 3E+13 
                 1 
                 3 
                 2 
               
               
                 5001 
                 TSS 
                 3E+13 
                 1 
                 0 
                 0 
               
               
                 5002 
                 TSS 
                 3E+13 
                 1 
                 0 
                 0 
               
               
                 5003 
                 TSS 
                 3E+13 
                 1 
                 0 
                 0 
               
               
                 6001 
                 G009862 
                 0 
                 0 
                 3 
                 2 
               
               
                 6002 
                 G009862 
                 0 
                 0 
                 3 
                 2 
               
               
                 6003 
                 G009862 
                 0 
                 0 
                 3 
                 2 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 hFIX expression 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Day 7 
                 Day 14 
                 Day 28 
                 Day 56 
               
               
                 Animal 
                 Factor IX 
                 Factor IX 
                 Factor IX 
                 Factor IX 
               
               
                 ID 
                 (ng/mL) 
                 (ng/mL) 
                 (ng/mL) 
                 (ng/mL) 
               
               
                   
               
               
                 4001 
                 122.84/+−2.85 
                  94.93/+−0.56 
                 105.65/+−1.94 
                  97.31/+−1.49 
               
               
                 4002 
                 149.77/+−13.5 
                 222.92/+−9.61 
                 252.49/+−6.46 
                 152.05/+−7.46 
               
               
                 4003 
                 134.06/+−6.17 
                 107.04/+−6.46 
                  95.30/+−3.18 
                  74.23/+−3.53 
               
               
                 5001 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 5002 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 5003 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 6001 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 6002 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 6003 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                   
               
            
           
         
       
     
     Example 14 In Vivo Testing of Factor IX Insertion in Non-Human Primates 
     In this example, a study was performed to evaluate the Factor IX gene insertion and hFIX protein expression in cynomolgus monkeys following administration of ssAAV derived from P00147 and/or CRISPR/Cas9 lipid nanoparticles (LNP) with various guides including G009860 and various LNP components. 
     Indel formation was measured by NGS, confirming that editing occurred. Total human Factor IX levels were determined from plasma samples by ELISA using a mouse mAB to human Factor IX antibody (HTI, Cat # AHIX-5041), sheep anti-human Factor 9 polyclonal antibody (Abcam, Cat # ab128048), and donkey anti-Sheep IgG pAbs with HRP (Abcam, Cat # ab97125), as described in Example 13. Human FIX protein levels &gt;3 fold higher than those achieved in the experiment of Example 13 were obtained from the bidirectional template using alternative CRISPR/Cas9 LNP. In the study, ELISA assay results indicate that circulating hFIX protein levels at or above the normal range of human FIX levels (3-5 ug/mL; Amiral et al., Clin. Chem., 30(9), 1512-16, 1984) were achieved using G009860 in the NHPs by at least the day 14 and 28 timepoints. Initial data indicated circulating human FIX protein levels of ˜3-4 μg/mL at day 14 after a single dose, with levels sustained through the first 28 days (˜3-5m/mL) of the study. The human FIX levels were measured at the conclusion of the study by the same method and data are presented in the Table 25. Additional guides G009847, G009862, and G009864 were also tested and shown to facilitate insertion of a FIX-expressing template in the NHP study. 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 Serum human Factor IX protein levels -ELISA Method of Example 13 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Day 7 
                   
                 Day 14 
                   
                 Day 28 
                   
                 Day 42 
                   
                 Day 56 
                   
               
               
                   
                 FIX 
                 STD 
                 FIX 
                 STD 
                 FIX 
                 STD 
                 FIX 
                 STD 
                 FIX 
                 STD 
               
               
                   
                 ng/mL 
                 DEV 
                 ng/mL 
                 DEV 
                 ng/mL 
                 DEV 
                 ng/mL 
                 DEV 
                 ng/mL 
                 DEV 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 3001 
                 2532.8 
                 145.6 
                 2562.6 
                 99.0 
                 3011.7 
                 62.7 
                 2936.7 
                 72.4 
                 2748.5 
                 86.0 
               
               
                 3002 
                 2211.4 
                 95.8 
                 2958.5 
                 119.2 
                 3350.2 
                 98.4 
                 3049.7 
                 112.7 
                 3036.7 
                 90.6 
               
               
                 3003 
                 3195.1 
                 475.6 
                 4433.9 
                 238.7 
                 3367.2 
                 157.7 
                 3746.1 
                 95.6 
                 3925.0 
                 157.4 
               
               
                   
               
            
           
         
       
     
     Circulating albumin levels were measured by ELISA, indicating that baseline albumin levels are maintained at 28 days. Tested albumin levels in untreated animals varied ±˜15% in the study. In treated animals, circulating albumin levels changed minimally and did not drop out of the normal range, and the levels recovered to baseline within one month. 
     Circulating human FIX protein levels were also determined by a sandwich immunoassay with a greater dynamic range. Briefly, an MSD GOLD 96-well Streptavidin SECTOR Plate (Meso Scale Diagnostics, Cat. L15SA-1) was blocked with 1% ECL Blocking Agent (Sigma, GERPN2125). After tapping out the blocking solution, biotinylated capture antibody (Sino Biological, 11503-R044) was immobilized on the plate. Recombinant human FIX protein (Enzyme Research Laboratories, HFIX 1009) was used to prepare a calibration standard in 0.5% ECL Blocking Agent. Following a wash, calibration standards and plasma samples were added to the plate and incubated. Following a wash, a detection antibody (Haematologic Technologies, AHIX-5041) conjugated with a sulfo-tag label was added to the wells and incubated. After washing away any unbound detection antibody, Read Buffer T was applied to the wells. Without any additional incubation, the plate was imaged with an MSD Quick Plex SQ120 instrument and data was analyzed with Discovery Workbench 4.0 software package (Meso Scale Discovery). Concentrations are expressed as mean calculated concentrations in ug/m. For the samples, N=3 unless indicated with an asterisk, in which case N=2. Expression of hFIX from the albumin locus in the treated study group as measured by the MSD ELISA is depicted in Table 26. 
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 Serum human Factor IX protein levels - MSD ELISA 
               
            
           
           
               
               
            
               
                   
                 Mean Calc. Conc. (ug/mL) 
               
            
           
           
               
               
               
               
            
               
                   
                 3001 
                 3002 
                 3003 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                   
                 Inter-Assay 
                   
                 Inter-Assay 
                   
                 Inter-Assay 
               
               
                 Point 
                 Conc. 
                 CV 
                 Conc. 
                 CV 
                 Conc. 
                 CV 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Day 7 
                 7.85 
                 20% 
                 5.63 
                 14% 
                 11.20 
                 26% 
               
               
                 Day 14 
                 8.65 
                 15% 
                 11.06 
                 18% 
                 14.70 
                 28% 
               
               
                 Day 28 
                 9.14 
                  7% 
                 14.12 
                  7% 
                 10.85 
                 25% 
               
               
                 Day 42 
                 9.03 
                 10% 
                 33.12* 
                  0% 
                 13.22 
                 13% 
               
               
                 Day 56 
                 10.24 
                 13% 
                 16.72 
                 12% 
                 33.84* 
                  4% 
               
               
                   
               
            
           
         
       
     
     Example 15—Off-Target Analysis of Albumin Human Guides 
     A biochemical method (See, e.g., Cameron et al.,  Nature Methods.  6, 600-606; 2017) was used to determine potential off-target genomic sites cleaved by Cas9 targeting Albumin. In this experiment, 13 sgRNA targeting human Albumin and two control guides with known off-target profiles were screened using isolated HEK293 genomic DNA. The number of potential off-target sites detected using a guide concentration of 16 nM in the biochemical assay were shown in Table 27. The assay identified potential off-target sites for the sgRNAs tested. 
                     TABLE 27                  Off-Target Analysis                                         Off-                   Target       gRNA       Guide Sequence   Site       ID   Target   (SEQ ID NO:)   Count               G012753   Albumin   GACUGAAACUUCACAGAAUA     62               (SEQ ID NO: 20)                   G012761   Albumin   AGUGCAAUGGAUAGGUCUUU     75               (SEQ ID NO: 28)                   G012752   Albumin   UGACUGAAACUUCACAGAAU    223               (SEQ ID NO: 19)                   G012764   Albumin   CCUCACUCUUGUCUGGGCAA   3985               (SEQ ID NO: 31)                   G012763   Albumin   UGGGCAAGGGAAGAAAAAAA   5443               (SEQ ID NO: 30)                   G009857   Albumin   AUUUAUGAGAUCAACAGCAC    131               (SEQ ID NO: 5)                   G009859   Albumin   UUAAAUAAAGCAUAGUGCAA     91               (SEQ ID NO: 7)                   G009860   Albumin   UAAAGCAUAGUGCAAUGGAU    133               (SEQ ID NO: 8)                   G012762   Albumin   UGAUUCCUACAGAAAAACUC     68               (SEQ ID NO: 29)                   G009844   Albumin   GAGCAACCUCACUCUUGUCU    107               (SEQ ID NO: 2)                   G012765   Albumin   ACCUCACUCUUGUCUGGGCA     41               (SEQ ID NO: 32)                   G012766   Albumin   UGAGCAACCUCACUCUUGUC     78               (SEQ ID NO: 33)                   G009874   Albumin   UAAUAAAAUUCAAACAUCCU     53               (SEQ ID NO: 13)                   G000644   EMX1   GAGUCCGAGCAGAAGAAGAA    304               (SEQ ID NO: 1129)                   G000645   VEGFA   GACCCCCUCCACCCCGCCUC   1641               (SEQ ID NO: 1130)                    
In known off-target detection assays such as the biochemical method used above, a large number of potential off-target sites are typically recovered, by design, so as to “cast a wide net” for potential sites that can be validated in other contexts, e.g., in a primary cell of interest. For example, the biochemical method typically overrepresents the number of potential off-target sites as the assay utilizes purified high molecular weight genomic DNA free of the cell environment and is dependent on the dose of Cas9 RNP used. Accordingly, potential off-target sites identified by these methods may be validated using targeted sequencing of the identified potential off-target sites.
 
     Example 16. Use of Humanized Albumin Mice to Screen Guide RNAs for Human F9 Insertion In Vivo 
     We aimed to identify effective guide RNAs for hF9 insertion into the human albumin locus. To this end, we utilized mice in which the mouse albumin locus was replaced with the corresponding human albumin genomic sequence, including the first intron (ALB hu/hu  mice). This allowed us to test the insertion efficiency of guide RNAs targeting the first intron of human albumin in the context of an adult liver in vivo. Two separate mouse experiments were set up using the ALB hu/hu  mice to screen a total of 11 guide RNAs, each targeting the first intron of the human albumin locus. All mice were weighed and injected via tail vein at day 0 of the experiment. Blood was collected at weeks 1, 3, 4, and 6 via tail bleed, and plasma was separated. Mice were terminated at week 7. Blood was collected via the vena cava, and plasma was separated. Livers and spleens were dissected as well. 
     In the first experiment, 6 LNPs comprising Cas9 mRNA and the following guides were prepared as in Example 1 and tested: G009852, G009859, G009860, G009864, G009874, and G012764. LNPs were diluted to 0.3 mg/kg (using an average weight of 30 grams) and co-injected with AAV8 packaged with the bi-directional hF9 insertion template at a dose of 3E11 viral genomes per mouse. Five ALB hu/hu  male mice between 12 and 14 weeks old were injected per group. Five mice from same cohort were injected with AAV8 packaged with a CAGG promoter operably linked to hF9, which leads to episomal expression of hF9 (at 3E11 viral genomes per mouse). There were three negative control groups with three mice per group that were injected with buffer alone, AAV8 packaged with the bi-directional hF9 insertion template alone, or LNP-G009874 alone. 
     In the experiment, the following LNPs comprising Cas9 mRNA and the following guides were prepared as in Example 1 and tested: G009860, G012764, G009844, G009857, G012752, G012753, and G012761. All were diluted to 0.3 mg/kg (using an average weight of 40 grams) and co-injected with AAV8 packaged with the bi-directional hF9 insertion template at a dose of 3E11 viral genomes per mouse. Five ALB hu/hu  male mice 30 weeks old were injected per group. Five mice from same cohort were injected with AAV8 packaged with a CAGG promoter operably linked to hF9, which leads to episomal expression of hF9 (at 3E11 viral genomes per mouse). There were three negative control groups with three mice per group that were injected with buffer alone, AAV8 packaged with the bi-directional hF9 insertion template alone, or LNP-G009874 alone. 
     For analysis, an ELISA was performed to measure levels of hFIX circulating in the mice at each timepoint. Human Factor IX ELISA Kits (ab188393) were used for this purpose, and all plates were run with human pooled normal plasma from George King Bio-Medical as a positive assay control. Human Factor IX expression levels in the plasma samples in each group at week 6 post-injection are shown in  FIG. 16A  and  FIG. 16B . Consistent with the in vitro insertion data, low to no Factor IX serum levels were detected when guide RNA G009852 was used. Consistent with the lack of an adjacent PAM sequence in human albumin, Factor IX serum levels were not detectable when guide RNA G009864 was used. Factor IX expression in the serum was observed for the groups using guide RNAs G009859, G009860, G009874, and G0012764. 
     Spleens and a portion of the left lateral lobe of all livers were submitted for next-generation sequencing (NGS) analysis. NGS was used to assess the percentage of liver cells with insertions/deletions (indels) at the humanized albumin locus at week 7 post-injection with AAV-hF9 donor and LNP-CRISPR/Cas9. Consistent with the lack of an adjacent PAM sequence in human albumin, no editing was detectable in the liver when guide RNA G009864 was used. Editing in the liver was observed for the groups using guide RNAs G009859, G009860, G009874, and G012764 (data not shown). 
     The remaining liver was fixed for 24 hours in 10% neutral buffered formalin and then transferred to 70% ethanol. Four to five samples from separate lobes were cut and shipped to HistoWisz and were processed and embedded in paraffin blocks. Five-micron sections were then cut from each paraffin block, and BASESCOPE™ was performed on the Ventana Ultra Discovery (Roche) using the universal BASESCOPE™ procedure and reagents by Advanced Cell Diagnostics and a custom designed probe that targets the unique mRNA junction formed between the human albumin signal sequence from the first intron of the ALB hu/hu  albumin locus and the hF9 transgene when successful integration and transcription is achieved. HALO imaging software (Indica Labs) was then used to quantify the percentage of positive cells in each sample. The average of percentage positive cells across the multiple lobes for each animal was then correlated to the hFIX levels in the serum at week 7. The results are shown in  FIG. 17  and Table 28. The week 7 serum levels and the % positive cells for the hALB-hFIX mRNA strongly correlated (r=0.89; R 2 =0.79). 
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 Week 7 hFIX and BASESCOPE ™ Data. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 hFIX 
                 % mRNA 
                 STD %  
                 Total  
               
               
                   
                   
                 ug/mL 
                 Probe 
                 mRNA 
                 Cells 
               
               
                 Mouse 
                 Guide 
                 (Week 7) 
                 (4-5 Sections) 
                 Probe 
                 Counted 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 1 
                 Buffer 
                 ND 
                 0.09 
                 0.03 
                 152833 
               
               
                 4 
                 AAV 
                 ND 
                 0.53 
                 0.67 
                 351084 
               
               
                   
                 Only 
                   
                   
                   
                   
               
               
                 7 
                 LNP 
                 ND 
                 0.48 
                 0.33 
                 75160 
               
               
                   
                 Only 
                   
                   
                   
                   
               
               
                 10 
                 CAG F9 
                 211.8 
                 0.20 
                 0.22 
                 190277 
               
               
                 15 
                 G009852 
                 ND 
                 0.30 
                 0.09 
                 144518 
               
               
                 20 
                 G009859 
                 0.5 
                 0.82 
                 0.45 
                 143817 
               
               
                 21 
                 G009859 
                 0.5 
                 0.88 
                 0.43 
                 160172 
               
               
                 22 
                 G009859 
                 2.3 
                 1.71 
                 1.54 
                 26015 
               
               
                 23 
                 G009859 
                 3.8 
                 2.74 
                 0.59 
                 183085 
               
               
                 24 
                 G009859 
                 0.6 
                 2.78 
                 1.96 
                 152424 
               
               
                 25 
                 G009860 
                 5.6 
                 12.46 
                 5.80 
                 78935 
               
               
                 26 
                 G009860 
                 10.6 
                 13.76 
                 5.32 
                 112252 
               
               
                 27 
                 G009860 
                 9.7 
                 14.80 
                 5.45 
                 201592 
               
               
                 28 
                 G009860 
                 2.1 
                 3.32 
                 0.76 
                 84710 
               
               
                 29 
                 G009860 
                 3.0 
                 1.52 
                 0.35 
                 203277 
               
               
                 30 
                 G009864 
                 ND 
                 1.94 
                 1.78 
                 145807 
               
               
                 35 
                 G009874 
                 1.7 
                 2.42 
                 1.14 
                 126665 
               
               
                 36 
                 G009874 
                 1.5 
                 1.08 
                 0.53 
                 195861 
               
               
                 37 
                 G009874 
                 2.1 
                 1.02 
                 1.29 
                 181679 
               
               
                 38 
                 G009874 
                 5.5 
                 0.40 
                 0.43 
                 175359 
               
               
                 39 
                 G009874 
                 1.5 
                 0.44 
                 0.18 
                 205417 
               
               
                 40 
                 G012764 
                 15.7 
                 28.85 
                 7.11 
                 167824 
               
               
                 41 
                 G012764 
                 19.6 
                 19.17 
                 8.23 
                 70081 
               
               
                 42 
                 G012764 
                 1.9 
                 1.95 
                 1.79 
                 154742 
               
               
                 43 
                 G012764 
                 7.7 
                 4.38 
                 0.68 
                 114060 
               
               
                 44 
                 G012764 
                 3.0 
                 1.64 
                 1.04 
                 238623 
               
               
                 43 
                 DapB (−) 
                 — 
                 0.12 
                 0.07 
                 144730 
               
               
                   
               
            
           
         
       
     
     Example 17—Use of Humanized Albumin Mice Crossed with F9 Knockout Mice to Assess Functionality of Inserted Human F9 In Vivo 
     For a next study, functionality of inserted hF9 was tested in male ALB ms/hu ×F9 −/−  mice. LNPs comprising Cas9 mRNA and the following guides were prepared as in Example 1 and tested: G009860 (targeting the first intron of the human albumin locus) and G000666 (targeting the first intron of the mouse albumin locus). G009860 was diluted to 0.3 mg/kg, and G000666 was diluted to 1.0 mg/kg (using an average weight of 31.2 grams), and both were co-injected with AAV8 packaged with the bi-directional hF9 insertion template at a dose of 3E11 viral genomes per mouse. Five ALB ms/hu ×F9 −/−  male mice (16 weeks old) were injected per group. Five mice from same cohort were injected with AAV8 packaged with a CAGG promoter operably linked to hF9, which leads to episomal expression of hF9 (at 3E11 viral genomes per mouse). There were six negative control animals with one mouse per group that was injected with buffer alone or AAV8 packaged with the bi-directional hF9 insertion template alone, and two mice per group that were injected with LNP-G009860 or LNP-G000666 alone at 0.3 mg/kg and 1.0 mg/kg, respectively. 
     For analysis, an ELISA was performed to measure levels of hFIX circulating in the mice at each timepoint. Human Factor IX ELISA Kits (ab188393) were used for this purpose, and all plates were run with human pooled normal plasma from George King Bio-Medical as a positive assay control. Spleens and a portion of the left lateral lobe of all livers were submitted for NGS analysis. 
     Human Factor IX expression levels in the plasma samples in each group at weeks 1, 2, and 4 post-injection are shown in  FIG. 18  and in Table 29. In addition, NGS results showing insertion and deletion (indel) levels at the albumin locus in the liver and spleen are shown in Table 29. As shown in  FIG. 18  and Table 29, hFIX was detected in the plasma of treated Alb +/hu F9 −/−  mice at 1, 3, and 4 weeks, with ELISA showing expression values of 0.5-10 μg/mL at 1, 3 and 4 weeks 
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 Human FIX Plasma Levels and NGS Results. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Week 1 
                 Week 3 
                 Week 4 
                 INDEL 
                 INDEL 
               
               
                 Sample 
                 (μg/mL) 
                 (μg/mL) 
                 (μg/mL) 
                 Liver 
                 Spleen 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 S1 PBS 
                 BLD 
                 BLD 
                 BLD 
                 6.12 
                 0.12 
               
               
                 S18 AAV8 only 
                 BLD 
                 BLD 
                 BLD 
                 0.73 
                 0.10 
               
               
                 S2 G000666 only 
                 BLD 
                 BLD 
                 BLD 
                 37.48 
                 0.92 
               
               
                 S4 G000666 only 
                 BLD 
                 BLD 
                 BLD 
                 30.67 
                 1.17 
               
               
                 S19 G009860 only 
                 BLD 
                 BLD 
                 BLD 
                 12.25 
                 0.31 
               
               
                 S20 G009860 only 
                 BLD 
                 BLD 
                 BLD 
                 10.73 
                 0.45 
               
               
                 S10 CAG 
                 42.60 
                 129.83 
                 117.74 
                 1.45 
                 0.12 
               
               
                 S14 CAG 
                 35.55 
                 82.25 
                 100.95 
                 0.08 
                 0.11 
               
               
                 S15 CAG 
                 37.30 
                 115.51 
                 107.26 
                 0.10 
                 0.05 
               
               
                 S16 CAG 
                 36.39 
                 81.27 
                 116.24 
                 0.05 
                 0.10 
               
               
                 S17 CAG 
                 40.50 
                 101.38 
                 124.15 
                 0.16 
                 0.06 
               
               
                 S5 AAV8 + G000666 
                 2.90 
                 5.00 
                 8.79 
                 41.46 
                 1.43 
               
               
                 S6 AAV8 + G000666 
                 4.67 
                 6.11 
                 10.29 
                 33.81 
                 1.59 
               
               
                 S7 AAV8 + G000666 
                 2.88 
                 3.15 
                 3.01 
                 33.47 
                 1.04 
               
               
                 S8 AAV8 + G000666 
                 0.94 
                 1.61 
                 No sample 
                 36.54 
                 1.34 
               
               
                 S9 AAV8 + G000666 
                 7.14 
                 7.53 
                 7.23 
                 30.63 
                 1.38 
               
               
                 S11 AAV8 + 
                 0.73 
                 0.62 
                 0.86 
                 11.15 
                 0.52 
               
               
                 G009860 
                   
                   
                   
                   
                   
               
               
                 S12 AAV8 + 
                 0.52 
                 0.43 
                 0.47 
                 7.05 
                 0.39 
               
               
                 G009860 
                   
                   
                   
                   
                   
               
               
                 S13 AAV8 + 
                 1.71 
                 1.89 
                 0.93 
                 18.38 
                 0.57 
               
               
                 G009860 
                   
                   
                   
                   
                   
               
               
                 S21 AAV8 + 
                 1.21 
                 2.79 
                 0.59 
                 13.44 
                 0.22 
               
               
                 G009860 
                   
                   
                   
                   
                   
               
               
                 S22 AAV8 + 
                 2.06 
                 1.03 
                 2.37 
                 18.06 
                 0.19 
               
               
                 G009860 
                   
                   
                   
                   
                   
               
               
                 Human 
                 4.00 
                 3.91 
                 4.12 
                 N/A 
                 N/A 
               
               
                   
               
            
           
         
       
     
     The remaining liver was fixed for 24 hours in 10% neutral buffered formalin and then transferred to 70% ethanol. Four to five samples from separate lobes were cut and shipped to HistoWiz and were processed and embedded in paraffin blocks. Five-micron sections were then cut from each paraffin block for analysis via BASESCOPE™ on the Ventana Ultra Discovery (Roche) using the universal BASESCOPE™ procedure and reagents by Advanced Cell Diagnostics and a custom designed probe that targets the unique mRNA junction formed between either the human or the mouse albumin signal sequence from the first intron of each respective albumin locus in the ALB ms/hu  mouse and the hF9 transgene when successful integration and transcription is achieved. HALO imaging software (Indica Labs) is used to quantify the percentage of positive cells in each sample. 
     Next, terminal blood was used for assessment of functional coagulation activity by activated partial thromboplastin time (aPTT) and Thrombin Generation Assay (TGA). Activated partial thromboplastin time (aPTT) is a clinical measurement of intrinsic pathway clotting activity in plasma. Plasma is induced to clot by the addition of ellagic acid or kaolin, both of which activate coagulation factor XII in the intrinsic pathway (as known as the contact pathway) of coagulation, that subsequently results in the generation of fibrin from fibrinogen once thrombin is activated. The aPTT assay provides an estimation of an individual&#39;s ability to generate a clot, and this information can be used to determine risk of bleeding or thrombosis. To test aPTT, a semi-automated benchtop system (Diagnostica Stago STart 4) with an electro-mechanical clot detection method (viscosity-based detection system) was used to assess clotting in plasma. To each cuvette with a steel ball, 50 μL of citrated plasma was added and incubated at 37° C. for 5 min, and then clotting was triggered with the addition of 50 μL of ellagic acid (final concentration of 30 μM) at 37° C. for 300 seconds. Following final activation of clotting by adding 50 μL of 0.025 M calcium chloride (final concentration of 8 mM) to each cuvette, the steal ball began to oscillate back and forth between the two drive coils. The movement of the ball was detected by the receiver coil. The generation of fibrin increased plasma viscosity until the ball ceased to move, which was recorded as the clotting time. The only parameter measured was clotting time. Runs were conducted in duplicate. 
     Thrombin generation assay (TGA) is a non-clinical assessment of the kinetics of thrombin generation in activated plasma. Thrombin generation is an essential process of coagulation because thrombin is responsible for activation of other coagulation factors and propagation of additional thrombin (via FXI activation) for the conversion of fibrinogen to fibrin. Thrombin generation assay provides an estimation of an individual&#39;s ability to generate thrombin, and this information can be used to determine risk of bleeding or thrombosis. To perform the TGA, a calibrated automated thrombogram was used to assess thrombin generation levels in a spectrophotometer (Thrombinograph™, Thermo Scientific). For high throughput experimentation, 96-well plates (Immulon II HB) were used. To each well, 55 μL of citrated plasma (4× diluted with saline for mouse plasma) was added and incubated at 37° C. for 30 min. Thrombin generation is triggered with the addition of 15 μL of 2 μM ellagic acid (final concentration of 0.33 μM) at 37° C. for 45 min. Thrombin generation was determined following the automated injection of 15 μL of the fluorogenic substrate with 16 mM CaCl 2 ) (FluCa; Thrombinoscope BV) into each well. The fluorogenic substrate reacted with the generated thrombin, which was measured continuously in the plasma every 33 sec for 90 min at 460 nm. The fluorescence intensity was proportional to the proteolytic activity of thrombin. The main parameters measured in the tracing were lag time, peak thrombin generation, time to peak thrombin generation, and endogenous thrombin potential (ETP). The lag time provides an estimation of time required for initial detection of thrombin in plasma. The peak is the maximum amount of thrombin generated at a given time after activation. Time to peak thrombin generation is time from initiation of the coagulation cascade to the peak generation of thrombin. ETP is the total amount of thrombin generated during the 60 minutes measured. Runs were conducted in duplicate. 
     As shown in  FIG. 19  and Table 30, insertion of the hF9 transgene using for example G000666 showed recovered clotting function in the aPTT assay. AAV only and LNP only negative control samples showed prolonged aPTT times of 45-60 seconds in saline. The positive control CAGG and test samples AAV8+LNP were closer to the normal human aPTT of 28-34 seconds. 
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 aPTT and TGA-EA. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Week 4  
                 Average  
                 TGA-EA  
               
               
                   
                   
                 F9 
                 aPTT 
                 Peak 
               
               
                 Sample # 
                 I.V. Injection 
                 μg/mL 
                 (sec) 
                 (nM) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 PBS 
                 BLD 
                 40.2 
                 11.13 
               
               
                 18 
                 AAV Only 
                 BLD 
                 62.5 
                 −1 
               
               
                 2 
                 LNP g666 only 
                 BLD 
                 53.9 
                 −1 
               
               
                 4 
                 LNP g666 only 
                 BLD 
                 65.0 
                 2.45 
               
               
                 19 
                 LNP G009860 only 
                 BLD 
                 34.1 
                 42.83 
               
               
                 20 
                 LNP G009860 only 
                 BLD 
                 56.7 
                 18.07 
               
               
                 10 
                 AAV + CAGG F9 
                 117.74 
                 41.1 
                 42.65 
               
               
                 14 
                 AAV + CAGG F9 
                 100.95 
                 34.1 
                 49.96 
               
               
                 15 
                 AAV + CAGG F9 
                 107.26 
                 42.2 
                 49.49 
               
               
                 16 
                 AAV + CAGG F9 
                 116.24 
                 37.9 
                 44.46 
               
               
                 17 
                 AAV + CAGG F9 
                 124.15 
                 44.1 
                 38.02 
               
               
                 5 
                 AAV + g666 
                 8.79 
                 31.3 
                 72.11 
               
               
                 6 
                 AAV + g666 
                 10.29 
                 32.6 
                 90.14 
               
               
                 7 
                 AAV + g666 
                 3.01 
                 33.5 
                 58.33 
               
               
                 8 
                 AAV + g666 
                 no sample 
                 NA 
                 NA 
               
               
                 9 
                 AAV + g666 
                 7.23 
                 25.9 
                 67.23 
               
               
                 11 
                 AAV + G009860 
                 0.86 
                 36.8 
                 56.92 
               
               
                 12 
                 AAV + G009860 
                 0.47 
                 37.7 
                 45.16 
               
               
                 13 
                 AAV + G009860 
                 0.93 
                 35.3 
                 60.45 
               
               
                 21 
                 AAV + G009860 
                 0.59 
                 36.1 
                 47.44 
               
               
                 22 
                 AAV + G009860 
                 2.37 
                 &gt;300 
                 Clots in tube 
               
               
                   
               
            
           
         
       
     
     As shown in  FIG. 20A ,  FIG. 20B , and  FIG. 21  and in Table 30, insertion of the hF9 transgene using for example G000666 showed increased thrombin generation in TGA-EA analysis. Thrombin concentrations were higher in the positive control CAGG and AAV8+LNP as compared to the negative control samples. 
     In conclusion, hFIX was detected in the plasma of Alb +/hu /F9 −/−  mice at 1, 3, and 4 weeks, and the expressed hFIX-R338L was found to be functional since thrombin was generated in a TGA assay, and aPTT clotting time was improved. 
     
       
         
           
               
            
               
                 Human albumin intron 1: 
               
               
                 (SEQ ID NO: 1) 
               
               
                 GTAAGAAATCCATTTTTCTATTGTTCAACTTTTATTCTATTTTCCCAG 
               
               
                   
               
               
                 TAAAATAAAGTTTTAGTAAACTCTGCATCTTTAAAGAATTATTTTGGC 
               
               
                   
               
               
                 ATTTATTTCTAAAATGGCATAGTATTTTGTATTTGTGAAGTCTTACAA 
               
               
                   
               
               
                 GGTTATCTTATTAATAAAATTCAAACATCCTAGGTAAAAAAAAAAAAA 
               
               
                   
               
               
                 GGTCAGAATTGTTTAGTGACTGTAATTTTCTTTTGCGCACTAAGGAAA 
               
               
                   
               
               
                 GTGCAAAGTAACTTAGAGTGACTGAAACTTCACAGAATAGGGTTGAAG 
               
               
                   
               
               
                 ATTGAATTCATAACTATCCCAAAGACCTATCCATTGCACTATGCTTTA 
               
               
                   
               
               
                 TTTAAAAACCACAAAACCTGTGCTGTTGATCTCATAAATAGAACTTGT 
               
               
                   
               
               
                 ATTTATATTTATTTTCATTTTAGTCTGTCTTCTTGGTTGCTGTTGATA 
               
               
                   
               
               
                 GACACTAAAAGAGTATTAGATATTATCTAAGTTTGAATATAAGGCTAT 
               
               
                   
               
               
                 AAATATTTAATAATTTTTAAAATAGTATTCTTGGTAATTGAATTATTC 
               
               
                   
               
               
                 TTCTGTTTAAAGGCAGAAGAAATAATTGAACATCATCCTGAGTTTTTC 
               
               
                   
               
               
                 TGTAGGAATCAGAGCCCAATATTTTGAAACAAATGCATAATCTAAGTC 
               
               
                   
               
               
                 AAATGGAAAGAAATATAAAAAGTAACATTATTACTTCTTGTTTTCTTC 
               
               
                   
               
               
                 AGTATTTAACAATCCTTTTTTTTCTTCCCTTGCCCAG 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Mouse albumin guide RNA 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
               
               
                 Guide 
                   
                 Mouse Genomic 
                 ID 
               
               
                 ID 
                 Guide Sequence 
                 Coordinates (mm10) 
                 NO: 
               
               
                   
               
               
                 G000551 
                 AUUUGCAUCUGAGAACCCUU 
                 chr5:90461148-90461168 
                  98 
               
               
                   
               
               
                 G000552 
                 AUCGGGAACUGGCAUCUUCA 
                 chr5:90461590-90461610 
                  99 
               
               
                   
               
               
                 G000553 
                 GUUACAGGAAAAUCUGAAGG 
                 chr5:90461569-90461589 
                 100 
               
               
                   
               
               
                 G000554 
                 GAUCGGGAACUGGCAUCUUC 
                 chr5:90461589-90461609 
                 101 
               
               
                   
               
               
                 G000555 
                 UGCAUCUGAGAACCCUUAGG 
                 chr5:90461151-90461171 
                 102 
               
               
                   
               
               
                 G000666 
                 CACUCUUGUCUGUGGAAACA 
                 chr5:90461709-90461729 
                 103 
               
               
                   
               
               
                 G000667 
                 AUCGUUACAGGAAAAUCUGA 
                 chr5:90461572-90461592 
                 104 
               
               
                   
               
               
                 G000668 
                 GCAUCUUCAGGGAGUAGCUU 
                 chr5:90461601-90461621 
                 105 
               
               
                   
               
               
                 G000669 
                 CAAUCUUUAAAUAUGUUGUG 
                 chr5:90461674-90461694 
                 106 
               
               
                   
               
               
                 G000670 
                 UCACUCUUGUCUGUGGAAAC 
                 chr5:90461710-90461730 
                 107 
               
               
                   
               
               
                 G011722 
                 UGCUUGUAUUUUUCUAGUAA 
                 chr5:90461039-90461059 
                 108 
               
               
                   
               
               
                 G011723 
                 GUAAAUAUCUACUAAGACAA 
                 chr5:90461425-90461445 
                 109 
               
               
                   
               
               
                 G011724 
                 UUUUUCUAGUAAUGGAAGCC 
                 chr5:90461047-90461067 
                 110 
               
               
                   
               
               
                 G011725 
                 UUAUAUUAUUGAUAUAUUUU 
                 chr5:90461174-90461194 
                 111 
               
               
                   
               
               
                 G011726 
                 GCACAGAUAUAAACACUUAA 
                 chr5:90461480-90461500 
                 112 
               
               
                   
               
               
                 G011727 
                 CACAGAUAUAAACACUUAAC 
                 chr5:90461481-90461501 
                 113 
               
               
                   
               
               
                 G011728 
                 GGUUUUAAAAAUAAUAAUGU 
                 chr5:90461502-90461522 
                 114 
               
               
                   
               
               
                 G011729 
                 UCAGAUUUUCCUGUAACGAU 
                 chr5:90461572-90461592 
                 115 
               
               
                   
               
               
                 G011730 
                 CAGAUUUUCCUGUAACGAUC 
                 chr5:90461573-90461593 
                 116 
               
               
                   
               
               
                 G011731 
                 CAAUGGUAAAUAAGAAAUAA 
                 chr5:90461408-90461428 
                 117 
               
               
                   
               
               
                 G013018 
                 GGAAAAUCUGAAGGUGGCAA 
                 chr5:90461563-90461583 
                 118 
               
               
                   
               
               
                 G013019 
                 GGCGAUCUCACUCUUGUCUG 
                 chr5:90461717-90461737 
                 119 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Mouse albumin sgRNAs and modification pattern 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                   
                 SEQ 
               
               
                 Guide 
                   
                 ID 
                   
                 ID 
               
               
                 ID 
                 Full Sequence 
                 NO: 
                 Full Sequence Modified 
                 NO: 
               
               
                   
               
               
                 G000551 
                 AUUUGCAUCUGAGAACCCUUGU 
                 120 
                 mA*mU*mU*UGCAUCUGAGAACCCUUGUUUUAGAm 
                 142 
               
               
                   
                 UUUAGAGCUAGAAAUAGCAAGU 
                   
                 GmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAU 
                   
               
               
                   
                 UAAAAUAAGGCUAGUCCGUUAU 
                   
                 AAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAm 
                   
               
               
                   
                 CAACUUGAAAAAGUGGCACCGA 
                   
                 AmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG 
                   
               
               
                   
                 GUCGGUGCUUUU 
                   
                 mGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G000552 
                 AUCGGGAACUGGCAUCUUCA 
                 121 
                 mA*mU*mC*GGGAACUGGCAUCUUCAGUUUUAGAm 
                 143 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 GmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAU 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 mGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G000553 
                 GUUACAGGAAAAUCUGAAGG 
                 122 
                 mG*mU*mU*ACAGGAAAAUCUGAAGGGUUUUAGA 
                 144 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 mGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G000554 
                 GAUCGGGAACUGGCAUCUUC 
                 123 
                 mG*mA*mU*CGGGAACUGGCAUCUUCGUUUUAGAm 
                 145 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 GmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAU 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 mGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G000555 
                 UGCAUCUGAGAACCCUUAGG 
                 124 
                 mU*mG*mC*AUCUGAGAACCCUUAGGGUUUUAGAm 
                 146 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 GmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAU 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 mGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G000666 
                 CACUCUUGUCUGUGGAAACA 
                 125 
                 mC*mA*mC*UCUUGUCUGUGGAAACAGUUUUAGAm 
                 147 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 GmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAU 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 mGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G000667 
                 AUCGUUACAGGAAAAUCUGA 
                 126 
                 mA*mU*mC*GUUACAGGAAAAUCUGAGUUUUAGA 
                 148 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 mGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G000668 
                 GCAUCUUCAGGGAGUAGCUU 
                 127 
                 mG*mC*mA*UCUUCAGGGAGUAGCUUGUUUUAGA 
                 149 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 mGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G000669 
                 CAAUCUUUAAAUAUGUUGUG 
                 128 
                 mC*mA*mA*UCUUUAAAUAUGUUGUGGUUUUAGA 
                 150 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 mGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G000670 
                 UCACUCUUGUCUGUGGAAAC 
                 129 
                 mU*mC*mA*CUCUUGUCUGUGGAAACGUUUUAGAm 
                 151 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 GmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAU 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 mGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G011722 
                 UGCUUGUAUUUUUCUAGUAA 
                 130 
                 mU*mG*mC*UUGUAUUUUUCUAGUAAGUUUUAGA 
                 152 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 mGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G011723 
                 GUAAAUAUCUACUAAGACAA 
                 131 
                 mG*mU*mA*AAUAUCUACUAAGACAAGUUUUAGA 
                 153 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 mGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G011724 
                 UUUUUCUAGUAAUGGAAGCC 
                 132 
                 mU*mU*mU*UUCUAGUAAUGGAAGCCGUUUUAGA 
                 154 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 mGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G011725 
                 UUAUAUUAUUGAUAUAUUUU 
                 133 
                 mU*mU*mA*UAUUAUUGAUAUAUUUUGUUUUAGA 
                 155 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 mGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G011726 
                 GCACAGAUAUAAACACUUAA 
                 134 
                 mG*mC*mA*CAGAUAUAAACACUUAAGUUUUAGA 
                 156 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 mGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G011727 
                 CACAGAUAUAAACACUUAAC 
                 135 
                 mC*mA*mC*AGAUAUAAACACUUAACGUUUUAGAm 
                 157 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 GmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAU 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 mGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G011728 
                 GGUUUUAAAAAUAAUAAUGU 
                 136 
                 mG*mG*mU*UUUAAAAAUAAUAAUGUGUUUUAGA 
                 158 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 mGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G011729 
                 UCAGAUUUUCCUGUAACGAU 
                 137 
                 mU*mC*mA*GAUUUUCCUGUAACGAUGUUUUAGA 
                 159 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 mGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G011730 
                 CAGAUUUUCCUGUAACGAUC 
                 138 
                 mC*mA*mG*AUUUUCCUGUAACGAUCGUUUUAGAm 
                 160 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 GmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAU 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 mGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G011731 
                 CAAUGGUAAAUAAGAAAUAA 
                 139 
                 mC*mA*mA*UGGUAAAUAAGAAAUAAGUUUUAGA 
                 161 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 mGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G013018 
                 GGAAAAUCUGAAGGUGGCAA 
                 140 
                 mG*mG*mA*AAAUCUGAAGGUGGCAAGUUUUAGA 
                 162 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 mGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmA 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 mAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G013019 
                 GGCGAUCUCACUCUUGUCUG 
                 141 
                 mG*mG*mC*GAUCUCACUCUUGUCUGGUUUUAGAm 
                 163 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 GmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAU 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 mGmUmGmCmU*mU*mU*mU 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Cyno albumin guide RNA 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
               
               
                 Guide 
                   
                 Cyno Genomic 
                 ID 
               
               
                 ID 
                 Guide Sequence 
                 Coordinates (mf5) 
                 NO: 
               
               
                   
               
               
                 G009844 
                 GAGCAACCUCACUCUUGUCU 
                 chr5:61198711-61198731 
                   2 
               
               
                   
               
               
                 G009845 
                 AGCAACCUCACUCUUGUCUG 
                 chr5:61198712-61198732 
                 165 
               
               
                   
               
               
                 G009846 
                 ACCUCACUCUUGUCUGGGGA 
                 chr5:61198716-61198736 
                 166 
               
               
                   
               
               
                 G009847 
                 CCUCACUCUUGUCUGGGGAA 
                 chr5:61198717-61198737 
                 167 
               
               
                   
               
               
                 G009848 
                 CUCACUCUUGUCUGGGGAAG 
                 chr5:61198718-61198738 
                 168 
               
               
                   
               
               
                 G009849 
                 GGGGAAGGGGAGAAAAAAAA 
                 chr5:61198731-61198751 
                 169 
               
               
                   
               
               
                 G009850 
                 GGGAAGGGGAGAAAAAAAAA 
                 chr5:61198732-61198752 
                 170 
               
               
                   
               
               
                 G009851 
                 AUGCAUUUGUUUCAAAAUAU 
                 chr5:61198825-61198845 
                   3 
               
               
                   
               
               
                 G009852 
                 UGCAUUUGUUUCAAAAUAUU 
                 chr5:61198826-61198846 
                 172 
               
               
                   
               
               
                 G009853 
                 UGAUUCCUACAGAAAAAGUC 
                 chr5:61198852-61198872 
                 173 
               
               
                   
               
               
                 G009854 
                 UACAGAAAAAGUCAGGAUAA 
                 chr5:61198859-61198879 
                 174 
               
               
                   
               
               
                 G009855 
                 UUUCUUCUGCCUUUAAACAG 
                 chr5:61198889-61198909 
                 175 
               
               
                   
               
               
                 G009856 
                 UUAUAGUUUUAUAUUCAAAC 
                 chr5:61198957-61198977 
                 176 
               
               
                   
               
               
                 G009857 
                 AUUUAUGAGAUCAACAGCAC 
                 chr5:61199062-61199082 
                   5 
               
               
                   
               
               
                 G009858 
                 GAUCAACAGCACAGGUUUUG 
                 chr5:61199070-61199090 
                   6 
               
               
                   
               
               
                 G009859 
                 UUAAAUAAAGCAUAGUGCAA 
                 chr5:61199096-61199116 
                   7 
               
               
                   
               
               
                 G009860 
                 UAAAGCAUAGUGCAAUGGAU 
                 chr5:61199101-61199121 
                   8 
               
               
                   
               
               
                 G009861 
                 UAGUGCAAUGGAUAGGUCUU 
                 chr5:61199108-61199128 
                   9 
               
               
                   
               
               
                 G009862 
                 AGUGCAAUGGAUAGGUCUUA 
                 chr5:61199109-61199129 
                 182 
               
               
                   
               
               
                 G009863 
                 UUACUUUGCACUUUCCUUAG 
                 chr5:61199186-61199206 
                 183 
               
               
                   
               
               
                 G009864 
                 UACUUUGCACUUUCCUUAGU 
                 chr5:61199187-61199207 
                 184 
               
               
                   
               
               
                 G009865 
                 UCUGACCUUUUAUUUUACCU 
                 chr5:61199238-61199258 
                 185 
               
               
                   
               
               
                 G009866 
                 UACUAAAACUUUAUUUUACU 
                 chr5:61199367-61199387 
                  10 
               
               
                   
               
               
                 G009867 
                 AAAGUUGAACAAUAGAAAAA 
                 chr5:61199401-61199421 
                  11 
               
               
                   
               
               
                 G009868 
                 AAUGCAUAAUCUAAGUCAAA 
                 chr5:61198812-61198832 
                  12 
               
               
                   
               
               
                 G009869 
                 AUUAUCCUGACUUUUUCUGU 
                 chr5:61198860-61198880 
                 189 
               
               
                   
               
               
                 G009870 
                 UGAAUUAUUCCUCUGUUUAA 
                 chr5:61198901-61198921 
                 190 
               
               
                   
               
               
                 G009871 
                 UAAUUUUCUUUUGCCCACUA 
                 chr5:61199203-61199223 
                 191 
               
               
                   
               
               
                 G009872 
                 AAAAGGUCAGAAUUGUUUAG 
                 chr5:61199229-61199249 
                 192 
               
               
                   
               
               
                 G009873 
                 AACAUCCUAGGUAAAAUAAA 
                 chr5:61199246-61199266 
                 193 
               
               
                   
               
               
                 G009874 
                 UAAUAAAAUUCAAACAUCCU 
                 chr5:61199258-61199278 
                  13 
               
               
                   
               
               
                 G009875 
                 UUGUCAUGUAUUUCUAAAAU 
                 chr5:61199322-61199342 
                 195 
               
               
                   
               
               
                 G009876 
                 UUUGUCAUGUAUUUCUAAAA 
                 chr5:61199323-61199343 
                 196 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Cyno sgRNA and modification patterns 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                   
                 SEQ 
               
               
                 Guide 
                   
                 ID 
                   
                 ID 
               
               
                 ID 
                 Full Sequence 
                 NO: 
                 Full Sequence Modified 
                 NO: 
               
               
                   
               
               
                 G009844 
                 GAGCAACCUCACUCUUGUCU 
                  34 
                 mG*mA*mG*CAACCUCACUCUUGUCUGUUUUAG 
                  66 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAAUAAGGCUAGUCCGUUAUCAmAmCmUmUm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 GmAmAmAmAmAmGmUmGmGmCmAmCmCmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 AmGmUmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009845 
                 AGCAACCUCACUCUUGUCUG 
                 198 
                 mA*mG*mC*AACCUCACUCUUGUCUGGUUUUAG 
                 231 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAAUAAGGCUAGUCCGUUAUCAmAmCmUmUm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 GmAmAmAmAmAmGmUmGmGmCmAmCmCmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 AmGmUmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009846 
                 ACCUCACUCUUGUCUGGGGA 
                 199 
                 mA*mC*mC*UCACUCUUGUCUGGGGAGUUUU 
                 232 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AGAmGmCmUmAmGmAmAmAmUmAmGmCAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 GUUAAAAUAAGGCUAGUCCGUUAUCAmAmCm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 UmUmGmAmAmAmAmAmGmUmGmGmCmAmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 CmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009847 
                 CCUCACUCUUGUCUGGGGAA 
                 200 
                 mC*mC*mU*CACUCUUGUCUGGGGAAGUUUUA 
                 233 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 GAmGmCmUmAmGmAmAmAmUmAmGmCAAGU 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 UAAAAUAAGGCUAGUCCGUUAUCAmAmCmUm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 UmGmAmAmAmAmAmGmUmGmGmCmAmCmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 GmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009848 
                 CUCACUCUUGUCUGGGGAAG 
                 201 
                 mC*mU*mC*ACUCUUGUCUGGGGAAGGUUUU 
                 234 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AGAmGmCmUmAmGmAmAmAmUmAmGmCAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 GUUAAAAUAAGGCUAGUCCGUUAUCAmAmCm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 UmUmGmAmAmAmAmAmGmUmGmGmCmAmCm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 CmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009849 
                 GGGGAAGGGGAGAAAAAAAA 
                 202 
                 mG*mG*mG*GAAGGGGAGAAAAAAAAGUUUUAG 
                 235 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009850 
                 GGGAAGGGGAGAAAAAAAAA 
                 203 
                 mG*mG*mG*AAGGGGAGAAAAAAAAAGUUUUAG 
                 236 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009851 
                 AUGCAUUUGUUUCAAAAUAU 
                  35 
                 mA*mU*mG*CAUUUGUUUCAAAAUAUGUUUUAG 
                  67 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009852 
                 UGCAUUUGUUUCAAAAUAUU 
                  36 
                 mU*mG*mC*AUUUGUUUCAAAAUAUUGUUUUAG 
                  68 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009853 
                 UGAUUCCUACAGAAAAAGUC 
                 206 
                 mU*mG*mA*UUCCUACAGAAAAAGUCGUUUUAG 
                 239 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009854 
                 UACAGAAAAAGUCAGGAUAA 
                 207 
                 mU*mA*mC*AGAAAAAGUCAGGAUAAGUUUUAG 
                 240 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009855 
                 UUUCUUCUGCCUUUAAACAG 
                 208 
                 mU*mU*mU*CUUCUGCCUUUAAACAGGUUUUAG 
                 241 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009856 
                 UUAUAGUUUUAUAUUCAAAC 
                 209 
                 mU*mU*mA*UAGUUUUAUAUUCAAACGUUUUAG 
                 242 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009857 
                 AUUUAUGAGAUCAACAGCAC 
                  37 
                 mA*mU*mU*UAUGAGAUCAACAGCACGUUUUAG 
                  69 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009858 
                 GAUCAACAGCACAGGUUUUG 
                  38 
                 mG*mA*mU*CAACAGCACAGGUUUUGGUUUUAG 
                  70 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009859 
                 UUAAAUAAAGCAUAGUGCAA 
                  39 
                 mU*mU*mA*AAUAAAGCAUAGUGCAAGUUUUAG 
                  71 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009860 
                 UAAAGCAUAGUGCAAUGGAU 
                  40 
                 mU*mA*mA*AGCAUAGUGCAAUGGAUGUUUUAG 
                  72 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009861 
                 UAGUGCAAUGGAUAGGUCUU 
                  41 
                 mU*mA*mG*UGCAAUGGAUAGGUCUUGUUUUAG 
                  73 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009862 
                 AGUGCAAUGGAUAGGUCUUA 
                 215 
                 mA*mG*mU*GCAAUGGAUAGGUCUUAGUUUUAG 
                 248 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009863 
                 UUACUUUGCACUUUCCUUAG 
                 216 
                 mU*mU*mA*CUUUGCACUUUCCUUAGGUUUUAG 
                 249 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009864 
                 UACUUUGCACUUUCCUUAGU 
                 217 
                 mU*mA*mC*UUUGCACUUUCCUUAGUGUUUUAG 
                 250 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009865 
                 UCUGACCUUUUAUUUUACCU 
                 218 
                 mU*mC*mU*GACCUUUUAUUUUACCUGUUUUAG 
                 251 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009866 
                 UACUAAAACUUUAUUUUACU 
                  42 
                 mU*mA*mC*UAAAACUUUAUUUUACUGUUUUAG 
                  74 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009867 
                 AAAGUUGAACAAUAGAAAAA 
                  43 
                 mA*mA*mA*GUUGAACAAUAGAAAAAGUUUUAG 
                  75 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009868 
                 AAUGCAUAAUCUAAGUCAAA 
                  44 
                 mA*mA*mU*GCAUAAUCUAAGUCAAAGUUUUAG 
                  76 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009869 
                 AUUAUCCUGACUUUUUCUGU 
                 222 
                 mA*mU*mU*AUCCUGACUUUUUCUGUGUUUUAG 
                 255 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009870 
                 UGAAUUAUUCCUCUGUUUAA 
                 223 
                 mU*mG*mA*AUUAUUCCUCUGUUUAAGUUUUAG 
                 256 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009871 
                 UAAUUUUCUUUUGCCCACUA 
                 224 
                 mU*mA*mA*UUUUCUUUUGCCCACUAGUUUUAG 
                 257 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 GmAmAmAmAmAmGmUmGmGmCmAmCmCmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 AmGmUmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009872 
                 AAAAGGUCAGAAUUGUUUAG 
                 225 
                 mA*mA*mA*AGGUCAGAAUUGUUUAGGUUUUAG 
                 258 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009873 
                 AACAUCCUAGGUAAAAUAAA 
                 226 
                 mA*mA*mC*AUCCUAGGUAAAAUAAAGUUUUAG 
                 259 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009874 
                 UAAUAAAAUUCAAACAUCCU 
                  45 
                 mU*mA*mA*UAAAAUUCAAACAUCCUGUUUUAG 
                  77 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009875 
                 UUGUCAUGUAUUUCUAAAAU 
                 228 
                 mU*mU*mG*UCAUGUAUUUCUAAAAUGUUUUAG 
                 261 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G009876 
                 UUUGUCAUGUAUUUCUAAAA 
                 229 
                 mU*mU*mU*GUCAUGUAUUUCUAAAAGUUUUAG 
                 262 
               
               
                   
                 GUUUUAGAGCUAGAAAUAGC 
                   
                 AmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAA 
                   
               
               
                   
                 AAGUUAAAAUAAGGCUAGUC 
                   
                 AAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGm 
                   
               
               
                   
                 CGUUAUCAACUUGAAAAAGU 
                   
                 AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm 
                   
               
               
                   
                 GGCACCGAGUCGGUGCUUUU 
                   
                 UmCmGmGmUmGmCmU*mU*mU*mU 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Vector Components and Sequences 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 1 st  orientation 
                 2 nd  orientation 
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Plasmid 
                 5′ 
                 Splice 
                   
                   
                   
                   
                 Splice 
                 3′ 
               
               
                 ID 
                 ITR 
                 Acceptor 
                 Transgene 
                 Poly-A 
                 Poly-A 
                 Transgene 
                 Acceptor 
                 ITR 
               
               
                   
               
               
                 P00147 
                 (SEQ 
                 Mouse 
                 Human 
                 SEQ 
                 SEQ 
                 Human 
                 Mouse 
                 (SEQ 
               
               
                   
                 ID 
                 Albumin 
                 Factor IX 
                 ID NO: 
                 ID NO: 
                 Factor IX 
                 Albumin 
                 ID 
               
               
                   
                 NO: 
                 Splice 
                 (R338L) 
                 266 
                 267 
                 (R338L) 
                 Splice 
                 NO: 
               
               
                   
                 263) 
                 Acceptor 
                 (SEQ ID 
                   
                   
                 (SEQ ID 
                 Acceptor 
                 270) 
               
               
                   
                   
                 (SEQ ID 
                 NO: 265) 
                   
                   
                 NO: 268) 
                 (SEQ ID 
                   
               
               
                   
                   
                 NO: 264) 
                   
                   
                   
                   
                 NO: 269) 
                   
               
               
                   
               
               
                 P00411 
                 (SEQ 
                 Human 
                 Human 
                 SEQ 
                 SEQ 
                 Human 
                 Human 
                 (SEQ 
               
               
                   
                 ID 
                 Factor 
                 Factor IX 
                 ID NO: 
                 ID NO: 
                 Factor IX 
                 Factor 
                 ID 
               
               
                   
                 NO: 
                 IX Splice 
                 (R338L)- 
                 266 
                 267 
                 (R338L)- 
                 IX Splice 
                 NO: 
               
               
                   
                 263) 
                 Acceptor 
                 HiBit 
                   
                   
                 HiBit (SEQ 
                 Acceptor 
                 270) 
               
               
                   
                   
                 (SEQ ID 
                 (SEQ ID 
                   
                   
                 ID NO: 
                 (SEQ ID 
                   
               
               
                   
                   
                 NO: 271) 
                 NO: 272) 
                   
                   
                 273) 
                 NO: 274) 
                   
               
               
                   
               
               
                 P00415 
                 (SEQ 
                 Mouse 
                 Nluc-P2A- 
                 SEQ 
                 SEQ 
                 Nluc-P2A- 
                 Mouse 
                 (SEQ 
               
               
                   
                 ID 
                 Albumin 
                 GFP (SEQ 
                 ID NO: 
                 ID NO: 
                 GFP (SEQ 
                 Albumin 
                 ID 
               
               
                   
                 NO: 
                 Splice 
                 ID NO: 
                 266 
                 267 
                 ID NO: 
                 Splice 
                 NO: 
               
               
                   
                 263) 
                 Acceptor 
                 275) 
                   
                   
                 276) 
                 Acceptor 
                 270) 
               
               
                   
                   
                 (SEQ ID 
                   
                   
                   
                   
                 (SEQ ID 
                   
               
               
                   
                   
                 NO: 264) 
                   
                   
                   
                   
                 NO: 269) 
                   
               
               
                   
               
               
                 P00418 
                 (SEQ 
                 Mouse 
                 Human 
                 SEQ 
                 SEQ 
                 Human 
                 Mouse 
                 (SEQ 
               
               
                   
                 ID 
                 Albumin 
                 Factor IX 
                 ID NO: 
                 ID NO: 
                 Factor IX 
                 Albumin 
                 ID 
               
               
                   
                 NO: 
                 Splice 
                 (R338L)- 
                 266 
                 267 
                 (R338L)- 
                 Splice 
                 NO: 
               
               
                   
                 263) 
                 Acceptor 
                 HiBit 
                   
                   
                 HiBit (SEQ 
                 Acceptor 
                 270) 
               
               
                   
                   
                 (SEQ ID 
                 (SEQ ID 
                   
                   
                 ID NO: 
                 (SEQ ID 
                   
               
               
                   
                   
                 NO: 264) 
                 NO: 272) 
                   
                   
                 273) 
                 NO: 269) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
            
               
                 5′ ITR Sequence (SEQ ID NO: 263): 
                   
               
               
                 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC 
               
               
                   
               
               
                 GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA 
               
               
                   
               
               
                 GGGAGTGGCCAACTCCATCACTAGGGGTTCCT 
               
               
                   
               
               
                 Mouse Albumin Splice Acceptor (1 st  orientation) (SEQ ID NO: 264): 
               
               
                 TAGGTCAGTGAAGAGAAGAACAAAAAGCAGCATATTACAGTTAGTTGTCTTCATCA 
               
               
                   
               
               
                 ATCTTTAAATATGTTGTGTGGTTTTTCTCTCCCTGTTTCCACAG 
               
               
                   
               
               
                 Human Factor IX (R338L), 1 st  Orientation (SEQ ID NO: 265): 
               
               
                 TTTCTTGATCATGAAAACGCCAACAAAATTCTGAATCGGCCAAAGAGGTATAATTCA 
               
               
                   
               
               
                 GGTAAATTGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGTATGGAAGAAAA 
               
               
                   
               
               
                 GTGTAGTTTTGAAGAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACTGAAT 
               
               
                   
               
               
                 TTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGCG 
               
               
                   
               
               
                 GCAGTTGCAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG 
               
               
                   
               
               
                 GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGCAG 
               
               
                   
               
               
                 TTTTGTAAAAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGA 
               
               
                   
               
               
                 CTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGAAGAGTT 
               
               
                   
               
               
                 TCTGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGATGTGGACT 
               
               
                   
               
               
                 ATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAAGCACCCAAT 
               
               
                   
               
               
                 CATTTAATGACTTCACTCGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAATTCC 
               
               
                   
               
               
                 CTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATCGTTA 
               
               
                   
               
               
                 ATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTGAAACTGGTGTTAAAATTACAG 
               
               
                   
               
               
                 TTGTCGCAGGTGAACATAATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT 
               
               
                   
               
               
                 GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTATTAATAAGTACAACCAT 
               
               
                   
               
               
                 GACATTGCCCTTCTGGAACTGGACGAACCCTTAGTGCTAAACAGCTACGTTACACCT 
               
               
                   
               
               
                 ATTTGCATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTGGATCTGGCTAT 
               
               
                   
               
               
                 GTAAGTGGCTGGGGAAGAGTCTTCCACAAAGGGAGATCAGCTTTAGTTCTTCAGTAC 
               
               
                   
               
               
                 CTTAGAGTTCCACTTGTTGACCGAGCCACATGTCTTCTATCTACAAAGTTCACCATCT 
               
               
                   
               
               
                 ATAACAACATGTTCTGTGCTGGCTTCCATGAAGGAGGTAGAGATTCATGTCAAGGAG 
               
               
                   
               
               
                 ATAGTGGGGGACCCCATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAATTA 
               
               
                   
               
               
                 TTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAATATGGAATATATACCAAGGTA 
               
               
                   
               
               
                 TCCCGGTATGTCAACTGGATTAAGGAAAAAACAAAGCTCACTTAA 
               
               
                   
               
               
                 Poly-A (1 st  orientation) (SEQ ID NO: 266): 
               
               
                 CCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTC 
               
               
                   
               
               
                 CTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGC 
               
               
                   
               
               
                 ATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAG 
               
               
                   
               
               
                 CAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTA 
               
               
                   
               
               
                 TGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCC 
               
               
                   
               
               
                 Poly-A (2 nd  orientation) (SEQ ID NO: 267): 
               
               
                 AAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGT 
               
               
                   
               
               
                 TGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAA 
               
               
                   
               
               
                 TTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATC 
               
               
                   
               
               
                 AATGTATCTTATCATGTCTG 
               
               
                   
               
               
                 Human Factor IX (R338L), 2 nd  Orientation (SEQ ID NO: 268): 
               
               
                 TTAGGTGAGCTTAGTCTTTTCTTTTATCCAATTCACGTAGCGAGAGACCTTCGTATAG 
               
               
                   
               
               
                 ATGCCATATTTCCCCTTCATCGCACATTCCTCCCCCCAACTTATTATCCCGGTCAAGA 
               
               
                   
               
               
                 AACTTGTTCCTTCGACTTCAGTGACGTGTGGTCCACCTGAATCACCTTGGCATGAGTC 
               
               
                   
               
               
                 GCGACCGCCCTCGTGAAACCCAGCACAAAACATGTTATTGTAAATCGTAAATTTCGT 
               
               
                   
               
               
                 GGACAGAAGACAGGTCGCTCTATCGACCAACGGGACGCGCAAATATTGCAGAACGA 
               
               
                   
               
               
                 GGGCTGATCGACCTTTGTGGAAGACCCGCCCCCACCCACTCACATATCCGCTCCCAA 
               
               
                   
               
               
                 ATTTCAAGAAGATATTTGTATATTCTTTATCGGCTATACAAATCGGGGTAACATAGG 
               
               
                   
               
               
                 AGTTAAGTACGAGTGGCTCGTCCAGCTCCAGGAGGGCTATATCATGGTTGTACTTGT 
               
               
                   
               
               
                 TTATAGCGGCATTATAATTGTGATGGGGTATGATCCTGATAACATTCCTTTTCTGTTC 
               
               
                   
               
               
                 AGTATGCTCAGTTTCTTCAATGTTGTGTTCGCCAGCCACGACCGTAATCTTAACCCCC 
               
               
                   
               
               
                 GTCTCGACACAGTGTGCGGCCGTTACAATCCACTTTTCATTGACTATGGAGCCCCCA 
               
               
                   
               
               
                 CAAAACGCGTCGACTTTTCCGTTGAGCACCACCTGCCATGGAAATTGGCCAGGTTTA 
               
               
                   
               
               
                 GCGTCCTCGCCCCCGACAACCCTAGTAAAGTCATTAAATGACTGTGTGGATTGTGTT 
               
               
                   
               
               
                 ATATTATCAAGAATCGTTTCGGCTTCAGTAGAGTTAACGTAGTCCACATCGGGAAAA 
               
               
                   
               
               
                 ACTGTCTCGGCCCTTGTCAACTTTGATGTCTGGGACACACTTACCCGACCGCACGGG 
               
               
                   
               
               
                 AAGGGCACCGCCGGTTCACAGCTCTTTTGATTCTCAGCGAGCCGGTAGCCCTCAGTG 
               
               
                   
               
               
                 CAACTACACACAACTTTGTTGTCGGCGGAATTTTTACAGAATTGCTCGCATCGTCCA 
               
               
                   
               
               
                 TTTTTAATGTTGCAGGTGACGTCCAACTCGCAGTTTTTTCCTTCAAAACCAAAAGGG 
               
               
                   
               
               
                 CACCAACACTCGTAGGAATTTATATCGTCTTTACAACTCCCCCCATTCAGACATGGA 
               
               
                   
               
               
                 TTAGATTCGCATTGGTCCCCATCGACATATTGCTTCCAGAACTCAGTGGTCCGTTCTG 
               
               
                   
               
               
                 TATTCTCAAACACCTCGCGCGCTTCTTCAAAACTGCATTTTTCCTCCATACACTCTCG 
               
               
                   
               
               
                 CTCCAAGTTCCCTTGCACGAATTCTTCAAGCTTTCCTGAGTTATACCTTTTAGGCCGG 
               
               
                   
               
               
                 TTAAGTATCTTATTCGCGTTTTCGTGGTCCAGAAA 
               
               
                   
               
               
                 Mouse Albumin Splice Acceptor (2 nd  orientation) (SEQ ID NO: 269): 
               
               
                 CTGTGGAAACAGGGAGAGAAAAACCACACAACATATTTAAAGATTGATGAAGACAA 
               
               
                   
               
               
                 CTAACTGTAATATGCTGCTTTTTGTTCTTCTCTTCACTGACCTA 
               
               
                   
               
               
                 3′ ITR Sequence (SEQ ID NO: 270): 
               
               
                 AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG 
               
               
                   
               
               
                 AGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTG 
               
               
                   
               
               
                 AGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA 
               
               
                   
               
               
                 Human Factor IX Splice Acceptor (1 st  Orientation) (SEQ ID NO: 271): 
               
               
                 GATTATTTGGATTAAAAACAAAGACTTTCTTAAGAGATGTAAAATTTTCATGATGTT 
               
               
                   
               
               
                 TTCTTTTTTGCTAAAACTAAAGAATTATTCTTTTACATTTCAG 
               
               
                   
               
               
                 Human Factor IX (R338L)-HiBit (1 st  Orientation) (SEQ ID NO: 272): 
               
               
                 TTTCTTGATCATGAAAACGCCAACAAAATTCTGAATCGGCCAAAGAGGTATAATTCA 
               
               
                   
               
               
                 GGTAAATTGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGTATGGAAGAAAA 
               
               
                   
               
               
                 GTGTAGTTTTGAAGAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACTGAAT 
               
               
                   
               
               
                 TTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGCG 
               
               
                   
               
               
                 GCAGTTGCAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAG 
               
               
                   
               
               
                 GAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGCAG 
               
               
                   
               
               
                 TTTTGTAAAAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGA 
               
               
                   
               
               
                 CTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGAAGAGTT 
               
               
                   
               
               
                 TCTGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGATGTGGACT 
               
               
                   
               
               
                 ATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAAGCACCCAAT 
               
               
                   
               
               
                 CATTTAATGACTTCACTCGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAATTCC 
               
               
                   
               
               
                 CTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATCGTTA 
               
               
                   
               
               
                 ATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTGAAACTGGTGTTAAAATTACAG 
               
               
                   
               
               
                 TTGTCGCAGGTGAACATAATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT 
               
               
                   
               
               
                 GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTATTAATAAGTACAACCAT 
               
               
                   
               
               
                 GACATTGCCCTTCTGGAACTGGACGAACCCTTAGTGCTAAACAGCTACGTTACACCT 
               
               
                   
               
               
                 ATTTGCATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTGGATCTGGCTAT 
               
               
                   
               
               
                 GTAAGTGGCTGGGGAAGAGTCTTCCACAAAGGGAGATCAGCTTTAGTTCTTCAGTAC 
               
               
                   
               
               
                 CTTAGAGTTCCACTTGTTGACCGAGCCACATGTCTTCTATCTACAAAGTTCACCATCT 
               
               
                   
               
               
                 ATAACAACATGTTCTGTGCTGGCTTCCATGAAGGAGGTAGAGATTCATGTCAAGGAG 
               
               
                   
               
               
                 ATAGTGGGGGACCCCATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAATTA 
               
               
                   
               
               
                 TTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAATATGGAATATATACCAAGGTC 
               
               
                   
               
               
                 TCCCGGTATGTCAACTGGATTAAGGAAAAAACAAAGCTCACTGTCAGCGGATGGAG 
               
               
                   
               
               
                 ACTGTTCAAGAAGATCAGCTAA 
               
               
                   
               
               
                 Human Factor IX (R338L)-HiBit (2 nd  Orientation) (SEQ ID NO: 273): 
               
               
                 TTAGGAAATCTTCTTAAACAGCCGCCAGCCGCTCACGGTGAGCTTAGTCTTTTCTTTT 
               
               
                   
               
               
                 ATCCAATTCACGTAGCGAGAGACCTTCGTATAGATGCCATATTTCCCCTTCATCGCA 
               
               
                   
               
               
                 CATTCCTCCCCCCAACTTATTATCCCGGTCAAGAAACTTGTTCCTTCGACTTCAGTGA 
               
               
                   
               
               
                 CGTGTGGTCCACCTGAATCACCTTGGCATGAGTCGCGACCGCCCTCGTGAAACCCAG 
               
               
                   
               
               
                 CACAAAACATGTTATTGTAAATCGTAAATTTCGTGGACAGAAGACAGGTCGCTCTAT 
               
               
                   
               
               
                 CGACCAACGGGACGCGCAAATATTGCAGAACGAGGGCTGATCGACCTTTGTGGAAG 
               
               
                   
               
               
                 ACCCGCCCCCACCCACTCACATATCCGCTCCCAAATTTCAAGAAGATATTTGTATAT 
               
               
                   
               
               
                 TCTTTATCGGCTATACAAATCGGGGTAACATAGGAGTTAAGTACGAGTGGCTCGTCC 
               
               
                   
               
               
                 AGCTCCAGGAGGGCTATATCATGGTTGTACTTGTTTATAGCGGCATTATAATTGTGA 
               
               
                   
               
               
                 TGGGGTATGATCCTGATAACATTCCTTTTCTGTTCAGTATGCTCAGTTTCTTCAATGT 
               
               
                   
               
               
                 TGTGTTCGCCAGCCACGACCGTAATCTTAACCCCCGTCTCGACACAGTGTGCGGCCG 
               
               
                   
               
               
                 TTACAATCCACTTTTCATTGACTATGGAGCCCCCACAAAACGCGTCGACTTTTCCGTT 
               
               
                   
               
               
                 GAGCACCACCTGCCATGGAAATTGGCCAGGTTTAGCGTCCTCGCCCCCGACAACCCT 
               
               
                   
               
               
                 AGTAAAGTCATTAAATGACTGTGTGGATTGTGTTATATTATCAAGAATCGTTTCGGC 
               
               
                   
               
               
                 TTCAGTAGAGTTAACGTAGTCCACATCGGGAAAAACTGTCTCGGCCCTTGTCAACTT 
               
               
                   
               
               
                 TGATGTCTGGGACACACTTACCCGACCGCACGGGAAGGGCACCGCCGGTTCACAGC 
               
               
                   
               
               
                 TCTTTTGATTCTCAGCGAGCCGGTAGCCCTCAGTGCAACTACACACAACTTTGTTGTC 
               
               
                   
               
               
                 GGCGGAATTTTTACAGAATTGCTCGCATCGTCCATTTTTAATGTTGCAGGTGACGTCC 
               
               
                   
               
               
                 AACTCGCAGTTTTTTCCTTCAAAACCAAAAGGGCACCAACACTCGTAGGAATTTATA 
               
               
                   
               
               
                 TCGTCTTTACAACTCCCCCCATTCAGACATGGATTAGATTCGCATTGGTCCCCATCGA 
               
               
                   
               
               
                 CATATTGCTTCCAGAACTCAGTGGTCCGTTCTGTATTCTCAAACACCTCGCGCGCTTC 
               
               
                   
               
               
                 TTCAAAACTGCATTTTTCCTCCATACACTCTCGCTCCAAGTTCCCTTGCACGAATTCT 
               
               
                   
               
               
                 TCAAGCTTTCCTGAGTTATACCTTTTAGGCCGGTTAAGTATCTTATTCGCGTTTTCGT 
               
               
                   
               
               
                 GGTCCAGAAA 
               
               
                   
               
               
                 Human Factor IX Splice Acceptor (2 nd  Orientation) (SEQ ID NO: 274): 
               
               
                 CTGAAATGTAAAAGAATAATTCTTTAGTTTTAGCAAAAAAGAAAACATCATGAAAA 
               
               
                   
               
               
                 TTTTACATCTCTTAAGAAAGTCTTTGTTTTTAATCCAAATAATC 
               
               
                   
               
               
                 Nluc-P2A-GFP (1 st  Orientation) (SEQ ID NO: 275): 
               
               
                 TTTCTTGATCATGAAAACGCCAACAAAATTCTGAATCGGCCAAAGAGGTATAATTCA 
               
               
                   
               
               
                 GGTAAATTGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGTATGGAAGAAAA 
               
               
                   
               
               
                 GTGTAGTTTTGAAGAAGCAGTATTCACTTTGGAGGACTTTGTCGGTGACTGGAGGCA 
               
               
                   
               
               
                 AACCGCTGGTTATAATCTCGACCAAGTACTGGAACAGGGCGGGGTAAGTTCCCTCTT 
               
               
                   
               
               
                 TCAGAATTTGGGTGTAAGCGTCACACCAATCCAGCGGATTGTGTTGTCTGGAGAGAA 
               
               
                   
               
               
                 CGGACTCAAAATTGACATCCATGTTATCATTCCATATGAAGGTCTCAGTGGAGACCA 
               
               
                   
               
               
                 AATGGGGCAGATCGAGAAGATTTTCAAGGTAGTTTACCCAGTCGACGATCACCACTT 
               
               
                   
               
               
                 CAAAGTCATTCTCCACTATGGCACACTTGTTATCGACGGAGTAACTCCTAATATGAT 
               
               
                   
               
               
                 TGATTACTTTGGTCGCCCGTATGAGGGCATCGCAGTGTTTGATGGCAAAAAGATCAC 
               
               
                   
               
               
                 CGTAACAGGAACGTTGTGGAATGGGAACAAGATAATCGACGAGAGATTGATAAATC 
               
               
                   
               
               
                 CAGACGGGTCACTCCTGTTCAGGGTTACAATTAACGGCGTCACAGGATGGAGACTCT 
               
               
                   
               
               
                 GTGAACGAATACTGGCCACAAATTTTTCACTCCTGAAGCAGGCCGGAGACGTGGAG 
               
               
                   
               
               
                 GAAAACCCAGGGCCCGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCAT 
               
               
                   
               
               
                 CCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGG 
               
               
                   
               
               
                 GCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGC 
               
               
                   
               
               
                 AAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGC 
               
               
                   
               
               
                 TTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCC 
               
               
                   
               
               
                 GAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGAC 
               
               
                   
               
               
                 CCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG 
               
               
                   
               
               
                 GCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTAC 
               
               
                   
               
               
                 AACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAA 
               
               
                   
               
               
                 CTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACC 
               
               
                   
               
               
                 AGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTG 
               
               
                   
               
               
                 AGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCT 
               
               
                   
               
               
                 GCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGG 
               
               
                   
               
               
                 GAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAA 
               
               
                   
               
               
                 Nluc-P2A-GFP (2 nd  Orientation) (SEQ ID NO: 276): 
               
               
                 TTACACCTTCCTCTTCTTCTTGGGGCTGCCGCCGCCCTTGTACAGCTCGTCCATGCCC 
               
               
                   
               
               
                 AGGGTGATGCCGGCGGCGGTCACGAACTCCAGCAGCACCATGTGGTCCCTCTTCTCG 
               
               
                   
               
               
                 TTGGGGTCCTTGCTCAGGGCGCTCTGGGTGCTCAGGTAGTGGTTGTCGGGCAGCAGC 
               
               
                   
               
               
                 ACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCCAGCTGCACGCTG 
               
               
                   
               
               
                 CCGTCCTCGATGTTGTGCCTGATCTTGAAGTTCACCTTGATGCCGTTCTTCTGCTTGT 
               
               
                   
               
               
                 CGGCCATGATGTACACGTTGTGGCTGTTGTAGTTGTACTCCAGCTTGTGGCCCAGGA 
               
               
                   
               
               
                 TGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATCCTGTTCACCAGGGTGTC 
               
               
                   
               
               
                 GCCCTCGAACTTCACCTCGGCCCTGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGAT 
               
               
                   
               
               
                 GGTCCTCTCCTGCACGTAGCCCTCGGGCATGGCGCTCTTGAAGAAGTCGTGCTGCTT 
               
               
                   
               
               
                 CATGTGGTCGGGGTACCTGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACCA 
               
               
                   
               
               
                 GGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGC 
               
               
                   
               
               
                 TTGCCGTAGGTGGCGTCGCCCTCGCCCTCGCCGCTCACGCTGAACTTGTGGCCGTTC 
               
               
                   
               
               
                 ACGTCGCCGTCCAGCTCCACCAGGATGGGCACCACGCCGGTGAACAGCTCCTCGCC 
               
               
                   
               
               
                 CTTGCTCACGGGGCCGGGGTTCTCCTCCACGTCGCCGGCCTGCTTCAGCAGGCTGAA 
               
               
                   
               
               
                 GTTGGTGGCCAGGATCCTCTCGCACAGCCTCCAGCCGGTCACGCCGTTGATGGTCAC 
               
               
                   
               
               
                 CCTGAACAGCAGGCTGCCGTCGGGGTTGATCAGCCTCTCGTCGATGATCTTGTTGCC 
               
               
                   
               
               
                 GTTCCACAGGGTGCCGGTCACGGTGATCTTCTTGCCGTCGAACACGGCGATGCCCTC 
               
               
                   
               
               
                 GTAGGGCCTGCCGAAGTAGTCGATCATGTTGGGGGTCACGCCGTCGATCACCAGGG 
               
               
                   
               
               
                 TGCCGTAGTGCAGGATCACCTTGAAGTGGTGGTCGTCCACGGGGTACACCACCTTGA 
               
               
                   
               
               
                 AAATCTTCTCGATCTGGCCCATCTGGTCGCCGCTCAGGCCCTCGTAGGGGATGATCA 
               
               
                   
               
               
                 CGTGGATGTCGATCTTCAGGCCGTTCTCGCCGCTCAGCACGATCCTCTGGATGGGGG 
               
               
                   
               
               
                 TCACGCTCACGCCCAGGTTCTGGAACAGGCTGCTCACGCCGCCCTGCTCCAGCACCT 
               
               
                   
               
               
                 GGTCCAGGTTGTAGCCGGCGGTCTGCCTCCAGTCGCCCACGAAGTCCTCCAGGGTGA 
               
               
                   
               
               
                 ACACGGCCTCCTCGAAGCTGCACTTCTCCTCCATGCACTCCCTCTCCAGGTTGCCCTG 
               
               
                   
               
               
                 CACGAACTCCTCCAGCTTGCCGCTGTTGTACCTCTTGGGCCTGTTCAGGATCTTGTTG 
               
               
                   
               
               
                 GCGTTCTCGTGGTCCAGGAA 
               
               
                   
               
               
                 P00147 full sequence (from ITR to ITR): (SEQ ID NO: 277) 
               
               
                 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC 
               
               
                   
               
               
                 GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA 
               
               
                   
               
               
                 GGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCTCTTAGGTCAGTGAAGAGA 
               
               
                   
               
               
                 AGAACAAAAAGCAGCATATTACAGTTAGTTGTCTTCATCAATCTTTAAATATGTTGT 
               
               
                   
               
               
                 GTGGTTTTTCTCTCCCTGTTTCCACAGTTTTTCTTGATCATGAAAACGCCAACAAAAT 
               
               
                   
               
               
                 TCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGA 
               
               
                   
               
               
                 ACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAAGAAGCACGAGAAGTT 
               
               
                   
               
               
                 TTTGAAAACACTGAAAGAACAACTGAATTTTGGAAGCAGTATGTTGATGGAGATCA 
               
               
                   
               
               
                 GTGTGAGTCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGACATTAATTCCTA 
               
               
                   
               
               
                 TGAATGTTGGTGTCCCTTTGGATTTGAAGGAAAGAACTGTGAATTAGATGTAACATG 
               
               
                   
               
               
                 TAACATTAAGAATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATAACAAGG 
               
               
                   
               
               
                 TGGTTTGCTCCTGTACTGAGGGATATCGACTTGCAGAAAACCAGAAGTCCTGTGAAC 
               
               
                   
               
               
                 CAGCAGTGCCATTTCCATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACCC 
               
               
                   
               
               
                 GTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATTCTACTGAAGCTGAAACCA 
               
               
                   
               
               
                 TTTTGGATAACATCACTCAAAGCACCCAATCATTTAATGACTTCACTCGGGTTGTTG 
               
               
                   
               
               
                 GTGGAGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGTTTTGAATGGTAAAG 
               
               
                   
               
               
                 TTGATGCATTCTGTGGAGGCTCTATCGTTAATGAAAAATGGATTGTAACTGCTGCCC 
               
               
                   
               
               
                 ACTGTGTTGAAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATTGAGG 
               
               
                   
               
               
                 AGACAGAACATACAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACCACAAC 
               
               
                   
               
               
                 TACAATGCAGCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGACGAA 
               
               
                   
               
               
                 CCCTTAGTGCTAAACAGCTACGTTACACCTATTTGCATTGCTGACAAGGAATACACG 
               
               
                   
               
               
                 AACATCTTCCTCAAATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCAC 
               
               
                   
               
               
                 AAAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTGACCGAGCC 
               
               
                   
               
               
                 ACATGTCTTCTATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTCC 
               
               
                   
               
               
                 ATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACTGAA 
               
               
                   
               
               
                 GTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGTGCAAT 
               
               
                   
               
               
                 GAAAGGCAAATATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGATTAAGG 
               
               
                   
               
               
                 AAAAAACAAAGCTCACTTAACCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGT 
               
               
                   
               
               
                 TTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCC 
               
               
                   
               
               
                 TAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGG 
               
               
                   
               
               
                 GGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATG 
               
               
                   
               
               
                 CTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCT 
               
               
                   
               
               
                 AGGGGGTATCCCCAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATG 
               
               
                   
               
               
                 AATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCA 
               
               
                   
               
               
                 ATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTT 
               
               
                   
               
               
                 GTCCAAACTCATCAATGTATCTTATCATGTCTGTTAGGTGAGCTTAGTCTTTTCTTTT 
               
               
                   
               
               
                 ATCCAATTCACGTAGCGAGAGACCTTCGTATAGATGCCATATTTCCCCTTCATCGCA 
               
               
                   
               
               
                 CATTCCTCCCCCCAACTTATTATCCCGGTCAAGAAACTTGTTCCTTCGACTTCAGTGA 
               
               
                   
               
               
                 CGTGTGGTCCACCTGAATCACCTTGGCATGAGTCGCGACCGCCCTCGTGAAACCCAG 
               
               
                   
               
               
                 CACAAAACATGTTATTGTAAATCGTAAATTTCGTGGACAGAAGACAGGTCGCTCTAT 
               
               
                   
               
               
                 CGACCAACGGGACGCGCAAATATTGCAGAACGAGGGCTGATCGACCTTTGTGGAAG 
               
               
                   
               
               
                 ACCCGCCCCCACCCACTCACATATCCGCTCCCAAATTTCAAGAAGATATTTGTATAT 
               
               
                   
               
               
                 TCTTTATCGGCTATACAAATCGGGGTAACATAGGAGTTAAGTACGAGTGGCTCGTCC 
               
               
                   
               
               
                 AGCTCCAGGAGGGCTATATCATGGTTGTACTTGTTTATAGCGGCATTATAATTGTGA 
               
               
                   
               
               
                 TGGGGTATGATCCTGATAACATTCCTTTTCTGTTCAGTATGCTCAGTTTCTTCAATGT 
               
               
                   
               
               
                 TGTGTTCGCCAGCCACGACCGTAATCTTAACCCCCGTCTCGACACAGTGTGCGGCCG 
               
               
                   
               
               
                 TTACAATCCACTTTTCATTGACTATGGAGCCCCCACAAAACGCGTCGACTTTTCCGTT 
               
               
                   
               
               
                 GAGCACCACCTGCCATGGAAATTGGCCAGGTTTAGCGTCCTCGCCCCCGACAACCCT 
               
               
                   
               
               
                 AGTAAAGTCATTAAATGACTGTGTGGATTGTGTTATATTATCAAGAATCGTTTCGGC 
               
               
                   
               
               
                 TTCAGTAGAGTTAACGTAGTCCACATCGGGAAAAACTGTCTCGGCCCTTGTCAACTT 
               
               
                   
               
               
                 TGATGTCTGGGACACACTTACCCGACCGCACGGGAAGGGCACCGCCGGTTCACAGC 
               
               
                   
               
               
                 TCTTTTGATTCTCAGCGAGCCGGTAGCCCTCAGTGCAACTACACACAACTTTGTTGTC 
               
               
                   
               
               
                 GGCGGAATTTTTACAGAATTGCTCGCATCGTCCATTTTTAATGTTGCAGGTGACGTCC 
               
               
                   
               
               
                 AACTCGCAGTTTTTTCCTTCAAAACCAAAAGGGCACCAACACTCGTAGGAATTTATA 
               
               
                   
               
               
                 TCGTCTTTACAACTCCCCCCATTCAGACATGGATTAGATTCGCATTGGTCCCCATCGA 
               
               
                   
               
               
                 CATATTGCTTCCAGAACTCAGTGGTCCGTTCTGTATTCTCAAACACCTCGCGCGCTTC 
               
               
                   
               
               
                 TTCAAAACTGCATTTTTCCTCCATACACTCTCGCTCCAAGTTCCCTTGCACGAATTCT 
               
               
                   
               
               
                 TCAAGCTTTCCTGAGTTATACCTTTTAGGCCGGTTAAGTATCTTATTCGCGTTTTCGT 
               
               
                   
               
               
                 GGTCCAGAAAAACTGTGGAAACAGGGAGAGAAAAACCACACAACATATTTAAAGA 
               
               
                   
               
               
                 TTGATGAAGACAACTAACTGTAATATGCTGCTTTTTGTTCTTCTCTTCACTGACCTAA 
               
               
                   
               
               
                 GAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG 
               
               
                   
               
               
                 CTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGC 
               
               
                   
               
               
                 CTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA 
               
               
                   
               
               
                 P00411 full sequence (form ITR to ITR): (SEQ ID NO: 278) 
               
               
                 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC 
               
               
                   
               
               
                 GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA 
               
               
                   
               
               
                 GGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCTCTGATTATTTGGATTAAAA 
               
               
                   
               
               
                 ACAAAGACTTTCTTAAGAGATGTAAAATTTTCATGATGTTTTCTTTTTTGCTAAAACT 
               
               
                   
               
               
                 AAAGAATTATTCTTTTACATTTCAGTTTTTCTTGATCATGAAAACGCCAACAAAATTC 
               
               
                   
               
               
                 TGAATCGGCCAAAGAGGTATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAAC 
               
               
                   
               
               
                 CTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAAGAAGCACGAGAAGTTT 
               
               
                   
               
               
                 TGAAAACACTGAAAGAACAACTGAATTTTGGAAGCAGTATGTTGATGGAGATCAGT 
               
               
                   
               
               
                 GTGAGTCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGACATTAATTCCTATG 
               
               
                   
               
               
                 AATGTTGGTGTCCCTTTGGATTTGAAGGAAAGAACTGTGAATTAGATGTAACATGTA 
               
               
                   
               
               
                 ACATTAAGAATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATAACAAGGTG 
               
               
                   
               
               
                 GTTTGCTCCTGTACTGAGGGATATCGACTTGCAGAAAACCAGAAGTCCTGTGAACCA 
               
               
                   
               
               
                 GCAGTGCCATTTCCATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACCCGT 
               
               
                   
               
               
                 GCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATTCTACTGAAGCTGAAACCATTT 
               
               
                   
               
               
                 TGGATAACATCACTCAAAGCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTG 
               
               
                   
               
               
                 GAGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGTTTTGAATGGTAAAGTTG 
               
               
                   
               
               
                 ATGCATTCTGTGGAGGCTCTATCGTTAATGAAAAATGGATTGTAACTGCTGCCCACT 
               
               
                   
               
               
                 GTGTTGAAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATTGAGGAGA 
               
               
                   
               
               
                 CAGAACATACAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACCACAACTAC 
               
               
                   
               
               
                 AATGCAGCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGACGAACCC 
               
               
                   
               
               
                 TTAGTGCTAAACAGCTACGTTACACCTATTTGCATTGCTGACAAGGAATACACGAAC 
               
               
                   
               
               
                 ATCTTCCTCAAATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAA 
               
               
                   
               
               
                 GGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTGACCGAGCCACA 
               
               
                   
               
               
                 TGTCTTCTATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTCCATG 
               
               
                   
               
               
                 AAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACTGAAGTG 
               
               
                   
               
               
                 GAAGGGACCAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGTGCAATGAA 
               
               
                   
               
               
                 AGGCAAATATGGAATATATACCAAGGTCTCCCGGTATGTCAACTGGATTAAGGAAA 
               
               
                   
               
               
                 AAACAAAGCTCACTGTCAGCGGATGGAGACTGTTCAAGAAGATCAGCTAACCTCGA 
               
               
                   
               
               
                 CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGAC 
               
               
                   
               
               
                 CCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCA 
               
               
                   
               
               
                 TTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGG 
               
               
                   
               
               
                 GGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCT 
               
               
                   
               
               
                 TCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCAAAAAACCTCCCA 
               
               
                   
               
               
                 CACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTT 
               
               
                   
               
               
                 ATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAA 
               
               
                   
               
               
                 GCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATC 
               
               
                   
               
               
                 ATGTCTGTTAGGAAATCTTCTTAAACAGCCGCCAGCCGCTCACGGTGAGCTTAGTCT 
               
               
                   
               
               
                 TTTCTTTTATCCAATTCACGTAGCGAGAGACCTTCGTATAGATGCCATATTTCCCCTT 
               
               
                   
               
               
                 CATCGCACATTCCTCCCCCCAACTTATTATCCCGGTCAAGAAACTTGTTCCTTCGACT 
               
               
                   
               
               
                 TCAGTGACGTGTGGTCCACCTGAATCACCTTGGCATGAGTCGCGACCGCCCTCGTGA 
               
               
                   
               
               
                 AACCCAGCACAAAACATGTTATTGTAAATCGTAAATTTCGTGGACAGAAGACAGGT 
               
               
                   
               
               
                 CGCTCTATCGACCAACGGGACGCGCAAATATTGCAGAACGAGGGCTGATCGACCTT 
               
               
                   
               
               
                 TGTGGAAGACCCGCCCCCACCCACTCACATATCCGCTCCCAAATTTCAAGAAGATAT 
               
               
                   
               
               
                 TTGTATATTCTTTATCGGCTATACAAATCGGGGTAACATAGGAGTTAAGTACGAGTG 
               
               
                   
               
               
                 GCTCGTCCAGCTCCAGGAGGGCTATATCATGGTTGTACTTGTTTATAGCGGCATTAT 
               
               
                   
               
               
                 AATTGTGATGGGGTATGATCCTGATAACATTCCTTTTCTGTTCAGTATGCTCAGTTTC 
               
               
                   
               
               
                 TTCAATGTTGTGTTCGCCAGCCACGACCGTAATCTTAACCCCCGTCTCGACACAGTG 
               
               
                   
               
               
                 TGCGGCCGTTACAATCCACTTTTCATTGACTATGGAGCCCCCACAAAACGCGTCGAC 
               
               
                   
               
               
                 TTTTCCGTTGAGCACCACCTGCCATGGAAATTGGCCAGGTTTAGCGTCCTCGCCCCC 
               
               
                   
               
               
                 GACAACCCTAGTAAAGTCATTAAATGACTGTGTGGATTGTGTTATATTATCAAGAAT 
               
               
                   
               
               
                 CGTTTCGGCTTCAGTAGAGTTAACGTAGTCCACATCGGGAAAAACTGTCTCGGCCCT 
               
               
                   
               
               
                 TGTCAACTTTGATGTCTGGGACACACTTACCCGACCGCACGGGAAGGGCACCGCCG 
               
               
                   
               
               
                 GTTCACAGCTCTTTTGATTCTCAGCGAGCCGGTAGCCCTCAGTGCAACTACACACAA 
               
               
                   
               
               
                 CTTTGTTGTCGGCGGAATTTTTACAGAATTGCTCGCATCGTCCATTTTTAATGTTGCA 
               
               
                   
               
               
                 GGTGACGTCCAACTCGCAGTTTTTTCCTTCAAAACCAAAAGGGCACCAACACTCGTA 
               
               
                   
               
               
                 GGAATTTATATCGTCTTTACAACTCCCCCCATTCAGACATGGATTAGATTCGCATTGG 
               
               
                   
               
               
                 TCCCCATCGACATATTGCTTCCAGAACTCAGTGGTCCGTTCTGTATTCTCAAACACCT 
               
               
                   
               
               
                 CGCGCGCTTCTTCAAAACTGCATTTTTCCTCCATACACTCTCGCTCCAAGTTCCCTTG 
               
               
                   
               
               
                 CACGAATTCTTCAAGCTTTCCTGAGTTATACCTTTTAGGCCGGTTAAGTATCTTATTC 
               
               
                   
               
               
                 GCGTTTTCGTGGTCCAGAAAAACTGAAATGTAAAAGAATAATTCTTTAGTTTTAGCA 
               
               
                   
               
               
                 AAAAAGAAAACATCATGAAAATTTTACATCTCTTAAGAAAGTCTTTGTTTTTAATCC 
               
               
                   
               
               
                 AAATAATCAGAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGC 
               
               
                   
               
               
                 GCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGG 
               
               
                   
               
               
                 TCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA 
               
               
                   
               
               
                 P00415 full sequence (from ITR to ITR): (SEQ ID NO: 279) 
               
               
                 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC 
               
               
                   
               
               
                 GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA 
               
               
                   
               
               
                 GGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCTCTTAGGTCAGTGAAGAGA 
               
               
                   
               
               
                 AGAACAAAAAGCAGCATATTACAGTTAGTTGTCTTCATCAATCTTTAAATATGTTGT 
               
               
                   
               
               
                 GTGGTTTTTCTCTCCCTGTTTCCACAGTTTTTCTTGATCATGAAAACGCCAACAAAAT 
               
               
                   
               
               
                 TCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGA 
               
               
                   
               
               
                 ACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAAGAAGCAGTATTCACT 
               
               
                   
               
               
                 TTGGAGGACTTTGTCGGTGACTGGAGGCAAACCGCTGGTTATAATCTCGACCAAGTA 
               
               
                   
               
               
                 CTGGAACAGGGCGGGGTAAGTTCCCTCTTTCAGAATTTGGGTGTAAGCGTCACACCA 
               
               
                   
               
               
                 ATCCAGCGGATTGTGTTGTCTGGAGAGAACGGACTCAAAATTGACATCCATGTTATC 
               
               
                   
               
               
                 ATTCCATATGAAGGTCTCAGTGGAGACCAAATGGGGCAGATCGAGAAGATTTTCAA 
               
               
                   
               
               
                 GGTAGTTTACCCAGTCGACGATCACCACTTCAAAGTCATTCTCCACTATGGCACACT 
               
               
                   
               
               
                 TGTTATCGACGGAGTAACTCCTAATATGATTGATTACTTTGGTCGCCCGTATGAGGG 
               
               
                   
               
               
                 CATCGCAGTGTTTGATGGCAAAAAGATCACCGTAACAGGAACGTTGTGGAATGGGA 
               
               
                   
               
               
                 ACAAGATAATCGACGAGAGATTGATAAATCCAGACGGGTCACTCCTGTTCAGGGTT 
               
               
                   
               
               
                 ACAATTAACGGCGTCACAGGATGGAGACTCTGTGAACGAATACTGGCCACAAATTT 
               
               
                   
               
               
                 TTCACTCCTGAAGCAGGCCGGAGACGTGGAGGAAAACCCAGGGCCCGTGAGCAAGG 
               
               
                   
               
               
                 GCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTA 
               
               
                   
               
               
                 AACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAA 
               
               
                   
               
               
                 GCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT 
               
               
                   
               
               
                 CGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAA 
               
               
                   
               
               
                 GCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCA 
               
               
                   
               
               
                 TCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGC 
               
               
                   
               
               
                 GACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA 
               
               
                   
               
               
                 CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGG 
               
               
                   
               
               
                 CCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAG 
               
               
                   
               
               
                 GACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGG 
               
               
                   
               
               
                 CCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAG 
               
               
                   
               
               
                 ACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGG 
               
               
                   
               
               
                 ATCACTCTCGGCATGGACGAGCTGTACAAGGGAGGAGGAAGCCCGAAGAAGAAGA 
               
               
                   
               
               
                 GAAAGGTCTAACCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCC 
               
               
                   
               
               
                 CCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATG 
               
               
                   
               
               
                 AGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGG 
               
               
                   
               
               
                 GGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGC 
               
               
                   
               
               
                 GGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATC 
               
               
                   
               
               
                 CCCAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGT 
               
               
                   
               
               
                 TGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCAC 
               
               
                   
               
               
                 AAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTC 
               
               
                   
               
               
                 ATCAATGTATCTTATCATGTCTGTTACACCTTCCTCTTCTTCTTGGGGCTGCCGCCGC 
               
               
                   
               
               
                 CCTTGTACAGCTCGTCCATGCCCAGGGTGATGCCGGCGGCGGTCACGAACTCCAGCA 
               
               
                   
               
               
                 GCACCATGTGGTCCCTCTTCTCGTTGGGGTCCTTGCTCAGGGCGCTCTGGGTGCTCAG 
               
               
                   
               
               
                 GTAGTGGTTGTCGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGT 
               
               
                   
               
               
                 AGTGGTCGGCCAGCTGCACGCTGCCGTCCTCGATGTTGTGCCTGATCTTGAAGTTCA 
               
               
                   
               
               
                 CCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATGTACACGTTGTGGCTGTTGTAGTT 
               
               
                   
               
               
                 GTACTCCAGCTTGTGGCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAG 
               
               
                   
               
               
                 CTCGATCCTGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCCCTGGTCTTGTAG 
               
               
                   
               
               
                 TTGCCGTCGTCCTTGAAGAAGATGGTCCTCTCCTGCACGTAGCCCTCGGGCATGGCG 
               
               
                   
               
               
                 CTCTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTACCTGCTGAAGCACTGCACG 
               
               
                   
               
               
                 CCGTAGGTCAGGGTGGTCACCAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGT 
               
               
                   
               
               
                 GCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCGTCGCCCTCGCCCTCGCCGCT 
               
               
                   
               
               
                 CACGCTGAACTTGTGGCCGTTCACGTCGCCGTCCAGCTCCACCAGGATGGGCACCAC 
               
               
                   
               
               
                 GCCGGTGAACAGCTCCTCGCCCTTGCTCACGGGGCCGGGGTTCTCCTCCACGTCGCC 
               
               
                   
               
               
                 GGCCTGCTTCAGCAGGCTGAAGTTGGTGGCCAGGATCCTCTCGCACAGCCTCCAGCC 
               
               
                   
               
               
                 GGTCACGCCGTTGATGGTCACCCTGAACAGCAGGCTGCCGTCGGGGTTGATCAGCCT 
               
               
                   
               
               
                 CTCGTCGATGATCTTGTTGCCGTTCCACAGGGTGCCGGTCACGGTGATCTTCTTGCCG 
               
               
                   
               
               
                 TCGAACACGGCGATGCCCTCGTAGGGCCTGCCGAAGTAGTCGATCATGTTGGGGGTC 
               
               
                   
               
               
                 ACGCCGTCGATCACCAGGGTGCCGTAGTGCAGGATCACCTTGAAGTGGTGGTCGTCC 
               
               
                   
               
               
                 ACGGGGTACACCACCTTGAAAATCTTCTCGATCTGGCCCATCTGGTCGCCGCTCAGG 
               
               
                   
               
               
                 CCCTCGTAGGGGATGATCACGTGGATGTCGATCTTCAGGCCGTTCTCGCCGCTCAGC 
               
               
                   
               
               
                 ACGATCCTCTGGATGGGGGTCACGCTCACGCCCAGGTTCTGGAACAGGCTGCTCACG 
               
               
                   
               
               
                 CCGCCCTGCTCCAGCACCTGGTCCAGGTTGTAGCCGGCGGTCTGCCTCCAGTCGCCC 
               
               
                   
               
               
                 ACGAAGTCCTCCAGGGTGAACACGGCCTCCTCGAAGCTGCACTTCTCCTCCATGCAC 
               
               
                   
               
               
                 TCCCTCTCCAGGTTGCCCTGCACGAACTCCTCCAGCTTGCCGCTGTTGTACCTCTTGG 
               
               
                   
               
               
                 GCCTGTTCAGGATCTTGTTGGCGTTCTCGTGGTCCAGGAA 
               
               
                   
               
               
                 P00418 full sequence (from ITR to ITR): (SEQ ID NO: 280) 
               
               
                 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC 
               
               
                   
               
               
                 GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA 
               
               
                   
               
               
                 GGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCTCTTAGGTCAGTGAAGAGA 
               
               
                   
               
               
                 AGAACAAAAAGCAGCATATTACAGTTAGTTGTCTTCATCAATCTTTAAATATGTTGT 
               
               
                   
               
               
                 GTGGTTTTTCTCTCCCTGTTTCCACAGTTTTTCTTGATCATGAAAACGCCAACAAAAT 
               
               
                   
               
               
                 TCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGA 
               
               
                   
               
               
                 ACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAAGAAGCACGAGAAGTT 
               
               
                   
               
               
                 TTTGAAAACACTGAAAGAACAACTGAATTTTGGAAGCAGTATGTTGATGGAGATCA 
               
               
                   
               
               
                 GTGTGAGTCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGACATTAATTCCTA 
               
               
                   
               
               
                 TGAATGTTGGTGTCCCTTTGGATTTGAAGGAAAGAACTGTGAATTAGATGTAACATG 
               
               
                   
               
               
                 TAACATTAAGAATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATAACAAGG 
               
               
                   
               
               
                 TGGTTTGCTCCTGTACTGAGGGATATCGACTTGCAGAAAACCAGAAGTCCTGTGAAC 
               
               
                   
               
               
                 CAGCAGTGCCATTTCCATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACCC 
               
               
                   
               
               
                 GTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATTCTACTGAAGCTGAAACCA 
               
               
                   
               
               
                 TTTTGGATAACATCACTCAAAGCACCCAATCATTTAATGACTTCACTCGGGTTGTTG 
               
               
                   
               
               
                 GTGGAGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGTTTTGAATGGTAAAG 
               
               
                   
               
               
                 TTGATGCATTCTGTGGAGGCTCTATCGTTAATGAAAAATGGATTGTAACTGCTGCCC 
               
               
                   
               
               
                 ACTGTGTTGAAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATTGAGG 
               
               
                   
               
               
                 AGACAGAACATACAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACCACAAC 
               
               
                   
               
               
                 TACAATGCAGCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGACGAA 
               
               
                   
               
               
                 CCCTTAGTGCTAAACAGCTACGTTACACCTATTTGCATTGCTGACAAGGAATACACG 
               
               
                   
               
               
                 AACATCTTCCTCAAATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCAC 
               
               
                   
               
               
                 AAAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTGACCGAGCC 
               
               
                   
               
               
                 ACATGTCTTCTATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTCC 
               
               
                   
               
               
                 ATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACTGAA 
               
               
                   
               
               
                 GTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGTGCAAT 
               
               
                   
               
               
                 GAAAGGCAAATATGGAATATATACCAAGGTCTCCCGGTATGTCAACTGGATTAAGG 
               
               
                   
               
               
                 AAAAAACAAAGCTCACTGTCAGCGGATGGAGACTGTTCAAGAAGATCAGCTAACCT 
               
               
                   
               
               
                 CGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTT 
               
               
                   
               
               
                 GACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATC 
               
               
                   
               
               
                 GCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCA 
               
               
                   
               
               
                 AGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATG 
               
               
                   
               
               
                 GCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCAAAAAACCTC 
               
               
                   
               
               
                 CCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTG 
               
               
                   
               
               
                 TTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAAT 
               
               
                   
               
               
                 AAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTT 
               
               
                   
               
               
                 ATCATGTCTGTTAGGAAATCTTCTTAAACAGCCGCCAGCCGCTCACGGTGAGCTTAG 
               
               
                   
               
               
                 TCTTTTCTTTTATCCAATTCACGTAGCGAGAGACCTTCGTATAGATGCCATATTTCCC 
               
               
                   
               
               
                 CTTCATCGCACATTCCTCCCCCCAACTTATTATCCCGGTCAAGAAACTTGTTCCTTCG 
               
               
                   
               
               
                 ACTTCAGTGACGTGTGGTCCACCTGAATCACCTTGGCATGAGTCGCGACCGCCCTCG 
               
               
                   
               
               
                 TGAAACCCAGCACAAAACATGTTATTGTAAATCGTAAATTTCGTGGACAGAAGACA 
               
               
                   
               
               
                 GGTCGCTCTATCGACCAACGGGACGCGCAAATATTGCAGAACGAGGGCTGATCGAC 
               
               
                   
               
               
                 CTTTGTGGAAGACCCGCCCCCACCCACTCACATATCCGCTCCCAAATTTCAAGAAGA 
               
               
                   
               
               
                 TATTTGTATATTCTTTATCGGCTATACAAATCGGGGTAACATAGGAGTTAAGTACGA 
               
               
                   
               
               
                 GTGGCTCGTCCAGCTCCAGGAGGGCTATATCATGGTTGTACTTGTTTATAGCGGCAT 
               
               
                   
               
               
                 TATAATTGTGATGGGGTATGATCCTGATAACATTCCTTTTCTGTTCAGTATGCTCAGT 
               
               
                   
               
               
                 TTCTTCAATGTTGTGTTCGCCAGCCACGACCGTAATCTTAACCCCCGTCTCGACACA 
               
               
                   
               
               
                 GTGTGCGGCCGTTACAATCCACTTTTCATTGACTATGGAGCCCCCACAAAACGCGTC 
               
               
                   
               
               
                 GACTTTTCCGTTGAGCACCACCTGCCATGGAAATTGGCCAGGTTTAGCGTCCTCGCC 
               
               
                   
               
               
                 CCCGACAACCCTAGTAAAGTCATTAAATGACTGTGTGGATTGTGTTATATTATCAAG 
               
               
                   
               
               
                 AATCGTTTCGGCTTCAGTAGAGTTAACGTAGTCCACATCGGGAAAAACTGTCTCGGC 
               
               
                   
               
               
                 CCTTGTCAACTTTGATGTCTGGGACACACTTACCCGACCGCACGGGAAGGGCACCGC 
               
               
                   
               
               
                 CGGTTCACAGCTCTTTTGATTCTCAGCGAGCCGGTAGCCCTCAGTGCAACTACACAC 
               
               
                   
               
               
                 AACTTTGTTGTCGGCGGAATTTTTACAGAATTGCTCGCATCGTCCATTTTTAATGTTG 
               
               
                   
               
               
                 CAGGTGACGTCCAACTCGCAGTTTTTTCCTTCAAAACCAAAAGGGCACCAACACTCG 
               
               
                   
               
               
                 TAGGAATTTATATCGTCTTTACAACTCCCCCCATTCAGACATGGATTAGATTCGCATT 
               
               
                   
               
               
                 GGTCCCCATCGACATATTGCTTCCAGAACTCAGTGGTCCGTTCTGTATTCTCAAACAC 
               
               
                   
               
               
                 CTCGCGCGCTTCTTCAAAACTGCATTTTTCCTCCATACACTCTCGCTCCAAGTTCCCT 
               
               
                   
               
               
                 TGCACGAATTCTTCAAGCTTTCCTGAGTTATACCTTTTAGGCCGGTTAAGTATCTTAT 
               
               
                   
               
               
                 TCGCGTTTTCGTGGTCCAGAAAAACTGTGGAAACAGGGAGAGAAAAACCACACAAC 
               
               
                   
               
               
                 ATATTTAAAGATTGATGAAGACAACTAACTGTAATATGCTGCTTTTTGTTCTTCTCTT 
               
               
                   
               
               
                 CACTGACCTAAGAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGC 
               
               
                   
               
               
                 GCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTT 
               
               
                   
               
               
                 GGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA 
               
               
                   
               
               
                 P00123 full sequence (from ITR to ITR): (SEQ ID NO: 281) 
               
               
                 GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGC 
               
               
                   
               
               
                 CCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG 
               
               
                   
               
               
                 GAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGATAGGTCAG 
               
               
                   
               
               
                 TGAAGAGAAGAACAAAAAGCAGCATATTACAGTTAGTTGTCTTCATCAATCTTTAAA 
               
               
                   
               
               
                 TATGTTGTGTGGTTTTTCTCTCCCTGTTTCCACAGTTTTTCTTGATCATGAAAACGCCA 
               
               
                   
               
               
                 ACAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATTGGAAGAGTTTGTTC 
               
               
                   
               
               
                 AAGGGAACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAAGAAGCACGA 
               
               
                   
               
               
                 GAAGTTTTTGAAAACACTGAAAGAACAACTGAATTTTGGAAGCAGTATGTTGATGG 
               
               
                   
               
               
                 AGATCAGTGTGAGTCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGACATTAA 
               
               
                   
               
               
                 TTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAGGAAAGAACTGTGAATTAGATGT 
               
               
                   
               
               
                 AACATGTAACATTAAGAATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATA 
               
               
                   
               
               
                 ACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGCAGAAAACCAGAAGTCCT 
               
               
                   
               
               
                 GTGAACCAGCAGTGCCATTTCCATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGC 
               
               
                   
               
               
                 TCACCCGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATTCTACTGAAGCTGA 
               
               
                   
               
               
                 AACCATTTTGGATAACATCACTCAAAGCACCCAATCATTTAATGACTTCACTCGGGT 
               
               
                   
               
               
                 TGTTGGTGGAGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGTTTTGAATGG 
               
               
                   
               
               
                 TAAAGTTGATGCATTCTGTGGAGGCTCTATCGTTAATGAAAAATGGATTGTAACTGC 
               
               
                   
               
               
                 TGCCCACTGTGTTGAAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATAT 
               
               
                   
               
               
                 TGAGGAGACAGAACATACAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACC 
               
               
                   
               
               
                 ACAACTACAATGCAGCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGG 
               
               
                   
               
               
                 ACGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGCATTGCTGACAAGGAAT 
               
               
                   
               
               
                 ACACGAACATCTTCCTCAAATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCT 
               
               
                   
               
               
                 TCCACAAAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTGACC 
               
               
                   
               
               
                 GAGCCACATGTCTTCTATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTG 
               
               
                   
               
               
                 GCTTCCATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTT 
               
               
                   
               
               
                 ACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTG 
               
               
                   
               
               
                 TGCAATGAAAGGCAAATATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGA 
               
               
                   
               
               
                 TTAAGGAAAAAACAAAGCTCACTTAACCTCGACTGTGCCTTCTAGTTGCCAGCCATC 
               
               
                   
               
               
                 TGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTC 
               
               
                   
               
               
                 CTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTC 
               
               
                   
               
               
                 TGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAG 
               
               
                   
               
               
                 GCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGG 
               
               
                   
               
               
                 GCTCTAGGGGGTATCCCCACTAGTCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG 
               
               
                   
               
               
                 AGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTG 
               
               
                   
               
               
                 AGCGAGCGAGCGCGCAGAGAGGGA 
               
               
                   
               
               
                 P00204 full sequence (from ITR to ITR): (SEQ ID NO: 282) 
               
               
                 GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGC 
               
               
                   
               
               
                 CCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG 
               
               
                   
               
               
                 GAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGACCTAGGTC 
               
               
                   
               
               
                 GTCTCCGGCTCTGCTTTTTCCAGGGGTGTGTTTCGCCGAGAAGCACGTAAGAGTTTT 
               
               
                   
               
               
                 ATGTTTTTTCATCTCTGCTTGTATTTTTCTAGTAATGGAAGCCTGGTATTTTAAAATA 
               
               
                   
               
               
                 GTTAAATTTTCCTTTAGTGCTGATTTCTAGATTATTATTACTGTTGTTGTTGTTATTAT 
               
               
                   
               
               
                 TGTCATTATTTGCATCTGAGAACTAGGTCAGTGAAGAGAAGAACAAAAAGCAGCAT 
               
               
                   
               
               
                 ATTACAGTTAGTTGTCTTCATCAATCTTTAAATATGTTGTGTGGTTTTTCTCTCCCTGT 
               
               
                   
               
               
                 TTCCACAGTTTTTCTTGATCATGAAAACGCCAACAAAATTCTGAATCGGCCAAAGAG 
               
               
                   
               
               
                 GTATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGTA 
               
               
                   
               
               
                 TGGAAGAAAAGTGTAGTTTTGAAGAAGCACGAGAAGTTTTTGAAAACACTGAAAGA 
               
               
                   
               
               
                 ACAACTGAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGT 
               
               
                   
               
               
                 TTAAATGGCGGCAGTTGCAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTT 
               
               
                   
               
               
                 GGATTTGAAGGAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAG 
               
               
                   
               
               
                 ATGCGAGCAGTTTTGTAAAAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGA 
               
               
                   
               
               
                 GGGATATCGACTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCAT 
               
               
                   
               
               
                 GTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCC 
               
               
                   
               
               
                 TGATGTGGACTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCA 
               
               
                   
               
               
                 AAGCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGGAGAAGATGCCAAACC 
               
               
                   
               
               
                 AGGTCAATTCCCTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCATTCTGTGGAGG 
               
               
                   
               
               
                 CTCTATCGTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTGAAACTGGTGT 
               
               
                   
               
               
                 TAAAATTACAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGAACATACAGAGC 
               
               
                   
               
               
                 AAAAGCGAAATGTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTATTAATA 
               
               
                   
               
               
                 AGTACAACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTAGTGCTAAACAGCT 
               
               
                   
               
               
                 ACGTTACACCTATTTGCATTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTG 
               
               
                   
               
               
                 GATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAAGGGAGATCAGCTTTA 
               
               
                   
               
               
                 GTTCTTCAGTACCTTAGAGTTCCACTTGTTGACCGAGCCACATGTCTTCTATCTACAA 
               
               
                   
               
               
                 AGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAGGAGGTAGAGATT 
               
               
                   
               
               
                 CATGTCAAGGAGATAGTGGGGGACCCCATGTTACTGAAGTGGAAGGGACCAGTTTC 
               
               
                   
               
               
                 TTAACTGGAATTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAATATGGAAT 
               
               
                   
               
               
                 ATATACCAAGGTATCCCGGTATGTCAACTGGATTAAGGAAAAAACAAAGCTCACTT 
               
               
                   
               
               
                 AACCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCT 
               
               
                   
               
               
                 TCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATT 
               
               
                   
               
               
                 GCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGAC 
               
               
                   
               
               
                 AGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCT 
               
               
                   
               
               
                 CTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCCTTAG 
               
               
                   
               
               
                 GTGGTTATATTATTGATATATTTTTGGTATCTTTGATGACAATAATGGGGGATTTTGA 
               
               
                   
               
               
                 AAGCTTAGCTTTAAATTTCTTTTAATTAAAAAAAAATGCTAGGCAGAATGACTCAAA 
               
               
                   
               
               
                 TTACGTTGGATACAGTTGAATTTATTACGGTCTCATAGGGCCTGCCTGCTCGACCAT 
               
               
                   
               
               
                 GCTATACTAAAAATTAAAAGTGTACTAGTCCACTCCCTCTCTGCGCGCTCGCTCGCT 
               
               
                   
               
               
                 CACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCT 
               
               
                   
               
               
                 CAGTGAGCGAGCGAGCGCGCAGAGAGGGA 
               
               
                   
               
               
                 P00353 full sequence (from ITR to ITR): (SEQ ID NO: 283) 
               
               
                 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC 
               
               
                   
               
               
                 GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA 
               
               
                   
               
               
                 GGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCTGATTTTGAAAGCTTAGCTT 
               
               
                   
               
               
                 TAAATTTCTTTTAATTAAAAAAAAATGCTAGGCAGAATGACTCAAATTACGTTGGAT 
               
               
                   
               
               
                 ACAGTTGAATTTATTACGGTCTCATAGGGCCTGCCTGCTCGACCATGCTATACTAAA 
               
               
                   
               
               
                 AATTAAAAGTGTGTGTTACTAATTTTATAAATGGAGTTTCCATTTATATTTACCTTTA 
               
               
                   
               
               
                 TTTCTTATTTACCATTGTCTTAGTAGATATTTACAAACATGACAGAAACACTAAATCT 
               
               
                   
               
               
                 TGAGTTTGAATGCACAGATATAAACACTTAACGGGTTTTAAAAATAATAATGTTGGT 
               
               
                   
               
               
                 GAAAAAATATAACTTTGAGTGTAGCAGAGAGGAACCATTGCCACCTTCAGATTTTCC 
               
               
                   
               
               
                 TGTAACGATCGGGAACTGGCATCTTCAGGGAGTAGCTTAGGTCAGTGAAGAGAAGA 
               
               
                   
               
               
                 ACAAAAAGCAGCATATTACAGTTAGTTGTCTTCATCAATCTTTAAATATGTTGTGTG 
               
               
                   
               
               
                 GTTTTTCTCTCCCTGTTTCCACAGTTTTTCTTGATCATGAAAACGCCAACAAAATTCT 
               
               
                   
               
               
                 GAATCGGCCAAAGAGGTTTCTTGATCATGAAAACGCCAACAAAATTCTGAATCGGC 
               
               
                   
               
               
                 CAAAGAGGTATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAACCTTGAGAGA 
               
               
                   
               
               
                 GAATGTATGGAAGAAAAGTGTAGTTTTGAAGAAGCACGAGAAGTTTTTGAAAACAC 
               
               
                   
               
               
                 TGAAAGAACAACTGAATTTTGGAAGCAGTATGTTGATGGAGATCAGTGTGAGTCCA 
               
               
                   
               
               
                 ATCCATGTTTAAATGGCGGCAGTTGCAAGGATGACATTAATTCCTATGAATGTTGGT 
               
               
                   
               
               
                 GTCCCTTTGGATTTGAAGGAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGA 
               
               
                   
               
               
                 ATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATAACAAGGTGGTTTGCTCCT 
               
               
                   
               
               
                 GTACTGAGGGATATCGACTTGCAGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCA 
               
               
                   
               
               
                 TTTCCATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGACTG 
               
               
                   
               
               
                 TTTTTCCTGATGTGGACTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACAT 
               
               
                   
               
               
                 CACTCAAAGCACCCAATCATTTAATGACTTCACTCGGGTTGTTGGTGGAGAAGATGC 
               
               
                   
               
               
                 CAAACCAGGTCAATTCCCTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCATTCTG 
               
               
                   
               
               
                 TGGAGGCTCTATCGTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTGAAAC 
               
               
                   
               
               
                 TGGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGAACATA 
               
               
                   
               
               
                 CAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACCACAACTACAATGCAGCT 
               
               
                   
               
               
                 ATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTAGTGCTA 
               
               
                   
               
               
                 AACAGCTACGTTACACCTATTTGCATTGCTGACAAGGAATACACGAACATCTTCCTC 
               
               
                   
               
               
                 AAATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAAGGGAGATC 
               
               
                   
               
               
                 AGCTTTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTGACCGAGCCACATGTCTTCTA 
               
               
                   
               
               
                 TCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAGGAGGT 
               
               
                   
               
               
                 AGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACTGAAGTGGAAGGGAC 
               
               
                   
               
               
                 CAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAAT 
               
               
                   
               
               
                 ATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGATTAAGGAAAAAACAAAG 
               
               
                   
               
               
                 CTCACTTAACCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCC 
               
               
                   
               
               
                 CGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA 
               
               
                   
               
               
                 GGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGG 
               
               
                   
               
               
                 GCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCG 
               
               
                   
               
               
                 GTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCC 
               
               
                   
               
               
                 CCGTGAGATCGCCCATCGGTATAATGATTTGGGAGAACAACATTTCAAAGGCCTGTA 
               
               
                   
               
               
                 AGTTATAATGCTGAAAGCCCACTTAATATTTCTGGTAGTATTAGTTAAAGTTTTAAA 
               
               
                   
               
               
                 ACACCTTTTTCCACCTTGAGTGTGAGAATTGTAGAGCAGTGCTGTCCAGTAGAAATG 
               
               
                   
               
               
                 TGTGCATTGACAGAAAGACTGTGGATCTGTGCTGAGCAATGTGGCAGCCAGAGATC 
               
               
                   
               
               
                 ACAAGGCTATCAAGCACTTTGCACATGGCAAGTGTAACTGAGAAGCACACATTCAA 
               
               
                   
               
               
                 ATAATAGTTAATTTTAATTGAATGTATCTAGCCATGTGTGGCTAGTAGCTCCTTTCCT 
               
               
                   
               
               
                 GGAGAGAGAATCTGGAGCCCACATCTAACTTGTTAAGTCTGGAATCTTATTTTTTAT 
               
               
                   
               
               
                 TTCTGGAAAGGTCTATGAACTATAGTTTTGGGGGCAGCTCACTTACTAACTTTTAAT 
               
               
                   
               
               
                 GCAATAAGAATCTCATGGTATCTTGAGAACATTATTTTGTCTCTTTGTAGATCTAGGA 
               
               
                   
               
               
                 ACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGC 
               
               
                   
               
               
                 CGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCG 
               
               
                   
               
               
                 AGCGAGCGCGCAGAGAGGGAGTGGCCAA 
               
               
                   
               
               
                 P00354 full sequence (from ITR to ITR): (SEQ ID NO: 284) 
               
               
                 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC 
               
               
                   
               
               
                 GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA 
               
               
                   
               
               
                 GGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCTTAGCCTCTGGCAAAATGAA 
               
               
                   
               
               
                 GTGGGTAACCTTTCTCCTCCTCCTCTTCGTCTCCGGCTCTGCTTTTTCCAGGGGTGTGT 
               
               
                   
               
               
                 TTCGCCGAGAAGCACGTAAGAGTTTTATGTTTTTTCATCTCTGCTTGTATTTTTCTAG 
               
               
                   
               
               
                 TAATGGAAGCCTGGTATTTTAAAATAGTTAAATTTTCCTTTAGTGCTGATTTCTAGAT 
               
               
                   
               
               
                 TATTATTACTGTTGTTGTTGTTATTATTGTCATTATTTGCATCTGAGAACCCTTAGGTG 
               
               
                   
               
               
                 GTTATATTATTGATATATTTTTGGTATCTTTGATGACAATAATGGGGGATTTTGAAAG 
               
               
                   
               
               
                 CTTAGCTTTAAATTTCTTTTAATTAAAAAAAAATGCTAGGCAGAATGACTCAAATTA 
               
               
                   
               
               
                 CGTTGGATACAGTTGAATTTATTACGGTCTCATAGGGCCTGCCTGCTCGACCATGCT 
               
               
                   
               
               
                 ATACTAAAAATTAAAAGTGTGTGTTACTAATTTTATAAATGGAGTTTCCATTTATATT 
               
               
                   
               
               
                 TACCTTTATTTCTTATTTACCATTGTCTTAGTAGATATTTACAAACATGACAGAAACA 
               
               
                   
               
               
                 CTAAATCTTGAGTTTGAATGCACAGATATAAACACTTAACGGGTTTTAAAAATAATA 
               
               
                   
               
               
                 ATGTTGGTGAAAAAATATAACTTTGAGTGTAGCAGAGAGGAACCATTGCCACCTTCA 
               
               
                   
               
               
                 GATTTTCCTGTAACGATCGGGAACTGGCATCTTCAGGGAGTAGCTTAGGTCAGTGAA 
               
               
                   
               
               
                 GAGAAGAACAAAAAGCAGCATATTACAGTTAGTTGTCTTCATCAATCTTTAAATATG 
               
               
                   
               
               
                 TTGTGTGGTTTTTCTCTCCCTGTTTCCACAGTTTTTCTTGATCATGAAAACGCCAACA 
               
               
                   
               
               
                 AAATTCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATTGGAAGAGTTTGTTCAA 
               
               
                   
               
               
                 GGGAACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAAGAAGCACGAGA 
               
               
                   
               
               
                 AGTTTTTGAAAACACTGAAAGAACAACTGAATTTTGGAAGCAGTATGTTGATGGAG 
               
               
                   
               
               
                 ATCAGTGTGAGTCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGACATTAATT 
               
               
                   
               
               
                 CCTATGAATGTTGGTGTCCCTTTGGATTTGAAGGAAAGAACTGTGAATTAGATGTAA 
               
               
                   
               
               
                 CATGTAACATTAAGAATGGCAGATGCGAGCAGTTTTGTAAAAATAGTGCTGATAAC 
               
               
                   
               
               
                 AAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGCAGAAAACCAGAAGTCCTGT 
               
               
                   
               
               
                 GAACCAGCAGTGCCATTTCCATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTC 
               
               
                   
               
               
                 ACCCGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAATTCTACTGAAGCTGAA 
               
               
                   
               
               
                 ACCATTTTGGATAACATCACTCAAAGCACCCAATCATTTAATGACTTCACTCGGGTT 
               
               
                   
               
               
                 GTTGGTGGAGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGTTTTGAATGGT 
               
               
                   
               
               
                 AAAGTTGATGCATTCTGTGGAGGCTCTATCGTTAATGAAAAATGGATTGTAACTGCT 
               
               
                   
               
               
                 GCCCACTGTGTTGAAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATT 
               
               
                   
               
               
                 GAGGAGACAGAACATACAGAGCAAAAGCGAAATGTGATTCGAATTATTCCTCACCA 
               
               
                   
               
               
                 CAACTACAATGCAGCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACTGGA 
               
               
                   
               
               
                 CGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGCATTGCTGACAAGGAATA 
               
               
                   
               
               
                 CACGAACATCTTCCTCAAATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTT 
               
               
                   
               
               
                 CCACAAAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTGACCG 
               
               
                   
               
               
                 AGCCACATGTCTTCTATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGC 
               
               
                   
               
               
                 TTCCATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTAC 
               
               
                   
               
               
                 TGAAGTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGTG 
               
               
                   
               
               
                 CAATGAAAGGCAAATATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGATT 
               
               
                   
               
               
                 AAGGAAAAAACAAAGCTCACTTAACCTCGACTGTGCCTTCTAGTTGCCAGCCATCTG 
               
               
                   
               
               
                 TTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCT 
               
               
                   
               
               
                 TTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCT 
               
               
                   
               
               
                 GGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGG 
               
               
                   
               
               
                 CATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGG 
               
               
                   
               
               
                 CTCTAGGGGGTATCCCCGTGAGATCGCCCATCGGTATAATGATTTGGGAGAACAACA 
               
               
                   
               
               
                 TTTCAAAGGCCTGTAAGTTATAATGCTGAAAGCCCACTTAATATTTCTGGTAGTATT 
               
               
                   
               
               
                 AGTTAAAGTTTTAAAACACCTTTTTCCACCTTGAGTGTGAGAATTGTAGAGCAGTGC 
               
               
                   
               
               
                 TGTCCAGTAGAAATGTGTGCATTGACAGAAAGACTGTGGATCTGTGCTGAGCAATGT 
               
               
                   
               
               
                 GGCAGCCAGAGATCACAAGGCTATCAAGCACTTTGCACATGGCAAGTGTAACTGAG 
               
               
                   
               
               
                 AAGCACACATTCAAATAATAGTTAATTTTAATTGAATGTATCTAGCCATGTGTGGCT 
               
               
                   
               
               
                 AGTAGCTCCTTTCCTGGAGAGAGAATCTGGAGCCCACATCTAACTTGTTAAGTCTGG 
               
               
                   
               
               
                 AATCTTATTTTTTATTTCTGGAAAGGTCTATGAACTATAGTTTTGGGGGCAGCTCACT 
               
               
                   
               
               
                 TACTAACTTTTAATGCAATAAGAATCTCATGGTATCTTGAGAACATTATTTTGTCTCT 
               
               
                   
               
               
                 TTGTAGTACTGAAACCTTATACATGTGAAGTAAGGGGTCTATACTTAAGTCACATCT 
               
               
                   
               
               
                 CCAACCTTAGTAATGTTTTAATGTAGTAAAAAAATGAGTAATTAATTTATTTTTAGA 
               
               
                   
               
               
                 AGGTCAATAGTATCATGTATTCCAAATAACAGAGGTATATGGTTAGAAAAGAAACA 
               
               
                   
               
               
                 ATTCAAAGGACTTATATAATATCTAGCCTTGACAATGAATAAATTTAGAGAGTAGTT 
               
               
                   
               
               
                 TGCCTGTTTGCCTCATGTTCATAAATCTATTGACACATATGTGCATCTGCACTTCAGC 
               
               
                   
               
               
                 ATGGTAGAAGTCCATATTCAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCC 
               
               
                   
               
               
                 TCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGC 
               
               
                   
               
               
                 GACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCA 
               
               
                   
               
               
                 A 
               
               
                   
               
               
                 P00350: The 300/600 bp HA F9 construct (for G551) (SEQ ID NO: 285) 
               
               
                 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC 
               
               
                   
               
               
                 GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA 
               
               
                   
               
               
                 GGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCTAAGTATATTAGAGCGAGTC 
               
               
                   
               
               
                 TTTCTGCACACAGATCACCTTTCCTATCAACCCCACTAGCCTCTGGCAAAATGAAGT 
               
               
                   
               
               
                 GGGTAACCTTTCTCCTCCTCCTCTTCGTCTCCGGCTCTGCTTTTTCCAGGGGTGTGTTT 
               
               
                   
               
               
                 CGCCGAGAAGCACGTAAGAGTTTTATGTTTTTTCATCTCTGCTTGTATTTTTCTAGTA 
               
               
                   
               
               
                 ATGGAAGCCTGGTATTTTAAAATAGTTAAATTTTCCTTTAGTGCTGATTTCTAGATTA 
               
               
                   
               
               
                 TTATTACTGTTGTTGTTGTTATTATTGTCATTATTTGCATCTGAGAACCTTTTTCTTGA 
               
               
                   
               
               
                 TCATGAAAACGCCAACAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGTAAAT 
               
               
                   
               
               
                 TGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAGT 
               
               
                   
               
               
                 TTTGAAGAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTTTGGAA 
               
               
                   
               
               
                 GCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGCGGCAGTTG 
               
               
                   
               
               
                 CAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAGGAAAGAA 
               
               
                   
               
               
                 CTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGCAGTTTTGTAA 
               
               
                   
               
               
                 AAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGCAGA 
               
               
                   
               
               
                 AAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGAAGAGTTTCTGTTTC 
               
               
                   
               
               
                 ACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAA 
               
               
                   
               
               
                 TTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAAGCACCCAATCATTTAA 
               
               
                   
               
               
                 TGACTTCACTCGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAATTCCCTTGGCA 
               
               
                   
               
               
                 GGTTGTTTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATCGTTAATGAAAA 
               
               
                   
               
               
                 ATGGATTGTAACTGCTGCCCACTGTGTTGAAACTGGTGTTAAAATTACAGTTGTCGC 
               
               
                   
               
               
                 AGGTGAACATAATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAATGTGATTC 
               
               
                   
               
               
                 GAATTATTCCTCACCACAACTACAATGCAGCTATTAATAAGTACAACCATGACATTG 
               
               
                   
               
               
                 CCCTTCTGGAACTGGACGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGCA 
               
               
                   
               
               
                 TTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTGGATCTGGCTATGTAAGTG 
               
               
                   
               
               
                 GCTGGGGAAGAGTCTTCCACAAAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAG 
               
               
                   
               
               
                 TTCCACTTGTTGACCGAGCCACATGTCTTCTATCTACAAAGTTCACCATCTATAACAA 
               
               
                   
               
               
                 CATGTTCTGTGCTGGCTTCCATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGG 
               
               
                   
               
               
                 GGGACCCCATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTG 
               
               
                   
               
               
                 GGGTGAAGAGTGTGCAATGAAAGGCAAATATGGAATATATACCAAGGTATCCCGGT 
               
               
                   
               
               
                 ATGTCAACTGGATTAAGGAAAAAACAAAGCTCACTTAACCTCGACTGTGCCTTCTAG 
               
               
                   
               
               
                 TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC 
               
               
                   
               
               
                 ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGG 
               
               
                   
               
               
                 TGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGA 
               
               
                   
               
               
                 AGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAA 
               
               
                   
               
               
                 GAACCAGCTGGGGCTCTAGGGGGTATCCCCCTTAGGTGGTTATATTATTGATATATT 
               
               
                   
               
               
                 TTTGGTATCTTTGATGACAATAATGGGGGATTTTGAAAGCTTAGCTTTAAATTTCTTT 
               
               
                   
               
               
                 TAATTAAAAAAAAATGCTAGGCAGAATGACTCAAATTACGTTGGATACAGTTGAAT 
               
               
                   
               
               
                 TTATTACGGTCTCATAGGGCCTGCCTGCTCGACCATGCTATACTAAAAATTAAAAGT 
               
               
                   
               
               
                 GTGTGTTACTAATTTTATAAATGGAGTTTCCATTTATATTTACCTTTATTTCTTATTTA 
               
               
                   
               
               
                 CCATTGTCTTAGTAGATATTTACAAACATGACAGAAACACTAAAGATCTAGGAACCC 
               
               
                   
               
               
                 CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCC 
               
               
                   
               
               
                 CGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCG 
               
               
                   
               
               
                 AGCGCGCAGAGAGGGAGTGGCCAA 
               
               
                   
               
               
                 P00356: The 300/2000 bp HA F9 construct (for G551) (SEQ ID NO: 286) 
               
               
                 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC 
               
               
                   
               
               
                 GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA 
               
               
                   
               
               
                 GGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCTAAGTATATTAGAGCGAGTC 
               
               
                   
               
               
                 TTTCTGCACACAGATCACCTTTCCTATCAACCCCACTAGCCTCTGGCAAAATGAAGT 
               
               
                   
               
               
                 GGGTAACCTTTCTCCTCCTCCTCTTCGTCTCCGGCTCTGCTTTTTCCAGGGGTGTGTTT 
               
               
                   
               
               
                 CGCCGAGAAGCACGTAAGAGTTTTATGTTTTTTCATCTCTGCTTGTATTTTTCTAGTA 
               
               
                   
               
               
                 ATGGAAGCCTGGTATTTTAAAATAGTTAAATTTTCCTTTAGTGCTGATTTCTAGATTA 
               
               
                   
               
               
                 TTATTACTGTTGTTGTTGTTATTATTGTCATTATTTGCATCTGAGAACCTTTTTCTTGA 
               
               
                   
               
               
                 TCATGAAAACGCCAACAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGTAAAT 
               
               
                   
               
               
                 TGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAGT 
               
               
                   
               
               
                 TTTGAAGAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTTTGGAA 
               
               
                   
               
               
                 GCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGCGGCAGTTG 
               
               
                   
               
               
                 CAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAGGAAAGAA 
               
               
                   
               
               
                 CTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGCAGTTTTGTAA 
               
               
                   
               
               
                 AAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGCAGA 
               
               
                   
               
               
                 AAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGAAGAGTTTCTGTTTC 
               
               
                   
               
               
                 ACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAA 
               
               
                   
               
               
                 TTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAAGCACCCAATCATTTAA 
               
               
                   
               
               
                 TGACTTCACTCGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAATTCCCTTGGCA 
               
               
                   
               
               
                 GGTTGTTTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATCGTTAATGAAAA 
               
               
                   
               
               
                 ATGGATTGTAACTGCTGCCCACTGTGTTGAAACTGGTGTTAAAATTACAGTTGTCGC 
               
               
                   
               
               
                 AGGTGAACATAATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAATGTGATTC 
               
               
                   
               
               
                 GAATTATTCCTCACCACAACTACAATGCAGCTATTAATAAGTACAACCATGACATTG 
               
               
                   
               
               
                 CCCTTCTGGAACTGGACGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGCA 
               
               
                   
               
               
                 TTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTGGATCTGGCTATGTAAGTG 
               
               
                   
               
               
                 GCTGGGGAAGAGTCTTCCACAAAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAG 
               
               
                   
               
               
                 TTCCACTTGTTGACCGAGCCACATGTCTTCTATCTACAAAGTTCACCATCTATAACAA 
               
               
                   
               
               
                 CATGTTCTGTGCTGGCTTCCATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGG 
               
               
                   
               
               
                 GGGACCCCATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTG 
               
               
                   
               
               
                 GGGTGAAGAGTGTGCAATGAAAGGCAAATATGGAATATATACCAAGGTATCCCGGT 
               
               
                   
               
               
                 ATGTCAACTGGATTAAGGAAAAAACAAAGCTCACTTAACCTCGACTGTGCCTTCTAG 
               
               
                   
               
               
                 TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC 
               
               
                   
               
               
                 ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGG 
               
               
                   
               
               
                 TGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGA 
               
               
                   
               
               
                 AGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAA 
               
               
                   
               
               
                 GAACCAGCTGGGGCTCTAGGGGGTATCCCCCTTAGGTGGTTATATTATTGATATATT 
               
               
                   
               
               
                 TTTGGTATCTTTGATGACAATAATGGGGGATTTTGAAAGCTTAGCTTTAAATTTCTTT 
               
               
                   
               
               
                 TAATTAAAAAAAAATGCTAGGCAGAATGACTCAAATTACGTTGGATACAGTTGAAT 
               
               
                   
               
               
                 TTATTACGGTCTCATAGGGCCTGCCTGCTCGACCATGCTATACTAAAAATTAAAAGT 
               
               
                   
               
               
                 GTGTGTTACTAATTTTATAAATGGAGTTTCCATTTATATTTACCTTTATTTCTTATTTA 
               
               
                   
               
               
                 CCATTGTCTTAGTAGATATTTACAAACATGACAGAAACACTAAATCTTGAGTTTGAA 
               
               
                   
               
               
                 TGCACAGATATAAACACTTAACGGGTTTTAAAAATAATAATGTTGGTGAAAAAATAT 
               
               
                   
               
               
                 AACTTTGAGTGTAGCAGAGAGGAACCATTGCCACCTTCAGATTTTCCTGTAACGATC 
               
               
                   
               
               
                 GGGAACTGGCATCTTCAGGGAGTAGCTTAGGTCAGTGAAGAGAAGAACAAAAAGCA 
               
               
                   
               
               
                 GCATATTACAGTTAGTTGTCTTCATCAATCTTTAAATATGTTGTGTGGTTTTTCTCTCC 
               
               
                   
               
               
                 CTGTTTCCACAGACAAGAGTGAGATCGCCCATCGGTATAATGATTTGGGAGAACAA 
               
               
                   
               
               
                 CATTTCAAAGGCCTGTAAGTTATAATGCTGAAAGCCCACTTAATATTTCTGGTAGTA 
               
               
                   
               
               
                 TTAGTTAAAGTTTTAAAACACCTTTTTCCACCTTGAGTGTGAGAATTGTAGAGCAGT 
               
               
                   
               
               
                 GCTGTCCAGTAGAAATGTGTGCATTGACAGAAAGACTGTGGATCTGTGCTGAGCAAT 
               
               
                   
               
               
                 GTGGCAGCCAGAGATCACAAGGCTATCAAGCACTTTGCACATGGCAAGTGTAACTG 
               
               
                   
               
               
                 AGAAGCACACATTCAAATAATAGTTAATTTTAATTGAATGTATCTAGCCATGTGTGG 
               
               
                   
               
               
                 CTAGTAGCTCCTTTCCTGGAGAGAGAATCTGGAGCCCACATCTAACTTGTTAAGTCT 
               
               
                   
               
               
                 GGAATCTTATTTTTTATTTCTGGAAAGGTCTATGAACTATAGTTTTGGGGGCAGCTCA 
               
               
                   
               
               
                 CTTACTAACTTTTAATGCAATAAGATCCATGGTATCTTGAGAACATTATTTTGTCTCT 
               
               
                   
               
               
                 TTGTAGTACTGAAACCTTATACATGTGAAGTAAGGGGTCTATACTTAAGTCACATCT 
               
               
                   
               
               
                 CCAACCTTAGTAATGTTTTAATGTAGTAAAAAAATGAGTAATTAATTTATTTTTAGA 
               
               
                   
               
               
                 AGGTCAATAGTATCATGTATTCCAAATAACAGAGGTATATGGTTAGAAAAGAAACA 
               
               
                   
               
               
                 ATTCAAAGGACTTATATAATATCTAGCCTTGACAATGAATAAATTTAGAGAGTAGTT 
               
               
                   
               
               
                 TGCCTGTTTGCCTCATGTTCATAAATCTATTGACACATATGTGCATCTGCACTTCAGC 
               
               
                   
               
               
                 ATGGTAGAAGTCCATATTCCTTTGCTTGGAAAGGCAGGTGTTCCCATTACGCCTCAG 
               
               
                   
               
               
                 AGAATAGCTGACGGGAAGAGGCTTTCTAGATAGTTGTATGAAAGATATACAAAATC 
               
               
                   
               
               
                 TCGCAGGTATACACAGGCATGATTTGCTGGTTGGGAGAGCCACTTGCCTCATACTGA 
               
               
                   
               
               
                 GGTTTTTGTGTCTGCTTTTCAGAGTCCTGATTGCCTTTTCCCAGTATCTCCAGAAATG 
               
               
                   
               
               
                 CTCATACGATGAGCATGCCAAATTAGTGCAGGAAGTAACAGACTTTGCAAAGACGT 
               
               
                   
               
               
                 GTGTTGCCGATGAGTCTGCCGCCAACTGTGACAAATCCCTTGTGAGTACCTTCTGAT 
               
               
                   
               
               
                 TTTGTGGATCTACTTTCCTGCTTTCTGGAACTCTGTTTCAAAGCCAATCATGACTCCA 
               
               
                   
               
               
                 TCACTTAAGGCCCCGGGAACACTGTGGCAGAGGGCAGCAGAGAGATTGATAAAGCC 
               
               
                   
               
               
                 AGGGTGATGGGAATTTTCTGTGGGACTCCATTTCATAGTAATTGCAGAAGCTACAAT 
               
               
                   
               
               
                 ACACTCAAAAAGTCTCACCACATGACTGCCCAAATGGGAGCTTGACAGTGACAGTG 
               
               
                   
               
               
                 ACAGTAGATATGCCAAAGTGGATGAGGGAAAGACCACAAGAGCTAAACCCTGTAAA 
               
               
                   
               
               
                 AAGAACTGTAGGCAACTAAGGAATGCAGAGAGAAAGATCTAGGAACCCCTAGTGAT 
               
               
                   
               
               
                 GGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAA 
               
               
                   
               
               
                 GCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCA 
               
               
                   
               
               
                 GAGAGGGAGTGGCCAA 
               
               
                   
               
               
                 P00362: The 300/1500 bp HA F9 construct (for G551) (SEQ ID NO: 287) 
               
               
                 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC 
               
               
                   
               
               
                 GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA 
               
               
                   
               
               
                 GGGAGTGGCCAACTCCATCACTAGGGGTTCCTAGATCTAAGTATATTAGAGCGAGTC 
               
               
                   
               
               
                 TTTCTGCACACAGATCACCTTTCCTATCAACCCCACTAGCCTCTGGCAAAATGAAGT 
               
               
                   
               
               
                 GGGTAACCTTTCTCCTCCTCCTCTTCGTCTCCGGCTCTGCTTTTTCCAGGGGTGTGTTT 
               
               
                   
               
               
                 CGCCGAGAAGCACGTAAGAGTTTTATGTTTTTTCATCTCTGCTTGTATTTTTCTAGTA 
               
               
                   
               
               
                 ATGGAAGCCTGGTATTTTAAAATAGTTAAATTTTCCTTTAGTGCTGATTTCTAGATTA 
               
               
                   
               
               
                 TTATTACTGTTGTTGTTGTTATTATTGTCATTATTTGCATCTGAGAACCTTTTTCTTGA 
               
               
                   
               
               
                 TCATGAAAACGCCAACAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGTAAAT 
               
               
                   
               
               
                 TGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAGT 
               
               
                   
               
               
                 TTTGAAGAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTTTGGAA 
               
               
                   
               
               
                 GCAGTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGCGGCAGTTG 
               
               
                   
               
               
                 CAAGGATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAGGAAAGAA 
               
               
                   
               
               
                 CTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGCAGTTTTGTAA 
               
               
                   
               
               
                 AAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGCAGA 
               
               
                   
               
               
                 AAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGAAGAGTTTCTGTTTC 
               
               
                   
               
               
                 ACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGATGTGGACTATGTAAA 
               
               
                   
               
               
                 TTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAAGCACCCAATCATTTAA 
               
               
                   
               
               
                 TGACTTCACTCGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAATTCCCTTGGCA 
               
               
                   
               
               
                 GGTTGTTTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATCGTTAATGAAAA 
               
               
                   
               
               
                 ATGGATTGTAACTGCTGCCCACTGTGTTGAAACTGGTGTTAAAATTACAGTTGTCGC 
               
               
                   
               
               
                 AGGTGAACATAATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAATGTGATTC 
               
               
                   
               
               
                 GAATTATTCCTCACCACAACTACAATGCAGCTATTAATAAGTACAACCATGACATTG 
               
               
                   
               
               
                 CCCTTCTGGAACTGGACGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGCA 
               
               
                   
               
               
                 TTGCTGACAAGGAATACACGAACATCTTCCTCAAATTTGGATCTGGCTATGTAAGTG 
               
               
                   
               
               
                 GCTGGGGAAGAGTCTTCCACAAAGGGAGATCAGCTTTAGTTCTTCAGTACCTTAGAG 
               
               
                   
               
               
                 TTCCACTTGTTGACCGAGCCACATGTCTTCTATCTACAAAGTTCACCATCTATAACAA 
               
               
                   
               
               
                 CATGTTCTGTGCTGGCTTCCATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGG 
               
               
                   
               
               
                 GGGACCCCATGTTACTGAAGTGGAAGGGACCAGTTTCTTAACTGGAATTATTAGCTG 
               
               
                   
               
               
                 GGGTGAAGAGTGTGCAATGAAAGGCAAATATGGAATATATACCAAGGTATCCCGGT 
               
               
                   
               
               
                 ATGTCAACTGGATTAAGGAAAAAACAAAGCTCACTTAACCTCGACTGTGCCTTCTAG 
               
               
                   
               
               
                 TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC 
               
               
                   
               
               
                 ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGG 
               
               
                   
               
               
                 TGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGA 
               
               
                   
               
               
                 AGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAA 
               
               
                   
               
               
                 GAACCAGCTGGGGCTCTAGGGGGTATCCCCCTTAGGTGGTTATATTATTGATATATT 
               
               
                   
               
               
                 TTTGGTATCTTTGATGACAATAATGGGGGATTTTGAAAGCTTAGCTTTAAATTTCTTT 
               
               
                   
               
               
                 TAATTAAAAAAAAATGCTAGGCAGAATGACTCAAATTACGTTGGATACAGTTGAAT 
               
               
                   
               
               
                 TTATTACGGTCTCATAGGGCCTGCCTGCTCGACCATGCTATACTAAAAATTAAAAGT 
               
               
                   
               
               
                 GTGTGTTACTAATTTTATAAATGGAGTTTCCATTTATATTTACCTTTATTTCTTATTTA 
               
               
                   
               
               
                 CCATTGTCTTAGTAGATATTTACAAACATGACAGAAACACTAAATCTTGAGTTTGAA 
               
               
                   
               
               
                 TGCACAGATATAAACACTTAACGGGTTTTAAAAATAATAATGTTGGTGAAAAAATAT 
               
               
                   
               
               
                 AACTTTGAGTGTAGCAGAGAGGAACCATTGCCACCTTCAGATTTTCCTGTAACGATC 
               
               
                   
               
               
                 GGGAACTGGCATCTTCAGGGAGTAGCTTAGGTCAGTGAAGAGAAGAACAAAAAGCA 
               
               
                   
               
               
                 GCATATTACAGTTAGTTGTCTTCATCAATCTTTAAATATGTTGTGTGGTTTTTCTCTCC 
               
               
                   
               
               
                 CTGTTTCCACAGACAAGAGTGAGATCGCCCATCGGTATAATGATTTGGGAGAACAA 
               
               
                   
               
               
                 CATTTCAAAGGCCTGTAAGTTATAATGCTGAAAGCCCACTTAATATTTCTGGTAGTA 
               
               
                   
               
               
                 TTAGTTAAAGTTTTAAAACACCTTTTTCCACCTTGAGTGTGAGAATTGTAGAGCAGT 
               
               
                   
               
               
                 GCTGTCCAGTAGAAATGTGTGCATTGACAGAAAGACTGTGGATCTGTGCTGAGCAAT 
               
               
                   
               
               
                 GTGGCAGCCAGAGATCACAAGGCTATCAAGCACTTTGCACATGGCAAGTGTAACTG 
               
               
                   
               
               
                 AGAAGCACACATTCAAATAATAGTTAATTTTAATTGAATGTATCTAGCCATGTGTGG 
               
               
                   
               
               
                 CTAGTAGCTCCTTTCCTGGAGAGAGAATCTGGAGCCCACATCTAACTTGTTAAGTCT 
               
               
                   
               
               
                 GGAATCTTATTTTTTATTTCTGGAAAGGTCTATGAACTATAGTTTTGGGGGCAGCTCA 
               
               
                   
               
               
                 CTTACTAACTTTTAATGCAATAAGATCCATGGTATCTTGAGAACATTATTTTGTCTCT 
               
               
                   
               
               
                 TTGTAGTACTGAAACCTTATACATGTGAAGTAAGGGGTCTATACTTAAGTCACATCT 
               
               
                   
               
               
                 CCAACCTTAGTAATGTTTTAATGTAGTAAAAAAATGAGTAATTAATTTATTTTTAGA 
               
               
                   
               
               
                 AGGTCAATAGTATCATGTATTCCAAATAACAGAGGTATATGGTTAGAAAAGAAACA 
               
               
                   
               
               
                 ATTCAAAGGACTTATATAATATCTAGCCTTGACAATGAATAAATTTAGAGAGTAGTT 
               
               
                   
               
               
                 TGCCTGTTTGCCTCATGTTCATAAATCTATTGACACATATGTGCATCTGCACTTCAGC 
               
               
                   
               
               
                 ATGGTAGAAGTCCATATTCCTTTGCTTGGAAAGGCAGGTGTTCCCATTACGCCTCAG 
               
               
                   
               
               
                 AGAATAGCTGACGGGAAGAGGCTTTCTAGATAGTTGTATGAAAGATATACAAAATC 
               
               
                   
               
               
                 TCGCAGGTATACACAGGCATGATTTGCTGGTTGGGAGAGCCACTTAGATCTAGGAAC 
               
               
                   
               
               
                 CCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCG 
               
               
                   
               
               
                 CCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAG 
               
               
                   
               
               
                 CGAGCGCGCAGAGAGGGAGTGGCCAA 
               
               
                   
               
               
                 Factor IX R338L polypeptide encoded in P00147 (SEQ ID NO: 702) 
               
               
                 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCK 
               
               
                   
               
               
                 DDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPA 
               
               
                   
               
               
                 VPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFP 
               
               
                   
               
               
                 WQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHN 
               
               
                   
               
               
                 YNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVL 
               
               
                   
               
               
                 QYLRVPLVDRATCLLSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEE 
               
               
                   
               
               
                 CAMKGKYGIYTKVSRYVNWIKEKTKLT 
               
               
                   
               
               
                 Cas9 ORF (SEQ ID NO: 703) 
               
               
                 ATGGATAAGAAGTACTCAATCGGGCTGGATATCGGAACTAATTCCGTGGGTTGGGC 
               
               
                   
               
               
                 AGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGTCCTGGGGAACA 
               
               
                   
               
               
                 CCGATAGACACAGCATCAAGAAAAATCTCATCGGAGCCCTGCTGTTTGACTCCGGC 
               
               
                   
               
               
                 GAAACCGCAGAAGCGACCCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGGCG 
               
               
                   
               
               
                 GAAGAATCGCATCTGCTATCTGCAAGAGATCTTTTCGAACGAAATGGCAAAGGTCG 
               
               
                   
               
               
                 ACGACAGCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAGGACAAGAAG 
               
               
                   
               
               
                 CATGAACGGCATCCTATCTTTGGAAACATCGTCGACGAAGTGGCGTACCACGAAAA 
               
               
                   
               
               
                 GTACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCG 
               
               
                   
               
               
                 ACCTCAGATTGATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGACACTTCC 
               
               
                   
               
               
                 TGATCGAAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTTTCATTCAAC 
               
               
                   
               
               
                 TGGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCTAGCGGCGTC 
               
               
                   
               
               
                 GATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCT 
               
               
                   
               
               
                 GATCGCACAGCTGCCGGGAGAGAAAAAGAACGGACTTTTCGGCAACTTGATCGCTC 
               
               
                   
               
               
                 TCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCGA 
               
               
                   
               
               
                 AGCTGCAACTCTCAAAGGACACCTACGACGACGACTTGGACAATTTGCTGGCACAA 
               
               
                   
               
               
                 ATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATC 
               
               
                   
               
               
                 TTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCC 
               
               
                   
               
               
                 TCGATGATTAAGCGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCGCT 
               
               
                   
               
               
                 CGTGAGACAGCAACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGA 
               
               
                   
               
               
                 ATGGGTACGCAGGGTACATCGATGGAGGCGCTAGCCAGGAAGAGTTCTATAAGTTC 
               
               
                   
               
               
                 ATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCTGAA 
               
               
                   
               
               
                 CAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACAACGGATCCATTCCCCACC 
               
               
                   
               
               
                 AGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCCAT 
               
               
                   
               
               
                 TCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATT 
               
               
                   
               
               
                 ACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCA 
               
               
                   
               
               
                 GAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGC 
               
               
                   
               
               
                 ACAAAGCTTCATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGG 
               
               
                   
               
               
                 TGCTTCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTA 
               
               
                   
               
               
                 AAGTGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTCCGGAGAACAG 
               
               
                   
               
               
                 AAGAAAGCAATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGTCAAGCA 
               
               
                   
               
               
                 GCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCG 
               
               
                   
               
               
                 GGGTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCA 
               
               
                   
               
               
                 TCAAGGACAAGGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGATATC 
               
               
                   
               
               
                 GTCCTGACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGACC 
               
               
                   
               
               
                 TACGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACT 
               
               
                   
               
               
                 GGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGG 
               
               
                   
               
               
                 TAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCA 
               
               
                   
               
               
                 ATTGATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAAAAAGCACAAGTGT 
               
               
                   
               
               
                 CCGGACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCCGGCG 
               
               
                   
               
               
                 ATTAAGAAGGGAATTCTGCAAACTGTGAAGGTGGTCGACGAGCTGGTGAAGGTCAT 
               
               
                   
               
               
                 GGGACGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAACCAGACTA 
               
               
                   
               
               
                 CCCAGAAGGGCCAGAAAAACTCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAAT 
               
               
                   
               
               
                 CAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTG 
               
               
                   
               
               
                 CAGAACGAGAAGCTCTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGA 
               
               
                   
               
               
                 CCAAGAGCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACA 
               
               
                   
               
               
                 GTCCTTTCTGAAGGATGACTCGATCGATAACAAGGTGTTGACTCGCAGCGACAAGA 
               
               
                   
               
               
                 ACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAA 
               
               
                   
               
               
                 TTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCT 
               
               
                   
               
               
                 CACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCAAAC 
               
               
                   
               
               
                 GGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCTTGGACTCC 
               
               
                   
               
               
                 CGCATGAACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGAT 
               
               
                   
               
               
                 TACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTTTACAAAGT 
               
               
                   
               
               
                 GAGAGAAATCAACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGGTCG 
               
               
                   
               
               
                 GTACCGCCCTGATCAAAAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACT 
               
               
                   
               
               
                 ACAAGGTCTACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGGAA 
               
               
                   
               
               
                 AGCAACTGCGAAATACTTCTTTTACTCAAACATCATGAACTTTTTCAAGACTGAAAT 
               
               
                   
               
               
                 TACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGAGAA 
               
               
                   
               
               
                 ACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCT 
               
               
                   
               
               
                 CTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTC 
               
               
                   
               
               
                 AAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGAAAG 
               
               
                   
               
               
                 ACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTC 
               
               
                   
               
               
                 CTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAAGCTCAAATCCGTCAAAG 
               
               
                   
               
               
                 AGCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGATT 
               
               
                   
               
               
                 TCCTCGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTCCCC 
               
               
                   
               
               
                 AAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGG 
               
               
                   
               
               
                 AGAACTCCAAAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTA 
               
               
                   
               
               
                 TCTTGCTTCGCACTACGAAAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGC 
               
               
                   
               
               
                 AGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCG 
               
               
                   
               
               
                 AGTTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCT 
               
               
                   
               
               
                 ACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTTG 
               
               
                   
               
               
                 TTCACCCTGACTAACCTGGGAGCCCCAGCCGCCTTCAAGTACTTCGATACTACTATC 
               
               
                   
               
               
                 GATCGCAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCA 
               
               
                   
               
               
                 AAGCATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGAT 
               
               
                   
               
               
                 U-dep Cas9 ORF (SEQ ID NO: 704) 
               
               
                 ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGC 
               
               
                   
               
               
                 AGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGAAACA 
               
               
                   
               
               
                 CAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGA 
               
               
                   
               
               
                 GAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAA 
               
               
                   
               
               
                 GAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAAGGTC 
               
               
                   
               
               
                 GACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAA 
               
               
                   
               
               
                 GCACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAA 
               
               
                   
               
               
                 AGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAGGCA 
               
               
                   
               
               
                 GACCTGAGACTGATCTACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTC 
               
               
                   
               
               
                 CTGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCCA 
               
               
                   
               
               
                 GCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGGAG 
               
               
                   
               
               
                 TCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAA 
               
               
                   
               
               
                 CCTGATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCG 
               
               
                   
               
               
                 CACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGAC 
               
               
                   
               
               
                 GCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCTGGACAACCTGCTGGC 
               
               
                   
               
               
                 ACAGATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACG 
               
               
                   
               
               
                 CAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCTG 
               
               
                   
               
               
                 AGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGACCTGACACTGCTGAA 
               
               
                   
               
               
                 GGCACTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGA 
               
               
                   
               
               
                 GCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTA 
               
               
                   
               
               
                 CAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCA 
               
               
                   
               
               
                 AGCTGAACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCAT 
               
               
                   
               
               
                 CCCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACT 
               
               
                   
               
               
                 TCTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGATCCTGACATTCAGA 
               
               
                   
               
               
                 ATCCCGTACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGAC 
               
               
                   
               
               
                 AAGAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAG 
               
               
                   
               
               
                 GGAGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGACAAGAACCTGCC 
               
               
                   
               
               
                 GAACGAAAAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACA 
               
               
                   
               
               
                 ACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTG 
               
               
                   
               
               
                 AGCGGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACAAACAGAAAGG 
               
               
                   
               
               
                 TCACAGTCAAGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGC 
               
               
                   
               
               
                 GTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACATACCACGA 
               
               
                   
               
               
                 CCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAAAACGAAGACA 
               
               
                   
               
               
                 TCCTGGAAGACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATCGAA 
               
               
                   
               
               
                 GAAAGACTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAA 
               
               
                   
               
               
                 GAGAAGAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCAACGGAATC 
               
               
                   
               
               
                 AGAGACAAGCAGAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGC 
               
               
                   
               
               
                 AAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAGGAAGACA 
               
               
                   
               
               
                 TCCAGAAGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACATCGCAAA 
               
               
                   
               
               
                 CCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCG 
               
               
                   
               
               
                 ACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCATCGAAATG 
               
               
                   
               
               
                 GCAAGAGAAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGAATG 
               
               
                   
               
               
                 AAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACC 
               
               
                   
               
               
                 CGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTGCAGAAC 
               
               
                   
               
               
                 GGAAGAGACATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGACTACGA 
               
               
                   
               
               
                 CGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGG 
               
               
                   
               
               
                 TCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAGCGAAGA 
               
               
                   
               
               
                 AGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTGATCA 
               
               
                   
               
               
                 CACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACT 
               
               
                   
               
               
                 GGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCACAAAG 
               
               
                   
               
               
                 CACGTCGCACAGATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGACA 
               
               
                   
               
               
                 AGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTC 
               
               
                   
               
               
                 AGAAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGCACA 
               
               
                   
               
               
                 CGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGC 
               
               
                   
               
               
                 TGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATC 
               
               
                   
               
               
                 GCAAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAGCAA 
               
               
                   
               
               
                 CATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGA 
               
               
                   
               
               
                 GACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAG 
               
               
                   
               
               
                 AGACTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAAGA 
               
               
                   
               
               
                 AGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAA 
               
               
                   
               
               
                 CAGCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGA 
               
               
                   
               
               
                 TTCGACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGG 
               
               
                   
               
               
                 AAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAA 
               
               
                   
               
               
                 AGAAGCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGA 
               
               
                   
               
               
                 AGTCAAGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAA 
               
               
                   
               
               
                 ACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACT 
               
               
                   
               
               
                 GGCACTGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGC 
               
               
                   
               
               
                 TGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAA 
               
               
                   
               
               
                 GCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCAAGAGAGTCATCC 
               
               
                   
               
               
                 TGGCAGACGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAGAGACAA 
               
               
                   
               
               
                 GCCGATCAGAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGG 
               
               
                   
               
               
                 GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAAGAGATACACA 
               
               
                   
               
               
                 AGCACAAAGGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGGACTGTA 
               
               
                   
               
               
                 CGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAG 
               
               
                   
               
               
                 AAGAAGAGAAAGGTCTAG 
               
               
                   
               
               
                 mRNA comprising U dep Cas9 (SEQ ID NO: 705) 
               
               
                 GGGUCCCGCAGUCGGCGUCCAGCGGCUCUGCUUGUUCGUGUGUGUGUCGUUGCAG 
               
               
                   
               
               
                 GCCUUAUUCGGAUCCGCCACCAUGGACAAGAAGUACAGCAUCGGACUGGACAUCG 
               
               
                   
               
               
                 GAACAAACAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGAGCAA 
               
               
                   
               
               
                 GAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGCAUCAAGAAGAACCUGAU 
               
               
                   
               
               
                 CGGAGCACUGCUGUUCGACAGCGGAGAAACAGCAGAAGCAACAAGACUGAAGAG 
               
               
                   
               
               
                 AACAGCAAGAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAGGA 
               
               
                   
               
               
                 AAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUUCUUCCACAGACUGGAA 
               
               
                   
               
               
                 GAAAGCUUCCUGGUCGAAGAAGACAAGAAGCACGAAAGACACCCGAUCUUCGGA 
               
               
                   
               
               
                 AACAUCGUCGACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCUGA 
               
               
                   
               
               
                 GAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGAGACUGAUCUACCUGGC 
               
               
                   
               
               
                 ACUGGCACACAUGAUCAAGUUCAGAGGACACUUCCUGAUCGAAGGAGACCUGAAC 
               
               
                   
               
               
                 CCGGACAACAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACAACC 
               
               
                   
               
               
                 AGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUCGACGCAAAGGCAAUCCU 
               
               
                   
               
               
                 GAGCGCAAGACUGAGCAAGAGCAGAAGACUGGAAAACCUGAUCGCACAGCUGCCG 
               
               
                   
               
               
                 GGAGAAAAGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGACUG 
               
               
                   
               
               
                 ACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGACGCAAAGCUGCAGCUGA 
               
               
                   
               
               
                 GCAAGGACACAUACGACGACGACCUGGACAACCUGCUGGCACAGAUCGGAGACCA 
               
               
                   
               
               
                 GUACGCAGACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCUGAGC 
               
               
                   
               
               
                 GACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCACCGCUGAGCGCAAGCAUGA 
               
               
                   
               
               
                 UCAAGAGAUACGACGAACACCACCAGGACCUGACACUGCUGAAGGCACUGGUCAG 
               
               
                   
               
               
                 ACAGCAGCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGAACGG 
               
               
                   
               
               
                 AUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAAGAAUUCUACAAGUUCAU 
               
               
                   
               
               
                 CAAGCCGAUCCUGGAAAAGAUGGACGGAACAGAAGAACUGCUGGUCAAGCUGAA 
               
               
                   
               
               
                 CAGAGAAGACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCGCAC 
               
               
                   
               
               
                 CAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAGACAGGAAGACUUCUACC 
               
               
                   
               
               
                 CGUUCCUGAAGGACAACAGAGAAAAGAUCGAAAAGAUCCUGACAUUCAGAAUCC 
               
               
                   
               
               
                 CGUACUACGUCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGACAA 
               
               
                   
               
               
                 GAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAGAAGUCGUCGACAAGG 
               
               
                   
               
               
                 GAGCAAGCGCACAGAGCUUCAUCGAAAGAAUGACAAACUUCGACAAGAACCUGCC 
               
               
                   
               
               
                 GAACGAAAAGGUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCUAC 
               
               
                   
               
               
                 AACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUGAGAAAGCCGGCAUUC 
               
               
                   
               
               
                 CUGAGCGGAGAACAGAAGAAGGCAAUCGUCGACCUGCUGUUCAAGACAAACAGA 
               
               
                   
               
               
                 AAGGUCACAGUCAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC 
               
               
                   
               
               
                 GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGCAAGCCUGGGAACA 
               
               
                   
               
               
                 UACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAAGAAA 
               
               
                   
               
               
                 ACGAAGACAUCCUGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA 
               
               
                   
               
               
                 AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCGACGACAAGGUCAU 
               
               
                   
               
               
                 GAAGCAGCUGAAGAGAAGAAGAUACACAGGAUGGGGAAGACUGAGCAGAAAGCU 
               
               
                   
               
               
                 GAUCAACGGAAUCAGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA 
               
               
                   
               
               
                 GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCACGACGACAGCCUG 
               
               
                   
               
               
                 ACAUUCAAGGAAGACAUCCAGAAGGCACAGGUCAGCGGACAGGGAGACAGCCUGC 
               
               
                   
               
               
                 ACGAACACAUCGCAAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCA 
               
               
                   
               
               
                 GACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAGACACAAGCCGGA 
               
               
                   
               
               
                 AAACAUCGUCAUCGAAAUGGCAAGAGAAAACCAGACAACACAGAAGGGACAGAA 
               
               
                   
               
               
                 GAACAGCAGAGAAAGAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAG 
               
               
                   
               
               
                 CCAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGAACGAAAAGCUG 
               
               
                   
               
               
                 UACCUGUACUACCUGCAGAACGGAAGAGACAUGUACGUCGACCAGGAACUGGACA 
               
               
                   
               
               
                 UCAACAGACUGAGCGACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAA 
               
               
                   
               
               
                 GGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGAACAGAGGAAA 
               
               
                   
               
               
                 GAGCGACAACGUCCCGAGCGAAGAAGUCGUCAAGAAGAUGAAGAACUACUGGAG 
               
               
                   
               
               
                 ACAGCUGCUGAACGCAAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAG 
               
               
                   
               
               
                 GCAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUCAAGAGACAG 
               
               
                   
               
               
                 CUGGUCGAAACAAGACAGAUCACAAAGCACGUCGCACAGAUCCUGGACAGCAGAA 
               
               
                   
               
               
                 UGAACACAAAGUACGACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCA 
               
               
                   
               
               
                 CACUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUUCUACAAGG 
               
               
                   
               
               
                 UCAGAGAAAUCAACAACUACCACCACGCACACGACGCAUACCUGAACGCAGUCGU 
               
               
                   
               
               
                 CGGAACAGCACUGAUCAAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGG 
               
               
                   
               
               
                 AGACUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAACAGGAAAU 
               
               
                   
               
               
                 CGGAAAGGCAACAGCAAAGUACUUCUUCUACAGCAACAUCAUGAACUUCUUCAAG 
               
               
                   
               
               
                 ACAGAAAUCACACUGGCAAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACA 
               
               
                   
               
               
                 AACGGAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCAACAGUC 
               
               
                   
               
               
                 AGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGUCAAGAAGACAGAAGUCCAG 
               
               
                   
               
               
                 ACAGGAGGAUUCAGCAAGGAAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUG 
               
               
                   
               
               
                 AUCGCAAGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAGCCCG 
               
               
                   
               
               
                 ACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCGAAAAGGGAAAGAGCAAG 
               
               
                   
               
               
                 AAGCUGAAGAGCGUCAAGGAACUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGC 
               
               
                   
               
               
                 UUCGAAAAGAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCAAG 
               
               
                   
               
               
                 AAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUCGAACUGGAAAACGGA 
               
               
                   
               
               
                 AGAAAGAGAAUGCUGGCAAGCGCAGGAGAACUGCAGAAGGGAAACGAACUGGCA 
               
               
                   
               
               
                 CUGCCGAGCAAGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUGA 
               
               
                   
               
               
                 AGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGUCGAACAGCACAAGCA 
               
               
                   
               
               
                 CUACCUGGACGAAAUCAUCGAACAGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUG 
               
               
                   
               
               
                 GCAGACGCAAACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAAGC 
               
               
                   
               
               
                 CGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCACACUGACAAACCUGGG 
               
               
                   
               
               
                 AGCACCGGCAGCAUUCAAGUACUUCGACACAACAAUCGACAGAAAGAGAUACACA 
               
               
                   
               
               
                 AGCACAAAGGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGACUGU 
               
               
                   
               
               
                 ACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGACGGAGGAGGAAGCCCGA 
               
               
                   
               
               
                 AGAAGAAGAGAAAGGUCUAGCUAGCCAUCACAUUUAAAAGCAUCUCAGCCUACC 
               
               
                   
               
               
                 AUGAGAAUAAGAGAAAGAAAAUGAAGAUCAAUAGCUUAUUCAUCUCUUUUUCUU 
               
               
                   
               
               
                 UUUCGUUGGUGUAAAGCCAACACCCUGUCUAAAAAACAUAAAUUUCUUUAAUCA 
               
               
                   
               
               
                 UUUUGCCUCUUUUCUCUGUGCUUCAAUUAAUAAAAAAUGGAAAGAACCUCGAGA 
               
               
                   
               
               
                 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 
               
               
                   
               
               
                 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA