Patent Publication Number: US-2023140670-A1

Title: Formulations

Description:
The present application claims the benefit of priority to U.S. Provisional Patent Application No. 62/566,240, filed Sep. 29, 2017, the contents of which are incorporated herein by reference in their entirety. 
    
    
     Lipid nanoparticle (“LNP”) compositions with improved properties for delivery of biologically active agents, in particular RNAs, mRNAs, and guide RNAs are provided herein. The LNP compositions facilitate delivery of RNA agents across cell membranes, and in particular embodiments, they introduce components and compositions for gene editing into living cells. 
     Biologically active agents that are particularly difficult to deliver to cells include proteins, nucleic acid-based drugs, and derivatives thereof. Compositions for delivery of promising gene editing technologies into cells, such as for delivery of CRISPR/Cas9 system components, are of particular interest. 
     A number of components and systems for editing genes in cells in vivo now exist, providing tremendous potential for treating diseases. CRISPR/Cas gene editing systems are active as ribonucleoprotein complexes in a cell. An RNA-directed nuclease binds to and directs cleavage of a DNA sequence in the cell. This site-specific nuclease activity facilitates gene editing through the cell&#39;s own natural processes. For example, the cell responds to double-stranded DNA breaks (DSBs) with an error-prone repair process known as non-homologous end joining (“NHEJ”). During NHEJ, nucleotides may be added or removed from the DNA ends by the cell, resulting in a sequence altered from the cleaved sequence. In other circumstances, cells repair DSBs by homology-directed repair (“HDR”) or homologous recombination (“HR”) mechanisms, in which an endogenous or exogenous template can be used to direct repair of the break. Several of these editing technologies take advantage of cellular mechanisms for repairing single-stranded breaks (SSBs) or DSBs. 
     Compositions for delivery of the protein and nucleic acid components of CRISPR/Cas to a cell, such as a cell in a patient, are needed. In particular, compositions for delivering mRNA encoding the CRISPR protein component, and for delivering CRISPR guide RNAs are of particular interest. Compositions with useful properties for in vitro and in vivo delivery that can stabilize and deliver RNA components, are also of particular interest. 
     We herein provide lipid nanoparticle-based compositions with useful properties, in particular for delivery of CRISPR/Cas gene editing components. 
     In certain embodiments, the LNP compositions comprise: an RNA component; and a lipid component, wherein the lipid component comprises: (1) about 50-60 mol-% amine lipid; (2) about 8-10 mol-% neutral lipid; and (3) about 2.5-4 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 6, In additional embodiments, the LNP compositions comprise (1) an RNA component; (2) about 50-60 mol-% amine lipid; (3) about 27-39.5 mol-% helper lipid; (4) about 8-10 mol-% neutral lipid; and (5) about 2.5-4 mol-% PEG lipid, wherein the N/P ratio of the LNP composition is about 5-7. 
     In other embodiments, the LNP compositions comprise an RNA component and a lipid component, wherein the lipid component comprises: (1) about 50-60 mol-% amine lipid; (2) about 5-15 mol-% neutral lipid; and (3) about 2.5-4 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-10. In additional embodiments, the LNP compositions comprise a lipid component that includes (1) about 40-60 mol-% amine lipid; (2) about 5-15 mol-% neutral lipid; and (3) about 2.5-4 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 6. In another embodiment, the LNP compositions comprise a lipid component that includes (1) about 50-60 mol-% amine, lipid; (2) about 5-15 mol-% neutral lipid; and (3) about 1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 6. 
     In some embodiments, the LNP compositions comprise an RNA component and a lipid component, wherein the lipid component comprises: (1) about 40-60 mol-% amine lipid; (2) about 0-5 mol-% neutral lipid, e,g., phospholipid; and (3) about 1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-10. In some embodiments, the LNP compositions comprise an RNA component and a lipid component, wherein the lipid component comprises: (1) about 40-60 mol-% amine lipid; (2) less than about 1 mol-% neutral lipid, e.g., phospholipid; and (3) about 1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-10. In certain embodiments, the LNP composition is essentially free of neutral lipid. In some embodiments, the LNP compositions comprise an RNA component and a lipid component, wherein the lipid component comprises: (1) about 40-60 mol-% amine lipid; and (2) about 1.5-10 mol-% PEG lipid, wherein the remainder of the lipid component is helper lipid. wherein the N/P ratio of the LNP composition is about 3-10, and wherein the LNP composition is free of neutral lipid, e.g., phospholipid. In certain embodiments, the LNP composition is essentially free of or free of a neutral phospholipid. In certain embodiments, the LNP composition is essentially free of or free of a neutral lipid, e.g., phospholipid. 
     In certain embodiments, the RNA component comprises an mRNA, such as an RNA-guided DNA-binding agent (e.g., a Cas nuclease or Class 2 Cas nuclease). In certain embodiments, the RNA component comprises a gRNA. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG.  1    shows the percentage of TTR gene editing achieved in mouse liver after delivery of CRISPR/Cas gene editing components Cas9 mRNA and gRNA in LNP compositions as indicated at a single dose of 1 mpk ( FIG.  1 A )) or 0.5 mpk ( FIG.  1 B ). 
         FIG.  2    shows particle distribution data for LNP compositions comprising Cas9 mRNA and gRNA. 
         FIG.  3    depicts physicochemical properties of LNP compositions, comparing log differential molar mass ( FIG.  3 A ) and average molecular weight measurements ( FIG.  3 B ) for the compositions. 
         FIG.  4    shows polydispersity calculations in  FIG.  4 A  and Burchard-Stockmeyer analysis in  FIG.  4 B , analyzing the LNP compositions of  FIG.  3   . 
         FIG.  5    provides the results of an experiment evaluating the effect of LNP compositions with increased PEG lipid concentrations on serum TTR knockdown, gene editing in the liver, and cytokine MCP-1 levels after a single dose administration in rats.  FIG.  5 A  graphs serum TTR levels;  FIG.  5 B  graphs percent editing in liver samples; and  FIG.  5 C  provides MCP-1 levels in pg/mL. 
         FIG.  6    shows that LNP compositions maintain potency for gene editing with various PEG lipids (as measured by serum TTR levels ( FIGS.  6 A and  6 B ) and percent editing ( FIG.  6 C ). 
         FIG.  7    shows that Lipid A analogs effectively deliver gene editing cargos in LNP compositions as measured by % liver editing after a single dose administration in mouse. 
         FIG.  8    shows a dose response curve of percent editing with various LNP compositions in primary cyno hepatocytes. 
         FIG.  9 A  and  FIG.  9 B  show serum TTR and percent editing results when the ratio of gRNA to mRNA varies, and  FIG.  9 C  and  FIG.  9 D  show serum TTR and percent editing results in liver when the amount of Cas9 mRNA is held constant and gRNA varies following a single dose administration in mouse. 
         FIG.  10 A  and  FIG.  10 B  show serum TTR and liver editing results after administration of LNP compositions with and without neutral lipid. 
     
    
    
     DETAILED DESCRIPTION 
     The present disclosure provides embodiments of lipid nanoparticle (LNP) compositions of RNAs, including CRISPR/Cas component RNAs (the “cargo”) for delivery to a cell and methods for their use. The LNP compositions may exhibit improved properties as compared to prior delivery technologies. The LNP composition may contain an RNA component and a lipid component, as defined herein. In certain embodiments, the RNA component includes a Cas nuclease, such as a Class 2 Cas nuclease. In certain embodiments, the cargo or RNA component includes an mRNA encoding a Class 2 Cas nuclease and a guide RNA or nucleic acids encoding guide RNAs. Methods of gene editing and methods of making engineered cells are also provided. 
     CRISPR/Cas Cargo 
     The CRISPR/Cas cargo delivered via LNP formulation may include an mRNA molecule encoding a protein of interest. For example, an mRNA for expressing a protein such as green fluorescent protein (GFP), and RNA-guided DNA-binding agent, or a Cas nuclease is included. LNP compositions that include a Cas nuclease snRNA, for example a Class 2 Cas nuclease mRNA that allows for expression in a cell of a Cas9 protein are provided. Further, the cargo may contain one or more guide RNAs or nucleic acids encoding guide RNAs. A template nucleic acid, e.g., for repair or recombination, may also be included in the composition or a template nucleic acid may be used in the methods described herein. 
     “mRNA” refers to a polynucleotide that 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. In general, mRNAs do not contain a substantial quantity of thymidine residues (e.g., 0 residues or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). An mRNA can contain modified uridines at some or all of its uridine positions. 
     CRISPR/Cas Nuclease Systems 
     One component of the disclosed formulations is an mRNA encoding RNA-guided DNA-binding agent, such as a Cas nuclease. 
     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. Exemplary RNA-guided DNA binding agents include Cas cleavases/nickases and inactivated forms thereof (“dCas DNA binding agents”). “Cas nuclease”, as used herein, encompasses Cas cleavases, Cas nickases, and dCas DNA binding agents. Cas cleavases/nickases and dCas DNA binding agents 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 1 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 H840A, D10A, or N863A variants), which further have RNA-guided DNA cleavase or nickase activity, and Class 2 dCas DNA binding agents, in which cleavase/nickase activity is inactivated. 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, K1003 A, 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), is homologous to Cas9, and 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). 
     In some embodiments, the RNA-guided DNA-binding agent is a Class 2 Cas nuclease. In some embodiments, the RNA-guided DNA-binding agent has cleavase activity, which can also be referred to as double-strand endonuclease activity. In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nuclease, such as a Class 2 Cas nuclease (which may be, e.g., a Cas nuclease of Type II, V, or VI). Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins and modifications thereof. Examples of Cas9 nucleases include those of the type 11 CRISPR systems of  S. pyogenes, S. aureus,  and other prokaryotes (see, e.g., the list in the next paragraph), and modified (e.g., engineered or mutant) versions thereof See, e.g., U.S. 2016/0312198 A1; U.S. 2016/0312199 A1. Other examples of Cas nucleases include a Csm or Cmr complex of a type III CRISPR system or the Cas10, Csm1, or Cmr2 subunit thereof; and a Cascade complex of a type I CRISPR system, or the Cas3 subunit thereof. In some embodiments, the Cas nuclease may be from a Type-IIA, Type-IIB, or Type-IIC system. For discussion of various CRISPR systems and Cas nucleases see, e.g., Makarova et al., Nat. Rev. Microbial. 9:467-477 (2011); Makarova et al., Nat. Rev. Microbial, 13: 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015). 
     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.,  Acetahalobilum arabaticum, Ammanifex degensii, Caldicelulosiruptor becscii, Candidatus desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiabacillus 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 proteoc/asticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Ccmdidatus Alethanopiastna termitum, Eubcicterium 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.    
     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 nuclease comprises more than one RuvC domain and/or more than one HNH domain. In some embodiments, the Cas9 nuclease is a wild type Cas9. In some embodiments, the Cas9 is capable of inducing a double strand break in target DNA. In certain embodiments, the Cas nuclease may cleave dsDNA, it may cleave one strand of dsDNA, or it may not have DNA cleavase or nickase activity. An exemplary Cas9 amino acid sequence is provided as SEQ ID NO: 3. An exemplary Cas9 mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 4. An exemplary Cas9 mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 10. 
     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 Fok1. 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-1 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. An exemplary Cas9 nickase amino acid sequence is provided as SEQ ID NO: 6. An exemplary Cas9 nickase mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 7. An exemplary Cas9 nickase mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 11. 
     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, an mRNA encoding 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, use of double nicking may improve specificity and reduce off-target effects. 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 lacks cleavase and nickase activity. In some embodiments, the RNA-guided DNA-binding agent comprises a dCas DNA-binding polypeptide. A dCas polypeptide has DNA-binding activity while essentially lacking catalytic (cleavase/nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-binding agent lacking cleavase and nickase activity or the dCas DNA-binding polypeptide is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which its endonucleolytic active sites are inactivated, e.g., by one or more alterations (e.g., point mutations) in its catalytic domains. See, e.g., U.S. 2014/0186958 A1; U.S. 2015/0166980 A1, An exemplary dCas9 amino acid sequence is provided as SEQ ID NO: 8. An exemplary Cas9 mRNA ORF sequence, which includes start and stop codons, is provided as SEQ ID NO: 9. An exemplary Cas9 mRNA coding sequence, suitable for inclusion in a fusion protein, is provided as SEQ ID NO: 12. 
     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 monoparticle sequence, such as, e.g., the SV40 NLS, PKKKRKV or PKKKRRV. In some embodiments, the NLS may be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK. In a specific embodiment, a single PKKKRKV 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. 
     In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA binding agent. In some embodiments, the half-life of the RNA-guided DNA binding agent may be increased. In some embodiments, the half-life of the RNA-guided DNA-binding agent may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA-binding agent may be modified by addition of ubiquitin or a polyubiquitin chain. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rub1 in  S. cerevislee ), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5). 
     In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may he a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire,), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and/or an epitope tag. Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6×His, 8×His, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins. 
     In additional embodiments, the heterologous functional domain may target the RNA-guided DNA-binding agent to a specific organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain may target the RNA-guided DNA-binding agent to mitochondria. 
     In further embodiments, the heterologous functional domain may be an effector domain. When the RNA-guided DNA-binding agent is directed to its target sequence, e.g., when a Cas nuclease is directed to a target sequence by a gRNA, the effector domain may modify or affect the target sequence. In some embodiments, the effector domain may be chosen from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, e.g., U.S. Pat. No. 9,023,649. In some embodiments, the heterologous functional domain is a transcriptional activator or repressor. See, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression,”  Cell  152:1173-83 (2013); Perez-Pinera et al., “RNA-guided gene activation by CRISPR-Cas9-based transcription factors,”  Nat. Methods  10:973-6 (2013); Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,”  Nat. Biotechnol.  31:833-8 (2013); Gilbert et al., “CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes,”  Cell  154:442-51 (2013). As such, the RNA-guided DNA-binding agent essentially becomes a transcription factor that can be directed to bind a desired target sequence using a guide RNA. In certain embodiments, the DNA modification domain is a methylation domain, such as a demethylation or methyltransferase domain. In certain embodiments, the effector domain is a DNA modification domain, such as a base-editing domain. In particular embodiments, the DNA modification domain is a nucleic acid editing domain that introduces a specific modification into the DNA, such as a deaminase domain, See, e.g., WO 2015/089406; U.S. 2016/0304846. The nucleic acid editing domains, deaminase domains, and Cas9 variants described in WO 2015/089406 and U.S. 2016/0304846 are hereby incorporated by reference. 
     The nuclease may comprise at least one domain that interacts with a guide RNA (“gRNA”). Additionally, the nuclease may be directed to a target sequence by a gRNA. In Class 2 Cas nuclease systems, the gRNA interacts with the nuclease as well as the target sequence, such that it directs binding to the target sequence. In some embodiments, the gRNA provides the specificity for the targeted cleavage, and the nuclease may be universal and paired with different gRNAs to cleave different target sequences. Class 2 Cas nuclease may pair with a gRNA scaffold structure of the types, orthologs, and exemplary species listed above. 
     Guide RNA (gRNA) 
     In some embodiments of the present disclosure, the cargo for the LNP formulation includes at least one gRNA. The gRNA may guide the Cas nuclease or Class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, a gRNA binds with and provides specificity of cleavage by a Class 2 Cas nuclease. In some embodiments, the gRNA and the Cas nuclease may form a ribonucleoprotein (RNP), e.g., a CRISPR/Cas complex such as a CRISPR/Cas9 complex which may be delivered by the LNP composition. In some embodiments, the CRISPR/Cas complex may be a Type-II CRISPR/Cas9 complex. In some embodiments, the CRISPR/Cas complex may be a Type-V CRISPR/Cas complex, such as a Cpf1/guide RNA complex. Cas nucleases and cognate gRNAs may be paired. The gRNA scaffold structures that pair with each Class 2 Cas nuclease vary with the specific CRISPR/Cas system. 
     “Guide RNA”, “gRNA”, and simply “guide” are used herein interchangeably to refer to either a 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 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. 
     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. 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%. 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 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 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 Cas proteins include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence&#39;s reverse compliment), as a nucleic acid substrate for a Cas protein 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 tareet sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence. 
     The length of the targeting sequence may depend on the CRISPR/Cas system and components used. For example, different Class 2 Cas nucleases from different bacterial species have varying optimal targeting sequence lengths. Accordingly, the targeting sequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the targeting sequence length is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the guide sequence of a naturally-occurring CRISPR/Cas system. In certain embodiments, the Cas nuclease and gRNA scaffold will be derived from the same CRISPR/Cas system. In some embodiments, the targeting sequence may comprise or consist of 18-24 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 19-21 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 20 nucleotides. 
     In some embodiments, the sgRNA is a “Cas9 sgRNA” capable of mediating RNA-guided DNA cleavage by a Cas9 protein. In some embodiments, the sgRNA is a “Cpf1 sgRNA” capable of mediating RNA-guided DNA cleavage by a Cpf1 protein. In certain embodiments, the gRNA comprises a crRNA and tracr RNA sufficient for forming an active complex with a Cas9 protein and mediating RNA-guided DNA cleavage. In certain embodiments, the gRNA comprises a crRNA sufficient for forming an active complex with a Cpf1 protein and mediating RNA-guided DNA cleavage. See Zetsche 2015. 
     Certain embodiments of the invention also provide nucleic acids, e.g., expression cassettes, encoding the gRNA described herein. A “guide RNA nucleic acid” is used herein to refer to a guide RNA (e.g. an sgRNA or a dgRNA) and a guide RNA expression cassette, which is a nucleic acid that encodes one or more guide RNAs. 
     In some embodiments, the nucleic acid may be a DNA molecule. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a targeting sequence flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR/Cas system. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a tracr RNA. In some embodiments, the crRNA and the tracr RNA may be encoded by two separate nucleic acids. In other embodiments, the crRNA and the tracr RNA may be encoded by a single nucleic acid. In some embodiments, the crRNA and the tracr RNA may be encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and the tracr RNA may be encoded by the same strand of a single nucleic acid. In some embodiments, the gRNA nucleic acid encodes an sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cas9 nuclease sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cpf1 nuclease sgRNA. 
     The nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or regulatory control sequence, such as a promoter, a 3′ UTR, or a 5′ UTR. In one example, 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 certain embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters also 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 some embodiments, the gRNA nucleic acid is a modified nucleic acid. In certain embodiments, the gRNA nucleic acid includes a modified nucleoside or nucleotide. In some embodiments, the gRNA nucleic acid includes a 5′ end modification, for example a modified nucleoside or nucleotide to stabilize and prevent integration of the nucleic acid. In some embodiments, the gRNA nucleic acid comprises a double-stranded DNA having a 5′ end modification on each strand. In certain embodiments, the gRNA nucleic acid includes an inverted dideoxy-T or an inverted abasic nucleoside or nucleotide as the 5′ end modification. In some embodiments, the gRNA nucleic acid includes a label such as biotin, desthiobioten-TEG, digoxigenin, and fluorescent markers, including, for example, FAM, ROX, TAMRA, and AlexaFluor. 
     In certain embodiments, more than one gRNA nucleic acid, such as a gRNA, can be used with a CRISPR/Cas nuclease system. Each gRNA nucleic acid may contain a different targeting sequence, such that the CRISPR/Cas system cleaves more than one target sequence. In some embodiments, one or more gRNAs may have the same or differing properties such as activity or stability within a CRISPR/Cas complex. Where more than one gRNA is used, each gRNA can be encoded on the same or on different gRNA nucleic acid. The promoters used to drive expression of the more than one gRNA may be the same or different. 
     Modified RNAs 
     In certain embodiments, the LNP compositions comprise modified RNAs. 
     Modified nucleosides or nucleotides can be present in an RNA, for example a gRNA or mRNA. A gRNA or mRNA comprising one or more modified nucleosides or nucleotides, for example, is called a “modified” RNA to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified RNA is synthesized with a non-canonical nucleoside or nucleotide, 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). Certain embodiments comprise a 5′ end modification to an mRNA, gRNA, or nucleic acid. Certain embodiments comprise a 3′ end modification to an mRNA, gRNA, or nucleic acid. A modified RNA can contain 5′ end and 3′ end modifications. A modified RNA can contain one or more modified residues at non-terminal locations. In certain embodiments, a gRNA includes at least one modified residue. In certain embodiments, an mRNA includes at least one modified residue. 
     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. 
     mRNAs 
     In some embodiments, 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, or Class 2 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 or Class 2 Cas nuclease, is provided, used, or administered. In some embodiments, the ORF encoding an RNA-guided DNA binding agent 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 in one or more of the following ways: (1) the modified ORF has a uridine content ranging from its minimum uridine content to 150% of the minimum uridine content; (2) the modified ORF has a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content; (3) the modified ORF has at least 90% identity to any one of SEQ ID NOs: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66; (4) the modified ORF consists of a set of codons of which at least 75% of the codons are minimal uridine codon(s) for a given amino acid, e.g. the codon(s) with the fewest uridines (usually 0 or 1 except for a codon for phenylalanine, where the minimal uridine codon has 2 uridines); or (5) the modified ORF comprises at least one modified uridine. In some embodiments, the modified ORF is modified in at least two, three, or four of the foregoing ways. In some embodiments, the modified ORF comprises at least one modified uridine and is modified in at least one, two, three, or all of (1)-(4) above. 
     “Modified uridine” is used herein to refer to a nucleoside other than thymidine with the same hydrogen bond acceptors as uridine and one or more structural differences from uridine. In some embodiments, a modified uridine is a substituted uridine, i.e., a uridine in which one or more non-proton substituents (e.g., alkoxy, such as methoxy) takes the place of a proton. In some embodiments, a modified uridine is pseudouridine. In some embodiments, a modified uridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more non-proton substituents (e.g., alkyl, such as methyl) takes the place of a proton. In some embodiments, a modified uridine is any of a substituted uridine, pseudouridine, or a substituted pseudouridine. 
     “Uridine position” as used herein refers to a position in a polynucleotide occupied by a uridine or a modified uridine. Thus, for example, a polynucleotide in which “100% of the uridine positions are modified uridines” contains a modified uridine at every position that would be a uridine in a conventional RNA (where all bases are standard A, U, C, or G bases) of the same sequence. Unless otherwise indicated, a U in a polynucleotide sequence of a sequence table or sequence listing in, or accompanying, this disclosure can be a uridine or a modified uridine. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Minimal Uridine Codons 
               
            
           
           
               
               
               
            
               
                   
                 Amino Acid 
                 Minimal uridine codon 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 A 
                 Alanine 
                 GCA or GCC or GCG 
               
               
                   
                 G 
                 Glycine 
                 GGA or GGC or GGG 
               
               
                   
                 V 
                 Valine 
                 GUC or GUA or GUG 
               
               
                   
                 D 
                 Aspartic acid 
                 GAC 
               
               
                   
                 E 
                 Glutamic acid 
                 GAA or GAG 
               
               
                   
                 I 
                 Isoleucine 
                 AUG or AUA or AUG 
               
               
                   
                 T 
                 Threonine 
                 ACA or ACC or ACG 
               
               
                   
                 N 
                 Asparagine 
                 AAC 
               
               
                   
                 K 
                 Lysine 
                 AAG or AAA 
               
               
                   
                 S 
                 Serine 
                 AGC 
               
               
                   
                 R 
                 Arginine 
                 AGA or AGG 
               
               
                   
                 L 
                 Leucine 
                 CUG or CUA or CUC 
               
               
                   
                 P 
                 Proline 
                 CCG or CCA or CCC 
               
               
                   
                 H 
                 Histidine 
                 CAC or CAA or CAG 
               
               
                   
                 Q 
                 Glutamine 
                 CAG or CAA 
               
               
                   
                 F 
                 Phenylalanine 
                 UUC 
               
               
                   
                 Y 
                 Tyrosine 
                 UAC 
               
               
                   
                 C 
                 Cysteine 
                 UGC 
               
               
                   
                 W 
                 Tryptophan 
                 UGG 
               
               
                   
                 M 
                 Methionine 
                 AUG 
               
               
                   
                   
               
            
           
         
       
     
     In any of the foregoing embodiments, the modified ORF may consist of a set of codons of which at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons are codons listed in the Table of Minimal Uridine Codons. In any of the foregoing embodiments, the modified ORF may comprise a sequence with at least 90%, 95%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66. 
     In any of the foregoing embodiments, the modified ORF may comprise a sequence with at least 90%, 95%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66. 
     In any of the foregoing embodiments, the modified ORF may have a uridine content ranging from its minimum uridine content to 150%, 145%, 140%, 135%, 130%, 125%, 120%,115%, 110%, 105%, 104%, 103%, 102%, or 101% of the minimum uridine content. 
     In any of the foregoing embodiments, the modified ORF may have a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of the minimum uridine dinucleotide content. 
     In any of the foregoing 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 I 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, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the uridine positions in an mRNA according to the disclosure are modified uridines. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are modified uridines, e.g., 5-methoxyuridine, 5-iodouridine, N1-methyl pseudouridine, pseudouridine, or a combination thereof. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA, according to the disclosure are 5-methoxyuridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are pseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are N1-methyl pseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-iodouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-methoxyuridine, and the remainder are N1-methyl pseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the disclosure are 5-iodouridine, and the remainder are N1-methyl pseudouridine. 
     In any of the foregoing embodiments, the modified ORF may comprise a reduced uridine dinucleotide content, such as the lowest possible uridine dinucleotide (UU) content, e.g. an ORF that (a) uses a minimal uridine codon (as discussed above) at every position and (b) encodes the same amino acid sequence as the given ORF. The uridine dinucleotide (UU) content can be expressed in absolute terms as the enumeration of UU dinucleotides in an ORF or on a rate basis as the percentage of positions occupied by the uridines of uridine dinucleotides (for example, AUUAU would have a uridine dinucleotide content of 40% because 2 of 5 positions are occupied by the uridines of a uridine dinucleotide). Modified uridine residues are considered equivalent to uridines for the purpose of evaluating minimum uridine dinucleotide content. 
     In some embodiments, the mRNA comprises at least one UTR from an expressed mammalian mRNA, such as a constitutively expressed mRNA. An mRNA is considered constitutively expressed in a mammal if it is continually transcribed in at least one tissue of a healthy adult mammal. In some embodiments, the mRNA comprises a 5′ UTR, 3′ UTR, or 5′ and 3′ UTRs from an expressed mammalian RNA, such as a constitutively expressed mammalian mRNA. Actin mRNA is an example of a constitutively expressed mRNA. 
     In some embodiments, the mRNA comprises at least one UTR from Hydroxysteroid 17-Beta Dehydrogenase 4 (HSD17B4 or HSD), e.g., a 5′ UTR from HSD. In some embodiments, the mRNA comprises at least one UTR from a globin mRNA, for example, human alpha globin (HBA) mRNA, human beta globin (HBB) mRNA, or  Xenopus laevis  beta globin (XBG) mRNA. In some embodiments, the mRNA comprises a 5′ UTR, 3′ UTR, or 5′ and 3′ UTRs from a globin mRNA, such as HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 5′ UTR from bovine growth hormone, cytomegalovirus (CMV), mouse Hba-a1, HSD, an albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 3′ UTR from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, an albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises 5′ and 3′ UTRs from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, an albumin gene, HBA, HBB, XBG, heat shock protein 90 (Hsp90), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin, alpha-tubulin, tumor protein (p53), or epidermal growth factor receptor (EGFR). 
     In some embodiments, the mRNA comprises 5′ and 3′ UTRs that are from the same source, e.g., a constitutively expressed mRNA such as actin, albumin, or a globin such as HBA, HBB, or XBG. 
     In some embodiments, the mRNA does not comprise a 5′ UTR, e.g., there are no additional nucleotides between the 5′ cap and the start codon. In some embodiments, the mRNA comprises a Kozak sequence (described below) between the 5′ cap and the start codon, but does not have any additional 5′ UTR. In some embodiments, the mRNA does not comprise a 3′ UTR, e.g., there are no additional nucleotides between the stop codon and the poly-A tail. 
     In some embodiments, the mRNA comprises a Kozak sequence. The Kozak sequence can affect translation initiation and the overall yield of a polypeptide translated from an mRNA. A Kozak sequence includes a methionine codon that can function as the start codon. A minimal Kozak sequence is NNNRUGN wherein at least one of the following is true: the first N is A or G and the second N is G. In the context of a nucleotide sequence, R means a purine (A or G). In some embodiments, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, or RNNAUGG. In some embodiments, the Kozak sequence is rccRUGg with zero mismatches or with up to one or two mismatches to positions in lowercase. In some embodiments, the Kozak sequence is rccAUGg with zero mismatches or with up to one or two mismatches to positions in lowercase. In some embodiments, the Kozak sequence is gccRccAUGG with zero mismatches or with up to one, two, or three mismatches to positions in lowercase. In some embodiments, the Kozak sequence is gccAccAUG with zero mismatches or with up to one, two, three, or four mismatches to positions in lowercase. In some embodiments, the Kozak sequence is GCCACCAUG. In some embodiments, the Kozak sequence is gccgccRccAUGG with zero mismatches or with up to one, two, three, or four mismatches to positions in lowercase. 
     In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 43, optionally wherein the ORF of SEQ ID NO: 43 (i.e., SEQ ID NO: 4) is substituted with an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66. 
     In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 44, optionally wherein the ORF of SEQ ID NO: 44 (i.e., SEQ ID NO: 4) is substituted with an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66. 
     In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 56, optionally wherein the ORF of SEQ ID NO: 56 (i.e., SEQ ID NO: 4) is substituted with an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66. 
     In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 57, optionally wherein the ORF of SEQ ID NO: 57 (i.e., SEQ ID NO: 4) is substituted with an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66. 
     In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: , optionally wherein the ORF of SEQ ID NO: 58 (i.e., SEQ ID NO: 4) is substituted with an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66. 
     In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 59, optionally wherein the ORE of SEQ ID NO: 59 (i.e., SEQ ID NO: 4) is substituted with art alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66. 
     In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 60, optionally wherein the ORF of SEQ ID NO: 60 (i.e., SEQ ID NO: 4) is substituted with an alternative ORE of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66. 
     In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding agent comprises a sequence having at least 90% identity to SEQ ID NO: 61, optionally wherein the ORF of SEQ ID NO: 61 (i.e., SEQ ID NO: 4) is substituted with an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66. 
     In some embodiments, the mRNA comprises an alternative ORF of any one of SEQ ID NO: 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66. 
     In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NOs 43, 44, or 56-61 is 95%. In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NOs 43, 44, or 56-61 is 98%. In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NOs 43, 44, or 56-61 is 99%. In some embodiments, the degree of identity to the optionally substituted sequences of SEQ ID NOs 43, 44, or 56-61 is 100%. 
     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 ClearCap™ 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 guanylyttransferase 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, 24172-24479. 
     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. In some instances, the poly-A tail is “interrupted” with one or more non-adenine nucleotide “anchors” at one or more locations within the poly-A tail. The poly-A tails may comprise at least 8 consecutive adenine nucleotides, but also comprise one or more non-adenine nucleotide. As used herein, “non-adenine nucleotides” refer to any natural or non-natural nucleotides that do not comprise adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly-A tails on the mRNA described herein may comprise consecutive adenine nucleotides located 3′ to nucleotides encoding an RNA-guided DNA-binding agent or a sequence of interest. In some instances, the poly-A tails on mRNA comprise non-consecutive adenine nucleotides located 3′ to nucleotides encoding an RNA-guided DNA-binding agent or a sequence of interest, wherein non-adenine nucleotides interrupt the adenine nucleotides at regular or irregularly spaced intervals. 
     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. In some instances, the poly-A tail is “interrupted” with one or more non-adenine nucleotide “anchors” at one or more locations within the poly-A tail. The poly-A tails may comprise at least 8 consecutive adenine nucleotides, but also comprise one or more non-adenine nucleotide. As used herein, “non-adenine nucleotides” refer to any natural or non-natural nucleotides that do not comprise adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly-A tails on the mRNA described herein may comprise consecutive adenine nucleotides located 3′ to nucleotides encoding an RNA-guided DNA-binding agent or a sequence of interest. In some instances, the poly-A tails on mRNA comprise non-consecutive adenine nucleotides located 3′ to nucleotides encoding an RNA-guided DNA-binding agent or a sequence of interest, wherein non-adenine nucleotides interrupt the adenine nucleotides at regular or irregularly spaced intervals. 
     In some embodiments, the one or more non-adenine nucleotides are positioned to interrupt the consecutive adenine nucleotides so that a poly(A) binding protein can bind to a stretch of consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotide(s) is located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotide is located after at least 8-50 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotide is located after at least 8-100 consecutive adenine nucleotides. In some embodiments, the non-adenine nucleotide is after one, two, three, four, five, six, or seven adenine nucleotides and is followed by at least 8 consecutive adenine nucleotides. 
     The poly-A tail may comprise one sequence of consecutive adenine nucleotides followed by one or more non-adenine nucleotides, optionally followed by additional adenine nucleotides. 
     In some embodiments, the poly-A tail comprises or contains one non-adenine nucleotide or one consecutive stretch of 2-10 non-adenine nucleotides. In some embodiments, the non-adenine nucleotide(s) is located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some instances, the one or more non-adenine nucleotides are located after at least 8-50 consecutive adenine nucleotides. In some embodiments, the one or more non-adenine nucleotides are located after at least 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, 41, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides. 
     In some embodiments, the non-adenine nucleotide is guanine, cytosine, or thymine. In some instances, the non-adenine nucleotide is a guanine nucleotide. In some embodiments, the non-adenine nucleotide is a cytosine nucleotide. In some embodiments, the non-adenine nucleotide is a thymine nucleotide. In some instances, where more than one non-adenine nucleotide is present, the non-adenine nucleotide may be selected from: a) guanine and thymine nucleotides; b) guanine and cytosine nucleotides; c) thymine and cytosine nucleotides; or d) guanine, thymine and cytosine nucleotides. An exemplary poly-A tail comprising non-adenine nucleotides is provided as SEQ ID NO: 62. 
     In some embodiments, the mRNA is purified. In some embodiments, the mRNA is purified using a precipation method (e.g., LiCl precipitation, alcohol precipitation, or an equivalent method, e.g., as described herein). In some embodiments, the mRNA is purified using a chromatography-based method, such as an HPLC-based method or an equivalent method (e.g., as described herein), In some embodiments, the mRNA is purified using both a precipitation method (e.g., LiCl precipitation) and an HPLC-based method. 
     In some embodiments, at least one gRNA is provided in combination with an mRNA disclosed herein. In some embodiments, a gRNA is provided as a separate molecule from the mRNA. In some embodiments, a gRNA is provided as a part, such as a part of a UTR, of an mRNA disclosed herein. 
     Chemically Modified gRNA 
     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 andlor backbone modification); and (vii) modification or replacement of the sugar (an exemplary sugar modification). 
     In some embodiments, a gRNA comprises a modified uridine at some or all uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5 position, e.g., with a halogen or C1-C6 alkoxy. In some embodiments, the modified undine is a pseudouridine modified at the I position, e.g., with a C1-C6 alkyl. 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-methoxy-oridine. 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, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the uridine positions in a gRNA according to the disclosure are modified uridines. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are modified uridines, e.g., 5-methoxyuridine, 5-iodouridine, NI-methyl pseudouridine, pseudouridine, or a combination thereof. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are 5-methoxyuridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are pseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are N1-methyl pseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are 5-iodouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are 5-methoxyuridine, and the remainder are N1-methyl pseudouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in a gRNA according to the disclosure are 5-iodouridine, and the remainder are N1-methyl pseudouridine. 
     Chemical modifications such as those listed above can be combined to provide modified gRNAs 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. 
     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 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. 
     Template Nucleic Acid 
     The compositions and methods disclosed herein may include a template nucleic acid. The template may be used to alter or insert a nucleic acid sequence at or near a target site for a Cas nuclease. In some embodiments, the methods comprise introducing a template to the cell. In some embodiments, a single template may be provided. In other embodiments, two or more templates may be provided such that editing may occur at two or more target sites. For example, different templates may be provided to edit a single gene in a cell, or two different genes in a cell. 
     In some embodiments, the template may be used in homologous recombination. In some embodiments, the homologous recombination may result in the integration of the template sequence or a portion of the template sequence into the target nucleic acid molecule. In other embodiments, the template may be used in homology-directed repair, which involves DNA strand invasion at the site of the cleavage in the nucleic acid. In some embodiments, the homology-directed repair may result in including the template sequence in the edited target nucleic acid molecule. In yet other embodiments, the template may be used in gene editing mediated by non-homologous end joining. In some embodiments, the template sequence has no similarity to the nucleic acid sequence near the cleavage site. In some embodiments, the template or a portion of the template sequence is incorporated. In some embodiments, the template includes flanking inverted terminal repeat (ITR) sequences. 
     In some embodiments, the template may comprise a first homology arm and a second homology arm (also called a first and second nucleotide sequence) that are complementary to sequences located upstream and downstream of the cleavage site, respectively. Where a template contains two homology arms, each arm can be the same length or different lengths, and the sequence between the homology arms can be substantially similar or identical to the target sequence between the homology arms, or it can be entirely unrelated. In some embodiments, the degree of complementarity or percent identity between the first nucleotide sequence on the template and the sequence upstream of the cleavage site, and between the second nucleotide sequence on the template and the sequence downstream of the cleavage site, may permit homologous recombination, such as, e.g., high-fidelity homologous recombination, between the template and the target nucleic acid molecule. In some embodiments, the degree of complementarity may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be about 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be at least 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be 100%. In some embodiments, the percent identity may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity may be about 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity may be at least 98%, 99%, or 100%. In some embodiments, the percent identity may be 100%. 
     In some embodiments, the template sequence may correspond to, comprise, or consist of an endogenous sequence of a target cell. It may also or alternatively correspond to, comprise, or consist of an exogenous sequence of a target cell. As used herein, the term “endogenous sequence” refers to a sequence that is native to the cell. The term “exogenous sequence” refers to a sequence that is not native to a cell, or a sequence whose native location in the genome of the cell is in a different location. In some embodiments, the endogenous sequence may be a genomic sequence of the cell. In some embodiments, the endogenous sequence may be a chromosomal or extrachromosomal sequence. In some embodiments, the endogenous sequence may be a plasmid sequence of the cell. In some embodiments, the template sequence may be substantially identical to a portion of the endogenous sequence in a cell at or near the cleavage site, but comprise at least one nucleotide change. In some embodiments, editing the cleaved target nucleic acid molecule with the template may result in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of the target nucleic acid molecule, In some embodiments, the mutation may result in one or more amino acid changes in a protein expressed from a gene comprising the target sequence. In some embodiments, the mutation may result in one or more nucleotide changes in an RNA expressed from the target gene. In some embodiments, the mutation may alter the expression level of the target gene. In some embodiments, the mutation may result in increased or decreased expression of the target gene. in some embodiments, the mutation may result in gene knock-down. In some embodiments, the mutation may result in gene knock-out. In some embodiments, the mutation may result in restored gene function. In some embodiments, editing of the cleaved target nucleic acid molecule with the template may result in a change in an exon sequence, an intron sequence, a regulatory sequence, a transcriptional control sequence, a translational control sequence, a splicing site, or a non-coding sequence of the target nucleic acid molecule, such as DNA. 
     In other embodiments, the template sequence may comprise an exogenous sequence. In some embodiments, the exogenous sequence may comprise a protein or RNA coding sequence operably linked to an exogenous promoter sequence such that, upon integration of the exogenous sequence into the target nucleic acid molecule, the cell is capable of expressing the protein or RNA encoded by the integrated sequence. In other embodiments, upon integration of the exogenous sequence into the target nucleic acid molecule, the expression of the integrated sequence may be regulated by an endogenous promoter sequence. In some embodiments, the exogenous sequence may provide a cDNA sequence encoding a protein or a portion of the protein. In yet other embodiments, the exogenous sequence may comprise or consist of an exon sequence, an intron sequence, a reaulatory sequence, a transcriptional control sequence, a translational control sequence, a splicing site, or a non-coding sequence. In some embodiments, the integration of the exogenous sequence may result in restored gene function. In some embodiments, the integration of the exogenous sequence may result in a gene knock-in. In some embodiments, the integration of the exogenous sequence may result in a gene knock-out. 
     The template may be of any suitable length. In some embodiments, the template may comprise 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or more nucleotides in length. The template may be a single-stranded nucleic acid. The template can be double-stranded or partially double-stranded nucleic acid. In certain embodiments, the single stranded template is 20, 30, 40, 50, 75, 100, 125, 150, 175, or 200 nucleotides in length. In some embodiments, the template may comprise a nucleotide sequence that is complementary to a portion of the target nucleic acid molecule comprising the target sequence (i.e., a “homology arm”). In some embodiments, the template may comprise a homology arm that is complementary to the sequence located upstream or downstream of the cleavage site on the target nucleic acid molecule. 
     In some embodiments, the template contains ssDNA or dsDNA containing flanking invert-terminal repeat (ITR) sequences. In some embodiments, the template is provided as a vector, plasmid, minicircle, nanocircle, or PCR product. 
     Purification of Nucleic Acids 
     In some embodiments, the nucleic acid is purified. In some embodiments, the nucleic acid is purified using a precipation method (e.g., LiCl precipitation, alcohol precipitation, or an equivalent method, e.g., as described herein). In some embodiments, the nucleic acid is purified using a chromatography-based method, such as an HPLC-based method or an equivalent method (e.g., as described herein). In some embodiments, the nucleic is purified using both a precipitation method (e.g., LiCl precipitation) and an HPLC-based method. 
     Target Sequences 
     In some embodiments, a CRISPR/Cas system of the present disclosure may be directed to and cleave a target sequence on a target nucleic acid molecule. For example, the target sequence may be recognized and cleaved by the Cas nuclease. In certain embodiments, a target sequence for a Cas nuclease is located near the nuclease&#39;s cognate PAM sequence. In some embodiments, a Class 2 Cas nuclease may be directed by a gRNA to a target sequence of a target nucleic acid molecule, where the gRNA hybridizes with and the Class 2 Cas protein cleaves the target sequence. In some embodiments, the guide RNA hybridizes with and a Class 2 Cas nuclease cleaves the target sequence adjacent to or comprising its cognate PAM. In some embodiments, the target sequence may be complementary to the targeting sequence of the guide RNA. In some embodiments, the degree of complementarity between a targeting sequence of a guide RNA and the portion of the corresponding target sequence that hybridizes to the guide RNA may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity between a targeting sequence of a guide RNA and the portion of the corresponding target sequence that hybridizes to the guide RNA may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the homology region of the target is adjacent to a cognate PAM sequence. In some embodiments, the target sequence may comprise a sequence 100% complementary with the targeting sequence of the guide RNA. In other embodiments, the target sequence may comprise at least one mismatch, deletion, or insertion, as compared to the targeting sequence of the guide RNA. 
     The length of the target sequence may depend on the nuclease system used. For example, the targeting sequence of a guide RNA for a CRISPRICas system may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length and the target sequence is a corresponding length, optionally adjacent to a PAM sequence. In some embodiments, the target sequence may comprise 15-24 nucleotides in length. In some embodiments, the target sequence may comprise 17-21 nucleotides in length. In some embodiments, the target sequence may comprise 20 nucleotides in length. When nickases are used, the target sequence may comprise a pair of target sequences recognized by a pair of nickases that cleave opposite strands of the DNA molecule. In some embodiments, the target sequence may comprise a pair of target sequences recognized by a pair of nickases that cleave the same strands of the DNA molecule. In some embodiments, the target sequence may comprise a part of target sequences recognized by one or more Cas nucleases. 
     The target nucleic acid molecule may be any DNA or RNA molecule that is endogenous or exogenous to a cell. In some embodiments, the target nucleic acid molecule may be an episomal DNA, a plasmid, a genomic DNA, viral genome, mitochondrial DNA, or chromosomal DNA from a cell or in the cell. In some embodiments, the target sequence of the target nucleic acid molecule may be a genomic sequence from a cell or in a cell, including a human cell. 
     In further embodiments, the target sequence may be a viral sequence. In further embodiments, the target sequence may be a pathogen sequence. In yet other embodiments, the target sequence may be a synthesized sequence. In further embodiments, the target sequence may be a chromosomal sequence. In certain embodiments, the target sequence may comprise a translocation junction, e.g., a translocation associated with a cancer. In some embodiments, the target sequence may be on a eukaryotic chromosome, such as a human chromosome. In certain embodiments, the target sequence is a liver-specific sequence, in that it is expressed in liver cells. 
     In some embodiments, the target sequence may be located in a coding sequence of a gene, an intron sequence of a gene, a regulatory sequence, a transcriptional control sequence of a gene, a translational control sequence of a gene, a splicing site or a non-coding sequence between genes. In some embodiments, the gene may be a protein coding gene. In other embodiments, the gene may be a non-coding RNA gene. In some embodiments, the target sequence may comprise all or a portion of a disease-associated gene. In some embodiments, the target sequence may be located in a non-genic functional site in the genome, for example a site that controls aspects of chromatin organization, such as a scaffold site or locus control region. 
     In embodiments involving a Cas nuclease, such as a Class 2 Cas nuclease, the target sequence may be adjacent to a protospacer adjacent motif (“PAM”). In some embodiments, the PAM may be adjacent to or within 1, 2, 3, or 4, nucleotides of the 3′ end of the target sequence. The length and the sequence of the PAM may depend on the Cas protein used. For example, the PAM may be selected from a consensus or a particular PAM sequence for a specific Cas9 protein or Cas9 ortholog, including those disclosed in  FIG.  1    of Ran et al.,  Nature,  520: 186-191 (2015), and FIG. S5 of Zetsche 2015, the relevant disclosure of each of which is incorporated herein by reference. In some embodiments, the PAM may be 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NGG, NGGNG, NG, NAAAAN, NNAAAAW, NNNNACA, GNNNCNNA, TTN, and NNNNGATT (wherein N is defined as any nucleotide, and W is defined as either A or T). In some embodiments, the PAM sequence may be NGG. In some embodiments, the PAM sequence may be NGGNG. In some embodiments, the PAM sequence may be TTN. In some embodiments, the PAM sequence may be NNAAAAW. 
     Lipid Formulation 
     Disclosed herein are various embodiments of LNP formulations for RNAs, including CRISPR/Cas camos. Such LNP formulations include an “amine lipid”, along with a helper lipid, a neutral lipid, and a PEG lipid. In some embodiments, such LNP formulations include an “amine lipid”, along with a helper lipid and a PEG lipid. In some embodiments, the LNP formulations include less than 1 percent neutral phospholipid. In some embodiments, the LNP formulations include less than 0.5 percent neutral phospholipid. By “lipid nanoparticle” is meant a particle that comprises a plurality of (i.e. more than one) lipid molecules physically associated with each other by intermolecular forces. 
     Amine Lipids 
     The LNP compositions for the delivery of biologically active agents comprise an “amine lipid”, which is defined as Lipid A or its equivalents, including acetal analogs of Lipid A. 
     In some embodiments, the amine lipid is Lipid A, which is (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. Lipid A can be depicted as: 
     
       
         
         
             
             
         
       
     
     Lipid A may be synthesized according to WO2015/095340 (e.g., pp. 84-86). In certain embodiments, the amine lipid is an equivalent to Lipid A. 
     In certain embodiments, an amine lipid is an analog of Lipid A. In certain embodiments, a Lipid A analog is an acetal analog of Lipid A. In particular LNP compositions, the acetal analog is a C4-C12 acetal analog. In some embodiments, the acetal analog is a C5-C12 acetal analog. In additional embodiments, the acetal analog is a C5-C10 acetal analog. In further embodiments, the acetal analog is chosen from a C4, C5, C6, C7, C9, C10, C11, and C 12 acetal analog. 
     Amine lipids suitable for use in the LNPs described herein are biodegradable in vivo and suitable for delivering a biologically active avent, such as an RNA to a cell. The amine lipids have low toxicity (e.g., are tolerated in an animal model without adverse effect in amounts of greater than or equal to 10 mg/kg of RNA cargo). In certain embodiments, LNPs comprising an amine lipid include those where at least 75% of the amine lipid is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. In certain embodiments, LNPs comprising an amine lipid include those where at least 50% of the mRNA or gRNA is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. In certain embodiments, LNPs comprising an amine lipid include those where at least 50% of the LNP is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days, for example by measuring a lipid (e.g., an amine lipid), RNA (e.g., mRNA), or another component. In certain embodiments, lipid-encapsulated versus free lipid, RNA, or nucleic acid component of the LNP is measured. 
     Lipid clearance may be measured as described in literature. See Maier, M. A., et al.  Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Tiler,  2013, 21(8), 1570-78 (“Maier”). For example, in Maier, LNP-siRNA systems containing luciferases-targeting siRNA were administered to six- to eight-week old male C57Bl/6 mice at 0.3 mg/kg by intravenous bolus injection via the lateral tail vein. Blood, liver, and spleen samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, and 168 hours post-dose. Mice were perfused with saline before tissue collection and blood samples were processed to obtain plasma. All samples were processed and analyzed by LC-MS. Further, Maier describes a procedure for assessing toxicity after administration of LNP-siRNA formulations. For example, a luciferase-targeting siRNA was administered at 0, 1, 3, 5, and 10 mg/kg (5 animals/group) via single intravenous bolus injection at a dose volume of 5 mL/kg to male Sprague-Dawley rats. After 24 hours, about 1 mL of blood was obtained from the jugular vein of conscious animals and the serum was isolated. At 72 hours post-dose, all animals were euthanized for necropsy. Assessments of clinical signs, body weight, serum chemistry, organ weights and histopathology were performed. Although Maier describes methods for assessing siRNA-LNP formulations, these methods may be applied to assess clearance, pharmacokinetics, and toxicity of administration of LNP compositions of the present disclosure. 
     The amine lipids may lead to an increased clearance rate. In some embodiments, the clearance rate is a lipid clearance rate, for example the rate at which a lipid is cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is an RNA clearance rate, for example the rate at which an mRNA or a gRNA is cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNP is cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNP is cleared from a tissue, such as liver tissue or spleen tissue. In certain embodiments, a high clearance rate leads to a safety profile with no substantial adverse effects. The amine lipids may reduce LNP accumulation in circulation and in tissues. In some embodiments, a reduction in LNP accumulation in circulation and in tissues leads to a safety profile with no substantial adverse effects. 
     The amine lipids of the present disclosure are ionizable (e.g., may form a salt) depending upon the pH of the medium they are in. For example, in a slightly acidic medium, the amine lipids may be protonated and thus bear a positive charge. Conversely, in a slightly basic medium, such as, for example, blood, where pH is approximately 7.35, the amine lipids may not be protonated and thus bear no charge. In some embodiments, the amine lipids of the present disclosure may be protonated at a pH of at least about 9. In some embodiments, the amine lipids of the present disclosure may be protonated at a pH of at least about 9. In some embodiments, the amine lipids of the present disclosure may be protonated at a pH of at least about 10. 
     The pH at which an amine lipid is predominantly protonated is related to its intrinsic pKa. In some embodiments, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.1 to about 7.4. In some embodiments, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.5 to about 6.6. In some embodiments, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.6 to about 6.4. In some embodiments, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.8 to about 6.2. For example, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.8 to about 6.5. The pKa of an amine lipid can be an important consideration in formulating LNPs as it has been found that cationic lipids with a pKa ranging from about 5.1 to about 7.4 are effective for delivery of cargo in vivo, e.g., to the liver. Furthermore, it has been found that cationic lipids with a pKa ranging from about 5.3 to about 6.4 are effective for delivery in vivo, e.g., to tumors. See, e.g., WO 2014/136086, 
     Additional Lipids 
     “Neutral lipids” suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoyiphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC). In another embodiment, the neutral phospholipid may be dipahnitoylphosphatidylcholine (DPPC). 
     “Helper lipids” include steroids, sterols, and alkyl resorcinois. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate, In one embodiment, the helper lipid may be cholesterol. In one embodiment, the helper lipid may be cholesterol hemisuccinate. 
     PEG lipids are stealth lipids that alter the length of time the nanoparticles can exist in vivo (e.g., in the blood). PEG lipids may assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. PEG lipids used herein may modulate phannacokinetic properties of the LNPs. Typically, the PEG lipid comprises a lipid moiety and a polymer moiety based on PEG. 
     In some embodiments, the lipid moiety may be derived from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. In some embodiments, the alkyl chain length comprises about C10 to C20. The dialkylglycerol or dialkylalyeamide group can further comprise one or more substituted alkyl groups. The chain lengths may be symmetrical or assymetric. 
     Unless otherwise indicated, the term “PEG” as used herein means any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, PEG moiety is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In certain embodiments, PEG moiety is Alternatively, the PEG moiety may be substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In one embodiment, the PEG moiety includes PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); alternatively, the PEG moiety does not include PEG copolymers, e.g., it may be a PEG monopolymer. In one embodiment, the PEG has a molecular weight of from about 130 to about 50,000, in a sub-embodiment, about 150 to about 30,000, in a sub-embodiment, about 150 to about 20,000, in a sub-embodiment about 150 to about 15,000, in a sub-embodiment, about 150 to about 10,000, in a sub-embodiment, about 150 to about 6,000, in a sub-embodiment, about 150 to about 5,000, in a sub-embodiment, about 150 to about 4,000, in a sub-embodiment, about 150 to about 3,000, in a sub-embodiment, about 300 to about 3,000, in a sub-embodiment, about 1,000 to about 3,000, and in a sub-embodiment, about 1,500 to about 2,500. 
     In certain embodiments, the PEG (e.g., conjugated to a lipid moiety or lipid, such as a stealth lipid), is a “PEG-2K,” also termed “PEG 2000,” which has an average molecular weight of about 2,000 daltons. PEG-2K is represented herein by the following formula (I), wherein n is 45, meaning that the number averaged degree of polymerization comprises about 45 subunits 
     
       
         
         
             
             
         
       
     
     However, other PEG embodiments known in the art may be used, including, e.g., those where the number-averaged degree of polymerization comprises about 23 subunits (n=23), and/or 68 subunits (n=68). In some embodiments, n may range from about 30 to about 60. In some embodiments, n may range from about 35 to about 55. In some embodiments, n may range from about 40 to about 50. In some embodiments, n may range from about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl. In some embodiments, R may be methyl. 
     In any of the embodiments described herein, the PEG lipid may be selected from PEG-dilauroylidycerol, PEG-dimyristoylglycerol (PEG-DMG) (catalog #GM-020 from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (catalog #DSPE-020CN, NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglyeamide, and PEG-distearoylglycamide, PEG-cholesterol (1-[8′-(Cholest-5-en-3[beta]-oxy)carboxamido-3′,6′-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol)ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG) (cat. #880150P from Avanti Polar Lipids, Alabaster, Ala., USA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (cat. #880120C from Avanti Polar Lipids, Alabaster, Ala., USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In one embodiment, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In one embodiment, the PEG lipid may be PEG2k-DSPE. In one embodiment, the PEG lipid may be PEG2k-DMA. In one embodiment, the PEG lipid may be PEG2k-C-DMA. In one embodiment, the PEG lipid may be compound S027, disclosed in WO2016/010840 at paragraphs [00240] to [00244]. In one embodiment, the PEG lipid may be PEG2k-DSA. En one embodiment, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16. In some embodiments, the PEG lipid may be PEG2k-C18. 
     LNP Formulations 
     Embodiments of the present disclosure provide lipid compositions described according to the respective molar ratios of the component lipids in the formulation. In one embodiment, the mol-% of the amine lipid may be from about 30 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 40 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 45 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 50 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 55 mol-% to about 60 mol-%. In one embodiment, the mol-% of the amine lipid may be from about 50 mol-% to about 55 mol-%. In one embodiment, the mol-% of the amine lipid may be about 50 mol-%. In one embodiment, the mol-% of the amine lipid may be about 55 mol-%. In some embodiments, the amine lipid mol-% of the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol-%. In some embodiments, the amine lipid mol-% of the LNP batch will be ±4 mol-%, ±3 mol-%, ±2 mol-%, ±1.5 mol-%, ±1 mol-%, ±0.5 mol-%, or ±0.25 mol-% of the target mol-%. All mol-% numbers are given as a fraction of the lipid component of the LNP compositions. In certain embodiments, LNP inter-lot variability of the amine lipid mol-% will be less than 15%, less than 10% or less than 5%. 
     In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be from about 5 mol-% to about 15 mol-%. In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be from about 7 mol-% to about 12 mol-%. In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be from about 0 mol-% to about 5 mol-%. In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be from about 0 mol-% to about 10 mol-%. In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be from about 5 mol-% to about 10 mol-%. In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be from about 8 mol-% to about 10 mol-%. 
     In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be about 5 mol-%, about 6 mol-%, about 7 mol-%, about 8 mol-%, about 9 mol-%, about 10 mol-%, about 11 mol-%, about 12 mol-%, about 13 mol-%, about 14 mol-%, or about 15 mol-%. In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be about 9 mol-%. 
     In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be from about 1 mol-% to about 5 mol-%. In one embodiment, the mol-% of the neutral lipid may be from about 0.1 mol-% to about 1 mol-%. In one embodiment, the mol-% of the neutral lipid such as neutral phospholipid may be about 0.1 mol-%, about 0.2 mol-%, about 0,5 mol-%, 1 mol-%, about 1.5 mol-%, about 2 mol-%, about 2.5 moi-%, about 3 mol-%, about 3.5 mol-%, about 4 mol-%, about 4.5 mol-%, or about 5 mol-%. 
     In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be less than about 1 mol-%. In one embodiment, the mol-% of the neutral lipid, neutral phospholipid, may be less than about 0.5 mol-%, In one embodiment, the mol-% of the neutral lipid, e.g., neutral phospholipid, may be about 0 mol-%, about 0.1 mol-%, about 0.2 mol-%, about 0.3 mol-%, about 0.4 mol-%, about 0.5 mol-%, about 0.6 mol-%, about 0.7 mol-%, about 0.8 mol-%, about 0.9 mol-%, or about 1 mol-%. In some embodiments, the formulations disclosed herein are free of neutral lipid (i.e., 0 mol-% neutral lipid). In some embodiments, the formulations disclosed herein are essentially free of neutral lipid (i.e., about 0 mol-% neutral lipid). In some embodiments, the formulations disclosed herein are free of neutral phospholipid (i.e., 0 mol-% neutral phospholipid). In some embodiments, the formulations disclosed herein are essentially free of neutral phospholipid (i.e., about 0 mol-% neutral phospholipid). 
     In some embodiments, the neutral lipid mol-% of the LNP batch will be ±30%, ±25%, ±20%, ±10%, ±5%., or ±12.5% of the target neutral lipid mol-%. In certain embodiments, LNP inter-lot variability will be less than 15%, less than 10% or less than 5%. 
     In one embodiment, the mol-% of the helper lipid may be from about 20 mol-% to about 60 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 25 mol-% to about 55 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 25 mol-% to about 50 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 25 mol-% to about 40 mol-%. In one embodiment, the mol-% of the helper lipid may he from about 30 mol-% to about 50 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 30 mol-% to about 40 mol-%. In one embodiment, the mol-% of the helper lipid is adjusted based on amine lipid, neutral lipid, and PEG lipid concentrations to bring the lipid component to 100 mol-%. In one embodiment, the mol-% of the helper lipid is adjusted based on amine lipid and PEG lipid concentrations to bring the lipid component to 100 mol-%. In one embodiment, the mol-% of the helper lipid is adjusted based on amine lipid and PEG lipid concentrations to bring the lipid component to at least 99 mol-%. In some embodiments, the helper mol-% of the LNP batch will be ±30%, ±25%, ±20%,±15%,±10%, ±5%, or ±12.5% of the target mol-%. In certain embodiments, LNP inter-lot variability will be less than 15%, less than 10% or less than 5%. 
     In one embodiment, the mol-% of the PEG lipid may be from about 1 mol-% to about 10 mol-%. In one embodiment, the mol-% of the PEG lipid may be from about 2 mol-% to about 10 mol-%. In one embodiment, the mol-% of the PEG lipid may be from about 2 mol-% to about 8 mol-%. In one embodiment, the mol-% of the PEG lipid may be from about 2 mol-% to about 4 mol-%. In one embodiment, the mol-% of the PEG lipid may be from about 2.5 mol-% to about 4 mol-%. In one embodiment, the mol-% of the PEG lipid may be about 3 mol-%. In one embodiment, the mol.-% of the PEG lipid may be about 2.5 mol-%. In some embodiments, the PEG lipid mol-% of the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±12.5% of the target PEG lipid mol-%. In certain embodiments, LNP inter-lot variability will be less than 15%, less than 10% or less than 5%. 
     In certain embodiments, the cargo includes an mRNA encoding an RNA-guided DNA-binding agent (e.g. a Cas nuclease, a Class 2 Cas nuclease, or Cas9), and a gRNA or a nucleic acid encoding a gRNA, or a combination of mRNA and gRNA. In one embodiment, an LNP composition may comprise a Lipid A or its equivalents. In some aspects, the amine lipid is Lipid A. In some aspects, the amine lipid is a Lipid A equivalent, e.g. an analog of Lipid A. In certain aspects, the amine lipid is an acetal analog of Lipid A. In various embodiments, an LNP composition comprises an amine lipid, a neutral lipid, a helper lipid, and a PEG lipid. In certain embodiments, the helper lipid is cholesterol. In certain embodiments, the neutral lipid is DSPC. In specific embodiments, PEG lipid is PEG2k-DMG. In some embodiments, an LNP composition may comprise a Lipid A, a helper lipid, a neutral lipid, and a PEG lipid. In some embodiments, an LNP composition comprises an amine lipid, DSPC, cholesterol, and a PEG lipid. In some embodiments, the LNP composition comprises a PEG lipid comprising DMG. In certain embodiments, the amine lipid is selected from Lipid A, and an equivalent of Lipid A, including an acetal analog of Lipid A. In additional embodiments, an LNP composition comprises Lipid A, cholesterol, DSPC, and PEG2k-DMG. 
     In various embodiments, an LNP composition comprises an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In various embodiments, an LNP composition comprises an amine lipid, a helper lipid, a neutral phospholipid, and a PEG lipid. In various embodiments, an LNP composition comprises a lipid component that consists of an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In various embodiments, an LNP composition comprises an amine lipid, a helper lipid, and a PEG lipid. In certain embodiments, an LNP composition does not comprise a neutral lipid, such as a neutral phospholipid. In various embodiments, an LNP composition comprises a lipid component that consists of an amine lipid, a helper lipid, and a PEG lipid. In certain embodiments, the neutral lipid is chosen from one or more of DSPC, DPPC, DAPC, DMPC, DOPC, DOPE, and DSPE. In certain embodiments, the neutral lipid is DSPC. In certain embodiments, the neutral lipid is DPPC. In certain embodiments, the neutral lipid is DAPC. In certain embodiments, the neutral lipid is DMPC. In certain embodiments, the neutral lipid is DOPC. In certain embodiments, the neutral lipid is DOPE. In certain embodiments, the neutral lipid is DSPE. In certain embodiments, the helper lipid is cholesterol. In specific embodiments, the PEG lipid is PEG2k-DMG. In some embodiments, an LNP composition may comprise a Lipid A, a helper lipid, and a PEG lipid. In some embodiments, an LNP composition may comprise a lipid component that consists of Lipid A, a helper lipid, and a PEG lipid. In some embodiments, an LNP composition comprises an amine lipid, cholesterol, and a PEG lipid. In some embodiments, an LNP composition comprises a lipid component that consists of an amine lipid, cholesterol, and a PEG lipid. In some embodiments, the LNP composition comprises a PEG lipid comprising DMG. In certain embodiments, the amine lipid is selected from Lipid A and an equivalent of Lipid A, including an acetal analog of Lipid A. In certain embodiments, the amine lipid is a C5-C12 or a C4-C12 acetal analog of Lipid A. In additional embodiments, an LNP composition comprises Lipid A, cholesterol, and PEG2k-DMG. 
     Embodiments of the present disclosure also provide lipid compositions described according to the molar ratio between the positively charged amine groups of the amine lipid (N) and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This may be mathematically represented by the equation N/P. In some embodiments, an LNP composition may comprise a lipid component that comprises an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid; and a nucleic acid component, wherein the N/P ratio is about 3 to 10. In some embodiments, an LNP composition may comprise a lipid component that comprises an amine lipid, a helper lipid, and a PEG lipid; and a nucleic acid component, wherein the N/P ratio is about 3 to 10. In some embodiments, an LNP composition may comprise a lipid component that comprises an amine lipid, a helper lipid, a neutral lipid, and a helper lipid; and an RNA component, wherein the N/P ratio is about 3 to 10. In some embodiments, an LNP composition may comprise a lipid component that comprises an amine lipid, a helper lipid, and a PEG lipid; and an RNA component, wherein the N/P ratio is about 3 to 10. In one embodiment, the N/P ratio may be about 5 to 7. In one embodiment, the N/P ration may be about 3 to 7. In one embodiment, the N/P ratio may be about 4.5 to 8. In one embodiment, the N/P ratio may be about 6. In one embodiment, the N/P ratio may he 6±1. In one embodiment, the N/P ratio may be 6±0.5. In some embodiments, the N/P ratio will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target N/P ratio. In certain embodiments, LNP inter-lot variability will be less than 15%. less than 10% or less than 5%. 
     In some embodiments, the nucleic acid component, e.g., an RNA component, may comprise an mRNA, such as an snRNA encoding a Cas nuclease. An RNA component includes RNA, optionally with additional nucleic acid and/or protein, e.g., RNP cargo. In one embodiment, RNA comprises a Cas9 mRNA. In some compositions comprising an mRNA encoding a Cas nuclease, the LNP further comprises a gRNA nucleic acid, such as a gRNA. In some embodiments, the RNA component comprises a Cas nuclease mRNA and a gRNA. In some embodiments, the RNA component comprises a Class 2 Cas nuclease mRNA and a sRNA. 
     In certain embodiments, an LNP composition may comprise an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In certain embodiments, an LNP composition may comprise an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, an amine lipid, a helper lipid, and a PEG lipid. In certain LNP compositions comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, the helper lipid is cholesterol, In other compositions comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, the neutral lipid is DSPC. In additional embodiments comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, the PEG lipid is PEG2k-DMG or PEG2k-C11. In specific compositions comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, the amine lipid is selected from Lipid A and its equivalents, such as an acetal analog of Lipid A. 
     In some embodiments, an LNP composition may comprise a gRNA. In certain embodiments, an LNP composition may comprise an amine lipid, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In certain embodiments, an LNP composition may comprise an amine lipid, a gRNA, a helper lipid, and a PEG lipid. In certain LNP compositions comprising a gRNA, the helper lipid is cholesterol. In some compositions comprising a gRNA, the neutral lipid is DSPC. In additional embodiments comprising a gRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the amine lipid is selected from Lipid A and its equivalents, such as an acetal analog of Lipid A. 
     In one embodiment, an LNP composition may comprise an sgRNA. In one embodiment, an LNP composition may comprise a Cas9 sgRNA. In one embodiment, an LNP composition may comprise a Cpf1 sgRNA. In some compositions comprising an sgRNA, the LNP includes an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In some compositions comprising an sgRNA, the LNP includes an amine lipid, a helper lipid, and a PEG lipid. In certain compositions comprising an sgRNA, the helper lipid is cholesterol. In other compositions comprising an sgRNA, the neutral lipid is DSPC. In additional embodiments comprising an sgRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the amine lipid is selected from Lipid A and its equivalents, such as acetal analogs of Lipid A. 
     In certain embodiments, an LNP composition comprises an mRNA encoding a Cas nuclease and a gRNA, which may be an sgRNA. In one embodiment, an LNP composition may comprise an amine lipid, an mRNA encoding a Cas nuclease, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In one embodiment, an LNP composition may comprise a lipid component consisting of an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid; and a nucleic acid component consisting of an mRNA encoding a Cas nuclease, and a gRNA. In one embodiment, an LNP composition may comprise a lipid component consisting of an amine lipid, a helper lipid, and a PEG lipid; and a nucleic acid component consisting of an mRNA encoding a Cas nuclease, and a gRNA. In certain compositions comprising an mRNA encoding a Cas nuclease and a gRNA, the helper lipid is cholesterol. In some compositions comprising an mRNA encoding a Cas nuclease and a gRNA, the neutral lipid is DSPC. Certain compositions comprising an mRNA encoding a Cas nuclease and a gRNA comprise less than about 1 mol-% neutral lipid, e.g. neutral phospholipid. Certain compositions comprising an mRNA encoding a Cas nuclease and a gRNA comprise less than about 0.5 mol-% neutral lipid, e.g. neutral phospholipid. In certain compositions, the LNP does not comprise a neutral lipid, e.g., neutral phospholipid. In additional embodiments comprising an mRNA encoding a Cas nuclease and a gRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the amine lipid is selected from Lipid A and its equivalents, such as acetal analogs of Lipid A. 
     In certain embodiments, the LNP compositions include a Cas nuclease mRNA, such as a Class 2 Cas mRNA and at least one gRNA. In certain embodiments, the LNP composition includes a ratio of gRNA to Gas nuclease mRNA, such as Class 2 Gas nuclease mRNA from about 25:1 to about 1:25, In certain embodiments, the LNP formulation includes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease mRNA from about 10:1 to about 1:10. In certain embodiments, the LNP formulation includes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease mRNA from about 8:1 to about 1:8. As measured herein, the ratios are by weight. In some embodiments, the LNP formulation includes a ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas mRNA from about 5:1 to about 1:5. In some embodiments, ratio range is about 3:1 to 1:3, about 2:1 to 1:2, about 5:1 to 1:2, about 5:1 to 1:1, about 3:1 to 1:2, about 3:1 to 1:1, about 3:1, about 2:1 to 1:1. In some embodiments, the gRNA to mRNA ratio is about 3:1 or about 2:1 In some embodiments the ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease is about 1:1. The ratio may be about 25:1, 10:1, 5:1, 3:1, 1:1, 1:3, 1:5, 1:0, or 1:25. 
     The LNP compositions disclosed herein may include a template nucleic acid. The template nucleic acid may be co-formulated with an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease mRNA. In some embodiments, the template nucleic acid may be co-formulated with a guide RNA. In some embodiments, the template nucleic acid may be co-formulated with both an mRNA encoding a Cas nuclease and a guide RNA. In some embodiments, the template nucleic acid may be formulated separately from an mRNA encoding a Cas nuclease or a guide RNA. The template nucleic acid may be delivered with, or separately from the LNP compositions. In some embodiments, the template nucleic acid may be single- or double-stranded, depending on the desired repair mechanism. The template may have regions of homology to the target DNA, or to sequences adjacent to the target DNA. 
     In some embodiments, LNPs are formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution, e.g., 100% ethanol. Suitable solutions or solvents include or may contain: water, PBS, Iris buffer, NaCl, citrate buffer, ethanol, chloroform, diethylether, cyclohexane, tetrahydrofuran, methanol, isopropanol. A pharmaceutically acceptable buffer, e.g., for in vivo administration of LNPs, may be used. In certain embodiments, a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 6.5. In certain embodiments, a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 7.0. In certain embodiments, the composition has a pH ranging from about 7.2 to about 7.7. In additional embodiments, the composition has a pH ranging from about 7.3 to about 7.7 or ranging from about 7.4 to about 7.6. In further embodiments, the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of a composition may be measured with a micro pH probe. In certain embodiments, a cryoprotectant is included in the composition. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may include up to 10% cryoprotectant, such as, for example, sucrose. In certain embodiments, the LNP composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% cryoprotectant. In certain embodiments, the LNP composition may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% sucrose. In some embodiments, the LNP composition may include a buffer. In some embodiments, the buffer may comprise a phosphate buffer (PBS), a Tris buffer, a citrate buffer, or mixtures thereof. In certain exemplary embodiments, the buffer comprises NaCl. In certain emboidments, NaCl is omitted. Exemplary amounts of NaCl may range from about 20 mM to about 45 mM. Exemplary amounts of NaCl may range from about 40 mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM. In some embodiments, the buffer is a Tris buffer. Exemplary amounts of Tris may range from about 20 mM to about 60 mM. Exemplary amounts of Tris may range from about 40 mM to about 60 mM. In some embodiments, the amount of Tris is about 50 mM. In some embodiments, the buffer comprises NaCl and Tris. Certain exemplary embodiments of the LNP compositions contain 5% sucrose and 45 mM NaCl in Tris buffer. In other exemplary embodiments, compositions contain sucrose in an amount of about 5% w/v, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The salt, buffer, and cryoprotectant amounts may be varied such that the osmolality of the overall formulation is maintained. For example, the final osmolality may he maintained at less than 450 mOsm/L. In further embodiments, the osmolality is between 350 and 250 mOsm/L. Certain embodiments have a final osmolality of 300+/−20 mOsm/L. 
     In some embodiments, inicrofluidic mixing, T-mixing, or cross-mixing is used. In certain aspects, flow rates, junction size, junction geometry, junction shape, tube diameter, solutions, and/or RNA and lipid concentrations may be varied. LNPs or LNP compositions may be concentrated or purified, e.g., via dialysis, tangential flow filtration, or chromatography. The LNPs may be stored as a suspension, an emulsion, or a lyophilized powder, for example. In some embodiments, an LNP composition is stored at 2-8° C., in certain aspects, the LNP compositions are stored at room temperature. In additional embodiments, an LNP composition is stored frozen, for example at −20′ C or −80° C. In other embodiments, an LNP composition is stored at a temperature ranging from about 0° C. to about −80° C. Frozen LNP compositions may be thawed before use, for example on ice, at room temperature, or at 25° C. 
     The LNPs may be, e.g., microspheres (including unilamellar and multilamellar vesicles, e.g., “liposomes”—lamellar phase lipid bilayers that, in some embodiments, are substantially spherical—and, in more particular embodiments, can comprise an aqueous core, e.g., comprising a substantial portion of RNA molecules), a dispersed phase in an emulsion, micelles, or an internal phase in a suspension. 
     Moreover, the LNP compositions are biodegradable, in that they do not accumulate to cytotoxic levels in vivo at a therapeutically effective dose. In some embodiments, the LNP compositions do not cause an innate immune response that leads to substantial adverse effects at a therapeutic dose level. In some embodiments, the LNP compositions provided herein do not cause toxicity at a therapeutic dose level. 
     In some embodiments, the pdi may range from about 0.005 to about 0.75. In some embodiments, the pdi may range from about 0.01 to about 0.5. In some embodiments, the pdi may range from about zero to about 0.4. In some embodiments, the pdi may range from about zero to about 0.35. In some embodiments, the pdi may range from about zero to about 0.35. In some embodiments, the pdi may range from about zero to about 0.3. In some embodiments, the pdi may range from about zero to about 0.25. In some embodiments, the pdi may range from about zero to about 0.2. In some embodiments, the pdi may be less than about 0.08, 0.1, 0.15, 0.2, or 0.4. 
     The LNPs disclosed herein have a size (e.g., Z-average diameter) of about 1 to about 250 nm. In some embodiments, the LNPs have a size of about 10 to about 200 nm. In further embodiments, the LNPs have a size of about 20 to about 150 nm. In some embodiments, the LNPs have a size of about 50 to about 150 nm. In some embodiments, the LNPs have a size of about 50 to about 100 nm. In some embodiments, the LNPs have a size of about 50 to about 120 nm. In some embodiments, the LNPs have a size of about 60 to about 100 nm. In some embodiments, the LNPs have a size of about 75 to about 150 nm. In some embodiments, the LNPs have a size of about 75 to about 120 nm. In some embodiments, the LNPs have a size of about 75 to about 100 nm. Unless indicated otherwise, all sizes referred to herein are the average sizes (diameters) of the fully formed nanoparticles, as measured by dynamic light scattering on a Malvern Zetasizer. The nanoparticle sample is diluted in phosphate buffered saline (PBS) so that the count rate is approximately 200-400 kcps. The data is presented as a weighted-average of the intensity measure (Z-average diameter). 
     In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 70%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 70% to about 90%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 90% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 75% to about 95%. 
     In some embodiments, the LNPs are formed with an average molecular weight ranging from about 1.00E+05 g/mol to about 1.00E+10 g/mol. In some embodiments, the LNPs are formed with an average molecular weight ranging from about 5.00E+05 g/mol to about 7.00E+07 g/mol. In some embodiments, the LNPs are formed with an average molecular weight ranging from about 1.00E+06 g/mol to about 1.00E+10 g/mol. In some embodiments, the LNPs are formed with an average molecular weight ranging from about 1.00E+07 g/mol to about 1.00E+09 g/mol. In some embodiments, the LNPs are formed with an average molecular weight ranging from about 5.00E+06 g/mol to about 5.00E+09 g/mol. 
     In some embodiments, the polydispersity (Mw/Mn; the ratio of the weight averaged molar mass (Mw) to the number averaged molar mass (Mn)) may range from about 1.000 to about 2.000. In some embodiments, the Mw/Mn may range from about 1.00 to about 1.500. In some embodiments, the Mw/Mn may range from about 1.020 to about 1.400. In some embodiments, the Mw/Mn may range from about 1.010 to about 1.100. In some embodiments, the Mw/Mn may range from about 1.100 to about 1.350. 
     Methods of Engineering Cells; Engineered Cells 
     The LNP compositions disclosed herein may be used in methods for engineering cells through gene editing, both in vivo and in vitro. In some embodiments, the methods involve contacting a cell with an LNP composition described herein. 
     In some embodiments, methods involve contacting a cell in a subject, such as a mammal, such as a human. In some embodiments, the cell is in an organ, such as a liver, such as a mammalian liver, such as a human liver. In some embodiments, the cell is a liver cell, such as a mammalian liver cell, such as a human liver cell. In some embodiments, the cell is a hepatocyte, such as a mammalian hepatocyte, such as a human hepatocyte. In some embodiments, the liver cell is a stem cell. In some embodiments, the human liver cell may be a liver sinusoidal endothelial cell (LSEC). In some embodiments, the human liver cell may be a Kupffer cell. In some embodiments, the human liver cell may be a hepatic stellate cell. In some embodiments, the human liver cell may be a tumor cell. In some embodiments, the human liver cell may be a liver stem cell. In additional embodiments, the cell comprises ApoE-binding receptors. In some embodiments, the liver cell such as a hepatocyte is in situ. In some embodiments, the Jiver cell such as a hepatocyte is isolated, e.g., in a culture, such as in a primary culture. Also provided are methods corresponding to the uses disclosed herein, which comprise administering the LNP compositions disclosed herein to a subject or contacting a cell such as those described above with the LNP compositions disclosed herein 
     In some embodiments, engineered cells are provided, for example an engineered cell derived from any one of the cell types in the preceding paragraph. Such engineered cells are produced according to the methods described herein. In some embodiments, the engineered cell resides within a tissue or organ, e.g., a liver within a subject. 
     In some of the methods and cells described herein, a cell comprises a modification, for example an insertion or deletion (“indel”) or substitution of nucleotides in a target sequence. In some embodiments, the modification comprises an insertion of 1, 2, 3, 4 or 5 or more nucleotides in a target sequence. In some embodiments, the modification comprises an insertion of either 1 or 2 nucleotides in a target sequence. In other embodiments, the modification comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides in a target sequence. In some embodiments, the modification comprises a deletion of either 1 or 2 nucleotides in a target sequence. In some embodiments, the modification comprises an indel which results in a frameshift mutation in a target sequence. In some embodiments, the modification comprises a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides in a target sequence. In some embodiments, the modification comprises a substitution of either 1 or 2 nucleotides in a target sequence. In some embodiments, the modification comprises one or more of an insertion, deletion, or substitution of nucleotides resulting from the incorporation of a template nucleic acid, for example any of the template nucleic acids described herein. 
     In some embodiments, a population of cells comprising engineered cells is provided, for example a population of cells comprising cells engineered according to the methods described herein. In some embodiments, the population comprises engineered cells cultured in vitro. In some embodiments, the population resides within a tissue or organ, e.g., a liver within a subject. In some embodiments, 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% or at least 95% or more of the cells within the population is engineered. In certain embodiments, a method disclosed herein results in 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% or at least 95% editing efficiency (or “percent editing”), defined by detetion of indels. In other embodiments, a method disclosed herein, results in 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% or at least 95% DNA modification efficiency, defined by detecting a change in sequence, whether by insertion, deletion, substitution or otherwise. In certain embodiments, a method disclosed herein results in an editing efficiency level or a DNA modification efficiency level of between about 5% to about 100%, about 10% to about 50%, about 20 to about 100%, about 20 to about 80%, about 40 to about 100%, or about 40 to about 80% in a cell population. 
     In some of the methods and cells described herein, cells within the population comprise a modification, e.g., an indel or substitution at a target sequence. In some embodiments, the modification comprises an insertion of 1, 2, 3, 4 or 5 or more nucleotides in a target sequence, In some embodiments, the modification comprises an insertion of either 1 or 2 nucleotides in a target sequence. In other embodiments, the modification comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides in a target sequence. In some embodiments, the modification comprises a deletion of either 1 or 2 nucleotides in a target sequence. In some embodiments, the modification results in a frameshift mutation in a target sequence. In some embodiments, the modification comprises an indel which results in a frameshift mutation in a target sequence. In some embodiments, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more of the engineered cells in the population comprise a frameshift mutation. In some embodiments, the modification comprises a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides in a target sequence. In some embodiments, the modification comprises a substitution of either 1 or 2 nucleotides in a target sequence. In some embodiments, the modification comprises one or more of an insertion, deletion, or substitution of nucleotides resulting from the incorporation of a template nucleic acid, for example any of the template nucleic acids described herein. 
     Methods of Gene Editing 
     The LNP compositions disclosed herein may be used for gene editing in vivo and in vitro. In one embodiment, one or more LNP compositions described herein may be administered to a subject in need thereof. In one embodiment, one or more LNP compositions described herein may contact a cell. In one embodiment, a therapeutically effective amount of a composition described herein may contact a cell of a subject in need thereof. In one embodiment, a genetically engineered cell may be produced by contacting a cell with an LNP composition described herein. In various embodiments, the methods comprise introducing a template nucleic acid to a cell or subject, as set forth above. 
     In some embodiments, the methods involve administering the LNP composition to a cell associated with a liver disorder. In some embodiments, the methods involve treating a liver disorder. In certain embodiments, the methods involve contacting a hepatic cell with the LNP composition. In certain embodiments, the methods involve contacting a hepatocyte with the LNP composition. In some embodiments, the methods involve contacting an ApoE binding cell with the LNP composition. 
     In one embodiment, an LNP composition comprising an mRNA encoding a Class 2 Cas nuclease and a gRNA may be administered to a cell, such as an ApoE binding cell. In additional embodiments, a template nucleic acid is also introduced to the cell. In certain instances, an LNP composition comprising, a Class 2 Cas nuclease and an sgRNA may be administered to a cell, such as an ApoE binding cell. In one embodiment, an LNP composition comprising an mRNA encoding a Class 2 Cas nuclease, a gRNA, and a template may be administered to a cell. In certain instances, an LNP composition comprising a Cas nuclease and an sgRNA may be administered to a liver cell. In some cases, the liver cell is in a subject. 
     In certain embodiments, a subject may receive a single dose of an LNP composition. In other examples, a subject may receive multiple doses of an LNP composition. In some embodiments, the LNP composition is administered 2-5 times. Where more than one dose is administered, the doses may be administered about 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days apart; about 2, 3, 4, 5, or 6 months apart; or about 1, 2, 3, 4, or 5 years apart. In certain embodiments, editing improves upon readministration of an LNP composition. 
     In one embodiment, an LNP composition comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease, may be administered to a cell, separately from the administration of a composition comprising a gRNA. In one embodiment, an LNP composition comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease and a gRNA may be administered to a cell, separately from the administration of a template nucleic acid to the cell. In one embodiment, an LNP composition comprising an mRNA encoding a Cas nuclease such as a Class 2 Cas nuclease may be administered to a cell, followed by the sequential administration of an LNP composition comprising a gRNA and then a template to the cell. In embodiments where an LNP composition comprising an mRNA encoding a Cas nuclease is administered before an LNP composition comprising a gRNA, the administrations may be separated by about 4, 6, 8, 12, or 24 hours; or 2, 3, 4, 5, 6, or 7 days. 
     In one embodiment, the LNP compositions may be used to edit a gene resulting in a gene knockout. In an embodiment, the LNP compositions may be used to edit a gene resulting in gene knockdown in a population of cells. In another embodiment, the LNP compositions may be used to edit a gene resulting in a gene correction. In a further embodiment, the LNP compositions may be used to edit a cell resulting in gene insertion. 
     In one embodiment, administration of the LNP compositions may result in gene editing which results in persistent response. For example, administration may result in a duration of response of a day, a month, a year, or longer. As used herein, “duration of response” means that, after cells have been edited using an LNP composition disclosed herein, the resulting modification is still present for a certain period of time after administration of the LNP composition. The modification may be detected by measuring target protein levels. The modification may be detected by detecting the target DNA. In some embodiments, the duration of response may be at least 1 week. In other embodiments, the duration of response may be at least 2 weeks. In one embodiment, the duration of response may be at least 1 month. In some embodiments, the duration of response may be at least 2 months. In one embodiment, the duration of response may be at least 4 months. In one embodiment, the duration of response may be at least 6 months. In certain embodiments, the duration of response may be about 26 weeks. In some embodiments, the duration of response may be at least 1 year. In some embodiments, the duration of response may be at least 5 years. In some embodiments, the duration of response may be at least 10 years. In some embodiments, a persistent response is detectable after at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 21, or 24 months, either by measuring target protein levels or by detection of the target DNA. In some embodiments, a persistent response is detectable after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 years, either by measuring target protein levels or by detection of the target DNA. 
     The LNP compositions can be administered parenterally. The LNP compositions may be administered directly into the blood stream, into tissue, into muscle, or into an internal organ. Administration may be systemic, e.g., to injection or infusion. Administration may be local. Suitable means for administration include intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, subretinal, intravitreal, intra-anterior chamber, intramuscular, intrasynovial, intradermal, and subcutaneous. Suitable devices for administration include needle (including microneedle) injectors, needle-free injectors, osmotic pumps, and infusion techniques. 
     The LNP compositions will generally, but not necessarily, be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term “excipient” includes any ingredient other than the compound(s) of the disclosure, the other lipid component(s) and the biologically active agent. An excipient may impart either a functional (e.g. drug release rate controlling) and/or a non-functional (e.g. processing aid or diluent) characteristic to the formulations. The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. 
     Parenteral formulations are typically aqueous or oily solutions or suspensions. Where the formulation is aqueous, excipients such as sugars (including but not restricted to glucose, mannitol, sorbitol, etc.) salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated with a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water (WFI). 
     While the invention is described in conjunction with the illustrated embodiments, it is 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, including equivalents of specific features, which may be included within the invention as defined by the appended claims. 
     Both the foregoing general description and detailed description, as well as the following examples, are exemplary and explanatory only and are not restrictive of the teachings. 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 literature incorporated by reference contradicts any term defined in this specification, this specification controls. All ranges given in the application encompass the endpoints unless stated otherwise. 
     It should be noted that, as used in this application, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes a plurality of compositions and reference to “a cell” includes a plurality of cells and the like. The use of “or” is inclusive and means “and/or” unless stated otherwise. 
     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. 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 use of a modifier such as “about” before a range or before a list of values, modifies each endpoint of the range or each value in the list. “About” also includes the value or enpoint. For example, “about 50-55” encompasses “about 50 to about 55”. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” is not limiting. 
     Unless specifically noted in the above 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; embodiments in the specification that recite “about” various components are also contemplated as “at” 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). 
     EXAMPLES 
     Example 1 
     LNP Compositions for In Vivo Editing in Mice 
     Small scale preparations of various LNP compositions were prepared to investigate their properties. In assays for percent liver editing in mice, Cas9 mRNA and chemically modified sgRNA targeting a mouse TTR sequence were formulated in LNPs with varying PEG mol-%, Lipid A mol-%, and N:P ratios as described in Table 2, below. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 LNP compositions. 
               
            
           
           
               
               
               
               
            
               
                 LNP # 
                 Lipid A mol-% 
                 PEG-DMG mol-% 
                 N:P ratio 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 (various) 
                 45 
                 2, 2.5, 3, 4, 5 
                 4.5 
               
               
                 (various) 
                 45 
                 2, 2.5, 3, 4, 5 
                 6 
               
               
                 (various) 
                 50 
                 2, 2.5, 3, 4, 5 
                 4.5 
               
               
                 (various) 
                 50 
                 2, 2.5, 3, 4, 5 
                 6 
               
               
                 (various) 
                 55 
                 2, 2.5, 3, 4, 5 
                 4.5 
               
               
                 (various) 
                 55 
                 2, 2.5, 3, 4, 5 
                 6 
               
               
                   
               
            
           
         
       
     
     In  FIG.  1   , LNP formulations are identified on the X-axis based on their Lipid A mol-% and N:P ratios, labeled “% CL; N:P”. As indicated in the legend to  FIG.  1   , PEG-2k-DMG concentrations of 2, 2.5, 3, 4, or 5 mol-% were formulated with (1) 45 mol-%, Lipid A; 4.5 N:P (“45; 4.5”); (2) 45 mol-% Lipid A; 6 N:P (“45; 6”); (3) 50 mol-% Lipid A; 4.5 N:P (“50; 4.5”); (4) 50 mol-% Lipid A; 6 N:P (“50; 6”); (5) 55 mol-% Lipid A; 4.5 N:P (“55; 4.5”); and (6) 55 mol-% Lipid A; 6 N:P (“55; 6”). The DSPC mol-% was kept constant at 9 mol-% and the cholesterol mol-% was added to bring the balance of each formulation lipid component to 100 mol-%. Each of the 30 formulations was formulated as described below, and administered as single dose at 1 mg per kg or 0.5 mg per kg doses of total RNA, ( FIG.  1 A  and  FIG.  1 B , respectively). 
     LNP Formulation—NanoAssemblr 
     The lipid nanoparticle components were dissolved in 100% ethanol with the lipid component molar ratios set forth above. The RNA cargos 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. The LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 4.5 or about 6, with the ratio of mRNA to gRNA at 1:1 by weight. 
     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. 
     Formulation Analytics 
     Dynamic Light Scattering (“DLS”) is used to characterize the polydispersity index (“pdi”) and size of the LNPs of the present disclosure. DLS measures the scattering of light that results from subjecting a sample to a light source. PDI, as determined from DLS measurements, represents the distribution of particle size (around the mean particle size) in a population, with a perfectly uniform population having a PDI of zero. 
     Electropheretic light scattering is used to characterize the surface charge of the LNP at a specified pH. The surface charge, or the zeta potential, is a measure of the magnitude of electrostatic repulsion/attraction between particles in the LNP suspension. 
     Assymetric-Flow Field Flow Fractionation—Multi-Angle Light Scattering (AF4-MALS) is used to separate particles in the formulation by hydrodynamic radius and then measure the molecular weights, hydrodynamic radii and root mean square radii of the fractionated particles. This allows the ability to assess molecular weight and size distributions as well as secondary characteristics such as the Burchard-Stockmeyer Plot (ratio of root mean square (“rms”) radius to hydrodynamic radius over time suggesting the internal core density of a particle) and the rms conformation plot (log of rms radius versus log of molecular weight where the slope of the resulting linear fit gives a degree of compactness versus elongation). 
     Nanoparticle tracking analysis (NTA, Malvern Nanosight) can be used to determine formulation particle size distribution as well as particle concentration. LNP samples are diluted appropriately and injected onto a microscope slide. A camera records the scattered light as the particles are slowly infused through field of view. After the movie is captured, the Nanoparticle Tracking Analysis processes the movie by tracking pixels and calculating a diffusion coefficient. This diffusion coefficient can be translated into the hydrodynamic radius of the particle. The instrument also counts the number of individual particles counted in the analysis to give particle concentration. 
     Cryo-electron microscopy (“cryo-EM”) can be used to determine the particle size, morphology, and structural characteristics of an LNP. 
     Lipid compositional analysis of the LNPs can be determined from liquid chromotography followed by charged aerosol detection (LC-CAD). This analysis can provide a comparison of the actual lipid content versus the theoretical lipid content. 
     LNP formulations are analyzed for average particle size, polydispersity index (pdi), total RNA content, encapsulation efficiency of RNA, and zeta potential. LNP formualtions may be further characterized by lipid analysis, AF4-MALS, NTA, and/or cryo-EM. Average particle size and polydispersity are measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument. LNP samples were diluted 30× in PBS prior to being measured by DLS. Z-average diameter which is an intensity-based measurement of average particle size was reported along with number average diameter and pdi. A Malvern Zetasizer instrument is also used to measure the zeta potential of the LNP. Samples are diluted 1:17 (50 μL into 800 μL) in 0.1× PBS, pH 7.4 prior to measurement. 
     A fluorescence-based assay (Ribogreen®, ThermoFisher Scientific) is used to determine total RNA concentration and free RNA. Encapsulation efficiency is calclulated as (Total RNA−Free RNA)/Total RNA. LNP samples are diluted appropriately with 1× TE buffer containing 0.2% Triton-X 100 to determine total RNA or 1× TE buffer to determine free RNA. Standard curves are prepared by utilizing the starting RNA solution used to make the formulations and diluted in 1× TE buffer+/−0.2% Triton-X 100. Diluted RiboGreen® dye (according to the manufacturer&#39;s instructions) is then added to each of the standards and samples and allowed to incubate for approximately 10 minutes at room temperature, in the absence of light. A SpectraMax M5 Microplate Reader (Molecular Devices) is used to read the samples with excitation, auto cutoff and emission wavelengths set to 488 nm, 515 nm, and 525 nm respectively. Total RNA and free RNA are determined from the appropriate standard curves. 
     Encapsulation efficiency is calclulated as (Total RNA−Free RNA)/Total RNA. The same procedure may be used for determining the encapsulation efficiency of a DNA-based or nucleic acid-containing cargo component. For single-strand DNA Oligreen Dye may be used, and for double-strand DNA, Picogreen Dye. 
     AF4-MALS is used to look at molecular weight and size distributions as well as secondary statistics from those calculations. LNPs are diluted as appropriate and injected into an AF4 separation channel using an HPLC autosampler where they are focused and then eluted with an exponential gradient in cross flow across the channel. All fluid is driven by an HPLC pump and Wyatt Eclipse Instrument. Particles eluting from the AF4 channel flow through a UV detector, multi-angle light scattering detector, quasi-elastic light scattering detector and differential refractive index detector. Raw data is processed by using a Debeye model to determine molecular weight and rms radius from the detector signals. 
     Lipid components in LNPs are analyzed quantitatively by HPLC coupled to a charged aerosol detector (CAD). Chromatographic separation of 4 lipid components is achieved by reverse phase HPLC. CAD is a destructive mass-based detector which detects all non-volatile compounds and the signal is consistent regardless of analyte structure. 
     Cas9 mRNA and gRNA Cargos 
     The Cas9 mRNA cargo was prepared by in vitro transcription. Capped and polyadenylated Cas9 mRNA comprising 1× NLS (SEQ ID NO:48) was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing a T7 promoter and a 100 nt poly(A/T) region was linearized by incubating at 37° C. for 2 hrs with XbaI with the following conditions: 200 ng/μL plasmid. 2 U/μL XbaI (NEB), and 1× reaction buffer. The XbaI was inactivated by heating the reaction at 65° C. for 20 min. The linearized plasmid was purified from enzyme and buffer salts using a silica maxi spin column (Epoch Life Sciences) and analyzed by agarose gel to confirm linearization. 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. After the 4 hr incubation, 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 from enzyme and nucleotides using a MegaClear Transcription Clean-up kit per the manufacturer&#39;s protocol (ThermoFisher). Alternatively, the Cas9 mRNA was purified with a LiCl precipitation method. 
     The sgRNA in this example was chemically synthesized and sourced from a commercial supplier. The sg282 sequence is provided below, with 2′-O-methyl modifications and phosphorothioate linkages as represented below (m=2′-OMe; *=phosphorothioate): 
     
       
         
           
               
            
               
                 (SEQ ID NO: 42) 
               
               
                 mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmAm 
               
               
                   
               
               
                 AmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAm 
               
               
                   
               
               
                 AmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU 
               
               
                   
               
               
                 *mU*mU.. 
               
            
           
         
       
     
     LNPs 
     The final LNPs were characterized to determine the encapsulation efficiency, polydispersity index, and average particle size according to the analytical methods provided above. 
     The LNPs were dosed to mice (single dose at 1 mg/kg or 0.5 mg/kg) and genomic DNA was isolated for NGS analysis as described below. 
     LNP Delivery In Vivo 
     CD-1 female mice, ranging from 6 to 10 weeks of age were used in each study. Animals were weighed and grouped according to body weight for preparing dosing solutions based on group average weight. LNPs were dosed via the lateral tail vein in a volume of 0.2 mL per animal (approximately 10 mL per kilogram body weight). The animals were observed at approximately 6 hours post dose for adverse effects. Body weight was measured at twenty-four hours post-administration, and animals were euthanized at various time points by exsanguination via cardiac puncture under isoflurane anesthesia. Blood was collected into serum separator tubes or into tubes containing buffered sodium citrate for plasma as described herein. For studies involving in vivo editing, liver tissue was collected from the median lobe or from three independent lobes (e.g., the right median, left median, and left lateral lobes) from each animal for DNA extraction and analysis. 
     Cohorts of mice were measured for liver editing by Next-Generation Sequencing (NGS) and serum TTR levels (data not shown). 
     Transthyretin (TTR) ELISA Analysis 
     Blood was collected and the serum was isolated as indicated. The total mouse TTR serum levels were determined using a Mouse Prealbumin (Transthyretin) ELISA Kit (Aviva Systems Biology, Cat. OKIA00111). Rat TTR serum levels were measured using a rat specific ELISA kit (Aviva Systems Biology catalog number OKIA00159) according to manufacture&#39;s protocol. Briefly, sera were serial diluted with kit sample diluent to a final dilution of 10,000-fold. This diluted sample was then added to the ELISA plates and the assay was then carried out according to directions. 
     NGS Sequencing 
     In brief, to quantitatively determine the efficiency of editing at the target location in the genome, genomic DNA was isolated and deep sequencing was utilized to identify the presence of insertions and deletions introduced by gene editing. 
     PCR primers were designed around the target site (e.g., TTR), and the genomic area of interest was amplified. Primer sequences are provided below. Additional PCR was performed according to the manufacturer&#39;s protocols (Illumina) to add the necessary chemistry for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument. The reads were aligned to the human reference genome (e.g., hg38) 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, substitution, or deletion 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 over the total number of sequence reads, including wild type. 
       FIG.  1    shows editing percentages in mouse liver as measured by NGS. As shown in  FIG.  1 A , when 1 mg per kg RNA is dosed, in vivo editing percentages range from about 20% to over 60% liver editing. At a 0.5 mg per kg dose,  FIG.  1 B , about 10% to 60% liver editing was observed. In this mouse in vivo testing, all compositions effectively delivered Cas9 mRNA and gRNA to the liver cells, with evidence of active CRISPR/Cas nuclease activity at the target site measured by NGS for each LNP composition. LNPs containing 5% PEG lipid had lower encapsulation (data not shown), and somewhat reduced potency. 
     Example 2 
     LNP Composition Analytics 
     Analytical characterization of LNPs shows improved physicochemical parameters in LNPs formulated with increasing amounts of Lipid A and PEG-lipid. Compositons that comprise either 2 mol-% or 3 mol-% PEG lipid (PEG2k-DMG) are provided in Table 3 below. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 LNP898 
                 LNP897 
                 LNP966 
                 LNP969 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 CL/chol./ 
                 45/44/9/2 
                 45/43/9/3 
                 50/38/9/3 
                 55/33/9/3 
               
               
                 DSPC/PEG 
               
               
                 (theoretical 
               
               
                 mol-%) 
               
               
                 mRNA 
                 Cas9 
                 Cas9 
                 Cas9 U-dep 
                 Cas9 U-dep 
               
               
                   
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
               
               
                   
                 NO: 48 
                 NO: 48 
                 NO: 43 
                 NO: 43 
               
               
                 gRNA 
                 G502 
                 G502 
                 G534 
                 G534 
               
               
                   
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
               
               
                   
                 NO: 70 
                 NO: 70 
                 NO: 72 
                 NO: 72 
               
               
                 N/P 
                 4.5 
                 4.5 
                 6.0 
                 6.0 
               
               
                   
               
            
           
         
       
     
     LNP Formulation—Cross Flow 
     The LNPs were formed by impinging jet mixing of the lipid in ethanol with two volumes of RNA solutions and one volume of water. The lipid in ethanol is mixed through a mixing cross with the two volumes of RNA solution. A fourth stream of water is mixed with the outlet stream of the cross through an inline tee. (See WO2016010840 at  FIG.  2   .) The LNPs were maintained at room temperature for 1 hour, and then 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. 
     Cas9 mRNA and sgRNA were prepared as in Example 1, except that capped and poly-adenylated Cas9 U-depleted (Cas9 Udep) mRNA comprises SEQ ID N:43. Sg282 is described in Example 1, and the sequence for sg534 (“G534”) is provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 72) 
               
               
                 mA*mC*mG*CAAAUAUCAGUCCAGCGGUUUUAGAmGmCmUmAmGmAmA 
               
               
                   
               
               
                 mAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUm 
               
               
                   
               
               
                 GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGm 
               
               
                   
               
               
                 CmU*mU*mU*mU 
               
            
           
         
       
     
     LMP formulations were analyzed for average particle size, polydispersity (pdi), total RNA content and encapsulation efficiency of RNA as described in Example 1. 
     Analysis of average, particle size, polydispersity (PDI), total RNA content and encapsulation efficiency of RNA are shown in Table 4. In addition to the theoretical lipid concentrations of the LNP compositions, lipid analysis demonstrated the actual mol-% lipid levels, as indicated in Table 5 below. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                   
                   
                 Z- 
                   
                 Number 
                 RNA 
                   
               
               
                 LNP 
                   
                 Ave. 
                   
                 Ave. 
                 Conc 
                 Eneaps. 
               
               
                 ID # 
                 N/P 
                 (nm) 
                 FBI 
                 (nm) 
                 (mg/mL) 
                 (%) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 LNP898 
                 4.5 
                 87.91 
                 0.030 
                 71.33 
                 1.53 
                 98 
               
               
                 LNP897 
                 4.5 
                 74.05 
                 0.036 
                 58.55 
                 1.43 
                 98 
               
               
                 LNP966 
                 6.0 
                 82.78 
                 0.010 
                 67.86 
                 2.12. 
                 98 
               
               
                 LNP969 
                 6.0 
                 92.97 
                 0.042 
                 75.52 
                 2.09 
                 97 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                   
                 Lipid Ratio 
                   
                   
                   
                   
               
               
                   
                 (Lipid A/Chol/ 
                 Lipid A 
                 Chol 
                 DSPC 
                 PEG 
               
               
                   
                 DSPC/PEG) 
                 mg/mL 
                 mg/mL 
                 mg/mL 
                 mg/mL 
               
               
                 LNP 
                 (theoretical 
                 (theoretical 
                 (theoretical 
                 (theoretical 
                 (theoretical 
               
               
                 ID # 
                 and actual) 
                 and actual) 
                 and actual) 
                 and actual) 
                 and actual) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 LNP898 
                 45/44/9/2 
                 18.0 
                 8.0 
                 3.3 
                 2.3 
               
               
                   
                 46.1/42.6/9.2/2 
                 18.3 
                 7.7 
                 3.4 
                 2.4 
               
               
                 LNP897 
                 45/43/9/3 
                 18.0 
                 7.8 
                 3.3 
                 3.5 
               
               
                   
                 44.8/42.9/9.2/3.1 
                 17.8 
                 7.7 
                 3.4 
                 3.6 
               
               
                 LNP966 
                 50/38/9/3 
                 33.4 
                 11.5 
                 5.6 
                 5.8 
               
               
                   
                 50.0/38.0/8.8/3.1 
                 35.6 
                 12.3 
                 5.8 
                 6.5 
               
               
                 LNP969 
                 55/33/9/3 
                 33.4 
                 9.1 
                 5.1 
                 5.3 
               
               
                   
                 54.8/33.2/8.8/3.2 
                 31.6 
                 8.7 
                 4.7 
                 5.4 
               
               
                   
               
            
           
         
       
     
     To further analyze the physicochemical properties, LNP897, LNP898, LNP966, and LNP969 were subjected to Asymmetric-Flow Field Flow Fractionation—Multi-Angle Light Scattering (AF4-MALS) analysis. The AF4-MALS instrument measures particle size and molecular weight distribtions, and provides information about particle conformation and density. 
     LNPs are injected into an AF4 separation channel using an HPLC autosampler where they are focused and then eluted with an exponential gradient in cross flow across the channel. All fluid is driven by an HPLC pump and Wyatt Eclipse Instrument. Particles eluting from the AF4 channel flow through a UV detector, Wyatt Heleos II multi-angle light scattering detector, quasi-elastic light scattering detector and Wyatt Optilab T-rEX differential refractive index detector. Raw data is processed in Wyatt Astra 7 Software by using a Debeye model to determine molecular weight and cats radius from the detector signals. 
     A log differential molar mass plot for the LNPs is provided as  FIG.  2 A . In brief, the X-axis indicates molar mass (g/mol), and the Y-axis indicates the differential number fraction. The log differential molar mass plot shows the distribution of the different molecular weights measured for a specific formulation. This gives data towards the mode of the molecular weights as well as the overall distribution of molecular weights within the formulation, which gives a better picture of particle heterogeniety than average molecular weight. 
     The heterogeniety of the different LNP formulations are determined by measuring the different molar mass moments and calculating the ratio of the weight averaged molar mass (Mw) to the number averaged molar mass (Mn) to give a polydispersity of Mw/Mn. The graph of the polydispersity for these different formulations is provided in  FIG.  2 B . 
     The data indicate tighter particle distributions with 3 mol-% PEG, and with 50 and 55 mol-% Lipid A at N/P 6.0 as shown in  FIG.  2 A . This is reflected in a tight polydispersity as shown in  FIG.  2 B   
     Example 3 
     AF4 MALS Data—Additional Formulations 
     Analytical characterization of LNPs shows improved physicochemical parameters in LNPs formulated with increasing amounts of Lipid A. Compositons that comprise either 45 mol-%, 50 mol-%, or 55 mol-% Lipid A with two different gRNA are provided in Table 6 below. 
     
       
         
           
               
               
               
               
               
               
             
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                 LNP1021 
                 LNP1022 
                 LNP1023 
                 LNP1024 
                 LNP1025 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 CL/chol./DSPC/PEG 
                 50/38/9/3 
                 55/33/9/3 
                 45/43/9/3 
                 50/38/9/3 
                 55/33/9/3 
               
               
                 (theoretical mol-%) 
               
               
                 mRNA 
                 Cas9 Udep 
                 Cas9 Udep 
                 Cas9 Udep 
                 Cas9 Udep 
                 Cas9 Udep 
               
               
                   
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
               
               
                   
                 NO: 43 
                 NO: 43 
                 NO: 43 
                 NO: 43 
                 NO: 43 
               
               
                 gRNA 
                 G502 
                 G502 
                 G502 
                 G509 
                 G509 
               
               
                   
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
               
               
                   
                 NO: 70 
                 NO: 70 
                 N0: 70 
                 NO: 71 
                 NO: 71 
               
               
                 N/P 
                 6.0 
                 6.0 
                 4.5 
                 6.0 
                 6.0 
               
               
                   
               
            
           
         
       
     
     The LNPs were formed as described in Example 2. 
     Cas9 mRNA and sgRNA were prepared as described above. 
     The LNPs compositions were characterized to determine the encapsulation efficiency, polydispersity index, and average particle size as described in Example 1. 
     Analysis of average particle size, polydispersity (PDI), total RNA content and encapsulation efficiency of RNA are shown in Table 7. In addition to the theoretical lipid concentrations of the LNP compositions, lipid analysis demonstrated the actual mol-% lipid levels, as indicated in Table 8, below. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                   
                   
                 Z- 
                   
                 Number 
                 RNA 
                   
               
               
                 LNP 
                   
                 Ave. 
                   
                 Ave. 
                 Conc. 
                 Encaps. 
               
               
                 ID # 
                 N/P 
                 (nm) 
                 PDI 
                 (nm) 
                 (mg/mL) 
                 (%) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 LNP1021 
                 6.0 
                 83.18 
                 0.027 
                 67.15 
                 1.63 
                 98 
               
               
                 LNP1022 
                 6.0 
                 94.08 
                 0.005 
                 78.28 
                 1.60 
                 97 
               
               
                 LNP1023 
                 4.5 
                 74.01 
                 0.017 
                 61.11 
                 1.61 
                 97 
               
               
                 LNP1024 
                 6.0 
                 85.37 
                 0.002 
                 70.42 
                 1.59 
                 97 
               
               
                 LNP1025 
                 6.0 
                 94.47 
                 0.018 
                 77.71 
                 1.60 
                 98 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                   
                 Lipid Ratio 
                   
                   
                   
                   
               
               
                   
                 (Lipid A/Chol/ 
                 Lipid A 
                 Chol. 
                 DSPC 
                 PEG 
               
               
                   
                 DSPC/PEG) 
                 mg/mL 
                 mg/mL 
                 mg/mL 
                 mg/mL 
               
               
                 LNP 
                 (theoretical 
                 (theoretical 
                 (theoretical 
                 (theoretical 
                 (theoretical 
               
               
                 ID # 
                 and actual) 
                 and actual) 
                 and actual) 
                 and actual) 
                 and actual) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 LNP1021 
                 50/38/9/3 
                 23.6 
                 8.1 
                 3.9 
                 4.1 
               
               
                   
                 50.9/37.4/8.6/3.1 
                 21.6 
                 7.2. 
                 3.4 
                 3.8 
               
               
                 LNP1022 
                 55/33/9/3 
                 23.6 
                 6.4 
                 3.6 
                 3.7 
               
               
                   
                 55.2/33.0/8.7/3.1 
                 20.4 
                 5.5 
                 3.0 
                 3.4 
               
               
                 LNP1023 
                 45/43/9/3 
                 17.7 
                 7.7 
                 3.3 
                 3.4 
               
               
                   
                 45.9/42.4/8.6/3.1 
                 15.3 
                 6.4 
                 2.7 
                 3.0 
               
               
                 LNP1024 
                 50/38/9/3 
                 23.6 
                 8.1 
                 3.9 
                 4.1 
               
               
                   
                 50.5/37.9/8.5/3.0 
                 22.4 
                 7.6 
                 3.5 
                 3.9 
               
               
                 LNP1024 
                 55/33/9/3 
                 23.6 
                 6.4 
                 3.6 
                 3.7 
               
               
                   
                 55.5/33.1/8.5/3.0 
                 21.3 
                 5.8 
                 3.0 
                 3.4 
               
               
                   
               
            
           
         
       
     
     To further analyze the physicochemical properties, LNP1021, LNP1022, LNP1023, LNP1024 and LNP1025 were subjected to Asymmetric-Flow Field Flow Fractionation—Multi-Angle Light Scattering (AF4-MALS) analysis. The AF4-MALS instrument measures particle size and molecular weight distribtions, and provides information about particle conformation and density. 
     LNPs were run on AF4-MALS as described in Example 1. 
     A log differential molar mass plot for the LNPs is provided as  FIG.  3 A . In brief, the X-axis indicates molar mass (g/mol), and the Y-axis indicates the differential number fraction. The log differential molar mass plot shows the distribution of the different molecular weights calculated for a specific formulation. This gives data towards the mode of the molecular weights as well as the overall distribution of molecular weights within the formulation, which gives a better picture of particle heterogeniety than average molecular weight. 
     Average molecular weight is plotted in  FIG.  3 B . The average molecular weight is the average of the entire distribution but gives no information about the shape of that distribution. LNP1022 and LNP1025 have the same average molecular weight but LNP1022 has a slightly broader distribution. 
     The heterogeniety of the different LNP formulations are calculated by look at the different molar mass moments and calculating the ratio of the weight averaged molar mass (Mw) to the number averaged molar mass (Mn) to give a polydispersity of Mw/Mn. The graph of the polydispersity for these different formulations is provided in  FIG.  4 A . 
     Additionally, a Burchard-Stockmeyer plot of the LNP formulations is provided as  FIG.  4 B . The Burchard-Stockmeyer plot shows the ratio of the rms radius versus the hydrodynamic radius across the elution of the formulation from the AF4 channel. This gives information towards the internal density of a lipid nanoparticle.  FIG.  4 B  shows that LNP1021, LNP1022 and LNP1023 have different profiles in this measurement. 
     Example 4 
     Increased PEG Lipid Maintains Potency with Reduced Cytokine Response 
     In another study, PEG DMG lipid was compared in LNP formulations comprising 2 mol-% or 3 mol-% of the PEG lipid. Compositons that comprise either 2 mol-%, or 3 mol-%, PEG DMG are provided in Table 9 below. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 9 
               
               
                   
                   
               
               
                   
                 LNP809 
                 LNP810 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 CL/chol./DSPC/PEG 
                 45/44/9/2 
                 45/43/9/3 
               
               
                   
                 (theoretical mol-%) 
               
               
                   
                 mRNA 
                 Cas9 
                 Cas9 
               
               
                   
                   
                 SEQ ID NO: 48 
                 SEQ ID NO: 48 
               
               
                   
                 gRNA 
                 G390 
                 G390 
               
               
                   
                   
                 SEQ ID NO: 69 
                 SEQ ID NO: 69 
               
               
                   
                 N/P 
                 4.5 
                 4.5 
               
               
                   
                   
               
            
           
         
       
     
     The LNPs were formed by the process described in Example 2. 
     Cas9 mRNA and sgRNA were prepared as in Example 1, with the sequence of sg390 (“G390”) provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 69) 
               
               
                 mG*mC*mC*GAGUCUGGAGAGCUGCAGUUUUAGAmGmCmUmAmGmAmAm 
               
               
                   
               
               
                 AmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAm 
               
               
                   
               
               
                 AmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU 
               
               
                   
               
               
                 *mU*mU. 
               
            
           
         
       
     
     LNP formulations were analyzed for average particle size, polydispersity (pdi), total RNA content and encapsulation efficiency of RNA as described in Example 1. 
     Analysis of average particle size, polydispersity (PDI), total RNA content and encapsulation efficiency of RNA are shown in Table 10. In addition to the theoretical lipid concentrations of the LNP compositions, lipid analysis demonstrated the actual mol-% lipid levels, as indicated in Table 11: below. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                   
                   
                 Z- 
                   
                 Number 
                 RNA 
                   
               
               
                 LNP 
                   
                 Ave. 
                   
                 Ave. 
                 Conc. 
                 Encaps. 
               
               
                 ID # 
                 N/P 
                 (nm) 
                 PDI 
                 (nm) 
                 (mg/mL) 
                 (%) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 LNP809 
                 4.5 
                 89.85 
                 0.060 
                 72.10 
                 2.45 
                 97 
               
               
                 LNP810 
                 4.5 
                 75.26 
                 0.025 
                 61.17 
                 2.14 
                 97 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 11 
               
               
                   
               
               
                   
                 Lipid Ratio 
                   
                   
                   
                   
               
               
                   
                 (Lipid A/Chol/ 
                 Lipid A 
                 Chol. 
                 DSPC 
                 PEG 
               
               
                   
                 DSPC/PEG) 
                 mg/mL 
                 mg/mL 
                 mg/mL 
                 mg/mL 
               
               
                 LNP 
                 (theoretical 
                 (theoretical 
                 (theoretical 
                 (theoretical 
                 (theoretical 
               
               
                 ID # 
                 and actual) 
                 and actual) 
                 and actual) 
                 and actual) 
                 and actual) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 LNP809 
                 45/44/9/2 
                 28.6 
                 12.7 
                 5.3 
                 3.7 
               
               
                   
                 45.7/43.3/9.0/2.1 
                 30.5 
                 13.1 
                 5.6 
                 4.0 
               
               
                 LNP810 
                 45/43/9/3 
                 25.2 
                 10.9 
                 4.7 
                 4.9 
               
               
                   
                 45.0/42.3/9.7/3.0 
                 24.7 
                 10.5 
                 4.9 
                 4.7 
               
               
                   
               
            
           
         
       
     
     Rat serum cytokines were evaluated using a Luminex magnetic bead multiplex assay (Milliplex MAP magnetic bead assay from Millipore Sigma, catalog number RECYTMAG-65K) analyzing MCP-1, IL-6, TNF-alpha and IFN-gamma. The assay beads were read on the BioRad BioPlex-200 and cytokine concentrations calculated off a standard curve using 4 parameter logistic fit with BioPlex Manager Software version 6.1. Data is graphed in  FIG.  5   . See  FIG.  5 A  (serum TTR),  FIG.  5 B  (liver editing), and  FIG.  5 C  (cytokine p MCP1). 
     Rat TTR serum levels were measured using a rat specific ELISA kit (Aviva Systems Biology catalog number OKIA00159) according to manufacture&#39;s protocol. Briefly, serums were serially diluted with kit sample diluent to a final dilution of 10,000-fold. This diluted sample was then added to the ELISA plates and the assay was then carried out according to directions. 
     Genomic DNA was isolated from approximately 10 mg of liver tissue and analyzed using NGS as described above. PCR primer sequences for amplification are described below. 
       FIG.  5 A  and  FIG.  5 B  show that serum TTR knockdown and liver editing were sufficient in the 2 mol-% and 3 mol-% PEG formulations.  FIG.  5 C  shows that MCP-1 response is reduced using 3 mol-% PEG formulations. 
     Example 5 
     LNP Delivery to Non-Human Primates 
     Three studies were conducted with LNP formulations prepared as described in Example 1. The particular molar amounts and cargos are provided in Tables 12-26. Each formulation containing Cas9 mRNA and guide RNA (gRNA) had a mRNA:gRNA ratio of 1:1 by weight. Doses of LNP (in mg/kg, total RNA content), route of administration and whether animals received pre-treatment of dexamethasone are indicated in the Tables. For animals receiving dexamethasone (Dex) pre-treatment, Dex was administered at 2 mg/kg by IV bolus injection, 1 hour prior to LNP or vehicle administration. 
     For blood chemistry analysis, blood was drawn from animals at times as indicated in the tables below for each factor that was measured. Cytokine induction was measured in pre- and post-treated NHPs. A minimum of 0.5 mL of whole blood was collected from a peripheral vein of restrained, conscious animals into a 4 mL serum separator tube. Blood was allowed to clot for a minimum of 30 minutes at room temperature followed by centrifugation at 2000×g for 15 minutes. Serum was aliquoted into 2 polypropylene microtubes of 120 μL each and stored at −60 to −86° C. until analysis. A non-human primate U-Plex Cytokine custom kit from Meso Scale Discovery (MSD) was used for analysis. The following parameters were included in the analysis INF-g, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, MCP-I and INF-a, with focus on IL-6 and MCP-1. Kit reagents and standards were prepared as directed in the manufacturer&#39;s protocol. NHP serum was used neat. The plates were run on an MSD Sector Imager 6000 with analysis performed with MSD Discovery work bench software Version 4012. 
     Complement levels were measured in pre- and post-treated animals by enzyme Immunoassay. Whole blood (0.5 mL) was collected from a peripheral vein of restrained, conscious animals into a tube containing 0.5 mL k 2 EDTA. Blood was centrifuged at 2000×g for 15 minutes. Plasma was aliquoted into 2 polypropylene microtubes of 120 μL each and stored at −60 to −86° C. until analysis. A Quidel MicroVue Complement Plus EIA kit (C3a-Cat #A031) or (Bb-Cat #A027) was used for analysis. Kit reagents and standards were prepared as directed in the manufacturer&#39;s protocol. The plates were run on an MSD Sector Imager 6000 at optical density at 450 nm. The results were analyzed using a 4-parameter curve fit. 
     The data for cytokine induction and complement activation are provided in the Tables below. “BLQ” means below the limit of quantification. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Study 1. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Molar Ratios (Lipid 
                   
                   
                   
                   
                   
                   
               
               
                   
                 A, Cholesterol, 
                   
                   
                   
                   
                 Dose level, 
               
               
                 Treatment 
                 DSPC, and PEG2k- 
                   
                   
                 sample 
                   
                 total RNA 
               
               
                 group 
                 DMG, respectively 
                 N:P 
                 Cargo 
                 size (n) 
                 Route 
                 content (mg/kg) 
                 Dex 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 (I) TSS 
                 n/a 
                 n/a 
                 n/a 
                 3 
                 IV - 
                 n/a 
                 no 
               
               
                 (vehicle) 
                   
                   
                   
                   
                 infusion 
               
               
                 (2) LNP699 
                 45/44/9/2 
                 4.5 
                 Cas9 mRNA 
                 3 
                 IV - 
                 3 
                 no 
               
               
                 G502 
                   
                   
                 (SEQ ID NO: 
                   
                 infusion 
               
               
                   
                   
                   
                 2); G000502 
               
               
                 (3) LNP688 
                 45/44/9/2 
                 4.5 
                 Cas9 mRNA 
                 3 
                 IV - 
                 3 
                 no 
               
               
                 G506 
                   
                   
                 (SEQ ID NO: 
                   
                 infusion 
               
               
                   
                   
                   
                 2); G000506 
               
               
                 (4) LNP689 
                 45/44/9/2 
                 4.5 
                 Cas9 mRNA 
                 3 
                 IV - 
                 3 
                 no 
               
               
                 G509 
                   
                   
                 (SEQ ID NO: 
                   
                 infusion 
               
               
                   
                   
                   
                 2); G000509 
               
               
                 (5) LNP690 
                 45/44/9/2 
                 4.5 
                 Cas9 mRNA 
                 3 
                 IV - 
                 3 
                 no 
               
               
                 G510 
                   
                   
                 (SEQ ID NO: 
                   
                 infusion 
               
               
                   
                   
                   
                 2); G000510 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Study 2. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Mohr Ratios (Lipid 
                   
                   
                   
                   
                   
                   
               
               
                   
                 A, Cholesterol, 
                   
                   
                   
                   
                 Dose level, 
               
               
                 Treatment 
                 DSPC, and PEG2k- 
                   
                   
                 sample 
                   
                 total RNA 
               
               
                 group 
                 DMG, respectively 
                 N:P 
                 Cargo 
                 size (n) 
                 Route 
                 content (mg/kg) 
                 Dex 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 (1) TSS 
                 n/a 
                 n/a 
                   
                 1 
                 IV- 
                 n/a 
                 yes 
               
               
                 (vehicle) 
                   
                   
                   
                   
                 bolus 
               
               
                 (2) TSS 
                 n/a 
                 n/a 
                   
                 1 
                 IV- 
                 n/a 
                 no 
               
               
                 (vehicle) 
                   
                   
                   
                   
                 bolus 
               
               
                 (3) LNP898 
                 45/44/9/2 
                 4.5 
                 Cas9 mRNA 
                 1 
                 IV - 
                 3 
                 yes 
               
               
                 G502 
                   
                   
                 (SEQ ID NO: 
                   
                 infusion 
               
               
                   
                   
                   
                 2); G000502 
               
               
                 (4) LNP898 
                 45/44/9/2 
                 4.5 
                 Cas9 mRNA 
                 1 
                 IV - 
                 3 
                 no 
               
               
                 G502 
                   
                   
                 (SEQ ID NO: 
                   
                 infusion 
               
               
                   
                   
                   
                 2); G000502 
               
               
                 (5) LNP897 
                 45/43/9/3 
                 4.5 
                 Cas9 mRNA 
                 1 
                 IV- 
                 3 
                 yes 
               
               
                 G502 
                   
                   
                 (SEQ ID NO: 
                   
                 bolus 
               
               
                   
                   
                   
                 2); G000502 
               
               
                 (6) LNP897 
                 45/43/9/3 
                 4.5 
                 Cas9 mRNA 
                 1 
                 IV- 
                 3 
                 no 
               
               
                 G502 
                   
                   
                 (SEQ ID NO: 
                   
                 bolos 
               
               
                   
                   
                   
                 2); G000502 
               
               
                 (7) LNP897 
                 45/43/9/3 
                 4.5 
                 Cas9 mRNA 
                 1 
                 IV - 
                 3 
                 yes 
               
               
                 G502 
                   
                   
                 (SEQ ID NO: 
                   
                 infusion 
               
               
                   
                   
                   
                 2); G000502 
               
               
                 (8) LNP897 
                 45/43/9/3 
                 4.5 
                 Cas9 mRNA 
                 1 
                 IV - 
                 3 
                 no 
               
               
                 G502 
                   
                   
                 (SEQ ID NO: 
                   
                 infusion 
               
               
                   
                   
                   
                 2); G000502 
               
               
                 (9) LNP916 
                 45/43/9/3 
                 4.5 
                 eGFP mRNA 
                 1 
                 IV - 
                 6 
                 yes 
               
               
                 GFP 
                   
                   
                 (SEQ ID NO: ) 
                   
                 infusion 
               
               
                 (10) LNP916 
                 45/43/9/3 
                 4.5 
                 eGFP mRNA 
                 1 
                 IV - 
                 6 
                 no 
               
               
                 GFP 
                   
                   
                 (SEQ ID NO: ) 
                   
                 infusion 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Study 3. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Molar Ratios (Lipid 
                   
                   
                   
                   
                   
                   
               
               
                   
                 A, Cholesterol, 
                   
                   
                   
                   
                 Dose level, 
               
               
                 Treatment 
                 DSPC, and PEG2k- 
                   
                   
                 sample 
                   
                 total RNA 
               
               
                 group 
                 DMG, respectively 
                 N:P 
                 Cargo 
                 size (n) 
                 Route 
                 content (mg/kg) 
                 Dex 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 (1) TSS 
                 n/a 
                 n/a 
                 n/a 
                 3 
                 IV- 
                 n/a 
                 no 
               
               
                   
                   
                   
                   
                   
                 bolus 
               
               
                 (2) LNP1021 
                 50/38/9/3 
                 6 
                 Cas9 mRNA 
                 3 
                 IV- 
                 1 
                 no 
               
               
                 G502 
                   
                   
                 (SEQ ID NO: 
                   
                 bolus 
               
               
                   
                   
                   
                 1); G000502 
               
               
                 (3) LNP1021 
                 50/38/9/4 
                 6 
                 Cas9 mRNA 
                 I 
                 IV- 
                 1 
                 yes 
               
               
                 G502 
                   
                   
                 (SEQ ID NO: 
                   
                 bolus 
               
               
                   
                   
                   
                 1); G000502 
               
               
                 (4) LNP1022 
                 55/33/9/3 
                 6 
                 Cas9 mRNA 
                 3 
                 IV- 
                 1 
                 no 
               
               
                 G502 
                   
                   
                 (SEQ ID NO: 
                   
                 bolus 
               
               
                   
                   
                   
                 1); G000502 
               
               
                 (5) LNP1023 
                 45/43/9/3 
                 4.5 
                 Cas9 mRNA 
                 3 
                 IV- 
                 3 
                 no 
               
               
                 G502 
                   
                   
                 (SEQ ID NO: 
                   
                 bolus 
               
               
                   
                   
                   
                 1); G000502 
               
               
                 (6) LNP1024 
                 50/38/9/3 
                 6 
                 Cas9 mRNA 
                 3 
                 IV- 
                 1 
                 no 
               
               
                 G509 
                   
                   
                 (SEQ ID NO: 
                   
                 bolus 
               
               
                   
                   
                   
                 1); G000509 
               
               
                 (7) LNP1024 
                 50/38/9/4 
                 6 
                 Cas9 mRNA 
                 1 
                 IV- 
                 1 
                 yes 
               
               
                 G509 
                   
                   
                 (SEQ ID NO: 
                   
                 bolus 
               
               
                   
                   
                   
                 1); G000509 
               
               
                 (8) LNP1025 
                 55/33/9/3 
                 6 
                 CasmRNA 
                 3 
                 IV- 
                 1 
                 no 
               
               
                 G509 
                   
                   
                 (SEQ ID NO: 
                   
                 bolus 
               
               
                   
                   
                   
                 1); G000509 
               
               
                 (9) LNP1021 
                 50/38/9/3 
                 6 
                 Cas9 mRNA 
                 1 
                 IV- 
                 3 
                 no 
               
               
                 G502 
                   
                   
                 (SEQ ID NO: 
                   
                 bolus 
               
               
                   
                   
                   
                 1); G000502 
               
               
                 (10) LNP1022 
                 50/38/9/3 
                 6 
                 Cas9 mRNA 
                 1 
                 IV- 
                 3 
                 no 
               
               
                 G502 
                   
                   
                 (SEQ ID NO: 
                   
                 bolus 
               
               
                   
                   
                   
                 1); G000502 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 IL-6 measurements from Study 1. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Treatment Group 
                 Pre-Bleed 
                 6 hour 
                 24 hour 
               
               
                   
                   
               
               
                   
                 (1) TSS (vehicle) 
                 5.71 ± 2.70 
                  29.1 ± 20.37 
                 7.05 ± 3.49 
               
               
                   
                 (2) LNP699 G502 
                 9.73 ± 8.34 
                 1296.41 ± 664.71 
                 5.43 ± 7.68 
               
               
                   
                 (3) LNP688 G506 
                 16.83 ± 4.08  
                  1749.47 ± 1727.22 
                 38.57 ± 39.39 
               
               
                   
                 (4) LNP689 G509 
                 18.11 ± 11.51 
                 1353.49 ± 766.66 
                 32.42 ± 18.40 
               
               
                   
                 (5) LNP690 G510 
                 13.95 ± 1.85  
                    11838 ± 17161.74 
                 90.07 ± 96.02 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 MCP-1 measurements from Study 1. 
               
            
           
           
               
               
               
               
            
               
                 Treatment Group 
                 Pre-Bleed 
                 6 hour 
                 24 hour 
               
               
                   
               
               
                 (1) TSS (vehicle) 
                 810.49 ± 178.27 
                 1351.16 ± 397.31  
                 745.25 ± 56.49 
               
               
                 (2) LNP699 G502 
                 842.31 ± 350.65 
                 19298.49 ± 11981.14 
                 2092.89 ± 171.21 
               
               
                 (3) LNP688 G506 
                 1190.79 ± 383.64  
                 13500.17 ± 12691.60 
                 1414.71 ± 422.43 
               
               
                 (4) LNP689 G509 
                 838.63 ± 284.42 
                 14427.7 ± 8715.48 
                   1590 ± 813.23 
               
               
                 (5) LNP690 G510 
                 785.32 ± 108.97 
                 52557.24 ± 48034.68 
                 6319.77 ± 983.37 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Complement C3a measurements from Study 1. 
               
            
           
           
               
               
               
               
            
               
                 Treatment Group 
                 Pre-Bleed 
                 6 hour 
                 day 7 
               
               
                   
               
               
                 (1) TSS (vehicle) 
                  23.9 ± 11.95 
                  25.51 ± 14.79 
                 30.67 ± 18.36 
               
               
                 (2) LNP699 G502 
                 32.36 ± 11.29 
                  94.33 ± 58.45 
                 38.50 ± 12.69 
               
               
                 (3) LNP688 G506 
                 22.30 ± 1.73  
                 127.00 ± 22.34 
                 37.80 ± 6.86  
               
               
                 (4) LNP689 G509 
                 35.83 ± 21.94 
                 174.00 ± 44.51 
                 50.83 ± 21.92 
               
               
                 (5) LNP690 G510 
                 36.30 ± 8.21  
                 163.00 ± 40.60 
                 42.50 ± 12.44 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Complement bb measurements from Study 1. 
               
            
           
           
               
               
               
               
               
            
               
                 Treatment Group 
                 04-bb 
                 Pre-Bleed 
                 6 hour 
                 day 7 
               
               
                   
               
               
                 (1) TSS (vehicle) 
                 Control 
                 1.53 ± 0.19 
                  3.37 ± 2.13 
                 1.43 ± 0.71 
               
               
                 (2) LNP699 G502 
                 G502 
                 1.45 ± 0.39 
                  9.01 ± 5.28 
                 1.57 ± 0.54 
               
               
                 (3) LNP688 G506 
                 G506 
                 1.45 ± 0.78 
                 11.78 ± 2.33 
                 1.78 ± 0.84 
               
               
                 (4) LNP689 G509 
                 G509 
                 1.95 ± 0.99 
                 15.73 ± 2.23 
                 2.83 ± 0.88 
               
               
                 (5) LNP690 G510 
                 G510 
                 2.12 ± 0.44 
                 13.57 ± 1.23 
                 2.21 ± 0.72 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 IL-6 measurements from Study 2. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Treatment group 
                 Pre-Bleed 
                 90 min 
                 6 hour 
                 24 hour 
                 Day 7 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 (1) TSS (vehicle) 
                 1.77 
                 11.46 
                 4.2 
                 2.76 
                 3.01 
               
               
                 (2) TSS (vehicle) 
                 5.23 
                 18.11 
                 20.36 
                 13.2 
                 6.36 
               
               
                 (3) LNP898 G502 
                 2.02 
                 1305.75 
                 1138.22 
                 383.32 
                 16.02 
               
               
                 (4) LNP898 G502 
                 2.34 
                 37.19 
                 91.59 
                 14.11 
                 3.07 
               
               
                 (5) LNP897 G502 
                 2.1 
                 55.79 
                 6.89 
                 2.26 
                 2.01 
               
               
                 (6) LNP897 G502 
                 6.8 
                 10.1 
                 44.72 
                 5.4 
                 2.01 
               
               
                 (7) LNP897 G502 
                 1.97 
                 44.87 
                 32.61 
                 2.97 
                 1.11 
               
               
                 (8) LNP897 G502 
                 3.14 
                 37.68 
                 73.41 
                 8.58 
                 2.22 
               
               
                 (9) LNP916 GFP 
                 1.6 
                 BLQ 
                 95.32 
                 27.58 
                 BLQ 
               
               
                 (10) LNP916 GFP 
                 2.43 
                 BLQ 
                 883.01 
                 66.71 
                 BLQ 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 MCP-1 measurements from Study 2. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Treatment group 
                 Pre-Bleed 
                 90 min 
                 6 hour 
                 24 hour 
                 Day 7 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 (1) TSS (vehicle) 
                 312.12 
                 197.24 
                 145.36 
                 177.02 
                 403.82 
               
               
                 (2) TSS (vehicle) 
                 232.44 
                 175.08 
                 187.72 
                 136.64 
                 325.69 
               
               
                 (3) LNP898 G502 
                 249.1 
                 2183.5 
                 1814.64 
                 1887.41 
                 372.38 
               
               
                 (4) LNP898 G502 
                 349.51 
                 430.49 
                 5635.55 
                 953.05 
                 236.6 
               
               
                 (5) LNP897 G502 
                 492.3 
                 989.98 
                 409.08 
                 302.97 
                 506.82 
               
               
                 (6) LNP897 G502 
                 283.79 
                 225.1 
                 1141.08 
                 484.59 
                 259.46 
               
               
                 (7) LNP897 G502 
                 223.16 
                 349.79 
                 398.57 
                 172.67 
                 287.09 
               
               
                 (8) LNP897 G502 
                 584.42 
                 853.51 
                 3880.81 
                 1588.46 
                 692.99 
               
               
                 (9) LNP916 GFP 
                 325.84 
                 BLQ 
                 1189.97 
                 2279.82 
                 BLQ 
               
               
                 (10) LNP916 GFP 
                 175.47 
                 BLQ 
                 3284.16 
                 2023.53 
                 BLQ 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Complement C3a measurements from Study 2. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Treatment group 
                 Pre-Bleed 
                 90 min 
                 6 hour 
                 24 hour 
                 Day 7 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 (1) TSS (vehicle) 
                 0.087 
                 0.096 
                 0.048 
                 0.033 
                 0.038 
               
               
                 (2) TSS (vehicle) 
                 0.369 
                 0.311 
                 0.146 
                 0.1 
                 0.106 
               
               
                 (3) LNP898 G502 
                 0.087 
                 0.953 
                 0.647 
                 0.277 
                 0.065 
               
               
                 (4) LNP898 G502 
                 0.099 
                 0.262 
                 0.123 
                 0.049 
                 0.044 
               
               
                 (5) LNP897 G502 
                 0.067 
                 0.479 
                 0.209 
                 0.036 
                 0.036 
               
               
                 (6) LNP897 G502 
                 0.141 
                 0.433 
                 0.34 
                 0.11 
                 0.074 
               
               
                 (7) LNP897 G502 
                 0.1 
                 0.345 
                 0.396 
                 0.096 
                 0.127 
               
               
                 (8) LNP897 G502 
                 0.261 
                 0.458 
                 0.409 
                 0.244 
                 0.313 
               
               
                 (9) LNP916 GFP 
                 0.149 
                 BLQ 
                 0.714 
                 0.382 
                 BLQ 
               
               
                 (10) LNP916 GFP 
                 0.117 
                 BLQ 
                 0.752 
                 0.723 
                 BLQ 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Complement bb measurements from Study 2. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Treatment group 
                 Pre-Bleed 
                 90 min 
                 6 hour 
                 24 hour 
                 Day 7 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 (1) TSS (vehicle) 
                 0.087 
                 0.096 
                 0.048 
                 0.033 
                 0.038 
               
               
                 (2) TSS (vehicle) 
                 0.369 
                 0.311 
                 0.146 
                 0.1 
                 0.106 
               
               
                 (3) LNP898 G502 
                 0.087 
                 0.953 
                 0.647 
                 0.277 
                 0.065 
               
               
                 (4) LNP898 G502 
                 0.099 
                 0.262 
                 0.123 
                 0.049 
                 0.044 
               
               
                 (5) LNP897 G502 
                 0.067 
                 0.479 
                 0.209 
                 0.036 
                 0.036 
               
               
                 (6) LNP897 G502 
                 0.141 
                 0.433 
                 0.34 
                 0.11 
                 0.074 
               
               
                 (7) LNP897 G502 
                 0.1 
                 0.345 
                 0.396 
                 0.096 
                 0.127 
               
               
                 (8) LNP897 G502 
                 0.261 
                 0.458 
                 0.409 
                 0.244 
                 0.313 
               
               
                 (9) LNP916 GFP 
                 0.149 
                 BLQ 
                 0.714 
                 0.382 
                 BLQ 
               
               
                 (10) LNP916 GFP 
                 0.117 
                 BLQ 
                 0.752 
                 0.723 
                 BLQ 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 IL-6 measurements from Study 3. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Treatment 
                   
                   
                   
                   
                   
               
               
                 group 
                 Pre-bleed 
                 90 min 
                 6 hour 
                 24 hour 
                 Day 7 
               
               
                   
               
               
                 (1) TSS 
                 1.89 ± 0.97 
                 2.56 ± 1.41 
                 0.90 ± 0.71 
                 BLQ 
                 0.08 
               
               
                 (2) LNP1021 
                  210 ± 0.35 
                 7.44 ± 5.16 
                 6.94 ± 8.45 
                 1.07 ± 1.11 
                 1.76 ± 0.98 
               
               
                 G502 
               
               
                 (3) LNP1021 
                 0.79 
                 2.96 
                  4.25 
                 0.67 
                 0.27 
               
               
                 G502 
               
               
                 (4) LNP1022 
                 1.54 ± 1.32 
                 20.42 ± 31.60 
                 13.94 ± 10.10 
                 0.98 ± 0.41 
                 2.04 ± 0.65 
               
               
                 G502 
               
               
                 (5) LNP1023 
                 2.92 ± 1.68 
                 6.28 ± 7.18 
                 6.06 ± 2.31 
                 3.62 ± 4.68 
                 2.00 ± 1.21 
               
               
                 G502 
               
               
                 (6) LNP1024 
                 1.43 ± 0.62 
                 2.64 ± 1.92 
                  7.72 ± 11.96 
                 0.45 ± 0.19 
                 0.88 ± 0.79 
               
               
                 G509 
               
               
                 (7) LNP1024 
                 1.35 ± 0.74 
                 2.64 ± 2.35 
                 1.71 ± 0.41 
                 0.36 ± 0.58 
                 0.51 ± 0.32 
               
               
                 G509 
               
               
                 (8) LNP1025 
                 1.64 
                 2.68 
                 25.65 
                 0.58 
                 2.00 
               
               
                 G509 
               
               
                 (9) LNP1021 
                 0.56 
                 6.15 
                 28.80 
                 0.85 
                 0.61 
               
               
                 G502 
               
               
                 (10) LNP1022 
                 1.76 
                 8.66 
                 2907.86  
                 11.26  
                 1.72 
               
               
                 G502 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 MCP-1 measurements from Study 2. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Treatment group 
                 Pre-bleed 
                 90 min 
                 6 hour 
                 24 hour 
                 Day 7 
               
               
                   
               
               
                 (1) TSS 
                 204.01 ± 46.39 
                 197.62 ± 19.54  
                 310.84 ± 45.87  
                 179.07 ± 20.77  
                 234.61 ± 71.79 
               
               
                 (2) LNP1021 
                 303.67 ± 36.37 
                 337.63 ± 195.18 
                 755.20 ± 581.45 
                 339.75 ± 206.20 
                 214.82 ± 40.81 
               
               
                 G502 
               
               
                 (3) LNP1021 
                 229.30 
                 358.10 
                 3182.00 
                 413.56 
                 178.30 
               
               
                 G502 
               
               
                 (4) LNP1022 
                  393.63 ± 187.81 
                 467.72 ± 221.61 
                 1852.94 ± 2199.66 
                 497.12 ± 412.30 
                 382.19 ± 67.27 
               
               
                 G502 
               
               
                 (5) LNP1023 
                 213.72 ± 8.85  
                 196.18 ± 62.81  
                 1722.18 ± 1413.90 
                 197.83 ± 74.01 
                 156.16 ± 18.87 
               
               
                 G502 
               
               
                 (6) LNP1024 
                 237.76 ± 96.36 
                 210.37 ± 95.17  
                 468.53 ± 250.42 
                 22.32 ± 69.06 
                 141.20 ± 71.90 
               
               
                 G509 
               
               
                 (7) LNP1024 
                 207.36 
                 183.07 
                 1885.66 
                 235.70 
                 163.11 
               
               
                 G509 
               
               
                 (8) LNP1025 
                  259.57 ± 112.98 
                 299.21 ± 304.89 
                 1193.10 ± 974.04  
                 258.82 ± 88.53  
                 219.86 ± 219.86 
               
               
                 G509 
               
               
                 (9) LNP1021 
                 199.29 
                 286.04 
                 2001.23 
                 197.57 
                 196.44 
               
               
                 G502 
               
               
                 (10) LNP1022 
                 305.81 
                 970.65 
                 7039.06 
                 8379.05  
                 203.47 
               
               
                 G502 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 Complement C3a measurements from Study 3. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Treatment group 
                 Pre-bleed 
                 90 min 
                 6 hour 
                 24 hour 
                 Day 7 
               
               
                   
               
               
                 (1) TSS 
                 42.47 ± 10.30 
                 55.40 ± 13.58 
                 29.30 ± 14.46 
                 41.70 ± 23.65 
                  27.43 ± 12.43 
               
               
                 (2) LNP1021 G502 
                 34.37 ± 0.50  
                 86.50 ± 3.66  
                 90.07 ± 4.85  
                 56.60 ± 2.25  
                 32.53 ± 0.93 
               
               
                 (3) LNP1021 G502 
                 34.30 
                 128.00  
                  93.30 
                 33.40 
                 28.20 
               
               
                 (4) LNP1022 G502 
                 41.55 ± 13.51 
                 151.37 ± 109.98 
                 82.00 ± 31.82 
                 45.57 ± 18.58 
                 32.77 ± 6.45 
               
               
                 (5) LNP1023 G502 
                 31.67 ± 3.19  
                 74.40 ± 22.08 
                 74.13 ± 48.61 
                 33.83 ± 9.75  
                 27.70 ± 8.05 
               
               
                 (6) LNP1024 G509 
                 56.60 ± 25.61 
                 100.37 ± 77.95  
                 74.73 ± 70.15 
                 55.20 ± 48.34 
                  49.97 ± 39.94 
               
               
                 (7) LNP1024 G509 
                 33.80 
                 33.90 
                  33.70 
                 26.10 
                 20.90 
               
               
                 (8) LNP1025 G509 
                 39.90 ± 13.01 
                 75.73 ± 1.38  
                 46.13 ± 30.56 
                 25.00 ± 3.80  
                 23.90 ± 7.18 
               
               
                 (9) LNP1021 G502 
                 34   
                 85.70 
                 133.00 
                 62.00 
                 25.50 
               
               
                 (10) LNP1022 G502 
                 29.8  
                 68.10 
                 113.00 
                 71.70 
                 23.30 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 Complement bb measurements from Study 3. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Treatment group 
                 Pre-bleed 
                 90 min 
                 6 hour 
                 24 hour 
                 Day 7 
               
               
                   
               
               
                 (1) TSS 
                 1.46 ± 0.70 
                 2.18 ± 0.78 
                 1.96 ± 0.64 
                 0.945 ± 0.15  
                 1.34 ± 0.50 
               
               
                 (2) LNP1021 G502 
                 1.77 ± 0.60 
                 6.51 ± 3.66 
                 11.00 ± 4.85  
                 3.59 ± 2.25 
                 2.07 ± 0.93 
               
               
                 (3) LNP1021 G502 
                 1.24 
                 2.90 
                 11.50 
                 2.97 
                 1.24 
               
               
                 (4) LNP1022 G502 
                 1.52 ± 0.34 
                 5.67 ± 2.28 
                 10.2 ± 3.36 
                 3.66 ± 1.68 
                 1.84 ± 0.24 
               
               
                 (5) LNP1023 G502 
                 1.65 ± 0.94 
                 4.4 ± 1     
                 7.68 ± 4.67 
                 2.64 ± 1.18 
                 2.08 ± 1.32 
               
               
                 (6) LNP1024 G509 
                 1.61 ± 0.13 
                 4.52 ± 1.81 
                 4.50 ± 3.22 
                 1.63 ± 0.84 
                 1.63 ± 0.32 
               
               
                 (7) LNP1024 G509 
                 0.96 
                 2.99 
                  2.64 
                 1.13 
                 1.07 
               
               
                 (8) LNP1025 G509 
                 1.37 ± 0.17 
                  4.9 ± 4.51 
                 3.79 ± 3.84 
                 1.66 ± 1.43 
                 1.35 ± 0.44 
               
               
                 (9) LNP1021 G502 
                 1.41 
                 5.67 
                 11.50 
                 4.64 
                 1.38 
               
               
                 (10) LNP1022 G502 
                 1.28 
                 5.22 
                 14.10 
                 5.64 
                 1.87 
               
               
                   
               
            
           
         
       
     
     Example 6 
     PEG Lipid Screen 
     In another study, alternative PEG lipids were compared in LNP formulations comprising 2 mol-% or 3 mol-% of the PEG lipid. 
     Three PEG lipids were used in the study: Lipid 1 (DMG-PEG2k; Nof), is depicted as: 
     
       
         
         
             
             
         
       
     
     Lipid 2, synthesized as described in Heyes, et al.,  J. Controlled Release,  107 (2005), pp, 278-279 (See “Synthesis of PEG2000-C-DMA”), can be depicted as: 
     
       
         
         
             
             
         
       
     
     and Lipid 3, disclosed in WO2016/010840 (see compound S027, paragraphs [00240] to [00244]) and WO2011/076807, can be depicted as: 
     
       
         
         
             
             
         
       
     
     Lipid A was formulated with each PEG lipid at 2 mol-% and 3 mol-%. The lipid nanoparticle components were dissolved in 100% ethanol with the lipid component molar ratios set forth above. In brief, the RNA cargos were prepared in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL. The LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 4.5 with the ratio of mRNA to gRNA at 1:1 by weight. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 27 
               
               
                   
               
               
                   
                 LNP 
                 LNP 
                 LNP 
                 LNP 
                 LNP 
                 LNP 
               
               
                 LNP # 
                 784 
                 785 
                 786 
                 787 
                 788 
                 789 
               
               
                   
               
             
            
               
                 CL/chol./DSPC/PEG 
                 45/44/9/2 
                 45/43/9/3 
                 45/44/9/2 
                 45/43/9/3 
                 45/44/9/2 
                 45/43/9/3 
               
               
                 (theoretical mol-%) 
               
               
                 mRNA 
                 Cas9 
                 Cas9 
                 Cas9 
                 Cas9 
                 Cas9 
                 Cas9 
               
               
                   
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
               
               
                   
                 NO: 48 
                 NO: 48 
                 NO: 48 
                 NO: 48 
                 NO: 48 
                 NO: 48 
               
               
                 gRNA 
                 G282 
                 G282 
                 G282 
                 G282, 
                 G282, 
                 G282 
               
               
                   
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
                 SEQ ID 
               
               
                   
                 NO: 42 
                 NO: 42 
                 NO: 42 
                 NO: 42 
                 NO: 42 
                 NO: 42 
               
               
                 PEG Type 
                 Lipid 1 
                 Lipid 1 
                 Lipid 2 
                 Lipid 2 
                 Lipid 3 
                 Lipid 3 
               
               
                 N/P 
                 4.5 
                 4.5 
                 4.5 
                 4.5 
                 4.5 
                 4.5 
               
               
                   
               
            
           
         
       
     
     Cas9 mRNA, sg282, and LNPs were prepared as described in Example 1. 
     LNP compositions with Lipid 1, Lipid 2, or Lipid 3 were were administered to female CD-1 mice and assessed as described in Example 1 at 1 mg/kg and 0.5 mg/kg of the body weight. Cohorts of mice were measured for liver editing by Next-Generation Sequencing (NGS) and serum TTR levels according to the methods of Example 1. 
       FIG.  6 A  and  FIG.  6 B  compare serum TTR levels between PEG lipid formulations.  FIG.  6 A  shows serum TTR in μg/mL, and  FIG.  6 B  shows the data as a percent knockdown (% TSS).  FIG.  6 C  shows percent editing achieved in the liver. The data indicate that LNP compositions with each of the tested PEG lipids tested potency at 2 mol-% and 3 mol-%, with Lipid 1 consistently performing slightly better than Lipid 2 and Lipid 3. 
     Example 7 
     Lipid A Analogs 
     A number of structural analogs of Lipid A were synthesized and tested in the LNP compositions described herein. 
     Synthesis: Lipid A is made by reacting 4,4-bis(octyloxy)butanoic acid (“Intermediate 13b” in Example 13 of WO2015/095340) with (9Z,12Z)-3-hydroxy-2-(hydroxymethyl)propyl octadeca-9,12-dienoate (“Intermediate 13c”), prior to addition of the head group by reacting the product of Intermediate 13b and Intermediate 13c with 3-diethylamino-1-propanol. (See pp. 84-86 of WO2015/095340.) 
     Intermediate 13b from WO2015/095340 (4,4 bis(octyloxy)butanoic acid) was synthesized via 4,4-bis(octyloxy)butanenitrile as follows: 
     Intermediate 13a: 4,4-bis(octyloxy)butanenitrile 
     
       
         
         
             
             
         
       
     
     To a mixture of 4,4-diethoxybutanenitrile (9,4 g, 60 mmol) and octan-1-ol (23.1 g, 178 mmol) was added pyridinium p-toluenesulfonate (748 mg, 3.0 mmol) at rt. The mixture was warmed to 1.05° C. and stirred for 18 hours with the reaction vessel open to air and not fitted with a refluxing condenser. The reaction mixture was then cooled to room temperature and purified on silica gel (0-5% gradient of ethyl acetate in hexanes) to provide 10.1 g (31.0 mmol) of Intermediate 13a as a clear oil.  1 H MAR (400 MHz, CDCl 3 ) δ 4.55 (t, J=5.3 Hz, 1H), 3.60 (dt, J=9.2, 6.6 Hz, 2H), 3.43 (dt, J=9.2, 6.6 Hz, 2H), 2.42 (t, J=7.4 Hz, 2H), 1.94 (td, J=7.4, 5.3 Hz, 2H), 1.63-1.50 (m, 4H), 1.38-1.19 (m, 20H), 0.93-0.82 (m, 6H) ppm. 
     Next, to a solution of Intermediate 13a (8.42 g, 31 mmol) in ethanol (30 mL) was added 31 mL of aqueous potassium hydroxide (2.5 M, 30.9 mL, 77.3 mmol) at room temperature. Upon fitting the vessel with a reflux condenser, the mixture was heated to 110° C. and stirred for 24 hours. The mixture was then cooled to room temperature, acidified with aqueous hydrochloride acid (1N) to pH 5, and extracted into hexanes three times. The combined organic extracts were washed with water (twice) and brine, dried over anhydrous magnesium sulfate, and concentrated in vacuo to afford 8.15 g (23.6 mmol) of Intermediate 13b as a clear oil, which was used without further purification.  1 H NMR (400 MHz, CDCl 3 ) δ 4.50 (t, J=5.5 Hz, 1H), 3.57 (dt, J=9.4, 6.7 Hz, 2H), 3.41 (dt, J=9.3, 6.7 Hz, 2H), 2.40 (t, J=7.4 Hz, 2H), 1.92 (td, J=7.4, 5.3 Hz, 2H), 1.56 (m, 4H), 1.37-1.21 (m, 20H), 0.92-0.83 (m, 6H) ppm (structure below). 
     Intermediate 13b 
     
       
         
         
             
             
         
       
     
     Using the methods described above, the C(5, 6, 7, 9, and 10)-acetal acid intermediates, called Intermediates B3-F3 and depicted below, were prepared using the appropriate alkan-1-ol reagents. 
     Intermediate B3 4,4-bis(pentyloxy)butanoic Acid 
     
       
         
         
             
             
         
       
     
       1 H NMR (400 MHz, CDCl 3 ) δ 4.52 (t, J=5.5 Hz, 1H), 3.58 (dt, J=9.3, 6.6 Hz, 2H), 3.41 (dt, J=9.3, 6.7 Hz, 2H), 2.45 (t, J=7.4 Hz, 2H), 1.94 (m, 2H), 1.57 (m, 4H), 1.32 (m, J=3.7 Hz, 8H), 0.95-0.83 (m, 6H) ppm. 
     Intermediate C3: 4,4-bis(hexyloxy)butanoic Acid 
     
       
         
         
             
             
         
       
     
       1 H NMR (400 MHz, CDCl 3 ) δ 4.44 (t, J=5,6 Hz, 1H), 3.49 (dt, J=9.3, 6.9 Hz, 2H), 3.39 (dt, J=9.3, 6.8 Hz, 2H), 2.12 (t, J=7.6 Hz, 2H), 1.79 (q, J=7.0 Hz, 2H), 1.54 (m, 4H), 1.29 (m, 12H), 0.94-0.82 (m, 6H) ppm. 
     Intermediate D3: 4,4-bis(heptyloxy)butanoic Acid 
     
       
         
         
             
             
         
       
     
       1 H NMR (400 MHz, CDCl 3 ) δ 8.85 (br s, 1H), 4.46 (t, J=5.6 Hz, 1H), 3.52 (dt, J=9.4, 6.8 Hz, 2H), 3.39 (dt, J=9.3, 6.8 Hz, 2H), 2.26 (t, J=7.6 Hz, 2H), 1.85 (q, J=7.0 Hz, 2H), 1.53 (m, 4H), 1.29 (m, 16H), 0.94-0.80 (m, 6H) ppm. 
     Intermediate E3: 4,4-bis(nonyloxy)butanoic Acid 
     
       
         
         
             
             
         
       
     
       1 H NMR (400 MHz, CDCl 3 ) δ 5.32 (br s, 1H), 4.44 (t, J=5.6 Hz, 1H), 3.49 (dt, J=9.3, 6.9 Hz, 2H), 3.38 (dt, J=9.4, 6.9 Hz, 2H), 2.10 (t, J=7.6 Hz, 2H), 1.78 (q, J=7.0 Hz, 2H), 1.53 (m, 4H), 1.27 (m, 24H), 0.88 (t, J=6.6 Hz, 6H) ppm. 
     Intermediate F3: 4,4-bis(decyloxy)butanoic Acid 
     
       
         
         
             
             
         
       
     
       1 H NMR, (400 MHz, CDCl 3 ) δ 4.48 (t, J=5.5 Hz, 1H), 3.55 (m, 2H), 3.42 (m, 2H), 2.29 (dd, J=10.8, 7.5 Hz, 2H), 1.90-1.82 (m, 2H), 1.55 (m, 4H), 1.27 (m, 28H), 0.88 (t, J=6.7 Hz, 6H) ppm. 
     Acetal analogs of Lipid A (C(8)) were synthesized by reacting the C(5, 6, 7, 9, or 10)-acetal acid intermediates (B3-F3) with Intermediate 13c, prior to reacting the product of that step with 3-diethylamino-1-propanol. (See pp 84-86 of WO2015/095340.) Each analog was synthesized and characterized by  1 H NMR (data not shown). 
     The C7, C9, and C10 analogs were formulated at 45 mol-% Lipid A Analog, 2 mol-% DMG-PEG2k, 9 mol-% DSPC, and 44 mol-% cholesterol, with an N:P ratio of 4.5. Each analog was also formulated at 55 mol-% Lipid A Analog, 2.5 mol-% DMG-PEG2k, 9 mol-% DSPC, and 38.5 mol-% cholesterol, with an NT ratio of 6. The lipid nanoparticle components were dissolved in 100% ethanol with the lipid component molar ratios set forth above. The RNA cargos were prepared in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL. 
     The RNA cargo included Cas9 mRNA comprising SEQ ID NO:43 and sg282, prepared as described above. The LNPs were formed as described in Example 1. 
     An expanded panel of acetal analogs, including LNP compositions comprising the C(5) and C(6) Lipid A analogs were tested alongside the prior panel. The two new analogs were formulated at 55 mol-% Lipid A Analog, 2.5 mol-% DMG-PEG2k, 9 mol-% DPSC, and 33,5 mol-% cholesterol, with an N/P ratio of 6, as described above. Analysis indicated that sizes for all LNPs is below 120 nm, PDT is below 0.2 and %-encapsulated RNA is higher than 80%. Analytical results for the formulations are in Table 28, below. 
     
       
         
           
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 28 
               
               
                   
               
               
                 LNP 
                 Lipid A 
                 Analog 
                   
                   
                 RNA Conc 
                   
                 Z-avg. 
                   
                 Number mean 
               
               
                 ID 
                 Analogs 
                 mol-% 
                 PEG % 
                 N/P 
                 (mg/mL) 
                 % EE 
                 (nm) 
                 PDI 
                 (nm) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 LNP 
                 C5 analog 
                 55 
                 2.5 
                 6 
                 0.063 
                 88 
                 118.8 
                 0.103 
                 88.17 
               
               
                 1122 
                 (LP000030-001) 
               
               
                 LNP 
                 C6 analog 
                 55 
                 2.5 
                 6 
                 0.067 
                 95 
                 107.6 
                 0.038 
                 88.1 
               
               
                 1123 
                 (LP000031-001) 
               
               
                 LNP 
                 C7 analog 
                 55 
                 2.5 
                 6 
                 0.068 
                 98 
                 100 
                 0.012 
                 81.55 
               
               
                 1004 
                 (LP000020-001) 
               
               
                 LNP 
                 LP000001-011 
                 55 
                 2.5 
                 6 
                 0.067 
                 98 
                 95.06 
                 0.01 
                 78.95 
               
               
                 1002 
               
               
                 LNP 
                 C9 analog 
                 55 
                 2.5 
                 6 
                 0.067 
                 97 
                 95.43 
                 0.022 
                 80.35 
               
               
                 1006 
                 (LP000021-001) 
               
               
                 LNP 
                 C10 analog 
                 55 
                 2.5 
                 6 
                 0.069 
                 95 
                 103.9 
                 0.008 
                 86.79 
               
               
                 1008 
                 (LP000022-001) 
               
               
                   
               
            
           
         
       
     
     The analogs were assessed for pKa using 6-(p-toluidino)-6-napthalene sulfonic acid (“TNS”) dissolved in water. In this assay 0.1 M phosphate buffer was prepared at different pH values ranging from 4.5 to 10.5. Each analog was individually prepared in 100% ethanol, The lipid and TNS were then added in individual pH buffer and transferred to a plate to analyze at 321-488 nm wavelength on the SpectraMax plate reader. Values were plotted to generate pKa, log IC 50  is used as pKa. 
     Female CD-1 mice were dosed as described in Example 1 with 0.3 mg/kg ( FIG.  7 A - FIG.  7 E ), or with 0.1 mg per kg ( FIG.  7 F - FIG.  7 G ), In brief, CD-1 female mice from Charles River Laboratories, n=5 per group, were administered the LNP compositions at varying doses. At necropsy (7 days post dose), serum was collected for TTR analysis and liver was collected for editing analysis. Serum TTR and percent editing assays were performed as described in Example 1. The serum TTR levels and liver editing from  FIG.  7 A - FIG.  7 E  indicate that all the analogs performed comparably to Lipid A at 0.3 milligrams per kilogram body weight.  FIG.  7 F - FIG.  7 G  indicate that while Lipid A had the highest potency, the newly synthesized analogs all have suitable TTR knockdown and liver editing. 
     Example 8 
     Dose Response Curve—Primary Cyno Hepatocytes 
     Primary liver hepatocytes. Primary cynomolgus liver hepatocytes (PCH) (Gibco) were thawed and resuspended in hepatocyte thawing medium with supplements (Gibco, Cat. CM7000) followed by centrifugation at 80 g for 4 minutes. The supernatant was discarded and the pelleted cells resuspended in hepatocyte plating medium plus supplement pack (Invitrogen, Cat. A1217601 and CM3000). Cells were counted and plated on Bio-coat collagen I coated 96-well plates (ThermoFisher, Cat. 877272) at a density of 50,000 cells/well. Plated cells were allowed to settle and adhere for 24 hours in a tissue culture incubator (37° C. and 5% CO 2  atmosphere) prior to LNP administration. After incubation cells were checked for monolayer formation and media was replaced with hepatocyte culture medium with serum-free supplement pack (Invitroven, Cat. A1217601 and CM4000). 
     LNP formulations for this study (LNP1021, LNP1022, LNP1023, LNP1024, LNP1025, and LNP897) were prepared as described above. 
     Various doses of lipid nanoparticle formulations containing modified sgRNAs were tested on primary cyno hepatocytes to generate a dose response curve. After plating and 24 hours in culture, LNPs were incubated in hepatocyte maintenance media containing 6% cyno serum at 37° C. for 5 minutes. Post-incubation the LNPs were added onto the primary cyno hepatocytes in an 8 point 2-fold dose response curve starting at 100 ng mRNA. The cells were lysed 72 hours post treatment for NGS analysis as described in Example 1. Percent editing was determined for various LNP compositions and the data are graphed in  FIG.  8 A . The % editing with Cas9 mRNA (SEQ ID NO 48) and U-depleted Cas9 mRNA (SEQ I NO:43) is presented in  FIG.  8 B . LNP compositions are described in Table 2 (LNP 897) and Table 5 (LNP 1021, 1022, 1023, 1024, and 1025). 
     The results show a quantitative assay for comparative potency assessements, demonstrating both mRNA and LNP composition affect potency. 
     Example 9 
     RNA Cargo: mRNA and gRNA Coformulations 
     This study evaluated in vivo efficacy in mice of different ratios of gRNA to mRNA. CleanCap™ capped Cas9 mRNAs with the ORF of SEQ ID NO: 4, HSD 5′ UTR, human albumin 3′ UTR, a Kozak sequence, and a poly-A tail were made by IVT synthesis as indicated in Example 1 with N1-methylpseudouridine triphosphate in place of uridine triphosphate. 
     LNP formulations prepared from the mRNA described and sg282 (SEQ ID NO: 42; G282) as described in Example 2 with Lipid A, cholesterol, DSPC, and PEG2k-DMG in a 50:38:9:3 molar ratio and with an N:P ratio of 6. The gRNA:Cas9 mRNA weight ratios of the formulations were as shown in Table 29. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 29 
               
               
                   
               
               
                   
                 Guide:Cas9 
                   
                   
                 Z-Ave 
                   
                 Number 
               
               
                 LNP 
                 mRNA Ratio 
                 RNA Conc 
                 EE 
                 Size 
                 Partick 
                 Ave 
               
               
                 ID 
                 (w/w) 
                 (mg/mL) 
                 (%) 
                 (nm) 
                 PDI 
                 (nm) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1110 
                 8:1 
                 0.92 
                 99 
                 69.52 
                 0.022 
                 56.47 
               
               
                 1111 
                 4:1 
                 0.86 
                 97 
                 76.65 
                 0.065 
                 57.36 
               
               
                 1112 
                 2:1 
                 0.90 
                 99 
                 76.58 
                 0.036 
                 63.11 
               
               
                 1113 
                 1:1 
                 0.97 
                 99 
                 76.60 
                 0.071 
                 58.92 
               
               
                 1114 
                 1:2 
                 1.05 
                 99 
                 76.34 
                 0.018 
                 62.82 
               
               
                 1115 
                 1:4 
                 0.65 
                 99 
                 82.64 
                 0.018 
                 66.63 
               
               
                 1116 
                 1:8 
                 0.75 
                 100 
                 82.01 
                 0.039 
                 65.05 
               
               
                   
               
            
           
         
       
     
     For in vivo characterization, the above LNPs were administered to mice at 0.1 mg total RNA (mg guide RNA+mg mRNA) per kg (n=5 per group). At 7 to 9 days post-dose, animals were sacrificed, blood and the liver were collected, and serum TTR and liver editing were measured as described in Example 1. Serum TTR and liver editing results are shown in  FIGS.  9 A and  9 B . Negative control mice were dosed with TSS vehicle. 
     In addition, the above LNPs were administered to mice at a constant mRNA dose of 0.05 mg mRNA per kg (n=5 per group), while varying the gRNA dose from 0.06 mg per kg to 0.4 mg per kg. At 7 to 9 days post-dose, animals were sacrificed, blood and the liver were collected, and serum TTR and liver editing were measured. Serum TTR and liver editing results are shown in  FIG.  9 C  and  FIG.  9 D . Negative control mice were dosed with TSS vehicle. 
     Example 10 
     Neutral Lipids 
     To evaluate the in vivo efficacy of LNPs, LNP formulations were prepared with the mRNA of Example 2 and sg534 (SEQ ID NO: 72; G534), as described in Example 2. The lipid nanoparticle components were dissolved in 100% ethanol with the lipid component molar ratios set forth below. In brief, the RNA cargos were prepared in a buffer of 25 mM citrate and 100 mM NaCl at pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL. The LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6 with the ratio of gRNA to mRNA at 1:2 by weight. 
     LNP formulations were analyzed for average particle size, polydispersity (pdi), total RNA content and encapsulation efficiency of RNA as described in Example 1. Analysis of average particle size, polydispersity (PDI), total RNA content and encapsulation efficiency of RNA are shown in Table 30. Molar ratios of lipid are provided as amine lipid (Lipid A)/neutral lipid/helper lipid (cholesterol)/PEG lipid (PEG2k-DMG). The neutral lipid was DSP, DPPC, or absent, as specified. 
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 LNP compositions and data. (Molar ratios of lipid are provided as amine lipid 
               
               
                 (Lipid A)/neutral lipid/helper lipid (cholesterol)/PEG lipid (PEG2k-DMG).) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Sample 
                 Neutral 
                 Molar 
                 RNA Conc 
                   
                 Z-avg. 
                   
                 Number mean 
                 % Liver 
                 Serum TTR 
                 % TTR 
               
               
                 ID 
                 Lipid 
                 Ratios 
                 (mg/mL) 
                 % EE 
                 (am) 
                 PDI 
                 (nm) 
                 Editing 
                 (μg/mL) 
                 KD 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 TSS 
                   
                   
                   
                   
                   
                   
                   
                 0.0 
                 1248.9 
                   
               
               
                 control 
               
               
                 CO241 
                 — 
                 50.0/0.0/ 
                 1.46 
                 94 
                 75.64 
                 0.090 
                 54.2.1 
                 1.8 
                 1070.2 
                 14.3 
               
               
                   
                   
                 47.0/3.0 
               
               
                 CO242 
                 — 
                 59.0/0.0/ 
                 1.51 
                 94 
                 92.25 
                 0.019 
                 75.56 
                 12.0 
                 819.6 
                 34.4 
               
               
                   
                   
                 38.0/3.0 
               
               
                 CO243 
                 — 
                 54.5/0.0/ 
                 1.62 
                 94 
                 78.90 
                 0.052 
                 61.49 
                 3.3 
                 1260.5 
                 −0.9 
               
               
                   
                   
                 42.5/3.0 
               
               
                 CO244 
                 DSPC 
                 50.0/9.0/ 
                 1.50 
                 93 
                 101.3 
                 0.044 
                 80.73 
                 27.4 
                 741.0 
                 40.7 
               
               
                   
                   
                 39.0/2.0 
               
               
                 CO034 
                 DSPC 
                 50.0/9.0/ 
                 1.48 
                 97 
                 84.23 
                 0.040 
                 66.96 
                 34.2 
                 630.1 
                 49.6 
               
               
                   
                   
                 38.0/3.0 
               
               
                 CO245 
                 DSPC 
                 52.5/4.0/ 
                 1.55 
                 95 
                 81.88 
                 0.054 
                 64.54 
                 5.8 
                 846.6 
                 32.2 
               
               
                   
                   
                 42.5/3.0 
               
               
                 CO246 
                 DPPC 
                 50.0/9.0/ 
                 1.52 
                 96 
                 87.11 
                 0.040 
                 70.04 
                 35.9 
                 528.6 
                 57.7 
               
               
                   
                   
                 38.0/3.0 
               
               
                 CO247 
                 DPPC 
                 52.5/4.0/ 
                 1.54 
                 97 
                 83.67 
                 0.050 
                 66.43 
                 18.3 
                 726.6 
                 41.8 
               
               
                   
                   
                 42.5/3.0 
               
               
                   
               
            
           
         
       
     
     For in vivo characterization, the above LNPs were administered intravenously to female Sprague Dawley rats at 0.3 mg total RNA (guide RNA and mRNA) per kg bodyweight. There were five rats per group. At seven days post-dosing, animals were sacrificed, blood and the liver were collected, and serum TTR and liver editing were measured as described in Example 1. Negative control animals were dosed with TSS vehicle. Serum TTR and liver editing results are shown in  FIGS.  10 A and  10 B , and in Table 30 (above). 
     Brief Description of Disclosed Sequences 
       
     
       
         
           
               
               
             
               
                   
               
               
                 SEQ ID NO 
                 Description 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 1 
                 DNA coding sequence of Cas9 using the thymidine analog of the 
               
               
                   
                 minimal uridine codons listed in Table 3, with start and stop codons 
               
               
                 2 
                 DNA coding sequence of Cas9 using codons with generally high 
               
               
                   
                 expression in humans 
               
               
                 3 
                 Amino acid sequence of Cas9 with one nuclear localization signal 
               
               
                   
                 (1xNLS) as the C-terminal 7 amino acids 
               
               
                 4 
                 Cas9 mRNA ORF using minimal uridine codons as listed in Table 3, 
               
               
                   
                 with start and stop codons 
               
               
                 5 
                 Cas9 mRNA ORF using codons with generally high expression in 
               
               
                   
                 humans, with start and stop codons 
               
               
                 6 
                 Amino acid sequence of Cas9 nickase with 1xNLS as the C-terminal 
               
               
                   
                 7 amino acids 
               
               
                 7 
                 Cas9 nickase mRNA ORF encoding SEQ ID NO: 6 using minimal 
               
               
                   
                 uridine codons as listed in Table 3, with start and stop codons 
               
               
                 8 
                 Amino acid sequence of dCas9 with 1xNLS as the C-terminal 7 
               
               
                   
                 amino acids 
               
               
                 9 
                 dCas9 mRNA ORF encoding SEQ ID NO: 8 using minimal uridine 
               
               
                   
                 codons as listed in Table 3, with start and stop codons 
               
               
                 10 
                 Cas9 mRNA coding sequence using minimal uridine codons as 
               
               
                   
                 listed in Table 3 (no start or stop codons; suitable for inclusion in 
               
               
                   
                 fusion protein coding sequence) 
               
               
                 11 
                 Cas9 nickase mRNA coding sequence using minimal uridine codons 
               
               
                   
                 as listed in Table 3 (no start or stop codons; suitable for inclusion in 
               
               
                   
                 fusion protein coding sequence) 
               
               
                 12 
                 dCas9 mRNA coding sequence using minimal uridine codons as 
               
               
                   
                 listed in Table 3 (no start or stop codons; suitable for inclusion in 
               
               
                   
                 fusion protein coding sequence) 
               
               
                 13 
                 Amino acid sequence of Cas9 (without NLS) 
               
               
                 14 
                 Cas9 mRNA ORF encoding SEQ ID NO: 13 using minimal uridine 
               
               
                   
                 codons as listed in Table 3, with start and stop codons 
               
               
                 15 
                 Cas9 coding sequence encoding SEQ ID NO: 13 using minimal 
               
               
                   
                 uridine codons as listed in Table 3 (no start or stop codons; suitable 
               
               
                   
                 for inclusion in fusion protein coding sequence) 
               
               
                 16 
                 Amino acid sequence of Cas9 nickase (without NLS) 
               
               
                 17 
                 Cas9 nickase mRNA ORF encoding SEQ ID NO: 16 using minimal 
               
               
                   
                 uridine codons as listed in Table 3, with start and stop codons 
               
               
                 18 
                 Cas9 nickase coding sequence encoding SEQ ID NO: 16 using 
               
               
                   
                 minimal uridine codons as listed in Table 3 (no start or stop codons; 
               
               
                   
                 suitable for inclusion in fusion protein coding sequence) 
               
               
                 19 
                 Amino acid sequence of dCas9 (without NLS) 
               
               
                 20 
                 dCas9 mRNA ORF encoding SEQ ID NO: 13 using minimal uridine 
               
               
                   
                 codons as listed in Table 3, with start and stop codons 
               
               
                 21 
                 dCas9 coding sequence encoding SEQ ID NO: 13 using minimal 
               
               
                   
                 uridine codons as listed in Table 3 (no start or stop codons; suitable 
               
               
                   
                 for inclusion in fusion protein coding sequence) 
               
               
                 22 
                 Amino acid sequence of Cas9 with two nuclear localization signals 
               
               
                   
                 (2xNLS) as the C-terminal amino acids 
               
               
                 23 
                 Cas9 mRNA ORF encoding SEQ ID NO: 13 using minimal uridine 
               
               
                   
                 codons as listed in Table 3, with start and stop codons 
               
               
                 24 
                 Cas9 coding sequence encoding SEQ ID NO: 13 using minimal 
               
               
                   
                 uridine codons as listed in Table 3 (no start or stop codons; suitable 
               
               
                   
                 for inclusion in fusion protein coding sequence) 
               
               
                 25 
                 Amino acid sequence of Cas9 nickase with two nuclear localization 
               
               
                   
                 signals as the C-terminal amino acids 
               
               
                 26 
                 Cas9 nickase mRNA ORF encoding SEQ ID NO: 16 using minimal 
               
               
                   
                 uridine codons as listed in Table 3, with start and stop codons 
               
               
                 27 
                 Cas9 nickase coding sequence encoding SEQ ID NO: 16 using 
               
               
                   
                 minimal uridine codons as listed in Table 3 (no start or stop codons; 
               
               
                   
                 suitable for inclusion in fusion protein coding sequence) 
               
               
                 28 
                 Amino acid sequence of dCas9 with two nuclear localization signals 
               
               
                   
                 as the C-terminal amino acids 
               
               
                 29 
                 dCas9 mRNA ORF encoding SEQ ID NO: 13 using minimal uridine 
               
               
                   
                 codons as listed in Table 3, with start and stop codons 
               
               
                 30 
                 dCas9 coding sequence encoding SEQ ID NO: 13 using minimal 
               
               
                   
                 uridine codons as listed in Table 3 (no start or stop codons; suitable 
               
               
                   
                 for inclusion in fusion protein coding sequence) 
               
               
                 31 
                 T7 Promoter 
               
               
                 32 
                 Human beta-globin 5′ UTR 
               
               
                 33 
                 Human beta-globin 3′ UTR 
               
               
                 34 
                 Human alpha-globin 5′ UTR 
               
               
                 35 
                 Human alpha-globin 3′ UTR 
               
               
                 36 
                   Xenopus laevis  beta-globin 5′ UTR 
               
               
                 37 
                   Xenopus laevis  beta-globin 3′ UTR 
               
               
                 38 
                 Bovine Growth Hormone 5′ UTR 
               
               
                 39 
                 Bovine Growth Hormone 3′ UTR 
               
               
                 40 
                   Mus musculus  hemoglobin alpha, adult chain 1 (Hba-a1), 3′UTR 
               
               
                 41 
                 HSD17B4 5′ UTR 
               
               
                 42 
                 G282 single guide RNA targeting the mouse TTR gene 
               
               
                 43 
                 Cas9 transcript with 5′ UTR of HSD, ORF corresponding to SEQ 
               
               
                   
                 ID NO: 4, Kozak sequence, and 3′ UTR of ALB 
               
               
                 44 
                 Cas9 transcript with 5′ UTR of HSD, ORF corresponding to SEQ 
               
               
                   
                 ID NO: 4, and 3′ UTR of ALB 
               
               
                 45 
                 Alternative Cas9 ORF with 19.36% U content 
               
               
                 46 
                 Cas9 transcript with 5′ UTR of HSD, ORF corresponding to SEQ 
               
               
                   
                 ID NO: 45, Kozak sequence, and 3′ UTR of ALB 
               
               
                 47 
                 Cas9 transcript with 5′ UTR of HSD, ORF corresponding to SEQ 
               
               
                   
                 ID NO: 45, and 3′ UTR of ALB 
               
               
                 48 
                 Cas9 transcript comprising Cas9 ORF using codons with generally 
               
               
                   
                 high expression in humans 
               
               
                 49 
                 Cas9 transcript comprising Kozak sequence with Cas9 ORF using 
               
               
                   
                 codons with generally high expression in humans 
               
               
                 50 
                 Cas9 ORF with splice junctions removed; 12.75% U content 
               
               
                 51 
                 Cas9 transcript with 5′ UTR of HSD, ORF corresponding to SEQ 
               
               
                   
                 ID NO: 50, Kozak sequence, and 3′ UTR of ALB 
               
               
                 52 
                 Cas9 ORF with minimal uridine codons frequently used in humans 
               
               
                   
                 in general; 12.75% U content 
               
               
                 53 
                 Cas9 transcript with 5′ UTR of HSD, ORF corresponding to SEQ 
               
               
                   
                 ID NO: 52, Kozak sequence, and 3′ UTR of ALB 
               
               
                 54 
                 Cas9 ORF with minimal uridine codons infrequently used in 
               
               
                   
                 humans in general; 12.75% U content 
               
               
                 55 
                 Cas9 transcript with 5′ UTR of HSD, ORF corresponding to SEQ 
               
               
                   
                 ID NO: 54, Kozak sequence, and 3′ UTR of ALB 
               
               
                 56 
                 Cas9 transcript with AGG as first three nucleotides for use with 
               
               
                   
                 CleanCap ™, 5′ UTR of HSD, ORF corresponding to SEQ ID NO: 
               
               
                   
                 4, Kozak sequence, and 3′ UTR of ALB 
               
               
                 57 
                 Cas9 transcript with 5′ UTR from CMV, ORF corresponding to 
               
               
                   
                 SEQ ID NO: 4, Kozak sequence, and 3′ UTR of ALB 
               
               
                 58 
                 Cas9 transcript with 5′ UTR from HBB, ORF corresponding to SEQ 
               
               
                   
                 ID NO: 4, Kozak sequence, and 3′ UTR of HBB 
               
               
                 59 
                 Cas9 transcript with 5′ UTR from XBG, ORF corresponding to SEQ 
               
               
                   
                 ID NO: 4, Kozak sequence, and 3′ UTR of XBG 
               
               
                 60 
                 Cas9 transcript with AGG as first three nucleotides for use with 
               
               
                   
                 CleanCap ™, 5′ UTR from XBG, ORF corresponding to SEQ ID 
               
               
                   
                 NO: 4, Kozak sequence, and 3′ UTR of XBG 
               
               
                 61 
                 Cas9 transcript with AGG as first three nucleotides for use with 
               
               
                   
                 CleanCap ™, 5′ UTR from HSD, ORF corresponding to SEQ ID 
               
               
                   
                 NO: 4, Kozak sequence, and 3′ UTR of ALB 
               
               
                 62 
                 30/30/39 poly-A sequence 
               
               
                 63 
                 poly-A 100 sequence 
               
               
                 64 
                 G209 single guide RNA targeting the mouse TTR gene 
               
               
                 65 
                 ORF encoding  Neisseria meningitidis  Cas9 using minimal uridine 
               
               
                   
                 codons as listed in Table 3, with start and stop codons 
               
               
                 66 
                 ORF encoding  Neisseria meningitidis  Cas9 using minimal uridine 
               
               
                   
                 codons as listed in Table 3 (no start or stop codons; suitable for 
               
               
                   
                 inclusion in fusion protein coding sequence) 
               
               
                 67 
                 Transcript comprising SEQ ID NO: 65 (encoding  Neisseria   
               
               
                   
                   meningitidis  Cas9) 
               
               
                 68 
                 Amino acid sequence of  Neisseria meningitidis  Cas9 
               
               
                 69 
                 G390 single guide RNA targeting the rat TTR gene 
               
               
                 70 
                 G502 single guide RNA targeting the cynomolgus monkey TTR 
               
               
                   
                 gene 
               
               
                 71 
                 G509 single guide RNA targeting the cynomolgus monkey TTR 
               
               
                   
                 gene 
               
               
                 72 
                 G534 single guide RNA targeting the rat TTR gene 
               
               
                   
               
            
           
         
       
     
     See the Sequence Table below for the sequences themselves. Transcript sequences generally include GGG as the first three nucleotides for use with ARCA or AGG as the first three nucleotides for use with CleanCap™. Accordingly, the first three nucleotides can be modified for use with other capping approaches, such as Vaccinia capping enzyme. Promoters and poly-A sequences are not included in the transcript sequences. A promoter such as a T7 promoter (SEQ ID NO: 31) and a poly-A sequence such as SEQ ID NO: 62 or 63 can be appended to the disclosed transcript sequences at the 5′ and 3′ ends, respectively. Most nucleotide sequences are provided as DNA but can be readily converted to RNA by changing Ts to Us. 
     The following sequence table provides a listing of sequences disclosed herein. It is understood that if a DNA sequence (comprising Ts) is referenced with respect to an RNA, then Ts should be replaced with Us (which may be modified or unmodified depending on the context), and vice versa. 
     
       
         
           
               
            
               
                   
               
               
                 Sequence Table 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ 
               
               
                 Description 
                 Sequence 
                 ID No. 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Cas9 DNA 
                 ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAAC 
                 1 
               
               
                 coding 
                 AGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCG 
                   
               
               
                 sequence 2 
                 AGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGC 
                   
               
               
                   
                 ATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGA 
                   
               
               
                   
                 GAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAG 
                   
               
               
                   
                 AAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGA 
                   
               
               
                   
                 AATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTC 
                   
               
               
                   
                 CACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAG 
                   
               
               
                   
                 CACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTC 
                   
               
               
                   
                 GCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG 
                   
               
               
                   
                 AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATC 
                   
               
               
                   
                 TACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCC 
                   
               
               
                   
                 TGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACA 
                   
               
               
                   
                 AGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGA 
                   
               
               
                   
                 AGAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAAT 
                   
               
               
                   
                 CCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCT 
                   
               
               
                   
                 GATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGG 
                   
               
               
                   
                 AAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAA 
                   
               
               
                   
                 GAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAG 
                   
               
               
                   
                 CAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACA 
                   
               
               
                   
                 GATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAA 
                   
               
               
                   
                 CCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAAC 
                   
               
               
                   
                 ACAGAAATCACAAAGGCACCCCTGAGCGCAAGCATGATCAAG 
                   
               
               
                   
                 AGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCA 
                   
               
               
                   
                 CTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTC 
                   
               
               
                   
                 TTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGA 
                   
               
               
                   
                 GGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATC 
                   
               
               
                   
                 CTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTG 
                   
               
               
                   
                 AACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAAC 
                   
               
               
                   
                 GGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCA 
                   
               
               
                   
                 ATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC 
                   
               
               
                   
                 AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCG 
                   
               
               
                   
                 TACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCA 
                   
               
               
                   
                 TGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAAC 
                   
               
               
                   
                 TTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTC 
                   
               
               
                   
                 ATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA 
                   
               
               
                   
                 AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAG 
                   
               
               
                   
                 TCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAA 
                   
               
               
                   
                 TGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAA 
                   
               
               
                   
                 TCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCA 
                   
               
               
                   
                 AGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCG 
                   
               
               
                   
                 ACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAA 
                   
               
               
                   
                 GCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACA 
                   
               
               
                   
                 AGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAG 
                   
               
               
                   
                 ACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGAT 
                   
               
               
                   
                 CGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAA 
                   
               
               
                   
                 GGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGG 
                   
               
               
                   
                 AAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCA 
                   
               
               
                   
                 GAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATT 
                   
               
               
                   
                 CGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCT 
                   
               
               
                   
                 GACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACA 
                   
               
               
                   
                 GGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG 
                   
               
               
                   
                 CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGT 
                   
               
               
                   
                 CGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAA 
                   
               
               
                   
                 CATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAA 
                   
               
               
                   
                 GGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAG 
                   
               
               
                   
                 AAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACC 
                   
               
               
                   
                 CGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGT 
                   
               
               
                   
                 ACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAAC 
                   
               
               
                   
                 TGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCG 
                   
               
               
                   
                 TCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGG 
                   
               
               
                   
                 TCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAAC 
                   
               
               
                   
                 GTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGG 
                   
               
               
                   
                 AGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTC 
                   
               
               
                   
                 GACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACT 
                   
               
               
                   
                 GGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAG 
                   
               
               
                   
                 ACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAAT 
                   
               
               
                   
                 GAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGT 
                   
               
               
                   
                 CAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAG 
                   
               
               
                   
                 AAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTA 
                   
               
               
                   
                 CCACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAAC 
                   
               
               
                   
                 AGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGT 
                   
               
               
                   
                 CTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGC 
                   
               
               
                   
                 AAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTT 
                   
               
               
                   
                 CTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACA 
                   
               
               
                   
                 CTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACA 
                   
               
               
                   
                 AACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGA 
                   
               
               
                   
                 CTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAA 
                   
               
               
                   
                 CATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAA 
                   
               
               
                   
                 GGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGC 
                   
               
               
                   
                 AAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCG 
                   
               
               
                   
                 ACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGT 
                   
               
               
                   
                 CGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAAC 
                   
               
               
                   
                 TGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGA 
                   
               
               
                   
                 ACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCA 
                   
               
               
                   
                 AGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCG 
                   
               
               
                   
                 AACTGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGA 
                   
               
               
                   
                 GAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTAC 
                   
               
               
                   
                 GTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGG 
                   
               
               
                   
                 GAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAAC 
                   
               
               
                   
                 AGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCG 
                   
               
               
                   
                 AATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACA 
                   
               
               
                   
                 AGGTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCA 
                   
               
               
                   
                 GAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAA 
                   
               
               
                   
                 ACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAA 
                   
               
               
                   
                 TCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACG 
                   
               
               
                   
                 CAACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAA 
                   
               
               
                   
                 GAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCC 
                   
               
               
                   
                 CGAAGAAGAAGAGAAAGGTCTAG 
                   
               
               
                   
               
               
                 Cas9 DNA 
                 ATGGATAAGAAGTACTCAATCGGGCTGGATATCGGAACTAATT 
                 2 
               
               
                 coding 
                 CCGTGGGTTGGGCAGTGATCACGGATGAATACAAAGTGCCGT 
                   
               
               
                 sequence 1 
                 CCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACACAGCA 
                   
               
               
                   
                 TCAAGAAAAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGA 
                   
               
               
                   
                 AACCGCAGAAGCGACCCGGCTCAAACGTACCGCGAGGCGACG 
                   
               
               
                   
                 CTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAGATC 
                   
               
               
                   
                 TTTTCGAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACC 
                   
               
               
                   
                 GCCTGGAAGAATCTTTCCTGGTGGAGGAGGACAAGAAGCATG 
                   
               
               
                   
                 AACGGCATCCTATCTTTGGAAACATCGTCGACGAAGTGGCGTA 
                   
               
               
                   
                 CCACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAGTT 
                   
               
               
                   
                 GGTTGACTCAACTGACAAGGCCGACCTCAGATTGATCTACTTG 
                   
               
               
                   
                 GCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATCG 
                   
               
               
                   
                 AAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTTT 
                   
               
               
                   
                 CATTCAACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAAC 
                   
               
               
                   
                 CCAATCAATGCTAGCGGCGTCGATGCCAAGGCCATCCTGTCCG 
                   
               
               
                   
                 CCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACA 
                   
               
               
                   
                 GCTGCCGGGAGAGAAAAAGAACGGACTTTTCGGCAACTTGAT 
                   
               
               
                   
                 CGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTG 
                   
               
               
                   
                 ACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCT 
                   
               
               
                   
                 ACGACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCA 
                   
               
               
                   
                 GTACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCA 
                   
               
               
                   
                 ATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCA 
                   
               
               
                   
                 AAGCGCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGAGC 
                   
               
               
                   
                 ATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCA 
                   
               
               
                   
                 ACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAAG 
                   
               
               
                   
                 AATGGGTACGCAGGGTACATCGATGGAGGCGCTAGCCAGGAA 
                   
               
               
                   
                 GAGTTCTATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACG 
                   
               
               
                   
                 GAACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGC 
                   
               
               
                   
                 TCCGGAAACAGAGAACCTTTGACAACGGATCCATTCCCCACCA 
                   
               
               
                   
                 GATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAG 
                   
               
               
                   
                 GACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAG 
                   
               
               
                   
                 AAAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGG 
                   
               
               
                   
                 CGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAG 
                   
               
               
                   
                 AGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAA 
                   
               
               
                   
                 GGGAGCTTCGGCACAAAGCTTCATCGAACGAATGACCAACTTC 
                   
               
               
                   
                 GACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAGC 
                   
               
               
                   
                 CTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAG 
                   
               
               
                   
                 TGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTC 
                   
               
               
                   
                 CGGAGAACAGAAGAAAGCAATTGTCGATCTGCTGTTCAAGAC 
                   
               
               
                   
                 CAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACn -   
                   
               
               
                   
                 CAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGGGT 
                   
               
               
                   
                 GGAGGACAGATTCAACGCTTCGCTGGGAACCTATCATGATCTC 
                   
               
               
                   
                 CTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAG 
                   
               
               
                   
                 AACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTT 
                   
               
               
                   
                 TCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACG 
                   
               
               
                   
                 CTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGGGCCG 
                   
               
               
                   
                 CCGGTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAAC 
                   
               
               
                   
                 GGTATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCC 
                   
               
               
                   
                 TCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAATTGAT 
                   
               
               
                   
                 CCACGACGACAGCCTGACCTTTAAGGAGGACATCCAAAAAGC 
                   
               
               
                   
                 ACAAGTGTCCGGACAGGGAGACTCACTCCATGAACACATCGC 
                   
               
               
                   
                 GAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATTCTGCA 
                   
               
               
                   
                 AACTGTGAAGGTGGTCGACGAGCTGGTGAAGGTCATGGGACG 
                   
               
               
                   
                 GCACAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAA 
                   
               
               
                   
                 CCAGACTACCCAGAAGGGCCAGAAAAACTCCCGCGAAAGGAT 
                   
               
               
                   
                 GAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGAT 
                   
               
               
                   
                 CCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGA 
                   
               
               
                   
                 GAAGCTCTACCTGTACTATTTGCAAAATGGACGGGACATGTAC 
                   
               
               
                   
                 GTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTACGACG 
                   
               
               
                   
                 TGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCGAT 
                   
               
               
                   
                 CGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAA 
                   
               
               
                   
                 GTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAA 
                   
               
               
                   
                 GAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAG 
                   
               
               
                   
                 AGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTC 
                   
               
               
                   
                 TCAGAGCTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTC 
                   
               
               
                   
                 GAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCTTGGAC 
                   
               
               
                   
                 TCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCATC 
                   
               
               
                   
                 CGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCG 
                   
               
               
                   
                 GACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCA 
                   
               
               
                   
                 ACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGGT 
                   
               
               
                   
                 CGGTACCGCCCTGATCAAAAAGTACCCTAAACTTGAATCGGAG 
                   
               
               
                   
                 TTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGATG 
                   
               
               
                   
                 ATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAAA 
                   
               
               
                   
                 TACTTCTTTTACTCAAACATCATGAACTTTTTCAAGACTGAAAT 
                   
               
               
                   
                 TACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGA 
                   
               
               
                   
                 AACTAACGGAGAAACGGGCGAAATCGTGTGGGACAAGGGCAG 
                   
               
               
                   
                 GGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTC 
                   
               
               
                   
                 AATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTCA 
                   
               
               
                   
                 AAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATT 
                   
               
               
                   
                 GCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTC 
                   
               
               
                   
                 GATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGG 
                   
               
               
                   
                 TGGAGAAGGGAAAGAGCAAAAAGCTCAAATCCGTCAAAGAGC 
                   
               
               
                   
                 TGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAGAA 
                   
               
               
                   
                 CCCGATTGATTTCCTCGAGGCGAAGGGTTACAAGGAGGTGAA 
                   
               
               
                   
                 GAAGGATCTGATCATCAAACTCCCCAAGTACTCACTGTTCGAA 
                   
               
               
                   
                 CTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAA 
                   
               
               
                   
                 CTCCAAAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTC 
                   
               
               
                   
                 AACTTCCTCTATCTTGCTTCGCACTACGAAAAACTCAAAGGGT 
                   
               
               
                   
                 CACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCAGC 
                   
               
               
                   
                 ACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTT 
                   
               
               
                   
                 TTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTC 
                   
               
               
                   
                 CTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAGAGAA 
                   
               
               
                   
                 CAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCTGG 
                   
               
               
                   
                 GAGCCCCAGCCGCCTTCAAGTACTTCGATACTACTATCGATCG 
                   
               
               
                   
                 CAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGACCCT 
                   
               
               
                   
                 GATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCGAT 
                   
               
               
                   
                 CTGTCGCAGCTGGGTGGCGATGGCGGTGGATCTCCGAAAAAG 
                   
               
               
                   
                 AAGAGAAAGGTGTAATGA 
                   
               
               
                   
               
               
                 Cas9 amino 
                 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK 
                 3 
               
               
                 acid 
                 KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE 
                   
               
               
                 sequence 
                 MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT 
                   
               
               
                   
                 IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS 
                   
               
               
                   
                 DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL 
                   
               
               
                   
                 IAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT 
                   
               
               
                   
                 YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA 
                   
               
               
                   
                 PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA 
                   
               
               
                   
                 GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF 
                   
               
               
                   
                 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV 
                   
               
               
                   
                 GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT 
                   
               
               
                   
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL 
                   
               
               
                   
                 SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED 
                   
               
               
                   
                 RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG 
                   
               
               
                   
                 KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 
                   
               
               
                   
                 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE 
                   
               
               
                   
                 NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL 
                   
               
               
                   
                 YLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK 
                   
               
               
                   
                 VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD 
                   
               
               
                   
                 NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY 
                   
               
               
                   
                 DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA 
                   
               
               
                   
                 YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK 
                   
               
               
                   
                 ATAKYFFYSNIMNFFKTEITLANGEIRKRPLITNGETGEIVWDKG 
                   
               
               
                   
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
                   
               
               
                   
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG 
                   
               
               
                   
                 ITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR 
                   
               
               
                   
                 MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ 
                   
               
               
                   
                 LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
                   
               
               
                   
                 REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 
                   
               
               
                   
                 HQSITGLYETRIDLSQLGGDGGGSPKKKRKV 
                   
               
               
                   
               
               
                 Cas9 mRNA 
                 AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAA 
                 4 
               
               
                 open reading 
                 CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC 
                   
               
               
                 frame (ORF) 
                 CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC 
                   
               
               
                 2 
                 AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG 
                   
               
               
                   
                 CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA 
                   
               
               
                   
                 GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG 
                   
               
               
                   
                 CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG 
                   
               
               
                   
                 CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG 
                   
               
               
                   
                 ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC 
                   
               
               
                   
                 GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA 
                   
               
               
                   
                 CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC 
                   
               
               
                   
                 UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC 
                   
               
               
                   
                 AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA 
                   
               
               
                   
                 CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU 
                   
               
               
                   
                 ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA 
                   
               
               
                   
                 GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG 
                   
               
               
                   
                 CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA 
                   
               
               
                   
                 AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG 
                   
               
               
                   
                 GGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGA 
                   
               
               
                   
                 AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG 
                   
               
               
                   
                 ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA 
                   
               
               
                   
                 GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU 
                   
               
               
                   
                 GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG 
                   
               
               
                   
                 CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC 
                   
               
               
                   
                 CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA 
                   
               
               
                   
                 GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA 
                   
               
               
                   
                 AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG 
                   
               
               
                   
                 GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU 
                   
               
               
                   
                 GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG 
                   
               
               
                   
                 ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC 
                   
               
               
                   
                 CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA 
                   
               
               
                   
                 AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA 
                   
               
               
                   
                 AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG 
                   
               
               
                   
                 UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG 
                   
               
               
                   
                 ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA 
                   
               
               
                   
                 AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG 
                   
               
               
                   
                 AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG 
                   
               
               
                   
                 GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU 
                   
               
               
                   
                 CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA 
                   
               
               
                   
                 UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA 
                   
               
               
                   
                 AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU 
                   
               
               
                   
                 CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU 
                   
               
               
                   
                 UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC 
                   
               
               
                   
                 GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA 
                   
               
               
                   
                 GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC 
                   
               
               
                   
                 UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA 
                   
               
               
                   
                 GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU 
                   
               
               
                   
                 CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA 
                   
               
               
                   
                 CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC 
                   
               
               
                   
                 AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA 
                   
               
               
                   
                 GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC 
                   
               
               
                   
                 ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA 
                   
               
               
                   
                 CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC 
                   
               
               
                   
                 AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC 
                   
               
               
                   
                 AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA 
                   
               
               
                   
                 AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGA 
                   
               
               
                   
                 AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA 
                   
               
               
                   
                 GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC 
                   
               
               
                   
                 CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA 
                   
               
               
                   
                 GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG 
                   
               
               
                   
                 ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC 
                   
               
               
                   
                 GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAG 
                   
               
               
                   
                 GACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAA 
                   
               
               
                   
                 GAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCG 
                   
               
               
                   
                 UCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCA 
                   
               
               
                   
                 AAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGC 
                   
               
               
                   
                 AGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCA 
                   
               
               
                   
                 UCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCAC 
                   
               
               
                   
                 GUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGA 
                   
               
               
                   
                 CGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC 
                   
               
               
                   
                 UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAG 
                   
               
               
                   
                 UUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACA 
                   
               
               
                   
                 CGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCA 
                   
               
               
                   
                 AGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGAC 
                   
               
               
                   
                 UACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGA 
                   
               
               
                   
                 ACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA 
                   
               
               
                   
                 GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCA 
                   
               
               
                   
                 AACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG 
                   
               
               
                   
                 AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCG 
                   
               
               
                   
                 CAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUC 
                   
               
               
                   
                 GUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGGAAGGA 
                   
               
               
                   
                 AAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGGAA 
                   
               
               
                   
                 GAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGAC 
                   
               
               
                   
                 AGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGU 
                   
               
               
                   
                 CGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAAC 
                   
               
               
                   
                 UGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG 
                   
               
               
                   
                 AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGU 
                   
               
               
                   
                 CAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGU 
                   
               
               
                   
                 UCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCA 
                   
               
               
                   
                 GGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAA 
                   
               
               
                   
                 GUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGC 
                   
               
               
                   
                 UGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC 
                   
               
               
                   
                 GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACA 
                   
               
               
                   
                 GAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAA 
                   
               
               
                   
                 ACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGAC 
                   
               
               
                   
                 AAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUU 
                   
               
               
                   
                 CACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACU 
                   
               
               
                   
                 UCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAG 
                   
               
               
                   
                 GAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGG 
                   
               
               
                   
                 ACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAG 
                   
               
               
                   
                 ACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGUCUAG 
                   
               
               
                   
               
               
                 Cas9 mRNA 
                 AUGGAUAAGAAGUACUCAAUCGGGCUGGAUAUCGGAACUAA 
                 5 
               
               
                 ORF 1 
                 UUCCGUGGGUUGGGCAGUGAUCACGGAUGAAUACAAAGUGC 
                   
               
               
                   
                 CGUCCAAGAAGUUCAAGGUCCUGGGGAACACCGAUAGACAC 
                   
               
               
                   
                 AGCAUCAAGAAAAAUCUCAUCGGAGCCCUGCUGUUUGACUC 
                   
               
               
                   
                 CGGCGAAACCGCAGAAGCGACCCGGCUCAAACGUACCGCGAG 
                   
               
               
                   
                 GCGACGCUACACCCGGCGGAAGAAUCGCAUCUGCUAUCUGC 
                   
               
               
                   
                 AAGAGAUCUUUUCGAACGAAAUGGCAAAGGUCGACGACAGC 
                   
               
               
                   
                 UUCUUCCACCGCCUGGAAGAAUCUUUCCUGGUGGAGGAGGA 
                   
               
               
                   
                 CAAGAAGCAUGAACGGCAUCCUAUCUUUGGAAACAUCGUCG 
                   
               
               
                   
                 ACGAAGUGGCGUACCACGAAAAGUACCCGACCAUCUACCAU 
                   
               
               
                   
                 CUGCGGAAGAAGUUGGUUGACUCAACUGACAAGGCCGACCU 
                   
               
               
                   
                 CAGAUUGAUCUACUUGGCCCUCGCCCAUAUGAUCAAAUUCC 
                   
               
               
                   
                 GCGGACACUUCCUGAUCGAAGGCGAUCUGAACCCUGAUAAC 
                   
               
               
                   
                 UCCGACGUGGAUAAGCUUUUCAUUCAACUGGUGCAGACCUA 
                   
               
               
                   
                 CAACCAACUGUUCGAAGAAAACCCAAUCAAUGCUAGCGGCG 
                   
               
               
                   
                 UCGAUGCCAAGGCCAUCCUGUCCGCCCGGCUGUCGAAGUCGC 
                   
               
               
                   
                 GGCGCCUCGAAAACCUGAUCGCACAGCUGCCGGGAGAGAAA 
                   
               
               
                   
                 AAGAACGGACUUUUCGGCAACUUGAUCGCUCUCUCACUGGG 
                   
               
               
                   
                 ACUCACUCCCAAUUUCAAGUCCAAUUUUGACCUGGCCGAGG 
                   
               
               
                   
                 ACGCGAAGCUGCAACUCUCAAAGGACACCUACGACGACGAC 
                   
               
               
                   
                 UUGGACAAUUUGCUGGCACAAAUUGGCGAUCAGUACGCGGA 
                   
               
               
                   
                 UCUGUUCCUUGCCGCUAAGAACCUUUCGGACGCAAUCUUGC 
                   
               
               
                   
                 UGUCCGAUAUCCUGCGCGUGAACACCGAAAUAACCAAAGCG 
                   
               
               
                   
                 CCGCUUAGCGCCUCGAUGAUUAAGCGGUACGACGAGCAUCA 
                   
               
               
                   
                 CCAGGAUCUCACGCUGCUCAAAGCGCUCGUGAGACAGCAAC 
                   
               
               
                   
                 UGCCUGAAAAGUACAAGGAGAUCUUCUUCGACCAGUCCAAG 
                   
               
               
                   
                 AAUGGGUACGCAGGGUACAUCGAUGGAGGCGCUAGCCAGGA 
                   
               
               
                   
                 AGAGUUCUAUAAGUUCAUCAAGCCAAUCCUGGAAAAGAUGG 
                   
               
               
                   
                 ACGGAACCGAAGAACUGCUGGUCAAGCUGAACAGGGAGGAU 
                   
               
               
                   
                 CUGCUCCGGAAACAGAGAACCUUUGACAACGGAUCCAUUCC 
                   
               
               
                   
                 CCACCAGAUCCAUCUGGGUGAGCUGCACGCCAUCUUGCGGCG 
                   
               
               
                   
                 CCAGGAGGACUUUUACCCAUUCCUCAAGGACAACCGGGAAA 
                   
               
               
                   
                 AGAUCGAGAAAAUUCUGACGUUCCGCAUCCCGUAUUACGUG 
                   
               
               
                   
                 GGCCCACUGGCGCGCGGCAAUUCGCGCUUCGCGUGGAUGAC 
                   
               
               
                   
                 UAGAAAAUCAGAGGAAACCAUCACUCCUUGGAAUUUCGAGG 
                   
               
               
                   
                 AAGUUGUGGAUAAGGGAGCUUCGGCACAAAGCUUCAUCGAA 
                   
               
               
                   
                 CGAAUGACCAACUUCGACAAGAAUCUCCCAAACGAGAAGGU 
                   
               
               
                   
                 GCUUCCUAAGCACAGCCUCCUUUACGAAUACUUCACUGUCU 
                   
               
               
                   
                 ACAACGAACUGACUAAAGUGAAAUACGUUACUGAAGGAAUG 
                   
               
               
                   
                 AGGAAGCCGGCCUUUCUGUCCGGAGAACAGAAGAAAGCAAU 
                   
               
               
                   
                 UGUCGAUCUGCUGUUCAAGACCAACCGCAAGGUGACCGUCA 
                   
               
               
                   
                 AGCAGCUUAAAGAGGACUACUUCAAGAAGAUCGAGUGUUUC 
                   
               
               
                   
                 GACUCAGUGGAAAUCAGCGGGGUGGAGGACAGAUUCAACGC 
                   
               
               
                   
                 UUCGCUGGGAACCUAUCAUGAUCUCCUGAAGAUCAUCAAGG 
                   
               
               
                   
                 ACAAGGACUUCCUUGACAACGAGGAGAACGAGGACAUCCUG 
                   
               
               
                   
                 GAAGAUAUCGUCCUGACCUUGACCCUUUUCGAGGAUCGCGA 
                   
               
               
                   
                 GAUGAUCGAGGAGAGGCUUAAGACCUACGCUCAUCUCUUCG 
                   
               
               
                   
                 ACGAUAAGGUCAUGAAACAACUCAAGCGCCGCCGGUACACU 
                   
               
               
                   
                 GGUUGGGGCCGCCUCUCCCGCAAGCUGAUCAACGGUAUUCG 
                   
               
               
                   
                 CGAUAAACAGAGCGGUAAAACUAUCCUGGAUUUCCUCAAAU 
                   
               
               
                   
                 CGGAUGGCUUCGCUAAUCGUAACUUCAUGCAAUUGAUCCAC 
                   
               
               
                   
                 GACGACAGCCUGACCUUUAAGGAGGACAUCCAAAAAGCACA 
                   
               
               
                   
                 AGUGUCCGGACAGGGAGACUCACUCCAUGAACACAUCGCGA 
                   
               
               
                   
                 AUCUGGCCGGUUCGCCGGCGAUUAAGAAGGGAAUUCUGCAA 
                   
               
               
                   
                 ACUGUGAAGGUGGUCGACGAGCUGGUGAAGGUCAUGGGACG 
                   
               
               
                   
                 OCACAAACCGGAGAAUAUCGUGAUUGAAAUGGCCCGAGAAA 
                   
               
               
                   
                 ACCAGACUACCCAGAAGGGCCAGAAAAACUCCCGCGAAAGG 
                   
               
               
                   
                 AUGAAGCGGAUCGAAGAAGGAAUCAAGGAGCUGGGCAGCCA 
                   
               
               
                   
                 GAUCCUGAAAGAGCACCCGGUGGAAAACACGCAGCUGCAGA 
                   
               
               
                   
                 ACGAGAAGCUCUACCUGUACUAUUUGCAAAAUGGACGGGAC 
                   
               
               
                   
                 AUGUACGUGGACCAAGAGCUGGACAUCAAUCGGUUGUCUGA 
                   
               
               
                   
                 UUACGACGUGGACCACAUCGUUCCACAGUCCUUUCUGAAGG 
                   
               
               
                   
                 AUGACUCGAUCGAUAACAAGGUGUUGACUCGCAGCGACAAG 
                   
               
               
                   
                 AACAGAGGGAAGUCAGAUAAUGUGCCAUCGGAGGAGGUCGU 
                   
               
               
                   
                 GAAGAAGAUGAAGAAUUACUGGCGGCAGCUCCUGAAUGCGA 
                   
               
               
                   
                 AGCUGAUUACCCAGAGAAAGUUUGACAAUCUCACUAAAGCC 
                   
               
               
                   
                 GAGCGCGGCGGACUCUCAGAGCUGGAUAAGGCUGGAUUCAU 
                   
               
               
                   
                 CAAACGGCAGCUGGUCGAGACUCGGCAGAUUACCAAGCACG 
                   
               
               
                   
                 UGGCGCAGAUCUUGGACUCCCGCAUGAACACUAAAUACGAC 
                   
               
               
                   
                 GAGAACGAUAAGCUCAUCCGGGAAGUGAAGGUGAUUACCCU 
                   
               
               
                   
                 GAAAAGCAAACUUGUGUCGGACUUUCGGAAGGACUUUCAGU 
                   
               
               
                   
                 UUUACAAAGUGAGAGAAAUCAACAACUACCAUCACGCGCAU 
                   
               
               
                   
                 GACGCAUACCUCAACGCUGUGGUCGGUACCGCCCUGAUCAA 
                   
               
               
                   
                 AAAGUACCCUAAACUUGAAUCGGAGUUUGUGUACGGAGACU 
                   
               
               
                   
                 ACAAGGUCUACGACGUGAGGAAGAUGAUAGCCAAGUCCGAA 
                   
               
               
                   
                 CAGGAAAUCGGGAAAGCAACUGCGAAAUACUUCUUUUACUC 
                   
               
               
                   
                 AAACAUCAUGAACUUUUUCAAGACUGAAAUUACGCUGGCCA 
                   
               
               
                   
                 AUGGAGAAAUCAGGAAGAGGCCACUGAUCGAAACUAACGGA 
                   
               
               
                   
                 GAAACGGGCGAAAUCGUGUGGGACAAGGGCAGGGACUUCGC 
                   
               
               
                   
                 AACUGUUCGCAAAGUGCUCUCUAUGCCGCAAGUCAAUAUUG 
                   
               
               
                   
                 UGAAGAAAACCGAAGUGCAAACCGGCGGAUUUUCAAAGGAA 
                   
               
               
                   
                 UCGAUCCUCCCAAAGAGAAAUAGCGACAAGCUCAUUGCACG 
                   
               
               
                   
                 CAAGAAAGACUGGGACCCGAAGAAGUACGGAGGAUUCGAUU 
                   
               
               
                   
                 CGCCGACUGUCGCAUACUCCGUCCUCGUGGUGGCCAAGGUG 
                   
               
               
                   
                 GAGAAGGGAAAGAGCAAAAAGCUCAAAUCCGUCAAAGAGCU 
                   
               
               
                   
                 GCUGGGGAUUACCAUCAUGGAACGAUCCUCGUUCGAGAAGA 
                   
               
               
                   
                 ACCCGAUUGAUUUCCUCGAGGCGAAGGGUUACAAGGAGGUG 
                   
               
               
                   
                 AAGAAGGAUCUGAUCAUCAAACUCCCCAAGUACUCACUGUU 
                   
               
               
                   
                 CGAACUGGAAAAUGGUCGGAAGCGCAUGCUGGCUUCGGCCG 
                   
               
               
                   
                 GAGAACUCCAAAAAGGAAAUGAGCUGGCCUUGCCUAGCAAG 
                   
               
               
                   
                 UACGUCAACUUCCUCUAUCUUGCUUCGCACUACGAAAAACU 
                   
               
               
                   
                 CAAAGGGUCACCGGAAGAUAACGAACAGAAGCAGCUUUUCG 
                   
               
               
                   
                 UGGAGCAGCACAAGCAUUAUCUGGAUGAAAUCAUCGAACAA 
                   
               
               
                   
                 AUCUCCGAGUUUUCAAAGCGCGUGAUCCUCGCCGACGCCAAC 
                   
               
               
                   
                 CUCGACAAAGUCCUGUCGGCCUACAAUAAGCAUAGAGAUAA 
                   
               
               
                   
                 GCCGAUCAGAGAACAGGCCGAGAACAUUAUCCACUUGUUCA 
                   
               
               
                   
                 CCCUGACUAACCUGGGAGCCCCAGCCGCCUUCAAGUACUUCG 
                   
               
               
                   
                 AUACUACUAUCGAUCGCAAAAGAUACACGUCCACCAAGGAA 
                   
               
               
                   
                 GUUCUGGACGCGACCCUGAUCCACCAAAGCAUCACUGGACUC 
                   
               
               
                   
                 UACGAAACUAGGAUCGAUCUGUCGCAGCUGGGUGGCGAUGG 
                   
               
               
                   
                 CGGUGGAUCUCCGAAAAAGAAGAGAAAGGUGUAAUGA 
                   
               
               
                   
               
               
                 Cas9 nickase 
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK 
                 6 
               
               
                 (D10A) 
                 KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE 
                   
               
               
                 amino acid 
                 MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT 
                   
               
               
                 sequence 
                 IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS 
                   
               
               
                   
                 DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL 
                   
               
               
                   
                 IAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT 
                   
               
               
                   
                 YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA 
                   
               
               
                   
                 PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFTDQSKNGYA 
                   
               
               
                   
                 GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF 
                   
               
               
                   
                 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV 
                   
               
               
                   
                 GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT 
                   
               
               
                   
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL 
                   
               
               
                   
                 SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED 
                   
               
               
                   
                 RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG 
                   
               
               
                   
                 KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 
                   
               
               
                   
                 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE 
                   
               
               
                   
                 NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL 
                   
               
               
                   
                 YLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK 
                   
               
               
                   
                 VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD 
                   
               
               
                   
                 NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY 
                   
               
               
                   
                 DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA 
                   
               
               
                   
                 YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK 
                   
               
               
                   
                 ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
                   
               
               
                   
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
                   
               
               
                   
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG 
                   
               
               
                   
                 ITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR 
                   
               
               
                   
                 MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ 
                   
               
               
                   
                 LFVEQHKHYLDEIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
                   
               
               
                   
                 REQAENIIHLFTLTNLGAPAAFKYPDTTIDRKRYTSTKEVLDATLI 
                   
               
               
                   
                 HQSITGLYETRIDLSQLGGDGGGSPKKKRKV 
                   
               
               
                   
               
               
                 Cas9 nickase 
                 AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAA 
                 7 
               
               
                 (D10A) 
                 CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC 
                   
               
               
                 mRNA ORF 
                 CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC 
                   
               
               
                   
                 AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG 
                   
               
               
                   
                 CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA 
                   
               
               
                   
                 GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG 
                   
               
               
                   
                 CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG 
                   
               
               
                   
                 CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG 
                   
               
               
                   
                 ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC 
                   
               
               
                   
                 GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA 
                   
               
               
                   
                 CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC 
                   
               
               
                   
                 UGAGACLGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC 
                   
               
               
                   
                 AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA 
                   
               
               
                   
                 CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU 
                   
               
               
                   
                 ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA 
                   
               
               
                   
                 GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG 
                   
               
               
                   
                 CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA 
                   
               
               
                   
                 AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG 
                   
               
               
                   
                 GGACUGACACCGAACUUCAAGAGCAACUVCGACCUGGCAGA 
                   
               
               
                   
                 AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG 
                   
               
               
                   
                 ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA 
                   
               
               
                   
                 GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU 
                   
               
               
                   
                 GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG 
                   
               
               
                   
                 CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC 
                   
               
               
                   
                 CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA 
                   
               
               
                   
                 GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA 
                   
               
               
                   
                 AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG 
                   
               
               
                   
                 GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU 
                   
               
               
                   
                 GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG 
                   
               
               
                   
                 ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC 
                   
               
               
                   
                 CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA 
                   
               
               
                   
                 AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA 
                   
               
               
                   
                 AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG 
                   
               
               
                   
                 UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG 
                   
               
               
                   
                 ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA 
                   
               
               
                   
                 AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG 
                   
               
               
                   
                 AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG 
                   
               
               
                   
                 GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU 
                   
               
               
                   
                 CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA 
                   
               
               
                   
                 UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA 
                   
               
               
                   
                 AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU 
                   
               
               
                   
                 CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU 
                   
               
               
                   
                 UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC 
                   
               
               
                   
                 GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA 
                   
               
               
                   
                 GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC 
                   
               
               
                   
                 UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA 
                   
               
               
                   
                 GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU 
                   
               
               
                   
                 CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA 
                   
               
               
                   
                 CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC 
                   
               
               
                   
                 AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA 
                   
               
               
                   
                 GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC 
                   
               
               
                   
                 ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA 
                   
               
               
                   
                 CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC 
                   
               
               
                   
                 AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC 
                   
               
               
                   
                 AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA 
                   
               
               
                   
                 AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGA 
                   
               
               
                   
                 AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA 
                   
               
               
                   
                 GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC 
                   
               
               
                   
                 CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA 
                   
               
               
                   
                 GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG 
                   
               
               
                   
                 ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC 
                   
               
               
                   
                 GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAG 
                   
               
               
                   
                 GACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAA 
                   
               
               
                   
                 GAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCG 
                   
               
               
                   
                 UCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCA 
                   
               
               
                   
                 AAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGC 
                   
               
               
                   
                 AGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCA 
                   
               
               
                   
                 UCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGGAC 
                   
               
               
                   
                 GUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGA 
                   
               
               
                   
                 CGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC 
                   
               
               
                   
                 UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAG 
                   
               
               
                   
                 UUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACA 
                   
               
               
                   
                 CGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCA 
                   
               
               
                   
                 AGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGAC 
                   
               
               
                   
                 UACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGA 
                   
               
               
                   
                 ACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA 
                   
               
               
                   
                 GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCA 
                   
               
               
                   
                 AACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG 
                   
               
               
                   
                 AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCG 
                   
               
               
                   
                 CAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUC 
                   
               
               
                   
                 GUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGA 
                   
               
               
                   
                 AAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAA 
                   
               
               
                   
                 GAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGAC 
                   
               
               
                   
                 AGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGU 
                   
               
               
                   
                 CGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAAC 
                   
               
               
                   
                 UGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG 
                   
               
               
                   
                 AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGU 
                   
               
               
                   
                 CAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGU 
                   
               
               
                   
                 UCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCA 
                   
               
               
                   
                 GGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAA 
                   
               
               
                   
                 GUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGC 
                   
               
               
                   
                 UGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGGUGUUC 
                   
               
               
                   
                 GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACA 
                   
               
               
                   
                 GAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAA 
                   
               
               
                   
                 ACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGAC 
                   
               
               
                   
                 AAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUU 
                   
               
               
                   
                 CACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACU 
                   
               
               
                   
                 UCGACACAACAAUCGACAGAAAGAGAUACACAAGGACAAAG 
                   
               
               
                   
                 GAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGG 
                   
               
               
                   
                 ACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAG 
                   
               
               
                   
                 ACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGUCUAG 
                   
               
               
                   
               
               
                 dCas9 
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK 
                 8 
               
               
                 (D10A 
                 KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE 
                   
               
               
                 H840A) 
                 MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT 
                   
               
               
                 amine acid 
                 IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS 
                   
               
               
                 sequence 
                 DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL 
                   
               
               
                   
                 IAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT 
                   
               
               
                   
                 YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA 
                   
               
               
                   
                 PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA 
                   
               
               
                   
                 GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF 
                   
               
               
                   
                 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV 
                   
               
               
                   
                 GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT 
                   
               
               
                   
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL 
                   
               
               
                   
                 SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED 
                   
               
               
                   
                 RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG 
                   
               
               
                   
                 KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 
                   
               
               
                   
                 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENTVIEMARE 
                   
               
               
                   
                 NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL 
                   
               
               
                   
                 YLYYLQNGRDMYVDQELDINRLSDYDVDATVPQSFLKDDSIDNK 
                   
               
               
                   
                 VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD 
                   
               
               
                   
                 NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY 
                   
               
               
                   
                 DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA 
                   
               
               
                   
                 YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK 
                   
               
               
                   
                 ATAKYFFYSNIMNFFKTEITLANGEIRKRPLDETNGETGEIVWDKG 
                   
               
               
                   
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
                   
               
               
                   
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG 
                   
               
               
                   
                 ITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR 
                   
               
               
                   
                 MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ 
                   
               
               
                   
                 LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
                   
               
               
                   
                 REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 
                   
               
               
                   
                 HQSITGLYETRIDLSQLGGDGGGSPKKKRKV 
                   
               
               
                   
               
               
                 dCas9 
                 AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAA 
                 9 
               
               
                 (D10A 
                 CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC 
                   
               
               
                 H840A) 
                 CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC 
                   
               
               
                 mRNA ORF 
                 AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG 
                   
               
               
                   
                 CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA 
                   
               
               
                   
                 GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG 
                   
               
               
                   
                 CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG 
                   
               
               
                   
                 CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG 
                   
               
               
                   
                 ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC 
                   
               
               
                   
                 GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA 
                   
               
               
                   
                 CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC 
                   
               
               
                   
                 UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC 
                   
               
               
                   
                 AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA 
                   
               
               
                   
                 CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU 
                   
               
               
                   
                 ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA 
                   
               
               
                   
                 GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG 
                   
               
               
                   
                 CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA 
                   
               
               
                   
                 AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG 
                   
               
               
                   
                 GGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGA 
                   
               
               
                   
                 AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG 
                   
               
               
                   
                 ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA 
                   
               
               
                   
                 GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU 
                   
               
               
                   
                 GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG 
                   
               
               
                   
                 CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC 
                   
               
               
                   
                 CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA 
                   
               
               
                   
                 GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA 
                   
               
               
                   
                 AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG 
                   
               
               
                   
                 GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU 
                   
               
               
                   
                 GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG 
                   
               
               
                   
                 ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC 
                   
               
               
                   
                 CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA 
                   
               
               
                   
                 AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA 
                   
               
               
                   
                 AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG 
                   
               
               
                   
                 UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG 
                   
               
               
                   
                 ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA 
                   
               
               
                   
                 AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG 
                   
               
               
                   
                 AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG 
                   
               
               
                   
                 GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU 
                   
               
               
                   
                 CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA 
                   
               
               
                   
                 UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA 
                   
               
               
                   
                 AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU 
                   
               
               
                   
                 CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU 
                   
               
               
                   
                 UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC 
                   
               
               
                   
                 GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA 
                   
               
               
                   
                 GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC 
                   
               
               
                   
                 UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA 
                   
               
               
                   
                 GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU 
                   
               
               
                   
                 CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA 
                   
               
               
                   
                 CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC 
                   
               
               
                   
                 AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA 
                   
               
               
                   
                 GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC 
                   
               
               
                   
                 ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA 
                   
               
               
                   
                 CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC 
                   
               
               
                   
                 AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC 
                   
               
               
                   
                 AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA 
                   
               
               
                   
                 AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGA 
                   
               
               
                   
                 AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA 
                   
               
               
                   
                 GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC 
                   
               
               
                   
                 CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA 
                   
               
               
                   
                 GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG 
                   
               
               
                   
                 ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC 
                   
               
               
                   
                 GACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAA 
                   
               
               
                   
                 GGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACA 
                   
               
               
                   
                 AGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUC 
                   
               
               
                   
                 GUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGC 
                   
               
               
                   
                 AAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGG 
                   
               
               
                   
                 CAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUC 
                   
               
               
                   
                 AUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCA 
                   
               
               
                   
                 CGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACG 
                   
               
               
                   
                 ACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA 
                   
               
               
                   
                 CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCA 
                   
               
               
                   
                 GUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCAC 
                   
               
               
                   
                 ACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUC 
                   
               
               
                   
                 AAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGA 
                   
               
               
                   
                 CUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCG 
                   
               
               
                   
                 AACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUAC 
                   
               
               
                   
                 AGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGC 
                   
               
               
                   
                 AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACG 
                   
               
               
                   
                 GAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUC 
                   
               
               
                   
                 GCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAU 
                   
               
               
                   
                 CGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGG 
                   
               
               
                   
                 AAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCA 
                   
               
               
                   
                 AGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGA 
                   
               
               
                   
                 CAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGG 
                   
               
               
                   
                 UCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAA 
                   
               
               
                   
                 CUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAA 
                   
               
               
                   
                 GAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAG 
                   
               
               
                   
                 UCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUG 
                   
               
               
                   
                 UUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGC 
                   
               
               
                   
                 AGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCA 
                   
               
               
                   
                 AGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAG 
                   
               
               
                   
                 CUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUU 
                   
               
               
                   
                 CGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAAC 
                   
               
               
                   
                 AGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCA 
                   
               
               
                   
                 AACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGA 
                   
               
               
                   
                 CAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGU 
                   
               
               
                   
                 UCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC 
                   
               
               
                   
                 UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAA 
                   
               
               
                   
                 GGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAG 
                   
               
               
                   
                 GACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGA 
                   
               
               
                   
                 GACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGUCUAG 
                   
               
               
                   
               
               
                 Cas9 bare 
                 GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAG 
                 10 
               
               
                 coding 
                 CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA 
                   
               
               
                 sequence 
                 GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC 
                   
               
               
                   
                 AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG 
                   
               
               
                   
                 AGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAA 
                   
               
               
                   
                 GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG 
                   
               
               
                   
                 GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU 
                   
               
               
                   
                 CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA 
                   
               
               
                   
                 AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC 
                   
               
               
                   
                 GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU 
                   
               
               
                   
                 GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA 
                   
               
               
                   
                 GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA 
                   
               
               
                   
                 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG 
                   
               
               
                   
                 CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA 
                   
               
               
                   
                 ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC 
                   
               
               
                   
                 GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG 
                   
               
               
                   
                 AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA 
                   
               
               
                   
                 AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA 
                   
               
               
                   
                 CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA 
                   
               
               
                   
                 CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC 
                   
               
               
                   
                 UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC 
                   
               
               
                   
                 CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU 
                   
               
               
                   
                 GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC 
                   
               
               
                   
                 CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC 
                   
               
               
                   
                 CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU 
                   
               
               
                   
                 GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA 
                   
               
               
                   
                 ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA 
                   
               
               
                   
                 GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA 
                   
               
               
                   
                 CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC 
                   
               
               
                   
                 UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG 
                   
               
               
                   
                 CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG 
                   
               
               
                   
                 ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA 
                   
               
               
                   
                 AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC 
                   
               
               
                   
                 GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC 
                   
               
               
                   
                 AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG 
                   
               
               
                   
                 AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA 
                   
               
               
                   
                 AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU 
                   
               
               
                   
                 CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU 
                   
               
               
                   
                 ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG 
                   
               
               
                   
                 AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU 
                   
               
               
                   
                 CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA 
                   
               
               
                   
                 AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC 
                   
               
               
                   
                 GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC 
                   
               
               
                   
                 AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG 
                   
               
               
                   
                 ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG 
                   
               
               
                   
                 GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA 
                   
               
               
                   
                 AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG 
                   
               
               
                   
                 ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA 
                   
               
               
                   
                 GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG 
                   
               
               
                   
                 AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA 
                   
               
               
                   
                 GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC 
                   
               
               
                   
                 GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACA 
                   
               
               
                   
                 GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA 
                   
               
               
                   
                 ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG 
                   
               
               
                   
                 ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG 
                   
               
               
                   
                 ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA 
                   
               
               
                   
                 ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA 
                   
               
               
                   
                 AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA 
                   
               
               
                   
                 GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA 
                   
               
               
                   
                 ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC 
                   
               
               
                   
                 AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA 
                   
               
               
                   
                 CUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGA 
                   
               
               
                   
                 CGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGA 
                   
               
               
                   
                 ACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUC 
                   
               
               
                   
                 AAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAA 
                   
               
               
                   
                 GCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAG 
                   
               
               
                   
                 AGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUC 
                   
               
               
                   
                 AAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU 
                   
               
               
                   
                 CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACG 
                   
               
               
                   
                 AAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUG 
                   
               
               
                   
                 AAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUU 
                   
               
               
                   
                 CUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACG 
                   
               
               
                   
                 ACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAG 
                   
               
               
                   
                 AAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUA 
                   
               
               
                   
                 CAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAAC 
                   
               
               
                   
                 AGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGC 
                   
               
               
                   
                 AACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAA 
                   
               
               
                   
                 CGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAG 
                   
               
               
                   
                 AAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCA 
                   
               
               
                   
                 ACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGU 
                   
               
               
                   
                 CAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAA 
                   
               
               
                   
                 GCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA 
                   
               
               
                   
                 AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAG 
                   
               
               
                   
                 CCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCG 
                   
               
               
                   
                 AAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUG 
                   
               
               
                   
                 CUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAA 
                   
               
               
                   
                 CCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCA 
                   
               
               
                   
                 AGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC 
                   
               
               
                   
                 GAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGG 
                   
               
               
                   
                 AGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGU 
                   
               
               
                   
                 ACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUG 
                   
               
               
                   
                 AAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGU 
                   
               
               
                   
                 CGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGA 
                   
               
               
                   
                 UCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAAC 
                   
               
               
                   
                 CUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA 
                   
               
               
                   
                 GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCA 
                   
               
               
                   
                 CACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC 
                   
               
               
                   
                 GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGA 
                   
               
               
                   
                 AGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGAC 
                   
               
               
                   
                 UGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC 
                   
               
               
                   
                 GGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGUC 
                   
               
               
                   
               
               
                 Cas9 nickase 
                 GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAG 
                 11 
               
               
                 bare coding 
                 CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA 
                   
               
               
                 sequence 
                 GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC 
                   
               
               
                   
                 AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG 
                   
               
               
                   
                 AGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAA 
                   
               
               
                   
                 GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG 
                   
               
               
                   
                 GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU 
                   
               
               
                   
                 CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA 
                   
               
               
                   
                 AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC 
                   
               
               
                   
                 GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU 
                   
               
               
                   
                 GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA 
                   
               
               
                   
                 GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA 
                   
               
               
                   
                 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG 
                   
               
               
                   
                 CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA 
                   
               
               
                   
                 ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC 
                   
               
               
                   
                 GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG 
                   
               
               
                   
                 AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA 
                   
               
               
                   
                 AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA 
                   
               
               
                   
                 CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA 
                   
               
               
                   
                 CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC 
                   
               
               
                   
                 UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC 
                   
               
               
                   
                 CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU 
                   
               
               
                   
                 GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC 
                   
               
               
                   
                 CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC 
                   
               
               
                   
                 CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU 
                   
               
               
                   
                 GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA 
                   
               
               
                   
                 ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA 
                   
               
               
                   
                 GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA 
                   
               
               
                   
                 CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC 
                   
               
               
                   
                 UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG 
                   
               
               
                   
                 CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG 
                   
               
               
                   
                 ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA 
                   
               
               
                   
                 AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC 
                   
               
               
                   
                 GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC 
                   
               
               
                   
                 AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG 
                   
               
               
                   
                 AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA 
                   
               
               
                   
                 AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU 
                   
               
               
                   
                 CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU 
                   
               
               
                   
                 ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG 
                   
               
               
                   
                 AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU 
                   
               
               
                   
                 CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA 
                   
               
               
                   
                 AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC 
                   
               
               
                   
                 GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC 
                   
               
               
                   
                 AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG 
                   
               
               
                   
                 ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG 
                   
               
               
                   
                 GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA 
                   
               
               
                   
                 AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG 
                   
               
               
                   
                 ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA 
                   
               
               
                   
                 GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG 
                   
               
               
                   
                 AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA 
                   
               
               
                   
                 GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC 
                   
               
               
                   
                 GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACA 
                   
               
               
                   
                 GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA 
                   
               
               
                   
                 ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG 
                   
               
               
                   
                 ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG 
                   
               
               
                   
                 ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA 
                   
               
               
                   
                 ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA 
                   
               
               
                   
                 AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA 
                   
               
               
                   
                 GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA 
                   
               
               
                   
                 ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC 
                   
               
               
                   
                 AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA 
                   
               
               
                   
                 CUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGA 
                   
               
               
                   
                 CGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGA 
                   
               
               
                   
                 ACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUC 
                   
               
               
                   
                 AAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAA 
                   
               
               
                   
                 GCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAG 
                   
               
               
                   
                 AGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUC 
                   
               
               
                   
                 AAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU 
                   
               
               
                   
                 CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACG 
                   
               
               
                   
                 AAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUG 
                   
               
               
                   
                 AAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUU 
                   
               
               
                   
                 CUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACG 
                   
               
               
                   
                 ACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAG 
                   
               
               
                   
                 AAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUA 
                   
               
               
                   
                 CAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAAC 
                   
               
               
                   
                 AGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGC 
                   
               
               
                   
                 AACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAA 
                   
               
               
                   
                 CGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAG 
                   
               
               
                   
                 AAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCA 
                   
               
               
                   
                 ACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGU 
                   
               
               
                   
                 CAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAA 
                   
               
               
                   
                 GCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA 
                   
               
               
                   
                 AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAG 
                   
               
               
                   
                 CCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCG 
                   
               
               
                   
                 AAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUG 
                   
               
               
                   
                 CUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAA 
                   
               
               
                   
                 CCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCA 
                   
               
               
                   
                 AGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC 
                   
               
               
                   
                 GAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGG 
                   
               
               
                   
                 AGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGU 
                   
               
               
                   
                 ACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUG 
                   
               
               
                   
                 AAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGU 
                   
               
               
                   
                 CGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGA 
                   
               
               
                   
                 UCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAAC 
                   
               
               
                   
                 CUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA 
                   
               
               
                   
                 GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCA 
                   
               
               
                   
                 CACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC 
                   
               
               
                   
                 GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGA 
                   
               
               
                   
                 AGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGAC 
                   
               
               
                   
                 UGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC 
                   
               
               
                   
                 GGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGUC 
                   
               
               
                   
               
               
                 dCas9 bare 
                 GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAG 
                 12 
               
               
                 coding 
                 CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA 
                   
               
               
                 sequence 
                 GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC 
                   
               
               
                   
                 AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG 
                   
               
               
                   
                 AGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAA 
                   
               
               
                   
                 GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG 
                   
               
               
                   
                 GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU 
                   
               
               
                   
                 CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA 
                   
               
               
                   
                 AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC 
                   
               
               
                   
                 GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU 
                   
               
               
                   
                 GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA 
                   
               
               
                   
                 GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA 
                   
               
               
                   
                 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG 
                   
               
               
                   
                 CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA 
                   
               
               
                   
                 ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC 
                   
               
               
                   
                 GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG 
                   
               
               
                   
                 AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA 
                   
               
               
                   
                 AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA 
                   
               
               
                   
                 CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA 
                   
               
               
                   
                 CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC 
                   
               
               
                   
                 UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC 
                   
               
               
                   
                 CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU 
                   
               
               
                   
                 GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC 
                   
               
               
                   
                 CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC 
                   
               
               
                   
                 CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU 
                   
               
               
                   
                 GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA 
                   
               
               
                   
                 ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA 
                   
               
               
                   
                 GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA 
                   
               
               
                   
                 CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC 
                   
               
               
                   
                 UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG 
                   
               
               
                   
                 CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG 
                   
               
               
                   
                 ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA 
                   
               
               
                   
                 AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC 
                   
               
               
                   
                 GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC 
                   
               
               
                   
                 AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG 
                   
               
               
                   
                 AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA 
                   
               
               
                   
                 AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU 
                   
               
               
                   
                 CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU 
                   
               
               
                   
                 ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG 
                   
               
               
                   
                 AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU 
                   
               
               
                   
                 CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA 
                   
               
               
                   
                 AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC 
                   
               
               
                   
                 GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC 
                   
               
               
                   
                 AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG 
                   
               
               
                   
                 ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG 
                   
               
               
                   
                 GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA 
                   
               
               
                   
                 AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG 
                   
               
               
                   
                 ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA 
                   
               
               
                   
                 GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG 
                   
               
               
                   
                 AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA 
                   
               
               
                   
                 GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC 
                   
               
               
                   
                 GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACA 
                   
               
               
                   
                 GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA 
                   
               
               
                   
                 ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG 
                   
               
               
                   
                 ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG 
                   
               
               
                   
                 ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA 
                   
               
               
                   
                 ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA 
                   
               
               
                   
                 AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA 
                   
               
               
                   
                 GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA 
                   
               
               
                   
                 ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC 
                   
               
               
                   
                 AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA 
                   
               
               
                   
                 CUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGG 
                   
               
               
                   
                 ACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAG 
                   
               
               
                   
                 AACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGU 
                   
               
               
                   
                 CAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAA 
                   
               
               
                   
                 AGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCA 
                   
               
               
                   
                 GAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAU 
                   
               
               
                   
                 CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACG 
                   
               
               
                   
                 UCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGAC 
                   
               
               
                   
                 GAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACU 
                   
               
               
                   
                 GAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGU 
                   
               
               
                   
                 UCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACAC 
                   
               
               
                   
                 GACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAA 
                   
               
               
                   
                 GAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACU 
                   
               
               
                   
                 ACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAA 
                   
               
               
                   
                 CAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG 
                   
               
               
                   
                 CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAA 
                   
               
               
                   
                 ACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGA 
                   
               
               
                   
                 GAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGC 
                   
               
               
                   
                 AACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCG 
                   
               
               
                   
                 UCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAA 
                   
               
               
                   
                 AGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAG 
                   
               
               
                   
                 AAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACA 
                   
               
               
                   
                 GCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUC 
                   
               
               
                   
                 GAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACU 
                   
               
               
                   
                 GCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGA 
                   
               
               
                   
                 ACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC 
                   
               
               
                   
                 AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUU 
                   
               
               
                   
                 CGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAG 
                   
               
               
                   
                 GAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAG 
                   
               
               
                   
                 UACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCU 
                   
               
               
                   
                 GAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCG 
                   
               
               
                   
                 UCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAG 
                   
               
               
                   
                 AUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAA 
                   
               
               
                   
                 CCUGGACAAGCUCCUGAGCGCAUACAACAAGCACAGAGACA 
                   
               
               
                   
                 AGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUC 
                   
               
               
                   
                 ACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUU 
                   
               
               
                   
                 CGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGG 
                   
               
               
                   
                 AAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGA 
                   
               
               
                   
                 CUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGA 
                   
               
               
                   
                 CGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGUC 
                   
               
               
                   
               
               
                 Amino acid 
                 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK 
                 13 
               
               
                 sequence of 
                 KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE 
                   
               
               
                 Cas9 
                 MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT 
                   
               
               
                 (without 
                 IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS 
                   
               
               
                 NLS) 
                 DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL 
                   
               
               
                   
                 IAQLPGEKKNGLFGNLIALSLGLTPNTKSNFDLAEDAKLQLSKDT 
                   
               
               
                   
                 YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA 
                   
               
               
                   
                 PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA 
                   
               
               
                   
                 GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF 
                   
               
               
                   
                 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV 
                   
               
               
                   
                 GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT 
                   
               
               
                   
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL 
                   
               
               
                   
                 SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED 
                   
               
               
                   
                 RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG 
                   
               
               
                   
                 KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 
                   
               
               
                   
                 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENTVIEMARE 
                   
               
               
                   
                 NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL 
                   
               
               
                   
                 YLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK 
                   
               
               
                   
                 VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD 
                   
               
               
                   
                 NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY 
                   
               
               
                   
                 DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA 
                   
               
               
                   
                 YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK 
                   
               
               
                   
                 ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
                   
               
               
                   
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
                   
               
               
                   
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG 
                   
               
               
                   
                 ITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR 
                   
               
               
                   
                 MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ 
                   
               
               
                   
                 LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
                   
               
               
                   
                 REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 
                   
               
               
                   
                 HQSITGLYETRIDLSQLGGD 
                   
               
               
                   
               
               
                 Cas9 mRNA 
                 AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAA 
                 14 
               
               
                 ORF 
                 CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC 
                   
               
               
                 encoding 
                 CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC 
                   
               
               
                 SEQ ID NO: 
                 AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG 
                   
               
               
                 13 using 
                 CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA 
                   
               
               
                 minimal 
                 GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG 
                   
               
               
                 uridine 
                 CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG 
                   
               
               
                 codons as 
                 CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG 
                   
               
               
                 listed in 
                 ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC 
                   
               
               
                 Table 3, with 
                 GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA 
                   
               
               
                 start and 
                 CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC 
                   
               
               
                 stop codons 
                 UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC 
                   
               
               
                   
                 AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA 
                   
               
               
                   
                 CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU 
                   
               
               
                   
                 ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA 
                   
               
               
                   
                 GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG 
                   
               
               
                   
                 CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA 
                   
               
               
                   
                 AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG 
                   
               
               
                   
                 GGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGA 
                   
               
               
                   
                 AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG 
                   
               
               
                   
                 ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA 
                   
               
               
                   
                 GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU 
                   
               
               
                   
                 GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG 
                   
               
               
                   
                 CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC 
                   
               
               
                   
                 CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA 
                   
               
               
                   
                 GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA 
                   
               
               
                   
                 AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG 
                   
               
               
                   
                 GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU 
                   
               
               
                   
                 GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG 
                   
               
               
                   
                 ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC 
                   
               
               
                   
                 CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA 
                   
               
               
                   
                 AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA 
                   
               
               
                   
                 AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG 
                   
               
               
                   
                 UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG 
                   
               
               
                   
                 ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA 
                   
               
               
                   
                 AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG 
                   
               
               
                   
                 AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG 
                   
               
               
                   
                 GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU 
                   
               
               
                   
                 CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA 
                   
               
               
                   
                 UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA 
                   
               
               
                   
                 AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU 
                   
               
               
                   
                 CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU 
                   
               
               
                   
                 UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC 
                   
               
               
                   
                 GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA 
                   
               
               
                   
                 GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC 
                   
               
               
                   
                 UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA 
                   
               
               
                   
                 GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU 
                   
               
               
                   
                 CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA 
                   
               
               
                   
                 CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC 
                   
               
               
                   
                 AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA 
                   
               
               
                   
                 GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC 
                   
               
               
                   
                 ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA 
                   
               
               
                   
                 CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC 
                   
               
               
                   
                 AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC 
                   
               
               
                   
                 AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA 
                   
               
               
                   
                 AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGA 
                   
               
               
                   
                 AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA 
                   
               
               
                   
                 GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC 
                   
               
               
                   
                 CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA 
                   
               
               
                   
                 GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG 
                   
               
               
                   
                 ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC 
                   
               
               
                   
                 GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAG 
                   
               
               
                   
                 GACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAA 
                   
               
               
                   
                 GAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCG 
                   
               
               
                   
                 UCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCA 
                   
               
               
                   
                 AAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGC 
                   
               
               
                   
                 AGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCA 
                   
               
               
                   
                 UCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCAC 
                   
               
               
                   
                 GUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGA 
                   
               
               
                   
                 CGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC 
                   
               
               
                   
                 UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAG 
                   
               
               
                   
                 UUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACA 
                   
               
               
                   
                 CGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCA 
                   
               
               
                   
                 AGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGAC 
                   
               
               
                   
                 UACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGA 
                   
               
               
                   
                 ACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA 
                   
               
               
                   
                 GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCA 
                   
               
               
                   
                 AACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG 
                   
               
               
                   
                 AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCG 
                   
               
               
                   
                 CAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUC 
                   
               
               
                   
                 GUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGA 
                   
               
               
                   
                 AAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAA 
                   
               
               
                   
                 GAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGAC 
                   
               
               
                   
                 AGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGU 
                   
               
               
                   
                 CGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAAC 
                   
               
               
                   
                 UGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG 
                   
               
               
                   
                 AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGU 
                   
               
               
                   
                 CAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGU 
                   
               
               
                   
                 UCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCA 
                   
               
               
                   
                 GGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAA 
                   
               
               
                   
                 GUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGC 
                   
               
               
                   
                 UGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC 
                   
               
               
                   
                 GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACA 
                   
               
               
                   
                 GAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAA 
                   
               
               
                   
                 ACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGAC 
                   
               
               
                   
                 AAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUU 
                   
               
               
                   
                 CACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACU 
                   
               
               
                   
                 UCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAG 
                   
               
               
                   
                 GAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGG 
                   
               
               
                   
                 ACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAG 
                   
               
               
                   
                 ACUAG 
                   
               
               
                   
               
               
                 Cas9 coding 
                 GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAG 
                 15 
               
               
                 sequence 
                 CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA 
                   
               
               
                 encoding 
                 GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC 
                   
               
               
                 SEQ ID NO: 
                 AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG 
                   
               
               
                 13 using 
                 AGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAA 
                   
               
               
                 minimal 
                 GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG 
                   
               
               
                 uridine 
                 GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU 
                   
               
               
                 codons as 
                 CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA 
                   
               
               
                 listed in 
                 AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC 
                   
               
               
                 Table 3 (no 
                 GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU 
                   
               
               
                 start or stop 
                 GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA 
                   
               
               
                 codons; 
                 GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA 
                   
               
               
                 suitable for 
                 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG 
                   
               
               
                 inclusion in 
                 CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA 
                   
               
               
                 fusion 
                 ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC 
                   
               
               
                 protein 
                 GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG 
                   
               
               
                 coding 
                 AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA 
                   
               
               
                 sequence) 
                 AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA 
                   
               
               
                   
                 CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA 
                   
               
               
                   
                 CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC 
                   
               
               
                   
                 UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC 
                   
               
               
                   
                 CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU 
                   
               
               
                   
                 GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC 
                   
               
               
                   
                 CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC 
                   
               
               
                   
                 CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU 
                   
               
               
                   
                 GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA 
                   
               
               
                   
                 ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA 
                   
               
               
                   
                 GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA 
                   
               
               
                   
                 CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC 
                   
               
               
                   
                 UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG 
                   
               
               
                   
                 CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG 
                   
               
               
                   
                 ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA 
                   
               
               
                   
                 AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC 
                   
               
               
                   
                 GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC 
                   
               
               
                   
                 AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG 
                   
               
               
                   
                 AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA 
                   
               
               
                   
                 AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU 
                   
               
               
                   
                 CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU 
                   
               
               
                   
                 ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG 
                   
               
               
                   
                 AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU 
                   
               
               
                   
                 CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA 
                   
               
               
                   
                 AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC 
                   
               
               
                   
                 GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC 
                   
               
               
                   
                 AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG 
                   
               
               
                   
                 ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG 
                   
               
               
                   
                 GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA 
                   
               
               
                   
                 AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG 
                   
               
               
                   
                 ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA 
                   
               
               
                   
                 GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG 
                   
               
               
                   
                 AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA 
                   
               
               
                   
                 GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC 
                   
               
               
                   
                 GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACA 
                   
               
               
                   
                 GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA 
                   
               
               
                   
                 ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG 
                   
               
               
                   
                 ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG 
                   
               
               
                   
                 ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA 
                   
               
               
                   
                 ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA 
                   
               
               
                   
                 AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA 
                   
               
               
                   
                 GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA 
                   
               
               
                   
                 ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC 
                   
               
               
                   
                 AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA 
                   
               
               
                   
                 CUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGA 
                   
               
               
                   
                 CGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGA 
                   
               
               
                   
                 ACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUC 
                   
               
               
                   
                 AAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAA 
                   
               
               
                   
                 GCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAG 
                   
               
               
                   
                 AGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUC 
                   
               
               
                   
                 AAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU 
                   
               
               
                   
                 CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACG 
                   
               
               
                   
                 AAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUG 
                   
               
               
                   
                 AAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUU 
                   
               
               
                   
                 CUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACG 
                   
               
               
                   
                 ACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAG 
                   
               
               
                   
                 AAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUA 
                   
               
               
                   
                 CAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAAC 
                   
               
               
                   
                 AGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGC 
                   
               
               
                   
                 AACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAA 
                   
               
               
                   
                 CGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAG 
                   
               
               
                   
                 AAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCA 
                   
               
               
                   
                 ACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGU 
                   
               
               
                   
                 CAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAA 
                   
               
               
                   
                 GCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA 
                   
               
               
                   
                 AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAG 
                   
               
               
                   
                 CCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCG 
                   
               
               
                   
                 AAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUG 
                   
               
               
                   
                 CUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAA 
                   
               
               
                   
                 CCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCA 
                   
               
               
                   
                 AGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC 
                   
               
               
                   
                 GAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGG 
                   
               
               
                   
                 AGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGU 
                   
               
               
                   
                 ACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUG 
                   
               
               
                   
                 AAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGU 
                   
               
               
                   
                 CGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGA 
                   
               
               
                   
                 UCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAAC 
                   
               
               
                   
                 CUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA 
                   
               
               
                   
                 GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCA 
                   
               
               
                   
                 CACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC 
                   
               
               
                   
                 GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGA 
                   
               
               
                   
                 AGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGAC 
                   
               
               
                   
                 UGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC 
                   
               
               
                   
               
               
                 Amino acid 
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK 
                 16 
               
               
                 sequence of 
                 KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE 
                   
               
               
                 Cas9 nickase 
                 MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT 
                   
               
               
                 (without 
                 IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS 
                   
               
               
                 NLS) 
                 DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL 
                   
               
               
                   
                 IAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT 
                   
               
               
                   
                 YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA 
                   
               
               
                   
                 PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA 
                   
               
               
                   
                 GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF 
                   
               
               
                   
                 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV 
                   
               
               
                   
                 GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT 
                   
               
               
                   
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL 
                   
               
               
                   
                 SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED 
                   
               
               
                   
                 RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG 
                   
               
               
                   
                 KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 
                   
               
               
                   
                 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE 
                   
               
               
                   
                 NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL 
                   
               
               
                   
                 YLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK 
                   
               
               
                   
                 VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD 
                   
               
               
                   
                 NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY 
                   
               
               
                   
                 DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA 
                   
               
               
                   
                 YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK 
                   
               
               
                   
                 ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
                   
               
               
                   
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
                   
               
               
                   
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG 
                   
               
               
                   
                 ITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR 
                   
               
               
                   
                 MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ 
                   
               
               
                   
                 LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
                   
               
               
                   
                 REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 
                   
               
               
                   
                 HQSITGLYETRIDLSQLGGD 
                   
               
               
                   
               
               
                 Cas9 nickase 
                 AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAA 
                 17 
               
               
                 mRNA ORF 
                 CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC 
                   
               
               
                 encoding 
                 CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC 
                   
               
               
                 SEQ ID NO: 
                 AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG 
                   
               
               
                 16 using 
                 CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA 
                   
               
               
                 minimal 
                 GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG 
                   
               
               
                 uridine 
                 CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG 
                   
               
               
                 codons as 
                 CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG 
                   
               
               
                 listed in 
                 ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC 
                   
               
               
                 Table 3, with 
                 GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA 
                   
               
               
                 start and 
                 CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC 
                   
               
               
                 stop codons 
                 UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC 
                   
               
               
                   
                 AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA 
                   
               
               
                   
                 CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU 
                   
               
               
                   
                 ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA 
                   
               
               
                   
                 GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG 
                   
               
               
                   
                 CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA 
                   
               
               
                   
                 AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG 
                   
               
               
                   
                 GGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGA 
                   
               
               
                   
                 AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG 
                   
               
               
                   
                 ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA 
                   
               
               
                   
                 GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU 
                   
               
               
                   
                 GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG 
                   
               
               
                   
                 CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC 
                   
               
               
                   
                 CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA 
                   
               
               
                   
                 GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA 
                   
               
               
                   
                 AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG 
                   
               
               
                   
                 GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU 
                   
               
               
                   
                 GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG 
                   
               
               
                   
                 ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC 
                   
               
               
                   
                 CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA 
                   
               
               
                   
                 AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA 
                   
               
               
                   
                 AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG 
                   
               
               
                   
                 UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG 
                   
               
               
                   
                 ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA 
                   
               
               
                   
                 AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG 
                   
               
               
                   
                 AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG 
                   
               
               
                   
                 GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU 
                   
               
               
                   
                 CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA 
                   
               
               
                   
                 UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA 
                   
               
               
                   
                 AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU 
                   
               
               
                   
                 CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU 
                   
               
               
                   
                 UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC 
                   
               
               
                   
                 GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA 
                   
               
               
                   
                 GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC 
                   
               
               
                   
                 UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA 
                   
               
               
                   
                 GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU 
                   
               
               
                   
                 CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA 
                   
               
               
                   
                 CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC 
                   
               
               
                   
                 AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA 
                   
               
               
                   
                 GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC 
                   
               
               
                   
                 ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA 
                   
               
               
                   
                 CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC 
                   
               
               
                   
                 AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC 
                   
               
               
                   
                 AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA 
                   
               
               
                   
                 AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGA 
                   
               
               
                   
                 AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA 
                   
               
               
                   
                 GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC 
                   
               
               
                   
                 CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA 
                   
               
               
                   
                 GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG 
                   
               
               
                   
                 ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC 
                   
               
               
                   
                 GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAG 
                   
               
               
                   
                 GACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAA 
                   
               
               
                   
                 GAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCG 
                   
               
               
                   
                 UCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCA 
                   
               
               
                   
                 AAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGC 
                   
               
               
                   
                 AGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCA 
                   
               
               
                   
                 UCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCAC 
                   
               
               
                   
                 GUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGA 
                   
               
               
                   
                 CGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC 
                   
               
               
                   
                 UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAG 
                   
               
               
                   
                 UUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACA 
                   
               
               
                   
                 CGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCA 
                   
               
               
                   
                 AGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGAC 
                   
               
               
                   
                 UACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGA 
                   
               
               
                   
                 ACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA 
                   
               
               
                   
                 GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCA 
                   
               
               
                   
                 AACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG 
                   
               
               
                   
                 AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCG 
                   
               
               
                   
                 CAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUC 
                   
               
               
                   
                 GUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGA 
                   
               
               
                   
                 AAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAA 
                   
               
               
                   
                 GAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGAC 
                   
               
               
                   
                 AGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGU 
                   
               
               
                   
                 CGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAAC 
                   
               
               
                   
                 UGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG 
                   
               
               
                   
                 AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGU 
                   
               
               
                   
                 CAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGU 
                   
               
               
                   
                 UCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCA 
                   
               
               
                   
                 GGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAA 
                   
               
               
                   
                 GUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGC 
                   
               
               
                   
                 UGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC 
                   
               
               
                   
                 GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACA 
                   
               
               
                   
                 GAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAA 
                   
               
               
                   
                 ACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGAC 
                   
               
               
                   
                 AAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUU 
                   
               
               
                   
                 CACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACU 
                   
               
               
                   
                 UCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAG 
                   
               
               
                   
                 GAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGG 
                   
               
               
                   
                 ACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAG 
                   
               
               
                   
                 ACUAG 
                   
               
               
                   
               
               
                 Cas9 nickase 
                 GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAG 
                 18 
               
               
                 coding 
                 CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA 
                   
               
               
                 sequence 
                 GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC 
                   
               
               
                 encoding 
                 AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG 
                   
               
               
                 SEQ ID NO: 
                 AGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAA 
                   
               
               
                 16 using 
                 GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG 
                   
               
               
                 minimal 
                 GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU 
                   
               
               
                 uridine 
                 CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA 
                   
               
               
                 codons as 
                 AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC 
                   
               
               
                 listed in 
                 GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU 
                   
               
               
                 Table 3 (no 
                 GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA 
                   
               
               
                 start or stop 
                 GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA 
                   
               
               
                 codons; 
                 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG 
                   
               
               
                 suitable for 
                 CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA 
                   
               
               
                 inclusion in 
                 ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC 
                   
               
               
                 fusion 
                 GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG 
                   
               
               
                 protein 
                 AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA 
                   
               
               
                 coding 
                 AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA 
                   
               
               
                 sequence) 
                 CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA 
                   
               
               
                   
                 CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC 
                   
               
               
                   
                 UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC 
                   
               
               
                   
                 CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU 
                   
               
               
                   
                 GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC 
                   
               
               
                   
                 CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC 
                   
               
               
                   
                 CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU 
                   
               
               
                   
                 GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA 
                   
               
               
                   
                 ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA 
                   
               
               
                   
                 GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA 
                   
               
               
                   
                 CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC 
                   
               
               
                   
                 UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG 
                   
               
               
                   
                 CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG 
                   
               
               
                   
                 ACAGGAAGACUUCUACCCGUBCCUGAAGGACAACAGAGAAA 
                   
               
               
                   
                 AGAUCGAAAAGAUCCUGACAUBCAGAAUCCCGUACUACGUC 
                   
               
               
                   
                 GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC 
                   
               
               
                   
                 AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG 
                   
               
               
                   
                 AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA 
                   
               
               
                   
                 AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU 
                   
               
               
                   
                 CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU 
                   
               
               
                   
                 ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG 
                   
               
               
                   
                 AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU 
                   
               
               
                   
                 CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA 
                   
               
               
                   
                 AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC 
                   
               
               
                   
                 GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC 
                   
               
               
                   
                 AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG 
                   
               
               
                   
                 ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG 
                   
               
               
                   
                 GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA 
                   
               
               
                   
                 AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG 
                   
               
               
                   
                 ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA 
                   
               
               
                   
                 GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG 
                   
               
               
                   
                 AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA 
                   
               
               
                   
                 GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC 
                   
               
               
                   
                 GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACA 
                   
               
               
                   
                 GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA 
                   
               
               
                   
                 ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG 
                   
               
               
                   
                 ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG 
                   
               
               
                   
                 ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA 
                   
               
               
                   
                 ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA 
                   
               
               
                   
                 AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA 
                   
               
               
                   
                 GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA 
                   
               
               
                   
                 ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC 
                   
               
               
                   
                 AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA 
                   
               
               
                   
                 CUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGA 
                   
               
               
                   
                 CGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGA 
                   
               
               
                   
                 ACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUC 
                   
               
               
                   
                 AAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAA 
                   
               
               
                   
                 GCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAG 
                   
               
               
                   
                 AGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUC 
                   
               
               
                   
                 AAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU 
                   
               
               
                   
                 CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACG 
                   
               
               
                   
                 AAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUG 
                   
               
               
                   
                 AAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUU 
                   
               
               
                   
                 CUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACG 
                   
               
               
                   
                 ACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAG 
                   
               
               
                   
                 AAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUA 
                   
               
               
                   
                 CAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAAC 
                   
               
               
                   
                 AGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGC 
                   
               
               
                   
                 AACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAA 
                   
               
               
                   
                 CGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAG 
                   
               
               
                   
                 AAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCA 
                   
               
               
                   
                 ACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGU 
                   
               
               
                   
                 CAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAA 
                   
               
               
                   
                 GCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA 
                   
               
               
                   
                 AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAG 
                   
               
               
                   
                 CCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCG 
                   
               
               
                   
                 AAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUG 
                   
               
               
                   
                 CUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAA 
                   
               
               
                   
                 CCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCA 
                   
               
               
                   
                 AGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC 
                   
               
               
                   
                 GAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGG 
                   
               
               
                   
                 AGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGU 
                   
               
               
                   
                 ACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUG 
                   
               
               
                   
                 AAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGU 
                   
               
               
                   
                 CGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGA 
                   
               
               
                   
                 UCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAAC 
                   
               
               
                   
                 CUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA 
                   
               
               
                   
                 GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCA 
                   
               
               
                   
                 CACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC 
                   
               
               
                   
                 GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGA 
                   
               
               
                   
                 AGUCCUGGACOCAACACUGAUCCACCAGAGCAUCACAGGAC 
                   
               
               
                   
                 UGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC 
                   
               
               
                   
               
               
                 Amino acid 
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK 
                 19 
               
               
                 sequence of 
                 KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE 
                   
               
               
                 dCas9 
                 MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT 
                   
               
               
                 (without 
                 IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS 
                   
               
               
                 NLS) 
                 DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL 
                   
               
               
                   
                 IAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT 
                   
               
               
                   
                 YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA 
                   
               
               
                   
                 PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA 
                   
               
               
                   
                 GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF 
                   
               
               
                   
                 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV 
                   
               
               
                   
                 GPLARGNSRFAWMTRKSEETTTPWNFEEVVDKGASAQSFIERMT 
                   
               
               
                   
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL 
                   
               
               
                   
                 SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED 
                   
               
               
                   
                 RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG 
                   
               
               
                   
                 KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 
                   
               
               
                   
                 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE 
                   
               
               
                   
                 NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL 
                   
               
               
                   
                 YLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK 
                   
               
               
                   
                 VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD 
                   
               
               
                   
                 NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY 
                   
               
               
                   
                 DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA 
                   
               
               
                   
                 YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK 
                   
               
               
                   
                 ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
                   
               
               
                   
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
                   
               
               
                   
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG 
                   
               
               
                   
                 ITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR 
                   
               
               
                   
                 MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ 
                   
               
               
                   
                 LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
                   
               
               
                   
                 REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 
                   
               
               
                   
                 HQSITGLYETRIDLSQLGGD 
                   
               
               
                   
               
               
                 dCas9 
                 AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAA 
                 20 
               
               
                 mRNA ORF 
                 CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC 
                   
               
               
                 encoding 
                 CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC 
                   
               
               
                 SEQ ID NO: 
                 AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG 
                   
               
               
                 19 using 
                 CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA 
                   
               
               
                 minimal 
                 GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG 
                   
               
               
                 uridine 
                 CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG 
                   
               
               
                 codons as 
                 CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG 
                   
               
               
                 listed in 
                 ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC 
                   
               
               
                 Table 3, with 
                 GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA 
                   
               
               
                 start and 
                 CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC 
                   
               
               
                 stop codons 
                 UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC 
                   
               
               
                   
                 AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA 
                   
               
               
                   
                 CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU 
                   
               
               
                   
                 ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA 
                   
               
               
                   
                 GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG 
                   
               
               
                   
                 CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA 
                   
               
               
                   
                 AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG 
                   
               
               
                   
                 GGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGA 
                   
               
               
                   
                 AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG 
                   
               
               
                   
                 ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA 
                   
               
               
                   
                 GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU 
                   
               
               
                   
                 GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG 
                   
               
               
                   
                 CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC 
                   
               
               
                   
                 CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA 
                   
               
               
                   
                 GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA 
                   
               
               
                   
                 AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG 
                   
               
               
                   
                 GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU 
                   
               
               
                   
                 GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG 
                   
               
               
                   
                 ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC 
                   
               
               
                   
                 CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA 
                   
               
               
                   
                 AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA 
                   
               
               
                   
                 AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG 
                   
               
               
                   
                 UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG 
                   
               
               
                   
                 ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA 
                   
               
               
                   
                 AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG 
                   
               
               
                   
                 AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG 
                   
               
               
                   
                 GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU 
                   
               
               
                   
                 CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA 
                   
               
               
                   
                 UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA 
                   
               
               
                   
                 AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU 
                   
               
               
                   
                 CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU 
                   
               
               
                   
                 UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC 
                   
               
               
                   
                 GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA 
                   
               
               
                   
                 GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC 
                   
               
               
                   
                 UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA 
                   
               
               
                   
                 GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU 
                   
               
               
                   
                 CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA 
                   
               
               
                   
                 CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC 
                   
               
               
                   
                 AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA 
                   
               
               
                   
                 GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC 
                   
               
               
                   
                 ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA 
                   
               
               
                   
                 CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC 
                   
               
               
                   
                 AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC 
                   
               
               
                   
                 AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA 
                   
               
               
                   
                 AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGA 
                   
               
               
                   
                 AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA 
                   
               
               
                   
                 GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC 
                   
               
               
                   
                 CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA 
                   
               
               
                   
                 GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG 
                   
               
               
                   
                 ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC 
                   
               
               
                   
                 GACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAA 
                   
               
               
                   
                 GGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACA 
                   
               
               
                   
                 AGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUC 
                   
               
               
                   
                 GUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGC 
                   
               
               
                   
                 AAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGG 
                   
               
               
                   
                 CAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUC 
                   
               
               
                   
                 AUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCA 
                   
               
               
                   
                 CGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACG 
                   
               
               
                   
                 ACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA 
                   
               
               
                   
                 CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCA 
                   
               
               
                   
                 GUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCAC 
                   
               
               
                   
                 ACGACGCAUACCUGAACGCAGUCGUCGGAACAGGACUGAUC 
                   
               
               
                   
                 AAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGA 
                   
               
               
                   
                 CUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCG 
                   
               
               
                   
                 AACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUAC 
                   
               
               
                   
                 AGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGC 
                   
               
               
                   
                 AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACG 
                   
               
               
                   
                 GAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUC 
                   
               
               
                   
                 GCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAU 
                   
               
               
                   
                 CGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGG 
                   
               
               
                   
                 AAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCA 
                   
               
               
                   
                 AGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGA 
                   
               
               
                   
                 CAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGG 
                   
               
               
                   
                 UCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAA 
                   
               
               
                   
                 CUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAA 
                   
               
               
                   
                 GAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAG 
                   
               
               
                   
                 UCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUG 
                   
               
               
                   
                 UUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGC 
                   
               
               
                   
                 AGGAGAACUGCAGAAGGGAAACGAACUGGCACUGGCGAGCA 
                   
               
               
                   
                 AGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAG 
                   
               
               
                   
                 CUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUU 
                   
               
               
                   
                 CGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAAC 
                   
               
               
                   
                 AGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCA 
                   
               
               
                   
                 AACCUGGACAAGGUCCUGAGCGCAUACAACAAGGACAGAGA 
                   
               
               
                   
                 CAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGU 
                   
               
               
                   
                 UCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC 
                   
               
               
                   
                 UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAA 
                   
               
               
                   
                 GGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAG 
                   
               
               
                   
                 GACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGA 
                   
               
               
                   
                 GACUAG 
                   
               
               
                   
               
               
                 dCas9 
                 GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAG 
                 21 
               
               
                 coding 
                 CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA 
                   
               
               
                 sequence 
                 GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC 
                   
               
               
                 encoding 
                 AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG 
                   
               
               
                 SEQ ID NO: 
                 AGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAA 
                   
               
               
                 19 using 
                 GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG 
                   
               
               
                 minimal 
                 GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU 
                   
               
               
                 uridine 
                 CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA 
                   
               
               
                 codons as 
                 AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC 
                   
               
               
                 listed in 
                 GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU 
                   
               
               
                 Table 3 (no 
                 GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA 
                   
               
               
                 start or stop 
                 GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA 
                   
               
               
                 codons; 
                 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG 
                   
               
               
                 suitable for 
                 CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA 
                   
               
               
                 inclusion in 
                 ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC 
                   
               
               
                 fusion 
                 GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG 
                   
               
               
                 protein 
                 AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA 
                   
               
               
                 coding 
                 AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA 
                   
               
               
                 sequence) 
                 CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA 
                   
               
               
                   
                 CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC 
                   
               
               
                   
                 UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC 
                   
               
               
                   
                 CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU 
                   
               
               
                   
                 GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC 
                   
               
               
                   
                 CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC 
                   
               
               
                   
                 CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU 
                   
               
               
                   
                 GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA 
                   
               
               
                   
                 ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA 
                   
               
               
                   
                 GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA 
                   
               
               
                   
                 CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC 
                   
               
               
                   
                 UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG 
                   
               
               
                   
                 CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG 
                   
               
               
                   
                 ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA 
                   
               
               
                   
                 AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC 
                   
               
               
                   
                 GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC 
                   
               
               
                   
                 AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG 
                   
               
               
                   
                 AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA 
                   
               
               
                   
                 AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU 
                   
               
               
                   
                 CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU 
                   
               
               
                   
                 ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG 
                   
               
               
                   
                 AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU 
                   
               
               
                   
                 CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA 
                   
               
               
                   
                 AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC 
                   
               
               
                   
                 GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC 
                   
               
               
                   
                 AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG 
                   
               
               
                   
                 ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG 
                   
               
               
                   
                 GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA 
                   
               
               
                   
                 AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG 
                   
               
               
                   
                 ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA 
                   
               
               
                   
                 GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG 
                   
               
               
                   
                 AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA 
                   
               
               
                   
                 GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC 
                   
               
               
                   
                 GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACA 
                   
               
               
                   
                 GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA 
                   
               
               
                   
                 ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG 
                   
               
               
                   
                 ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG 
                   
               
               
                   
                 ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA 
                   
               
               
                   
                 ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA 
                   
               
               
                   
                 AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA 
                   
               
               
                   
                 GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA 
                   
               
               
                   
                 ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC 
                   
               
               
                   
                 AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA 
                   
               
               
                   
                 CUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGG 
                   
               
               
                   
                 ACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAG 
                   
               
               
                   
                 AACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGU 
                   
               
               
                   
                 CAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAA 
                   
               
               
                   
                 AGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCA 
                   
               
               
                   
                 GAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAU 
                   
               
               
                   
                 CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACG 
                   
               
               
                   
                 UCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGAC 
                   
               
               
                   
                 GAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACU 
                   
               
               
                   
                 GAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGU 
                   
               
               
                   
                 UCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACAC 
                   
               
               
                   
                 GACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAA 
                   
               
               
                   
                 GAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACU 
                   
               
               
                   
                 ACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAA 
                   
               
               
                   
                 CAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG 
                   
               
               
                   
                 CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAA 
                   
               
               
                   
                 ACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGA 
                   
               
               
                   
                 GAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGC 
                   
               
               
                   
                 AACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCG 
                   
               
               
                   
                 UCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAA 
                   
               
               
                   
                 AGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAG 
                   
               
               
                   
                 AAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACA 
                   
               
               
                   
                 GCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUC 
                   
               
               
                   
                 GAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACU 
                   
               
               
                   
                 GCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGA 
                   
               
               
                   
                 ACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC 
                   
               
               
                   
                 AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUU 
                   
               
               
                   
                 CGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAG 
                   
               
               
                   
                 GAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAG 
                   
               
               
                   
                 UACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCU 
                   
               
               
                   
                 GAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCG 
                   
               
               
                   
                 UCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAG 
                   
               
               
                   
                 AUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAA 
                   
               
               
                   
                 CCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACA 
                   
               
               
                   
                 AGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUC 
                   
               
               
                   
                 ACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUU 
                   
               
               
                   
                 CGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGG 
                   
               
               
                   
                 AAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGA 
                   
               
               
                   
                 CUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGA 
                   
               
               
                   
                 CGGAGGAGGAAGC 
                   
               
               
                   
               
               
                 Amino acid 
                 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK 
                 22 
               
               
                 sequence of 
                 KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE 
                   
               
               
                 Cas9 with 
                 MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT 
                   
               
               
                 two nuclear 
                 IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS 
                   
               
               
                 localization 
                 DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL 
                   
               
               
                 signals as the 
                 IAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT 
                   
               
               
                 C-terminal 
                 YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA 
                   
               
               
                 amino acids 
                 PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA 
                   
               
               
                   
                 GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF 
                   
               
               
                   
                 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV 
                   
               
               
                   
                 GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT 
                   
               
               
                   
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL 
                   
               
               
                   
                 SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED 
                   
               
               
                   
                 RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG 
                   
               
               
                   
                 KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 
                   
               
               
                   
                 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE 
                   
               
               
                   
                 NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL 
                   
               
               
                   
                 YLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK 
                   
               
               
                   
                 VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD 
                   
               
               
                   
                 NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY 
                   
               
               
                   
                 DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA 
                   
               
               
                   
                 YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK 
                   
               
               
                   
                 ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
                   
               
               
                   
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
                   
               
               
                   
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG 
                   
               
               
                   
                 ITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR 
                   
               
               
                   
                 MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ 
                   
               
               
                   
                 LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
                   
               
               
                   
                 REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 
                   
               
               
                   
                 HQSITGLYETRIDLSQLGGD GSGSPKKKRKVDGSPKKKRKVDSG 
                   
               
               
                   
               
               
                 Cas9 mRNA 
                 AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAA 
                 23 
               
               
                 ORF 
                 CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC 
                   
               
               
                 encoding 
                 CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC 
                   
               
               
                 SEQ ID NO: 
                 AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG 
                   
               
               
                 22 using 
                 CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA 
                   
               
               
                 minimal 
                 GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG 
                   
               
               
                 uridine 
                 CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG 
                   
               
               
                 codons as 
                 CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG 
                   
               
               
                 listed in 
                 ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC 
                   
               
               
                 Table 3, with 
                 GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA 
                   
               
               
                 start and 
                 CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC 
                   
               
               
                 stop codons 
                 UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC 
                   
               
               
                   
                 AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA 
                   
               
               
                   
                 CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU 
                   
               
               
                   
                 ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA 
                   
               
               
                   
                 GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG 
                   
               
               
                   
                 CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA 
                   
               
               
                   
                 AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG 
                   
               
               
                   
                 GGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGA 
                   
               
               
                   
                 AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG 
                   
               
               
                   
                 ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA 
                   
               
               
                   
                 GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU 
                   
               
               
                   
                 GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG 
                   
               
               
                   
                 CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC 
                   
               
               
                   
                 CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA 
                   
               
               
                   
                 GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA 
                   
               
               
                   
                 AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG 
                   
               
               
                   
                 GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU 
                   
               
               
                   
                 GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG 
                   
               
               
                   
                 ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC 
                   
               
               
                   
                 CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA 
                   
               
               
                   
                 AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA 
                   
               
               
                   
                 AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG 
                   
               
               
                   
                 UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG 
                   
               
               
                   
                 ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA 
                   
               
               
                   
                 AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG 
                   
               
               
                   
                 AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG 
                   
               
               
                   
                 GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU 
                   
               
               
                   
                 CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA 
                   
               
               
                   
                 UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA 
                   
               
               
                   
                 AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU 
                   
               
               
                   
                 CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU 
                   
               
               
                   
                 UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC 
                   
               
               
                   
                 GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA 
                   
               
               
                   
                 GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC 
                   
               
               
                   
                 UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA 
                   
               
               
                   
                 GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU 
                   
               
               
                   
                 CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA 
                   
               
               
                   
                 CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC 
                   
               
               
                   
                 AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA 
                   
               
               
                   
                 GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC 
                   
               
               
                   
                 ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA 
                   
               
               
                   
                 CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC 
                   
               
               
                   
                 AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC 
                   
               
               
                   
                 AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA 
                   
               
               
                   
                 AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGA 
                   
               
               
                   
                 AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA 
                   
               
               
                   
                 GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC 
                   
               
               
                   
                 CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA 
                   
               
               
                   
                 GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG 
                   
               
               
                   
                 ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC 
                   
               
               
                   
                 GACUACGACGUCGACCACAUCGUCCCOCAGAGCUUCCUGAAG 
                   
               
               
                   
                 GACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAA 
                   
               
               
                   
                 GAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCG 
                   
               
               
                   
                 UCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCA 
                   
               
               
                   
                 AAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGC 
                   
               
               
                   
                 AGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCA 
                   
               
               
                   
                 UCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCAC 
                   
               
               
                   
                 GUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGA 
                   
               
               
                   
                 CGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC 
                   
               
               
                   
                 UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAG 
                   
               
               
                   
                 UUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACA 
                   
               
               
                   
                 CGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCA 
                   
               
               
                   
                 AGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGAC 
                   
               
               
                   
                 UACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGA 
                   
               
               
                   
                 ACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA 
                   
               
               
                   
                 GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCA 
                   
               
               
                   
                 AACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG 
                   
               
               
                   
                 AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCG 
                   
               
               
                   
                 CAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUC 
                   
               
               
                   
                 GUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGA 
                   
               
               
                   
                 AAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAA 
                   
               
               
                   
                 GAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGAC 
                   
               
               
                   
                 AGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGU 
                   
               
               
                   
                 CGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAAC 
                   
               
               
                   
                 UGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG 
                   
               
               
                   
                 AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGU 
                   
               
               
                   
                 CAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGU 
                   
               
               
                   
                 UCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCA 
                   
               
               
                   
                 GGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAA 
                   
               
               
                   
                 GUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGC 
                   
               
               
                   
                 UGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC 
                   
               
               
                   
                 GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACA 
                   
               
               
                   
                 GAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAA 
                   
               
               
                   
                 ACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGAC 
                   
               
               
                   
                 AAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUU 
                   
               
               
                   
                 CACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACU 
                   
               
               
                   
                 UCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAG 
                   
               
               
                   
                 GAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGG 
                   
               
               
                   
                 ACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAG 
                   
               
               
                   
                 ACGGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGA 
                   
               
               
                   
                 AGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGAUAG 
                   
               
               
                   
               
               
                 Cas9 coding 
                 GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAG 
                 24 
               
               
                 sequence 
                 CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA 
                   
               
               
                 encoding 
                 GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC 
                   
               
               
                 SEQ ID NO: 
                 AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG 
                   
               
               
                 23 using 
                 AGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAA 
                   
               
               
                 minimal 
                 GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG 
                   
               
               
                 uridine 
                 GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU 
                   
               
               
                 codons as 
                 CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA 
                   
               
               
                 listed in 
                 AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC 
                   
               
               
                 Table 3 (no 
                 GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU 
                   
               
               
                 start or stop 
                 GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA 
                   
               
               
                 codons; 
                 GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA 
                   
               
               
                 suitable for 
                 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG 
                   
               
               
                 inclusion in 
                 CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA 
                   
               
               
                 fusion 
                 ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC 
                   
               
               
                 protein 
                 GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG 
                   
               
               
                 coding 
                 AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA 
                   
               
               
                 sequence) 
                 AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA 
                   
               
               
                   
                 CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA 
                   
               
               
                   
                 CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC 
                   
               
               
                   
                 UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC 
                   
               
               
                   
                 CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU 
                   
               
               
                   
                 GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC 
                   
               
               
                   
                 CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC 
                   
               
               
                   
                 CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU 
                   
               
               
                   
                 GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA 
                   
               
               
                   
                 ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA 
                   
               
               
                   
                 GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA 
                   
               
               
                   
                 CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC 
                   
               
               
                   
                 UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG 
                   
               
               
                   
                 CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG 
                   
               
               
                   
                 ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA 
                   
               
               
                   
                 AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC 
                   
               
               
                   
                 GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC 
                   
               
               
                   
                 AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG 
                   
               
               
                   
                 AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA 
                   
               
               
                   
                 AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU 
                   
               
               
                   
                 CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU 
                   
               
               
                   
                 ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG 
                   
               
               
                   
                 AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU 
                   
               
               
                   
                 CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA 
                   
               
               
                   
                 AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC 
                   
               
               
                   
                 GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC 
                   
               
               
                   
                 AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG 
                   
               
               
                   
                 ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG 
                   
               
               
                   
                 GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA 
                   
               
               
                   
                 AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG 
                   
               
               
                   
                 ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA 
                   
               
               
                   
                 GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG 
                   
               
               
                   
                 AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA 
                   
               
               
                   
                 GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC 
                   
               
               
                   
                 GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACA 
                   
               
               
                   
                 GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA 
                   
               
               
                   
                 ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG 
                   
               
               
                   
                 ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG 
                   
               
               
                   
                 ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA 
                   
               
               
                   
                 ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA 
                   
               
               
                   
                 AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA 
                   
               
               
                   
                 GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA 
                   
               
               
                   
                 ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC 
                   
               
               
                   
                 AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA 
                   
               
               
                   
                 CUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGA 
                   
               
               
                   
                 CGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGA 
                   
               
               
                   
                 ACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUC 
                   
               
               
                   
                 AAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAA 
                   
               
               
                   
                 GCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAG 
                   
               
               
                   
                 AGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUC 
                   
               
               
                   
                 AAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU 
                   
               
               
                   
                 CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACG 
                   
               
               
                   
                 AAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUG 
                   
               
               
                   
                 AAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUU 
                   
               
               
                   
                 CUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACG 
                   
               
               
                   
                 ACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAG 
                   
               
               
                   
                 AAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUA 
                   
               
               
                   
                 CAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAAC 
                   
               
               
                   
                 AGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGC 
                   
               
               
                   
                 AACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAA 
                   
               
               
                   
                 CGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAG 
                   
               
               
                   
                 AAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCA 
                   
               
               
                   
                 ACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGU 
                   
               
               
                   
                 CAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAA 
                   
               
               
                   
                 GCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA 
                   
               
               
                   
                 AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAG 
                   
               
               
                   
                 CCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCG 
                   
               
               
                   
                 AAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUG 
                   
               
               
                   
                 CUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAA 
                   
               
               
                   
                 CCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCA 
                   
               
               
                   
                 AGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC 
                   
               
               
                   
                 GAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGG 
                   
               
               
                   
                 AGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGU 
                   
               
               
                   
                 ACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUG 
                   
               
               
                   
                 AAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGU 
                   
               
               
                   
                 CGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGA 
                   
               
               
                   
                 UCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAAC 
                   
               
               
                   
                 CUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA 
                   
               
               
                   
                 GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCA 
                   
               
               
                   
                 CACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC 
                   
               
               
                   
                 GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGA 
                   
               
               
                   
                 AGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGAC 
                   
               
               
                   
                 UGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC 
                   
               
               
                   
                 GGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAG 
                   
               
               
                   
                 CCCGAAGAAGAAGAGAAAGGUCGACAGCGGA 
                   
               
               
                   
               
               
                 Amino acid 
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK 
                 25 
               
               
                 sequence of 
                 KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE 
                   
               
               
                 Cas9 nickase 
                 MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT 
                   
               
               
                 with two 
                 IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS 
                   
               
               
                 nuclear 
                 DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL 
                   
               
               
                 localization 
                 IAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT 
                   
               
               
                 signals as the 
                 YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA 
                   
               
               
                 C- ter min al 
                 PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA 
                   
               
               
                 amino acids 
                 GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF 
                   
               
               
                   
                 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV 
                   
               
               
                   
                 GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT 
                   
               
               
                   
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL 
                   
               
               
                   
                 SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED 
                   
               
               
                   
                 RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG 
                   
               
               
                   
                 KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 
                   
               
               
                   
                 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE 
                   
               
               
                   
                 NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL 
                   
               
               
                   
                 YLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK 
                   
               
               
                   
                 VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD 
                   
               
               
                   
                 NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY 
                   
               
               
                   
                 DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA 
                   
               
               
                   
                 YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK 
                   
               
               
                   
                 ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
                   
               
               
                   
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
                   
               
               
                   
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG 
                   
               
               
                   
                 ITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR 
                   
               
               
                   
                 MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ 
                   
               
               
                   
                 LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
                   
               
               
                   
                 REQAENIIHLFTLTNLGAPAAFKYPDTTIDRKRYTSTKEVLDATLI 
                   
               
               
                   
                 HQSITGLYETRIDLSQLGGDGSGSPKKKRKVDGSPKKKRKVDSG 
                   
               
               
                   
               
               
                 Cas9 nickase 
                 AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAA 
                 26 
               
               
                 mRNA ORF 
                 CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC 
                   
               
               
                 encoding 
                 CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC 
                   
               
               
                 SEQ ID NO: 
                 AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG 
                   
               
               
                 25 using 
                 CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA 
                   
               
               
                 minimal 
                 GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG 
                   
               
               
                 uridine 
                 CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG 
                   
               
               
                 codons as 
                 CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG 
                   
               
               
                 listed in 
                 ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC 
                   
               
               
                 Table 3, with 
                 GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA 
                   
               
               
                 start and 
                 CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC 
                   
               
               
                 stop codons 
                 UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC 
                   
               
               
                   
                 AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA 
                   
               
               
                   
                 CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU 
                   
               
               
                   
                 ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA 
                   
               
               
                   
                 GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG 
                   
               
               
                   
                 CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA 
                   
               
               
                   
                 AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG 
                   
               
               
                   
                 GGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGA 
                   
               
               
                   
                 AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG 
                   
               
               
                   
                 ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA 
                   
               
               
                   
                 GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU 
                   
               
               
                   
                 GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG 
                   
               
               
                   
                 CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC 
                   
               
               
                   
                 CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA 
                   
               
               
                   
                 GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA 
                   
               
               
                   
                 AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG 
                   
               
               
                   
                 GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU 
                   
               
               
                   
                 GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG 
                   
               
               
                   
                 ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC 
                   
               
               
                   
                 CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA 
                   
               
               
                   
                 AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA 
                   
               
               
                   
                 AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG 
                   
               
               
                   
                 UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG 
                   
               
               
                   
                 ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA 
                   
               
               
                   
                 AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG 
                   
               
               
                   
                 AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG 
                   
               
               
                   
                 GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU 
                   
               
               
                   
                 CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA 
                   
               
               
                   
                 UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA 
                   
               
               
                   
                 AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU 
                   
               
               
                   
                 CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU 
                   
               
               
                   
                 UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC 
                   
               
               
                   
                 GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA 
                   
               
               
                   
                 GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC 
                   
               
               
                   
                 UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA 
                   
               
               
                   
                 GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU 
                   
               
               
                   
                 CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA 
                   
               
               
                   
                 CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC 
                   
               
               
                   
                 AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA 
                   
               
               
                   
                 GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC 
                   
               
               
                   
                 ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA 
                   
               
               
                   
                 CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC 
                   
               
               
                   
                 AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC 
                   
               
               
                   
                 AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA 
                   
               
               
                   
                 AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGA 
                   
               
               
                   
                 AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA 
                   
               
               
                   
                 GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC 
                   
               
               
                   
                 CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA 
                   
               
               
                   
                 GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG 
                   
               
               
                   
                 ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC 
                   
               
               
                   
                 GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAG 
                   
               
               
                   
                 GACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAA 
                   
               
               
                   
                 GAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCG 
                   
               
               
                   
                 UCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCA 
                   
               
               
                   
                 AAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGC 
                   
               
               
                   
                 AGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCA 
                   
               
               
                   
                 UCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCAC 
                   
               
               
                   
                 GUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGA 
                   
               
               
                   
                 CGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC 
                   
               
               
                   
                 UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAG 
                   
               
               
                   
                 UUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACA 
                   
               
               
                   
                 CGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCA 
                   
               
               
                   
                 AGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGAC 
                   
               
               
                   
                 UACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGA 
                   
               
               
                   
                 ACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA 
                   
               
               
                   
                 GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCA 
                   
               
               
                   
                 AACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG 
                   
               
               
                   
                 AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCG 
                   
               
               
                   
                 CAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUC 
                   
               
               
                   
                 GUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGA 
                   
               
               
                   
                 AAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAA 
                   
               
               
                   
                 GAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGAC 
                   
               
               
                   
                 AGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGU 
                   
               
               
                   
                 CGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAAC 
                   
               
               
                   
                 UGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG 
                   
               
               
                   
                 AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGU 
                   
               
               
                   
                 CAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGU 
                   
               
               
                   
                 UCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCA 
                   
               
               
                   
                 GGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAA 
                   
               
               
                   
                 GUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGC 
                   
               
               
                   
                 UGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC 
                   
               
               
                   
                 GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACA 
                   
               
               
                   
                 GAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAA 
                   
               
               
                   
                 ACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGAC 
                   
               
               
                   
                 AAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUU 
                   
               
               
                   
                 CACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACU 
                   
               
               
                   
                 UCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAG 
                   
               
               
                   
                 GAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGG 
                   
               
               
                   
                 ACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAG 
                   
               
               
                   
                 ACGGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGA 
                   
               
               
                   
                 AGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGAUAG 
                   
               
               
                   
               
               
                 Cas9 nickase 
                 GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAG 
                 27 
               
               
                 coding 
                 CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA 
                   
               
               
                 sequence 
                 GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC 
                   
               
               
                 encoding 
                 AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG 
                   
               
               
                 SEQ ID NO: 
                 AGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAA 
                   
               
               
                 25 using 
                 GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG 
                   
               
               
                 minimal 
                 GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU 
                   
               
               
                 uridine 
                 CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA 
                   
               
               
                 codons as 
                 AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC 
                   
               
               
                 listed in 
                 GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU 
                   
               
               
                 Table 3 (no 
                 GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA 
                   
               
               
                 start or stop 
                 GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA 
                   
               
               
                 codons; 
                 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG 
                   
               
               
                 suitable for 
                 CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA 
                   
               
               
                 inclusion in 
                 ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC 
                   
               
               
                 fusion 
                 GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG 
                   
               
               
                 protein 
                 AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA 
                   
               
               
                 coding 
                 AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA 
                   
               
               
                 sequence) 
                 CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA 
                   
               
               
                   
                 CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC 
                   
               
               
                   
                 UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC 
                   
               
               
                   
                 CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU 
                   
               
               
                   
                 GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC 
                   
               
               
                   
                 CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC 
                   
               
               
                   
                 CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU 
                   
               
               
                   
                 GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA 
                   
               
               
                   
                 ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA 
                   
               
               
                   
                 GAAUUCUACAAGUVCAUCAAGCCGAUCCUGGAAAAGAUGGA 
                   
               
               
                   
                 CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC 
                   
               
               
                   
                 UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG 
                   
               
               
                   
                 CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG 
                   
               
               
                   
                 ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA 
                   
               
               
                   
                 AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC 
                   
               
               
                   
                 GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC 
                   
               
               
                   
                 AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG 
                   
               
               
                   
                 AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA 
                   
               
               
                   
                 AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU 
                   
               
               
                   
                 CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU 
                   
               
               
                   
                 ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG 
                   
               
               
                   
                 AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU 
                   
               
               
                   
                 CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA 
                   
               
               
                   
                 AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC 
                   
               
               
                   
                 GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC 
                   
               
               
                   
                 AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG 
                   
               
               
                   
                 ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG 
                   
               
               
                   
                 GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA 
                   
               
               
                   
                 AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG 
                   
               
               
                   
                 ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA 
                   
               
               
                   
                 GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG 
                   
               
               
                   
                 AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA 
                   
               
               
                   
                 GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC 
                   
               
               
                   
                 GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACA 
                   
               
               
                   
                 GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA 
                   
               
               
                   
                 ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG 
                   
               
               
                   
                 ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG 
                   
               
               
                   
                 ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA 
                   
               
               
                   
                 ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA 
                   
               
               
                   
                 AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA 
                   
               
               
                   
                 GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA 
                   
               
               
                   
                 ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC 
                   
               
               
                   
                 AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA 
                   
               
               
                   
                 CUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGA 
                   
               
               
                   
                 CGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGA 
                   
               
               
                   
                 ACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUC 
                   
               
               
                   
                 AAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAA 
                   
               
               
                   
                 GCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAG 
                   
               
               
                   
                 AGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUC 
                   
               
               
                   
                 AAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU 
                   
               
               
                   
                 CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACG 
                   
               
               
                   
                 AAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUG 
                   
               
               
                   
                 AAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUU 
                   
               
               
                   
                 CUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACG 
                   
               
               
                   
                 ACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAG 
                   
               
               
                   
                 AAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUA 
                   
               
               
                   
                 CAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAAC 
                   
               
               
                   
                 AGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGC 
                   
               
               
                   
                 AACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAA 
                   
               
               
                   
                 CGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAG 
                   
               
               
                   
                 AAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCA 
                   
               
               
                   
                 ACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGU 
                   
               
               
                   
                 CAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAA 
                   
               
               
                   
                 GCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA 
                   
               
               
                   
                 AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAG 
                   
               
               
                   
                 CCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCG 
                   
               
               
                   
                 AAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUG 
                   
               
               
                   
                 CUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAA 
                   
               
               
                   
                 CCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCA 
                   
               
               
                   
                 AGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC 
                   
               
               
                   
                 GAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGG 
                   
               
               
                   
                 AGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGU 
                   
               
               
                   
                 ACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUG 
                   
               
               
                   
                 AAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGU 
                   
               
               
                   
                 CGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGA 
                   
               
               
                   
                 UCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAAC 
                   
               
               
                   
                 CUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA 
                   
               
               
                   
                 GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCA 
                   
               
               
                   
                 CACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC 
                   
               
               
                   
                 GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGA 
                   
               
               
                   
                 AGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGAC 
                   
               
               
                   
                 UGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC 
                   
               
               
                   
                 GGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAG 
                   
               
               
                   
                 CCCGAAGAAGAAGAGAAAGGUCGACAGCGGA 
                   
               
               
                   
               
               
                 Amino acid 
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK 
                 28 
               
               
                 sequence of 
                 KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE 
                   
               
               
                 dCas9 with 
                 MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT 
                   
               
               
                 two nuclear 
                 IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS 
                   
               
               
                 localization 
                 DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL 
                   
               
               
                 signals as the 
                 IAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT 
                   
               
               
                 C-terminal 
                 YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA 
                   
               
               
                 amino acids 
                 PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA 
                   
               
               
                   
                 GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF 
                   
               
               
                   
                 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV 
                   
               
               
                   
                 GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT 
                   
               
               
                   
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL 
                   
               
               
                   
                 SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED 
                   
               
               
                   
                 RFNASLGTYHDLLKDKDKDFLDNEENEDILEDIVLTLTLFEDREM 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG 
                   
               
               
                   
                 KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 
                   
               
               
                   
                 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE 
                   
               
               
                   
                 NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL 
                   
               
               
                   
                 YLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK 
                   
               
               
                   
                 VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD 
                   
               
               
                   
                 NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY 
                   
               
               
                   
                 DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA 
                   
               
               
                   
                 YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK 
                   
               
               
                   
                 ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
                   
               
               
                   
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
                   
               
               
                   
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG 
                   
               
               
                   
                 ITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR 
                   
               
               
                   
                 MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ 
                   
               
               
                   
                 LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
                   
               
               
                   
                 REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 
                   
               
               
                   
                 HQSITGLYETRIDLSQLGGDGSGSPKKKRKVDGSPKKKRKVDSG 
                   
               
               
                   
               
               
                 dCas9 
                 AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAA 
                 29 
               
               
                 mRNA ORF 
                 CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC 
                   
               
               
                 encoding 
                 CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC 
                   
               
               
                 SEQ ID NO: 
                 AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG 
                   
               
               
                 28 using 
                 CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA 
                   
               
               
                 minimal 
                 GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG 
                   
               
               
                 uridine 
                 CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG 
                   
               
               
                 codons as 
                 CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG 
                   
               
               
                 listed in 
                 ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC 
                   
               
               
                 Table 3, with 
                 GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA 
                   
               
               
                 start and 
                 CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC 
                   
               
               
                 stop codons 
                 UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC 
                   
               
               
                   
                 AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA 
                   
               
               
                   
                 CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU 
                   
               
               
                   
                 ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA 
                   
               
               
                   
                 GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG 
                   
               
               
                   
                 CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA 
                   
               
               
                   
                 AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG 
                   
               
               
                   
                 GGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGA 
                   
               
               
                   
                 AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG 
                   
               
               
                   
                 ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA 
                   
               
               
                   
                 GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU 
                   
               
               
                   
                 OCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG 
                   
               
               
                   
                 CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC 
                   
               
               
                   
                 CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA 
                   
               
               
                   
                 GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA 
                   
               
               
                   
                 AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG 
                   
               
               
                   
                 GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU 
                   
               
               
                   
                 GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG 
                   
               
               
                   
                 ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC 
                   
               
               
                   
                 CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA 
                   
               
               
                   
                 AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA 
                   
               
               
                   
                 AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG 
                   
               
               
                   
                 UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG 
                   
               
               
                   
                 ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA 
                   
               
               
                   
                 AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG 
                   
               
               
                   
                 AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG 
                   
               
               
                   
                 GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU 
                   
               
               
                   
                 CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA 
                   
               
               
                   
                 UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA 
                   
               
               
                   
                 AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU 
                   
               
               
                   
                 CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU 
                   
               
               
                   
                 UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC 
                   
               
               
                   
                 GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA 
                   
               
               
                   
                 GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC 
                   
               
               
                   
                 UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA 
                   
               
               
                   
                 GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU 
                   
               
               
                   
                 CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA 
                   
               
               
                   
                 CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC 
                   
               
               
                   
                 AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA 
                   
               
               
                   
                 GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC 
                   
               
               
                   
                 ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA 
                   
               
               
                   
                 CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC 
                   
               
               
                   
                 AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC 
                   
               
               
                   
                 AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA 
                   
               
               
                   
                 AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGA 
                   
               
               
                   
                 AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA 
                   
               
               
                   
                 GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC 
                   
               
               
                   
                 CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA 
                   
               
               
                   
                 GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG 
                   
               
               
                   
                 ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC 
                   
               
               
                   
                 GACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAA 
                   
               
               
                   
                 GGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACA 
                   
               
               
                   
                 AGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUC 
                   
               
               
                   
                 GUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGC 
                   
               
               
                   
                 AAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGG 
                   
               
               
                   
                 CAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUC 
                   
               
               
                   
                 AUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCA 
                   
               
               
                   
                 CGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACG 
                   
               
               
                   
                 ACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA 
                   
               
               
                   
                 CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCA 
                   
               
               
                   
                 GUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCAC 
                   
               
               
                   
                 ACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUC 
                   
               
               
                   
                 AAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGA 
                   
               
               
                   
                 CUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCG 
                   
               
               
                   
                 AACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUAC 
                   
               
               
                   
                 AGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGC 
                   
               
               
                   
                 AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACG 
                   
               
               
                   
                 GAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUC 
                   
               
               
                   
                 GCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAU 
                   
               
               
                   
                 CGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGG 
                   
               
               
                   
                 AAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCA 
                   
               
               
                   
                 AGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGA 
                   
               
               
                   
                 CAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGG 
                   
               
               
                   
                 UCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAA 
                   
               
               
                   
                 CUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAA 
                   
               
               
                   
                 GAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAG 
                   
               
               
                   
                 UCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUG 
                   
               
               
                   
                 UUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGC 
                   
               
               
                   
                 AGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCA 
                   
               
               
                   
                 AGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAG 
                   
               
               
                   
                 CUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUU 
                   
               
               
                   
                 CGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAAC 
                   
               
               
                   
                 AGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCA 
                   
               
               
                   
                 AACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGA 
                   
               
               
                   
                 CAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGU 
                   
               
               
                   
                 UCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC 
                   
               
               
                   
                 UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAA 
                   
               
               
                   
                 GGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAG 
                   
               
               
                   
                 GACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGA 
                   
               
               
                   
                 GAC 
                   
               
               
                   
                 GGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAG 
                   
               
               
                   
                 CCCGAAGAAGAAGAGAAAGGUCGACAGCGGAUAG 
                   
               
               
                   
               
               
                 dCas9 
                 GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAG 
                 30 
               
               
                 coding 
                 CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA 
                   
               
               
                 sequence 
                 GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC 
                   
               
               
                 encoding 
                 AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG 
                   
               
               
                 SEQ ID NO: 
                 AGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAA 
                   
               
               
                 28 using 
                 GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG 
                   
               
               
                 minimal 
                 GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU 
                   
               
               
                 uridine 
                 CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA 
                   
               
               
                 codons as 
                 AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC 
                   
               
               
                 listed in 
                 GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU 
                   
               
               
                 Table 3 (no 
                 GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA 
                   
               
               
                 start or stop 
                 GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA 
                   
               
               
                 codons; 
                 GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG 
                   
               
               
                 suitable for 
                 CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA 
                   
               
               
                 inclusion in 
                 ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC 
                   
               
               
                 fusion 
                 GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG 
                   
               
               
                 protein 
                 AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA 
                   
               
               
                 coding 
                 AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA 
                   
               
               
                 sequence) 
                 CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA 
                   
               
               
                   
                 CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC 
                   
               
               
                   
                 UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC 
                   
               
               
                   
                 CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU 
                   
               
               
                   
                 GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC 
                   
               
               
                   
                 CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC 
                   
               
               
                   
                 CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU 
                   
               
               
                   
                 GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA 
                   
               
               
                   
                 ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA 
                   
               
               
                   
                 GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA 
                   
               
               
                   
                 CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC 
                   
               
               
                   
                 UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG 
                   
               
               
                   
                 CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG 
                   
               
               
                   
                 ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA 
                   
               
               
                   
                 AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC 
                   
               
               
                   
                 GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC 
                   
               
               
                   
                 AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG 
                   
               
               
                   
                 AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA 
                   
               
               
                   
                 AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU 
                   
               
               
                   
                 CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU 
                   
               
               
                   
                 ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG 
                   
               
               
                   
                 AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU 
                   
               
               
                   
                 CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA 
                   
               
               
                   
                 AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC 
                   
               
               
                   
                 GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC 
                   
               
               
                   
                 AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG 
                   
               
               
                   
                 ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG 
                   
               
               
                   
                 GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA 
                   
               
               
                   
                 AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG 
                   
               
               
                   
                 ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA 
                   
               
               
                   
                 GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG 
                   
               
               
                   
                 AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA 
                   
               
               
                   
                 GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC 
                   
               
               
                   
                 GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACA 
                   
               
               
                   
                 GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA 
                   
               
               
                   
                 ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG 
                   
               
               
                   
                 ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG 
                   
               
               
                   
                 ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA 
                   
               
               
                   
                 ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA 
                   
               
               
                   
                 AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA 
                   
               
               
                   
                 GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA 
                   
               
               
                   
                 ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC 
                   
               
               
                   
                 AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA 
                   
               
               
                   
                 CUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGG 
                   
               
               
                   
                 ACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAG 
                   
               
               
                   
                 AACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGU 
                   
               
               
                   
                 CAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAA 
                   
               
               
                   
                 AGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCA 
                   
               
               
                   
                 GAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAU 
                   
               
               
                   
                 CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACG 
                   
               
               
                   
                 UCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGAC 
                   
               
               
                   
                 GAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACU 
                   
               
               
                   
                 GAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGU 
                   
               
               
                   
                 UCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACAC 
                   
               
               
                   
                 GACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAA 
                   
               
               
                   
                 GAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACU 
                   
               
               
                   
                 ACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAA 
                   
               
               
                   
                 CAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG 
                   
               
               
                   
                 CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAA 
                   
               
               
                   
                 ACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGA 
                   
               
               
                   
                 GAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGC 
                   
               
               
                   
                 AACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCG 
                   
               
               
                   
                 UCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAA 
                   
               
               
                   
                 AGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAG 
                   
               
               
                   
                 AAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACA 
                   
               
               
                   
                 GCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUC 
                   
               
               
                   
                 GAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACU 
                   
               
               
                   
                 GCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGA 
                   
               
               
                   
                 ACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC 
                   
               
               
                   
                 AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUU 
                   
               
               
                   
                 CGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAG 
                   
               
               
                   
                 GAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAG 
                   
               
               
                   
                 UACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCU 
                   
               
               
                   
                 GAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCG 
                   
               
               
                   
                 UCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAG 
                   
               
               
                   
                 AUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAA 
                   
               
               
                   
                 CCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACA 
                   
               
               
                   
                 AGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUC 
                   
               
               
                   
                 ACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUU 
                   
               
               
                   
                 CGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGG 
                   
               
               
                   
                 AAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGA 
                   
               
               
                   
                 CUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGA 
                   
               
               
                   
                 C 
                   
               
               
                   
                 GGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAG 
                   
               
               
                   
                 CCCGAAGAAGAAGAGAAAGGUCGACAGCGGA 
                   
               
               
                   
               
               
                 T7 promoter 
                 TAATACGACTCACTATA 
                 31 
               
               
                   
               
               
                 Human beta- 
                 ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAAC 
                 32 
               
               
                 globin 5′ 
                 AGACACC 
                   
               
               
                 UTR 
                   
                   
               
               
                   
               
               
                 Human beta- 
                 GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTT 
                 33 
               
               
                 globin 3′ 
                 CCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCT 
                   
               
               
                 UTR 
                 TGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTG 
                   
               
               
                   
                 C 
                   
               
               
                   
               
               
                 Human 
                 CATAAACCCTGGCGCGCTCGCGGCCCGGCACTCTTCTGGTCCC 
                 34 
               
               
                 alpha-globin 
                 CACAGACTCAGAGAGAACCCACC 
                   
               
               
                 5′ UTR 
                   
                   
               
               
                   
               
               
                 Human 
                 GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCC 
                 35 
               
               
                 alpha-globin 
                 CCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTT 
                   
               
               
                 3′ UTR 
                 TGAATAAAGTCTGAGTGGGCGGC 
                   
               
               
                   
               
               
                 
                   Xenopus 
                 
                 AAGCTCAGAATAAACGCTCAACTTTGGCC 
                 36 
               
               
                   laevis  beta- 
                   
                   
               
               
                 giobin 5′ 
                   
                   
               
               
                 UTR 
                   
                   
               
               
                   
               
               
                 
                   Xenopus 
                 
                 ACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATA 
                 37 
               
               
                   laevis  beta- 
                 ATACCAACTTACACTTTACAAAATGTTGTCCCCCAAAATGTAG 
                   
               
               
                 giobin 3′ 
                 CCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCACATTC 
                   
               
               
                 UTR 
                 T 
                   
               
               
                   
               
               
                 Bovine 
                 CAGGGTCCTGTGGACAGCTCACCAGCT 
                 38 
               
               
                 Growth 
                   
                   
               
               
                 Hormone 5′ 
                   
                   
               
               
                 UTR 
                   
                   
               
               
                   
               
               
                 Bovine 
                 TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGA 
                 39 
               
               
                 Growth 
                 CCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA 
                   
               
               
                 Hormone 3′ 
                 GGAAATTGCATCGCA 
                   
               
               
                 UTR 
                   
                   
               
               
                   
               
               
                 
                   Mus 
                 
                 GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTC 
                 40 
               
               
                 
                   musculus 
                 
                 TCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTA 
                   
               
               
                 hemoglobin 
                 GGAAG 
                   
               
               
                 alpha, adult 
                   
                   
               
               
                 chain 1 
                   
                   
               
               
                 (Hba-a1), 
                   
                   
               
               
                 3′UTR 
                   
                   
               
               
                   
               
               
                 HSD17B4 5′ 
                 TCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGT 
                 41 
               
               
                 UTR 
                 CGTTGCAGGCCTTATTC 
                   
               
               
                   
               
               
                 G2S2 guide 
                 mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGm 
                 42 
               
               
                 RNA 
                 CmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGG 
                   
               
               
                 targeting 
                 CUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAm 
                   
               
               
                 TTR 
                 GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmC 
                   
               
               
                   
                 mU*mU*mU*mU 
                   
               
               
                   
               
               
                 Cas9 
                 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT 
                 43 
               
               
                 transcript 
                 GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAGA 
                   
               
               
                 with 5′ UTR 
                 AGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT 
                   
               
               
                 ofHSD, 
                 GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGT 
                   
               
               
                 ORF 
                 TCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGA 
                   
               
               
                 corresponding 
                 ACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAG 
                   
               
               
                 to SEQ 
                 AAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACA 
                   
               
               
                 ID NO: 4, 
                 AGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC 
                   
               
               
                 Kozak 
                 AACGAAATOGCAAAGGTCGACGACAGCTTCTTCCACAGACTG 
                   
               
               
                 sequence, 
                 GAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGA 
                   
               
               
                 and 3′ UTR 
                 CACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACG 
                   
               
               
                 of ALB 
                 AAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCG 
                   
               
               
                   
                 ACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC 
                   
               
               
                   
                 TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGG 
                   
               
               
                   
                 AGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCAT 
                   
               
               
                   
                 CCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCC 
                   
               
               
                   
                 GATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGC 
                   
               
               
                   
                 AAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACA 
                   
               
               
                   
                 GCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGAT 
                   
               
               
                   
                 CGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTC 
                   
               
               
                   
                 GACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACA 
                   
               
               
                   
                 TACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGAC 
                   
               
               
                   
                 CAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGAC 
                   
               
               
                   
                 GCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATC 
                   
               
               
                   
                 ACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGAC 
                   
               
               
                   
                 GAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGA 
                   
               
               
                   
                 CAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAG 
                   
               
               
                   
                 AGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGC 
                   
               
               
                   
                 CAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAG 
                   
               
               
                   
                 ATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAA 
                   
               
               
                   
                 GACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATC 
                   
               
               
                   
                 CCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGA 
                   
               
               
                   
                 AGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAA 
                   
               
               
                   
                 AAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCG 
                   
               
               
                   
                 GACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAA 
                   
               
               
                   
                 GAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAG 
                   
               
               
                   
                 TCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAA 
                   
               
               
                   
                 TGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGC 
                   
               
               
                   
                 CGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA 
                   
               
               
                   
                 ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCC 
                   
               
               
                   
                 GGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCT 
                   
               
               
                   
                 GCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAA 
                   
               
               
                   
                 GGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGA 
                   
               
               
                   
                 AATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAAC 
                   
               
               
                   
                 ATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTG 
                   
               
               
                   
                 GACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTG 
                   
               
               
                   
                 ACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGA 
                   
               
               
                   
                 CTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAG 
                   
               
               
                   
                 CAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAG 
                   
               
               
                   
                 CAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAA 
                   
               
               
                   
                 AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACA 
                   
               
               
                   
                 GAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAA 
                   
               
               
                   
                 GGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACA 
                   
               
               
                   
                 GCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAA 
                   
               
               
                   
                 TCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAAC 
                   
               
               
                   
                 TGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCA 
                   
               
               
                   
                 TCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAG 
                   
               
               
                   
                 AAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAAT 
                   
               
               
                   
                 CAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGA 
                   
               
               
                   
                 AAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT 
                   
               
               
                   
                 GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACAT 
                   
               
               
                   
                 CAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCA 
                   
               
               
                   
                 GAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGAC 
                   
               
               
                   
                 AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGA 
                   
               
               
                   
                 GCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGC 
                   
               
               
                   
                 TGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACC 
                   
               
               
                   
                 TGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAG 
                   
               
               
                   
                 GCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATC 
                   
               
               
                   
                 ACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACA 
                   
               
               
                   
                 AAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTC 
                   
               
               
                   
                 ATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC 
                   
               
               
                   
                 TTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACG 
                   
               
               
                   
                 CACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGA 
                   
               
               
                   
                 TCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAG 
                   
               
               
                   
                 ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCG 
                   
               
               
                   
                 AACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA 
                   
               
               
                   
                 GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA 
                   
               
               
                   
                 ACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGA 
                   
               
               
                   
                 GAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCA 
                   
               
               
                   
                 ACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTC 
                   
               
               
                   
                 AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAG 
                   
               
               
                   
                 CATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAA 
                   
               
               
                   
                 GAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCC 
                   
               
               
                   
                 GACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAA 
                   
               
               
                   
                 GGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGG 
                   
               
               
                   
                 GAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGA 
                   
               
               
                   
                 TCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG 
                   
               
               
                   
                 ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGA 
                   
               
               
                   
                 AAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGC 
                   
               
               
                   
                 AGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACT 
                   
               
               
                   
                 TCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCC 
                   
               
               
                   
                 CGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACA 
                   
               
               
                   
                 AGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA 
                   
               
               
                   
                 GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCC 
                   
               
               
                   
                 TGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC 
                   
               
               
                   
                 AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGG 
                   
               
               
                   
                 GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA 
                   
               
               
                   
                 GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACAC 
                   
               
               
                   
                 TGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCG 
                   
               
               
                   
                 ACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAG 
                   
               
               
                   
                 AAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATC 
                   
               
               
                   
                 TCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAT 
                   
               
               
                   
                 AGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACA 
                   
               
               
                   
                 CCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTT 
                   
               
               
                   
                 TCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG 
                   
               
               
                   
               
               
                 Cas9 
                 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGT 
                 44 
               
               
                 transcript 
                 GTGTGTCGTTGCAGGCCTTATTCGGATCCATGGACAAGAAGTA 
                   
               
               
                 with 5′ UTR 
                 CAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGC 
                   
               
               
                 of HSD, 
                 AGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAA 
                   
               
               
                 ORF 
                 GGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGAACCT 
                   
               
               
                 corresponding 
                 GATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAGC 
                   
               
               
                 to SEQ 
                 AACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGAA 
                   
               
               
                 ID NO: 4, 
                 GAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACG 
                   
               
               
                 and 3′ UTR 
                 AAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAG 
                   
               
               
                 of ALB 
                 AAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACC 
                   
               
               
                   
                 CGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAA 
                   
               
               
                   
                 AGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACA 
                   
               
               
                   
                 GCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGG 
                   
               
               
                   
                 CACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAG 
                   
               
               
                   
                 ACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCC 
                   
               
               
                   
                 AGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGA 
                   
               
               
                   
                 TCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAA 
                   
               
               
                   
                 GACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGC 
                   
               
               
                   
                 TGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCG 
                   
               
               
                   
                 CACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCG 
                   
               
               
                   
                 ACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACAT 
                   
               
               
                   
                 ACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACC 
                   
               
               
                   
                 AGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACG 
                   
               
               
                   
                 CAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA 
                   
               
               
                   
                 CAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGACG 
                   
               
               
                   
                 AACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGAC 
                   
               
               
                   
                 AGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGA 
                   
               
               
                   
                 GCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGCC 
                   
               
               
                   
                 AGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGA 
                   
               
               
                   
                 TGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAAG 
                   
               
               
                   
                 ACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCC 
                   
               
               
                   
                 CGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAA 
                   
               
               
                   
                 GACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAA 
                   
               
               
                   
                 AGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG 
                   
               
               
                   
                 ACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAG 
                   
               
               
                   
                 AAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGT 
                   
               
               
                   
                 CGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAAT 
                   
               
               
                   
                 GACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCC 
                   
               
               
                   
                 GAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAA 
                   
               
               
                   
                 CTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCG 
                   
               
               
                   
                 GCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTG 
                   
               
               
                   
                 CTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAG 
                   
               
               
                   
                 GAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAA 
                   
               
               
                   
                 ATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACA 
                   
               
               
                   
                 TACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGG 
                   
               
               
                   
                 ACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGA 
                   
               
               
                   
                 CACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGAC 
                   
               
               
                   
                 TGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGC 
                   
               
               
                   
                 AGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGC 
                   
               
               
                   
                 AGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAAA 
                   
               
               
                   
                 GACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAG 
                   
               
               
                   
                 AAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAG 
                   
               
               
                   
                 GAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAG 
                   
               
               
                   
                 CCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAAT 
                   
               
               
                   
                 CAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACT 
                   
               
               
                   
                 GGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCAT 
                   
               
               
                   
                 CGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGA 
                   
               
               
                   
                 AGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATC 
                   
               
               
                   
                 AAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAA 
                   
               
               
                   
                 AACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTG 
                   
               
               
                   
                 CAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATC 
                   
               
               
                   
                 AACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAG 
                   
               
               
                   
                 AGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACA 
                   
               
               
                   
                 AGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGAG 
                   
               
               
                   
                 CGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT 
                   
               
               
                   
                 GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCT 
                   
               
               
                   
                 GACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGG 
                   
               
               
                   
                 CAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCA 
                   
               
               
                   
                 CAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAA 
                   
               
               
                   
                 AGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTCA 
                   
               
               
                   
                 TCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACT 
                   
               
               
                   
                 TCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGC 
                   
               
               
                   
                 ACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGAT 
                   
               
               
                   
                 CAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGA 
                   
               
               
                   
                 CTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGA 
                   
               
               
                   
                 ACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAG 
                   
               
               
                   
                 CAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAAC 
                   
               
               
                   
                 GGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGA 
                   
               
               
                   
                 AACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAAC 
                   
               
               
                   
                 AGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAA 
                   
               
               
                   
                 GAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCAT 
                   
               
               
                   
                 CCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAA 
                   
               
               
                   
                 GGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGAC 
                   
               
               
                   
                 AGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGG 
                   
               
               
                   
                 AAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAAT 
                   
               
               
                   
                 CACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGA 
                   
               
               
                   
                 CTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCT 
                   
               
               
                   
                 GATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAA 
                   
               
               
                   
                 CGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGA 
                   
               
               
                   
                 AGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCC 
                   
               
               
                   
                 TGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGG 
                   
               
               
                   
                 AAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGC 
                   
               
               
                   
                 ACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCAGCA 
                   
               
               
                   
                 AGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGA 
                   
               
               
                   
                 GCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGG 
                   
               
               
                   
                 CAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGC 
                   
               
               
                   
                 ACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAA 
                   
               
               
                   
                 GAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGAT 
                   
               
               
                   
                 CCACCAGAGCATCACAGGACTGTACGAAACAAGAATCGACCT 
                   
               
               
                   
                 GAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGA 
                   
               
               
                   
                 AGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCTCAGC 
                   
               
               
                   
                 CTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTT 
                   
               
               
                   
                 ATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTG 
                   
               
               
                   
                 TCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCT 
                   
               
               
                   
                 GTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG 
                   
               
               
                   
               
               
                 Alternative 
                 ATGGATAAGAAGTACTCGATCGGGCTGGATATCGGAACTAATT 
                 45 
               
               
                 Cas9 ORF 
                 CCGTGGGTTGGGCAGTGATCACGGATGAATACAAAGTGCCGT 
                   
               
               
                 with 19.36% 
                 CCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACACAGCA 
                   
               
               
                 Ucontent 
                 TCAAGAAGAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGA 
                   
               
               
                   
                 AACCGCAGAAGCGACCCGGCTCAAACGTACCGCGAGGCGACG 
                   
               
               
                   
                 CTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAAATC 
                   
               
               
                   
                 TTTTCGAACGAAATGGCAAAGGTGGACGACAGCTTCTTCCACC 
                   
               
               
                   
                 GCCTGGAAGAATCTTTCCTGGTGGAGGAGGACAAGAAGCATG 
                   
               
               
                   
                 AACGGCATCCTATCTTTGGAAACATCGTGGACGAAGTGGCGTA 
                   
               
               
                   
                 CCACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAGTT 
                   
               
               
                   
                 GGTTGACTCAACTGACAAGGCCGACCTCAGATTGATCTACTTG 
                   
               
               
                   
                 GCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATCG 
                   
               
               
                   
                 AAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTGTT 
                   
               
               
                   
                 CATTCAACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAAC 
                   
               
               
                   
                 CCAATCAATGCCAGCGGCGTCGATGCCAAGGCCATCCTGTCCG 
                   
               
               
                   
                 CCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACA 
                   
               
               
                   
                 GCTGCCGGGAGAGAAGAAGAACGGACTTTTCGGCAACTTGAT 
                   
               
               
                   
                 CGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTG 
                   
               
               
                   
                 ACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCT 
                   
               
               
                   
                 ACGACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCA 
                   
               
               
                   
                 GTACGCGGATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCA 
                   
               
               
                   
                 ATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCA 
                   
               
               
                   
                 AAGCGCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGAGC 
                   
               
               
                   
                 ATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCA 
                   
               
               
                   
                 ACTGCCTGAAAAGTACAAGGAGATTTTCTTCGACCAGTCCAAG 
                   
               
               
                   
                 AATGGGTACGCAGGGTACATCGATGGAGGCGCCAGCCAGGAA 
                   
               
               
                   
                 GAGrrCTATAAGITCATCAAGCCAATCCTGGAAAAGATGGACG 
                   
               
               
                   
                 GAACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGC 
                   
               
               
                   
                 TCCGCAAACAGAGAACCTTTGACAACGGAAGCATTCCACACC 
                   
               
               
                   
                 AGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGA 
                   
               
               
                   
                 GGACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGA 
                   
               
               
                   
                 GAAAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTG 
                   
               
               
                   
                 GCGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCA 
                   
               
               
                   
                 GAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATA 
                   
               
               
                   
                 AGGGAGCTTCGGCACAATCCTTCATCGAACGAATGACCAACTT 
                   
               
               
                   
                 CGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAG 
                   
               
               
                   
                 CCTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAA 
                   
               
               
                   
                 GTGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTG 
                   
               
               
                   
                 AGCGGAGAACAGAAGAAAGCGATTGTCGATCTGCTGTTCAAG 
                   
               
               
                   
                 ACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTAC 
                   
               
               
                   
                 TTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGA 
                   
               
               
                   
                 GTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCATGATC 
                   
               
               
                   
                 TCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGG 
                   
               
               
                   
                 AGAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCT 
                   
               
               
                   
                 TTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTA 
                   
               
               
                   
                 CGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGC 
                   
               
               
                   
                 CGCCGGTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCA 
                   
               
               
                   
                 ACGGTATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTT 
                   
               
               
                   
                 CCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAGTTG 
                   
               
               
                   
                 ATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAGAAA 
                   
               
               
                   
                 GCACAAGTGAGCGGACAGGGAGACTCACTCCATGAACACATC 
                   
               
               
                   
                 GCGAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATCCTG 
                   
               
               
                   
                 CAAACTGTGAAGGTGGTGGACGAGCTGGTGAAGGTCATGGGA 
                   
               
               
                   
                 CGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAA 
                   
               
               
                   
                 AACCAGACTACCCAGAAGGGCCAGAAGAACTCCCGCGAAAGG 
                   
               
               
                   
                 ATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAG 
                   
               
               
                   
                 ATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAAC 
                   
               
               
                   
                 GAGAAGCTCTACCTGTACTATTTGCAAAATGGACGGGACATGT 
                   
               
               
                   
                 ACGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTACGA 
                   
               
               
                   
                 CGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCC 
                   
               
               
                   
                 ATCGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGG 
                   
               
               
                   
                 AAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATG 
                   
               
               
                   
                 AAGAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCC 
                   
               
               
                   
                 AGAGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGAC 
                   
               
               
                   
                 TCTCAGAGCTGGATAAGGCTGGATTCATCAAACGGGAGCTGGT 
                   
               
               
                   
                 CGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCCTGGA 
                   
               
               
                   
                 CTCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCAT 
                   
               
               
                   
                 CCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTC 
                   
               
               
                   
                 GGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATC 
                   
               
               
                   
                 AACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGG 
                   
               
               
                   
                 TCGGCACCGCCCTGATCAAGAAGTACCCTAAACTTGAATCGGA 
                   
               
               
                   
                 GTTTGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGAT 
                   
               
               
                   
                 GATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAA 
                   
               
               
                   
                 ATACTTCTTTTACTCAAACATCATGAACTTCTTCAAGACTGAA 
                   
               
               
                   
                 ATTACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATC 
                   
               
               
                   
                 GAAACTAACGGAGAAACGGGCGAAATCGTGTGGGACAAGGGC 
                   
               
               
                   
                 AGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAG 
                   
               
               
                   
                 TCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTC 
                   
               
               
                   
                 AAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCAT 
                   
               
               
                   
                 TGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATT 
                   
               
               
                   
                 CGATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAG 
                   
               
               
                   
                 GTGGAGAAGGGAAAGAGCAAGAAGCTCAAATCCGTCAAAGA 
                   
               
               
                   
                 GCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAG 
                   
               
               
                   
                 AACCCGATTGATTTCCTGGAGGCGAAGGGTTACAAGGAGGTG 
                   
               
               
                   
                 AAGAAGGATCTGATCATCAAACTGCCCAAGTACTCACTGTTCG 
                   
               
               
                   
                 AACTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAG 
                   
               
               
                   
                 AACTCCAGAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACG 
                   
               
               
                   
                 TCAACTTCCTCTATCTTGCTTCGCACTACGAGAAACTCAAAGG 
                   
               
               
                   
                 GTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCA 
                   
               
               
                   
                 GCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAG 
                   
               
               
                   
                 TTTTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAG 
                   
               
               
                   
                 TCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAGAG 
                   
               
               
                   
                 AACAGGCCGAGAACATTATCCACTTGTTCACCCTGACTAACCT 
                   
               
               
                   
                 GGGAGCTCCAGCCGCCTTCAAGTACTTCGATACTACTATCGAC 
                   
               
               
                   
                 CGCAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGACC 
                   
               
               
                   
                 CTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCG 
                   
               
               
                   
                 ATCTGTCGCAGCTGGGTGGCGATGGTGGCGGTGGATCCTACCC 
                   
               
               
                   
                 ATACGACGTGCCTGACTACGCCTCCGGAGGTGGTGGCCCCAAG 
                   
               
               
                   
                 AAGAAACGGAAGGTGTGATAG 
                   
               
               
                   
               
               
                 Cas9 
                 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT 
                 46 
               
               
                 transcript 
                 GTGTCGTTGCAGGCCTTATTCGGATCTGCCACCATGGATAAGA 
                   
               
               
                 with 5′ UTR 
                 AGTACTCGATCGGGCTGGATATCGGAACTAATTCCGTGGGTTG 
                   
               
               
                 of HSD, 
                 GGCAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTT 
                   
               
               
                 ORF 
                 CAAGGTCCTGGGGAACACCGATAGACACAGCATCAAGAAGAA 
                   
               
               
                 corresponding 
                 TCTCATCGGAGCCCTGCTGTTTGACTCCGGCGAAACCGCAGAA 
                   
               
               
                 to SEQ 
                 GCGACCCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGG 
                   
               
               
                 ID NO: 45, 
                 CGGAAGAATCGCATCTGCTATCTGCAAGAAATCTTTTCGAACG 
                   
               
               
                 Kozak 
                 AAATGGCAAAGGTGGACGACAGCTTCTTCCACCGCCTGGAAG 
                   
               
               
                 sequence, 
                 AATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATC 
                   
               
               
                 and 3′ UTR 
                 CTATCTTTGGAAACATCGTGGACGAAGTGGCGTACCACGAAA 
                   
               
               
                 of ALB 
                 AGTACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTC 
                   
               
               
                   
                 AACTGACAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCC 
                   
               
               
                   
                 CATATGATCAAATTCCGCGGACACTTCCTGATCGAAGGCGATC 
                   
               
               
                   
                 TGAACCCTGATAACTCCGACGTGGATAAGCTGTTCATTCAACT 
                   
               
               
                   
                 GGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAA 
                   
               
               
                   
                 TGCCAGCGGCGTCGATGCCAAGGCCATCCTGTCCGGCCGGCTG 
                   
               
               
                   
                 TCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCG 
                   
               
               
                   
                 GGAGAGAAGAAGAACGGACTTTTCGGCAACTTGATCGCTCTCT 
                   
               
               
                   
                 CACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGC 
                   
               
               
                   
                 CGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGACGA 
                   
               
               
                   
                 CGACTTGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCG 
                   
               
               
                   
                 GATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCT 
                   
               
               
                   
                 GTCCGATATCCTGCGCGTGAACACCGAAATAACCAAAGCGCC 
                   
               
               
                   
                 GCTTAGCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAG 
                   
               
               
                   
                 GATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTG 
                   
               
               
                   
                 AAAAGTACAAGGAGATTTTCTTCGACCAGTCCAAGAATGGGT 
                   
               
               
                   
                 ACGCAGGGTACATCGATGGAGGCGCCAGCCAGGAAGAGTTCT 
                   
               
               
                   
                 ATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCG 
                   
               
               
                   
                 AAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCCGCA 
                   
               
               
                   
                 AACAGAGAACCTTTGACAACGGAAGCATTCCACACCAGATCC 
                   
               
               
                   
                 ATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTT 
                   
               
               
                   
                 TTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAAT 
                   
               
               
                   
                 TCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGC 
                   
               
               
                   
                 GGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAA 
                   
               
               
                   
                 ACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAG 
                   
               
               
                   
                 CTTCGGCACAATCCTTCATCGAACGAATGACCAACTTCGACAA 
                   
               
               
                   
                 GAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTT 
                   
               
               
                   
                 TACGAATACTTCACTGTCTACAACGAACTGACTAAAGTGAAAT 
                   
               
               
                   
                 ACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGAGCGGAG 
                   
               
               
                   
                 AACAGAAGAAAGCGATTGTCGATCTGCTGTTCAAGACCAACC 
                   
               
               
                   
                 GCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGA 
                   
               
               
                   
                 AGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGAGTGGAGG 
                   
               
               
                   
                 ACAGATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTGAA 
                   
               
               
                   
                 GATCATCAAGGACAAGGACTTCCTTGACAACGAGGAGAACGA 
                   
               
               
                   
                 GGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAG 
                   
               
               
                   
                 GATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCAT 
                   
               
               
                   
                 CTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGT 
                   
               
               
                   
                 ACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTAT 
                   
               
               
                   
                 TCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAA 
                   
               
               
                   
                 TCGGATGGCTTCGCTAATCGTAACTTCATGCAGTTGATCCACG 
                   
               
               
                   
                 ACGACAGCCTGACCTTTAAGGAGGACATCCAGAAAGCACAAG 
                   
               
               
                   
                 TGAGCGGACAGGGAGACTCACTCCATGAACACATCGCGAATC 
                   
               
               
                   
                 TGGCCGGTTCGCCGGCGATTAAGAAGGGAATCCTGCAAACTGT 
                   
               
               
                   
                 GAAGGTGGTGGACGAGCTGGTGAAGGTCATGGGACGGCACAA 
                   
               
               
                   
                 ACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAACCAGAC 
                   
               
               
                   
                 TACCCAGAAGGGCCAGAAGAACTCCCGCGAAAGGATGAAGCG 
                   
               
               
                   
                 GATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAA 
                   
               
               
                   
                 AGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCT 
                   
               
               
                   
                 CTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGAC 
                   
               
               
                   
                 CAAGAGCTGGACATCAATCGGTTGTCTGATTACGACGTGGACC 
                   
               
               
                   
                 ACATCGTTCCACAGTCCTTTCTGAAGGATGACTCCATCGATAA 
                   
               
               
                   
                 CAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGA 
                   
               
               
                   
                 TAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTA 
                   
               
               
                   
                 CTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAA 
                   
               
               
                   
                 GTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAG 
                   
               
               
                   
                 CTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTC 
                   
               
               
                   
                 GGCAGATTACCAAGCACGTGGCGCAGATCCTGGACTCCCGCAT 
                   
               
               
                   
                 GAACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGT 
                   
               
               
                   
                 GAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGG 
                   
               
               
                   
                 AAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACC 
                   
               
               
                   
                 ATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGCACCGC 
                   
               
               
                   
                 CCTGATCAAGAAGTACCCTAAACTTGAATCGGAGTTTGTGTAC 
                   
               
               
                   
                 GGAGACTACAAGGTCTACGACGTGAGGAAGATGATAGCCAAG 
                   
               
               
                   
                 TCCGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTCTTTT 
                   
               
               
                   
                 ACTCAAACATCATGAACTTCTTCAAGACTGAAATTACGCTGGC 
                   
               
               
                   
                 CAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGG 
                   
               
               
                   
                 AGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGC 
                   
               
               
                   
                 AACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTG 
                   
               
               
                   
                 AAGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCG 
                   
               
               
                   
                 ATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAG 
                   
               
               
                   
                 AAAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCG 
                   
               
               
                   
                 ACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGG 
                   
               
               
                   
                 GAAAGAGCAAGAAGCTCAAATCCGTCAAAGAGCTGCTGGGGA 
                   
               
               
                   
                 TTACCATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGA 
                   
               
               
                   
                 TTTCCTGGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCT 
                   
               
               
                   
                 GATCATCAAACTGCCCAAGTACTCACTGTTCGAACTGGAAAAT 
                   
               
               
                   
                 GGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAGAAA 
                   
               
               
                   
                 GGAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCT 
                   
               
               
                   
                 ATCTTGCTTCGCACTACGAGAAACTCAAAGGGTCACCGGAAG 
                   
               
               
                   
                 ATAACGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATT 
                   
               
               
                   
                 ATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCG 
                   
               
               
                   
                 CGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCC 
                   
               
               
                   
                 TACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAG 
                   
               
               
                   
                 AACATTATCCACTTGTTCACCCTGACTAACCTGGGAGCTCCAG 
                   
               
               
                   
                 CCGCCTTCAAGTACTTCGATACTACTATCGACCGCAAAAGATA 
                   
               
               
                   
                 CACGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAA 
                   
               
               
                   
                 AGCATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCAGC 
                   
               
               
                   
                 TGGGTGGCGATGGTGGCGGTGGATCCTACCCATACGACGTGCC 
                   
               
               
                   
                 TGACTACGCCTCCGGAGGTGGTGGCCCCAAGAAGAAACGGAA 
                   
               
               
                   
                 GGTGTGATAGCTAGCCATCACATTTAAAAGCATCTCAGCCTAC 
                   
               
               
                   
                 CATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTC 
                   
               
               
                   
                 ATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTA 
                   
               
               
                   
                 AAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGC 
                   
               
               
                   
                 TTCAATTAATAAAAAATGGAAAGAACCTCGAG 
                   
               
               
                   
               
               
                 Cas9 
                 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT 
                 47 
               
               
                 transcript 
                 GTGTCGTTGCAGGCCTTATTCGGATCTATGGATAAGAAGTACT 
                   
               
               
                 with 5′ UTR 
                 CGATCGGGCTGGATATCGGAACTAATTCCGTGGGTTGGGCAGT 
                   
               
               
                 of HSD, 
                 GATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGT 
                   
               
               
                 ORF 
                 CCTGGGGAACACCGATAGACACAGCATCAAGAAGAATCTCAT 
                   
               
               
                 corresponding 
                 CGGAGCCCTGCTGTTTGACTCCGGCGAAACCGCAGAAGCGAC 
                   
               
               
                 to SEQ 
                 CCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGGCGGAA 
                   
               
               
                 ID NO: 45, 
                 GAATCGCATCTGCTATCTGCAAGAAATCTTTTCGAACGAAATG 
                   
               
               
                 and 3′ UTR 
                 GCAAAGGTGGACGACAGCTTCTTCCACCGCCTGGAAGAATCTT 
                   
               
               
                 of ALB 
                 TCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATCCTATCT 
                   
               
               
                   
                 TTGGAAACATCGTGGACGAAGTGGCGTACCACGAAAAGTACC 
                   
               
               
                   
                 CGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTCAACTGA 
                   
               
               
                   
                 CAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCCCATATG 
                   
               
               
                   
                 ATCAAATTCCGCGGACACTTCCTGATCGAAGGCGATCTGAACC 
                   
               
               
                   
                 CTGATAACTCCGACGTGGATAAGCTGTTCATTCAACTGGTGCA 
                   
               
               
                   
                 GACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCCAG 
                   
               
               
                   
                 CGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAG 
                   
               
               
                   
                 TCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGAG 
                   
               
               
                   
                 AAGAAGAACGGACTTTTCGGCAACTTGATCGCTCTCTCACTGG 
                   
               
               
                   
                 GACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGA 
                   
               
               
                   
                 CGCGAAGCTGCAACTCTCAAAGGACACCTACGACGACGACTT 
                   
               
               
                   
                 GGACAATTTGCTGGCACAAATTGGCGATCAGTACGCGGATCTG 
                   
               
               
                   
                 TTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCG 
                   
               
               
                   
                 ATATCCTGCGCGTGAACACCGAAATAACCAAAGCGCCGCTTA 
                   
               
               
                   
                 GCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAGGATCT 
                   
               
               
                   
                 CACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTGAAAA 
                   
               
               
                   
                 GTACAAGGAGATTTTCTTCGACCAGTCCAAGAATGGGTACGCA 
                   
               
               
                   
                 GGGTACATCGATGGAGGCGCCAGCCAGGAAGAGTTCTATAAG 
                   
               
               
                   
                 TTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAA 
                   
               
               
                   
                 CTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCCGCAAACAG 
                   
               
               
                   
                 AGAACCTTTGACAACGGAAGCATTCCACACCAGATCCATCTGG 
                   
               
               
                   
                 GTGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTTTTACCC 
                   
               
               
                   
                 ATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTCTGAC 
                   
               
               
                   
                 GTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAAT 
                   
               
               
                   
                 TCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATC 
                   
               
               
                   
                 ACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGG 
                   
               
               
                   
                 CACAATCCTTCATCGAACGAATGACCAACTTCGACAAGAATCT 
                   
               
               
                   
                 CCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAA 
                   
               
               
                   
                 TACTTCACTGTCTACAACGAACTGACTAAAGTGAAATACGTTA 
                   
               
               
                   
                 CTGAAGGAATGAGGAAGCCGGCCTTTCTGAGCGGAGAACAGA 
                   
               
               
                   
                 AGAAAGCGATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGT 
                   
               
               
                   
                 GACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGA 
                   
               
               
                   
                 GTGTTTCGACTCAGTGGAAATCAGCGGAGTGGAGGACAGATT 
                   
               
               
                   
                 CAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCATC 
                   
               
               
                   
                 AAGGACAAGGACTTCCTTGACAACGAGGAGAACGAGGACATC 
                   
               
               
                   
                 CTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGATCGCG 
                   
               
               
                   
                 AGATGATCGAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGA 
                   
               
               
                   
                 CGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGG 
                   
               
               
                   
                 TTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGAT 
                   
               
               
                   
                 AAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAATCGGATG 
                   
               
               
                   
                 GCTTCGCTAATCGTAACTTCATGCAGTTGATCCACGACGACAG 
                   
               
               
                   
                 CCTGACCTTTAAGGAGGACATCCAGAAAGCACAAGTGAGCGG 
                   
               
               
                   
                 ACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGG 
                   
               
               
                   
                 TTCGCCGGCGATTAAGAAGGGAATCCTGCAAACTGTGAAGGT 
                   
               
               
                   
                 GGTGGACGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGA 
                   
               
               
                   
                 GAATATCGTGATTGAAATGGCCCGAGAAAACCAGACTACCCA 
                   
               
               
                   
                 GAAGGGCCAGAAGAACTCCCGCGAAAGGATGAAGCGGATCGA 
                   
               
               
                   
                 AGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCA 
                   
               
               
                   
                 CCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCT 
                   
               
               
                   
                 GTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGA 
                   
               
               
                   
                 GCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCACATC 
                   
               
               
                   
                 GTTCCACAGTCCTTTCTGAAGGATGACTCCATCGATAACAAGG 
                   
               
               
                   
                 TGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGATAATG 
                   
               
               
                   
                 TGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGC 
                   
               
               
                   
                 GGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTG 
                   
               
               
                   
                 ACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAGCTGG 
                   
               
               
                   
                 ATAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGC 
                   
               
               
                   
                 AGATTACCAAGCACGTGGCGCAGATCCTGGACTCCCGCATGA 
                   
               
               
                   
                 ACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGTGA 
                   
               
               
                   
                 AGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAA 
                   
               
               
                   
                 GGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCAT 
                   
               
               
                   
                 CACGCGCATGACGCATACCTCAACGCTGTGGTCGGCACCGCCC 
                   
               
               
                   
                 TGATCAAGAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGG 
                   
               
               
                   
                 AGACTACAAGGTCTACGACGTGAGGAAGATGATAGCCAAGTC 
                   
               
               
                   
                 CGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTCTTTTA 
                   
               
               
                   
                 CTCAAACATCATGAACTTCTTCAAGACTGAAATTACGCTGGCC 
                   
               
               
                   
                 AATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGA 
                   
               
               
                   
                 GAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCA 
                   
               
               
                   
                 ACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGA 
                   
               
               
                   
                 AGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGA 
                   
               
               
                   
                 TCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGA 
                   
               
               
                   
                 AAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGA 
                   
               
               
                   
                 CTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGG 
                   
               
               
                   
                 AAAGAGCAAGAAGCTCAAATCCGTCAAAGAGCTGCTGGGGAT 
                   
               
               
                   
                 TACCATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGAT 
                   
               
               
                   
                 TTCCTGGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTG 
                   
               
               
                   
                 ATCATCAAACTGCCCAAGTACTCACTGTTCGAACTGGAAAATG 
                   
               
               
                   
                 GTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAGAAAG 
                   
               
               
                   
                 GAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTA 
                   
               
               
                   
                 TCTTGCTTCGCACTACGAGAAACTCAAAGGGTCACCGGAAGAT 
                   
               
               
                   
                 AACGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGGATTAT 
                   
               
               
                   
                 CTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCGCG 
                   
               
               
                   
                 TGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCTA 
                   
               
               
                   
                 CAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAGAA 
                   
               
               
                   
                 CATTATCCACTTGTTCACCCTGACTAACCTGGGAGCTCCAGCC 
                   
               
               
                   
                 GCCTTCAAGTACTTCGATACTACTATCGACCGCAAAAGATACA 
                   
               
               
                   
                 CGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAA 
                   
               
               
                   
                 GCATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCAGCT 
                   
               
               
                   
                 GGGTGGCGATGGTGGCGGTGGATCCTACCCATACGACGTGCCT 
                   
               
               
                   
                 GACTACGCCTCCGGAGGTGGTGGCCCCAAGAAGAAACGGAAG 
                   
               
               
                   
                 GTGTGATAGCTAGCCATCACATTTAAAAGCATCTCAGCCTACC 
                   
               
               
                   
                 ATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCA 
                   
               
               
                   
                 TCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAA 
                   
               
               
                   
                 AAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCT 
                   
               
               
                   
                 TCAATTAATAAAAAATGGAAAGAACCTCGAG 
                   
               
               
                   
               
               
                 Cas9 
                 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT 
                 48 
               
               
                 transcript 
                 GTGTCGTTGCAGGCCTTATTCGGATCCATGCCTAAGAAAAAGC 
                   
               
               
                 comprising 
                 GGAAGGTCGACGGGGATAAGAAGTACTCAATCGGGCTGGATA 
                   
               
               
                 Cas9 ORF 
                 TCGGAACTAATTCCGTGGGTTGGGCAGTGATCACGGATGAATA 
                   
               
               
                 using codons 
                 CAAAGTGCCGTCCAAGAAGTTCAAGGTCCTGGGGAACACCGA 
                   
               
               
                 with 
                 TAGACACAGCATCAAGAAAAATCTCATCGGAGCCCTGCTGTTT 
                   
               
               
                 generally 
                 GACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTACC 
                   
               
               
                 high 
                 GCGAGGCGACGCTACACCCGGCGGAAGAATCGCATCTGCTAT 
                   
               
               
                 expression in 
                 CTGCAAGAGATCTTTTCGAACGAAATGGCAAAGGTCGACGAC 
                   
               
               
                 humans 
                 AGCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAGG 
                   
               
               
                   
                 ACAAGAAGCATGAACGGCATCCTATCTTTGGAAACATCGTCGA 
                   
               
               
                   
                 CGAAGTGGCGTACCACGAAAAGTACCCGACCATCTACCATCTG 
                   
               
               
                   
                 CGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGA 
                   
               
               
                   
                 TTGATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGAC 
                   
               
               
                   
                 ACTTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGACGT 
                   
               
               
                   
                 GGATAAGCTTTTCATTCAACTGGTGCAGACCTACAACCAACTG 
                   
               
               
                   
                 TTCGAAGAAAACCCAATCAATGCTAGCGGCGTCGATGCCAAG 
                   
               
               
                   
                 GCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAA 
                   
               
               
                   
                 ACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGGACTTT 
                   
               
               
                   
                 TCGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTC 
                   
               
               
                   
                 AAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCT 
                   
               
               
                   
                 CAAAGGACACCTACGACGACGACTTGGACAATTTGCTGGCAC 
                   
               
               
                   
                 AAATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAA 
                   
               
               
                   
                 CCTTTCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAAC 
                   
               
               
                   
                 ACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAG 
                   
               
               
                   
                 CGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCG 
                   
               
               
                   
                 CTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGAGATCTTCT 
                   
               
               
                   
                 TCGACCAGTCCAAGAATGGGTACGCAGGGTACATCGATGGAG 
                   
               
               
                   
                 GCGCTAGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCT 
                   
               
               
                   
                 GGAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCTGAA 
                   
               
               
                   
                 CAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACAACGG 
                   
               
               
                   
                 ATCCATTCCCCACCAGATCCATCTGGGTGAGCTGCACGCCATC 
                   
               
               
                   
                 TTGCGGCGCCAGGAGGACTTTTACCCATTCCTCAAGGACAACC 
                   
               
               
                   
                 GGGAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATTA 
                   
               
               
                   
                 CGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATG 
                   
               
               
                   
                 ACTAGAAAATCAGAGGAAACCATCACTCCTTGGAATTTCGAG 
                   
               
               
                   
                 GAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTTCATCGAA 
                   
               
               
                   
                 CGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTG 
                   
               
               
                   
                 CTTCCTAAGCACAGCCTCCTTTACGAATACTTCACTGTCTACAA 
                   
               
               
                   
                 CGAACTGACTAAAGTGAAATACGTTACTGAAGGAATGAGGAA 
                   
               
               
                   
                 GCCGGCCTTTCTGTCCGGAGAACAGAAGAAAGCAATTGTCGAT 
                   
               
               
                   
                 CTGCTGTTCAAGACCAACCGCAAGGTGACCGTCAAGCAGCTTA 
                   
               
               
                   
                 AAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGG 
                   
               
               
                   
                 AAATCAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGAA 
                   
               
               
                   
                 CCTATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCT 
                   
               
               
                   
                 TGACAACGAGGAGAACGAGGACATCCTGGAAGATATCGTCCT 
                   
               
               
                   
                 GACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAG 
                   
               
               
                   
                 GCTTAAGACCTACGCTCATCTCTTCGACGATAAGGTCATGAAA 
                   
               
               
                   
                 CAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTCCC 
                   
               
               
                   
                 GCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAA 
                   
               
               
                   
                 CTATCCTGGATTTCCTCAAATCGGATGGCTTCGCTAATCGTAA 
                   
               
               
                   
                 CTTCATGCAATTGATCCACGACGACAGCCTGACCTTTAAGGAG 
                   
               
               
                   
                 GACATCCAAAAAGCACAAGTGTCCGGACAGGGAGACTCACTC 
                   
               
               
                   
                 CATGAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTAAGA 
                   
               
               
                   
                 AGGGAATTCTGCAAACTGTGAAGGTGGTCGACGAGCTGGTGA 
                   
               
               
                   
                 AGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGAAA 
                   
               
               
                   
                 TGGCCCGAGAAAACCAGACTACCCAGAAGGGCCAGAAAAACT 
                   
               
               
                   
                 CCCGCGAAAGGATGAAGCGGATCGAAGAAGGAATCAAGGAG 
                   
               
               
                   
                 CTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAAACACG 
                   
               
               
                   
                 CAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTGCAAAATG 
                   
               
               
                   
                 GACGGGACATGTACGTGGACCAAGAGCTGGACATCAATCGGT 
                   
               
               
                   
                 TGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTTCTG 
                   
               
               
                   
                 AAGGATGACTCGATCGATAACAAGGTGTTGACTCGCAGCGAC 
                   
               
               
                   
                 AAGAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTC 
                   
               
               
                   
                 GTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTGAATGCG 
                   
               
               
                   
                 AAGCTGATTACCCAGAGAAAGTTTGACAATCTCACTAAAGCCG 
                   
               
               
                   
                 AGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCA 
                   
               
               
                   
                 AACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGG 
                   
               
               
                   
                 CGCAGATCTTGGACTCCCGCATGAACACTAAATACGACGAGA 
                   
               
               
                   
                 ACGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAA 
                   
               
               
                   
                 GCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTTTACAA 
                   
               
               
                   
                 AGTGAGAGAAATCAACAACTACCATCACGCGCATGACGCATA 
                   
               
               
                   
                 CCTCAACGCTGTGGTCGGTACCGCCCTGATCAAAAAGTACCCT 
                   
               
               
                   
                 AAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACG 
                   
               
               
                   
                 ACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGGA 
                   
               
               
                   
                 AAGCAACTGCGAAATACTTCTTTTACTCAAACATCATGAACTT 
                   
               
               
                   
                 TTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCAGGAA 
                   
               
               
                   
                 GAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAAATCGT 
                   
               
               
                   
                 GTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCT 
                   
               
               
                   
                 CTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTGCA 
                   
               
               
                   
                 AACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAA 
                   
               
               
                   
                 TAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGACCCGAA 
                   
               
               
                   
                 GAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTC 
                   
               
               
                   
                 CTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAAGCTC 
                   
               
               
                   
                 AAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGA 
                   
               
               
                   
                 TCCTCGTTCGAGAAGAACCCGATTGATTTCCTCGAGGCGAAGG 
                   
               
               
                   
                 GTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTCCCCA 
                   
               
               
                   
                 AGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCT 
                   
               
               
                   
                 GGCTTCGGCCGGAGAACTCCAAAAAGGAAATGAGCTGGCCTT 
                   
               
               
                   
                 GCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACG 
                   
               
               
                   
                 AAAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGC 
                   
               
               
                   
                 TTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGA 
                   
               
               
                   
                 ACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGACGCC 
                   
               
               
                   
                 AACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGAT 
                   
               
               
                   
                 AAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTTGTTC 
                   
               
               
                   
                 ACCCTGACTAACCTGGGAGCCCCAGCCGCCTTCAAGTACTTCG 
                   
               
               
                   
                 ATACTACTATCGATCGCAAAAGATACACGTCCACCAAGGAAG 
                   
               
               
                   
                 TTCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACTCTA 
                   
               
               
                   
                 CGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATTGATAG 
                   
               
               
                   
                 TCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGAAT 
                   
               
               
                   
                 AAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTT 
                   
               
               
                   
                 CTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATA 
                   
               
               
                   
                 AATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAAT 
                   
               
               
                   
                 AAAAAATGGAAAGAACCTCGAG 
                   
               
               
                   
               
               
                 Cas9 
                 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT 
                 49 
               
               
                 transcript 
                 GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGCCTAAGA 
                   
               
               
                 comprising 
                 AAAAGCGGAAGGTCGACGGGGATAAGAAGTACTCAATCGGGC 
                   
               
               
                 Kozak 
                 TGGATATCGGAACTAATTCCGTGGGTTGGGCAGTGATCACGGA 
                   
               
               
                 sequence 
                 TGAATACAAAGTGCCGTCCAAGAAGTTCAAGGTCCTGGGGAA 
                   
               
               
                 with Cas9 
                 CACCGATAGACACAGCATCAAGAAAAATCTCATCGGAGCCCT 
                   
               
               
                 ORF using 
                 GCTGTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAA 
                   
               
               
                 codons with 
                 ACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAATCGCAT 
                   
               
               
                 generally 
                 CTGCTATCTGCAAGAGATCTTTTCGAACGAAATGGCAAAGGTC 
                   
               
               
                 high 
                 GACGACAGCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGG 
                   
               
               
                 expression in 
                 AGGAGGACAAGAAGCATGAACGGCATCCTATCTTTGGAAACA 
                   
               
               
                 humans 
                 TCGTCGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCT 
                   
               
               
                   
                 ACCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCG 
                   
               
               
                   
                 ACCTCAGATTGATCTACTTGGCCCTCGCCCATATGATCAAATT 
                   
               
               
                   
                 CCGCGGACACTTCCTGATCGAAGGCGATCTGAACCCTGATAAC 
                   
               
               
                   
                 TCCGACGTGGATAAGCTTTTCATTCAACTGGTGCAGACCTACA 
                   
               
               
                   
                 ACCAACTGTTCGAAGAAAACCCAATCAATGCTAGCGGCGTCG 
                   
               
               
                   
                 ATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCG 
                   
               
               
                   
                 CCTCGAAAACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAA 
                   
               
               
                   
                 CGGACTTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACTC 
                   
               
               
                   
                 CCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCT 
                   
               
               
                   
                 GCAACTCTCAAAGGACACCTACGACGACGACTTGGACAATTTG 
                   
               
               
                   
                 CTGGCACAAATTGGCGATCAGTACGCGGATCTGTTCCTTGCCG 
                   
               
               
                   
                 CTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTGCG 
                   
               
               
                   
                 CGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGAT 
                   
               
               
                   
                 GATTAAGCGGTACGACGAGCATCACCAGGATCTCACGCTGCTC 
                   
               
               
                   
                 AAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGAG 
                   
               
               
                   
                 ATCTTCTTCGACCAGTCCAAGAATGGGTACGCAGGGTACATCG 
                   
               
               
                   
                 ATGGAGGCGCTAGCCAGGAAGAGTTCTATAAGTTCATCAAGC 
                   
               
               
                   
                 CAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTCA 
                   
               
               
                   
                 AGCTGAACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTG 
                   
               
               
                   
                 ACAACGGATCCATTCCCCACCAGATCCATCTGGGTGAGCTGCA 
                   
               
               
                   
                 CGCCATCTTGCGGCGCCAGGAGGACTTTTACCCATTCCTCAAG 
                   
               
               
                   
                 GACAACCGGGAAAAGATCGAGAAAATTCTGACGTTCCGCATC 
                   
               
               
                   
                 CCGTATrACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCG 
                   
               
               
                   
                 CGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGGA 
                   
               
               
                   
                 ATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTT 
                   
               
               
                   
                 CATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAACGA 
                   
               
               
                   
                 GAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAATACTTCACT 
                   
               
               
                   
                 GTCTACAACGAACTGACTAAAGTGAAATACGTTACTGAAGGA 
                   
               
               
                   
                 ATGAGGAAGCCGGCCTTTCTGTCCGGAGAACAGAAGAAAGCA 
                   
               
               
                   
                 ATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGTCA 
                   
               
               
                   
                 AGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCG 
                   
               
               
                   
                 ACTCAGTGGAAATCAGCGGGGTGGAGGACAGATTCAACGCTT 
                   
               
               
                   
                 CGCTGGGAACCTATCATGATCTCCTGAAGATCATCAAGGACAA 
                   
               
               
                   
                 GGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGA 
                   
               
               
                   
                 TATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAGATGATC 
                   
               
               
                   
                 GAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGATAAGG 
                   
               
               
                   
                 TCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCC 
                   
               
               
                   
                 GCCTCTCCCGCAAGCTGATCAACGGTATTCGCGATAAACAGAG 
                   
               
               
                   
                 CGGTAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCT 
                   
               
               
                   
                 AATCGTAACTTCATGCAATTGATCCACGACGACAGCCTGACCT 
                   
               
               
                   
                 TTAAGGAGGACATCCAAAAAGCACAAGTGTCCGGACAGGGAG 
                   
               
               
                   
                 ACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCCGGC 
                   
               
               
                   
                 GATTAAGAAGGGAATTCTGCAAACTGTGAAGGTGGTCGACGA 
                   
               
               
                   
                 GCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGT 
                   
               
               
                   
                 GATTGAAATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCA 
                   
               
               
                   
                 GAAAAACTCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAAT 
                   
               
               
                   
                 CAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGA 
                   
               
               
                   
                 AAACACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTG 
                   
               
               
                   
                 CAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGACATC 
                   
               
               
                   
                 AATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGT 
                   
               
               
                   
                 CCTTTCTGAAGGATGACTCGATCGATAACAAGGTGTTGACTCG 
                   
               
               
                   
                 CAGCGACAAGAACAGAGGGAAGTCAGATAATGTGCCATCGGA 
                   
               
               
                   
                 GGAGGTCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCT 
                   
               
               
                   
                 GAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACT 
                   
               
               
                   
                 AAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGA 
                   
               
               
                   
                 TTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGC 
                   
               
               
                   
                 ACGTGGCGCAGATCTTGGACTCCCGCATGAACACTAAATACGA 
                   
               
               
                   
                 CGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCT 
                   
               
               
                   
                 GAAAAGCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTT 
                   
               
               
                   
                 TACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGAC 
                   
               
               
                   
                 GCATACCTCAACGCTGTGGTCGGTACCGCCCTGATCAAAAAGT 
                   
               
               
                   
                 ACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGT 
                   
               
               
                   
                 CTACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAAT 
                   
               
               
                   
                 CGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACATCATG 
                   
               
               
                   
                 AACTTTTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCA 
                   
               
               
                   
                 GGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAA 
                   
               
               
                   
                 ATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAA 
                   
               
               
                   
                 GTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAG 
                   
               
               
                   
                 TGCAAACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGA 
                   
               
               
                   
                 GAAATAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGACC 
                   
               
               
                   
                 CGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTC 
                   
               
               
                   
                 CGTCCTCGTGGTGOCCAAGGTGGAGAAGGGAAAGAGCAAAAA 
                   
               
               
                   
                 GCTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAA 
                   
               
               
                   
                 CGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTCGAGGCGA 
                   
               
               
                   
                 AGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTCC 
                   
               
               
                   
                 CCAAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCAT 
                   
               
               
                   
                 GCTGGCTTCGGCCGGAGAACTCCAAAAAGGAAATGAGCTGGC 
                   
               
               
                   
                 CTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACT 
                   
               
               
                   
                 ACGAAAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGC 
                   
               
               
                   
                 AGCTTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAATCAT 
                   
               
               
                   
                 CGAACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGAC 
                   
               
               
                   
                 GCCAACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGA 
                   
               
               
                   
                 GATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTTG 
                   
               
               
                   
                 TTCACCCTGACTAACCTGGGAGCCCCAGCCGCCTTCAAGTACT 
                   
               
               
                   
                 TCGATACTACTATCGATCGCAAAAGATACACGTCCACCAAGGA 
                   
               
               
                   
                 AGTTCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACTC 
                   
               
               
                   
                 TACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATTGAT 
                   
               
               
                   
                 AGTCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGA 
                   
               
               
                   
                 ATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTT 
                   
               
               
                   
                 TTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACA 
                   
               
               
                   
                 TAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTA 
                   
               
               
                   
                 ATAAAAAATGGAAAGAACCTCGAG 
                   
               
               
                   
               
               
                 Cas9 ORF 
                 ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAAC 
                 50 
               
               
                 with splice 
                 AGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCG 
                   
               
               
                 junctions 
                 AGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGC 
                   
               
               
                 removed; 
                 ATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGA 
                   
               
               
                 12.75% U 
                 GAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAG 
                   
               
               
                 content 
                 AAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGA 
                   
               
               
                   
                 AATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTC 
                   
               
               
                   
                 CACcggCTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGC 
                   
               
               
                   
                 ACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCG 
                   
               
               
                   
                 CATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGA 
                   
               
               
                   
                 AGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCT 
                   
               
               
                   
                 ACCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCT 
                   
               
               
                   
                 GATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAA 
                   
               
               
                   
                 GCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAA 
                   
               
               
                   
                 GAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATC 
                   
               
               
                   
                 CTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTG 
                   
               
               
                   
                 ATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGA 
                   
               
               
                   
                 AACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAG 
                   
               
               
                   
                 AGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGC 
                   
               
               
                   
                 AAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAG 
                   
               
               
                   
                 ATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC 
                   
               
               
                   
                 CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAAC 
                   
               
               
                   
                 ACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAG 
                   
               
               
                   
                 AGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCA 
                   
               
               
                   
                 CTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTC 
                   
               
               
                   
                 TTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGA 
                   
               
               
                   
                 GGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATC 
                   
               
               
                   
                 CTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTG 
                   
               
               
                   
                 AACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAAC 
                   
               
               
                   
                 GGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCA 
                   
               
               
                   
                 ATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC 
                   
               
               
                   
                 AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCG 
                   
               
               
                   
                 TACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCA 
                   
               
               
                   
                 TGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAAC 
                   
               
               
                   
                 TTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTC 
                   
               
               
                   
                 ATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA 
                   
               
               
                   
                 AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAG 
                   
               
               
                   
                 TCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAA 
                   
               
               
                   
                 TGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAA 
                   
               
               
                   
                 TCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCA 
                   
               
               
                   
                 AGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCG 
                   
               
               
                   
                 ACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAA 
                   
               
               
                   
                 GCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACA 
                   
               
               
                   
                 AGGACITCCTGGACAACGAAGAAAACGAAGACATCCTGGAAG 
                   
               
               
                   
                 ACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGAT 
                   
               
               
                   
                 CGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAA 
                   
               
               
                   
                 GGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGG 
                   
               
               
                   
                 AAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCA 
                   
               
               
                   
                 GAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATT 
                   
               
               
                   
                 CGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCT 
                   
               
               
                   
                 GACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACA 
                   
               
               
                   
                 GGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG 
                   
               
               
                   
                 CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGT 
                   
               
               
                   
                 CGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAA 
                   
               
               
                   
                 CATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAA 
                   
               
               
                   
                 GGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAG 
                   
               
               
                   
                 AAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACC 
                   
               
               
                   
                 CGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGT 
                   
               
               
                   
                 ACTACCTGCAaAACGGAAGAGACATGTACGTCGACCAGGAACT 
                   
               
               
                   
                 GGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGT 
                   
               
               
                   
                 CCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGT 
                   
               
               
                   
                 CCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACG 
                   
               
               
                   
                 TCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGA 
                   
               
               
                   
                 GACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCG 
                   
               
               
                   
                 ACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTG 
                   
               
               
                   
                 GACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGA 
                   
               
               
                   
                 CAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATG 
                   
               
               
                   
                 AACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTC 
                   
               
               
                   
                 AAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGA 
                   
               
               
                   
                 AAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTAC 
                   
               
               
                   
                 CACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACA 
                   
               
               
                   
                 GCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC 
                   
               
               
                   
                 TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCA 
                   
               
               
                   
                 AAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTC 
                   
               
               
                   
                 TTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACAC 
                   
               
               
                   
                 TGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAA 
                   
               
               
                   
                 ACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC 
                   
               
               
                   
                 TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAAC 
                   
               
               
                   
                 ATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAG 
                   
               
               
                   
                 GAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCA 
                   
               
               
                   
                 AGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGAC 
                   
               
               
                   
                 AGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCG 
                   
               
               
                   
                 AAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTG 
                   
               
               
                   
                 CTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAAC 
                   
               
               
                   
                 CCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAG 
                   
               
               
                   
                 AAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAAC 
                   
               
               
                   
                 TGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAA 
                   
               
               
                   
                 CTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTC 
                   
               
               
                   
                 AACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGA 
                   
               
               
                   
                 AGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAG 
                   
               
               
                   
                 CACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAA 
                   
               
               
                   
                 TTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAG 
                   
               
               
                   
                 GTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGA 
                   
               
               
                   
                 GAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAAC 
                   
               
               
                   
                 CTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATC 
                   
               
               
                   
                 GACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCA 
                   
               
               
                   
                 ACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGA 
                   
               
               
                   
                 ATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCG 
                   
               
               
                   
                 AAGAAGAAGAGAAAGGTCTAG 
                   
               
               
                   
               
               
                 Cas9 
                 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT 
                 51 
               
               
                 transcript 
                 GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAGA 
                   
               
               
                 with 5′ UTR 
                 AGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT 
                   
               
               
                 ofHSD. 
                 GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGT 
                   
               
               
                 ORF 
                 TCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGA 
                   
               
               
                 corresponding 
                 ACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAG 
                   
               
               
                 to SEQ 
                 AAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACA 
                   
               
               
                 ID NO: 50, 
                 AGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC 
                   
               
               
                 Kozak 
                 AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACcggCTGG 
                   
               
               
                 sequence, 
                 AAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGAC 
                   
               
               
                 and 3′ UTR 
                 ACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGA 
                   
               
               
                 of ALB 
                 AAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGA 
                   
               
               
                   
                 CAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACT 
                   
               
               
                   
                 GGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGG 
                   
               
               
                   
                 AGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCAT 
                   
               
               
                   
                 CCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCC 
                   
               
               
                   
                 GATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGC 
                   
               
               
                   
                 AAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACA 
                   
               
               
                   
                 GCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGAT 
                   
               
               
                   
                 CGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTC 
                   
               
               
                   
                 GACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACA 
                   
               
               
                   
                 TACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGAC 
                   
               
               
                   
                 CAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGAC 
                   
               
               
                   
                 GCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATC 
                   
               
               
                   
                 ACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGAC 
                   
               
               
                   
                 GAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGA 
                   
               
               
                   
                 CAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAG 
                   
               
               
                   
                 AGCAAGAACGGATACGCAGGATACATCGACGGAGOAGCAAGC 
                   
               
               
                   
                 CAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAG 
                   
               
               
                   
                 ATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAA 
                   
               
               
                   
                 GACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATC 
                   
               
               
                   
                 CCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGA 
                   
               
               
                   
                 AGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAA 
                   
               
               
                   
                 AAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCG 
                   
               
               
                   
                 GACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAA 
                   
               
               
                   
                 GAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAG 
                   
               
               
                   
                 TCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAA 
                   
               
               
                   
                 TGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGC 
                   
               
               
                   
                 CGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA 
                   
               
               
                   
                 ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCC 
                   
               
               
                   
                 GGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCT 
                   
               
               
                   
                 GCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAA 
                   
               
               
                   
                 GGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGA 
                   
               
               
                   
                 AATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAAC 
                   
               
               
                   
                 ATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTG 
                   
               
               
                   
                 GACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTG 
                   
               
               
                   
                 ACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGA 
                   
               
               
                   
                 CTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAG 
                   
               
               
                   
                 CAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAG 
                   
               
               
                   
                 CAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAA 
                   
               
               
                   
                 AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACA 
                   
               
               
                   
                 GAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAA 
                   
               
               
                   
                 GGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACA 
                   
               
               
                   
                 GCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAA 
                   
               
               
                   
                 TCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAAC 
                   
               
               
                   
                 TGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCA 
                   
               
               
                   
                 TCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAG 
                   
               
               
                   
                 AAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAAT 
                   
               
               
                   
                 CAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGA 
                   
               
               
                   
                 AAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT 
                   
               
               
                   
                 GCAaAACGGAAGAGACATGTACGTCGACCAGGAACTGGACAT 
                   
               
               
                   
                 CAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCA 
                   
               
               
                   
                 GAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGAC 
                   
               
               
                   
                 AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGA 
                   
               
               
                   
                 GCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGC 
                   
               
               
                   
                 TGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACC 
                   
               
               
                   
                 TGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAG 
                   
               
               
                   
                 GCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATC 
                   
               
               
                   
                 ACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACA 
                   
               
               
                   
                 AAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTC 
                   
               
               
                   
                 ATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC 
                   
               
               
                   
                 TTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACG 
                   
               
               
                   
                 CACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGA 
                   
               
               
                   
                 TCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAG 
                   
               
               
                   
                 ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCG 
                   
               
               
                   
                 AACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA 
                   
               
               
                   
                 GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA 
                   
               
               
                   
                 ACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGA 
                   
               
               
                   
                 GAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCA 
                   
               
               
                   
                 ACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTC 
                   
               
               
                   
                 AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAG 
                   
               
               
                   
                 CATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAA 
                   
               
               
                   
                 GAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCC 
                   
               
               
                   
                 GACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAA 
                   
               
               
                   
                 GGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGG 
                   
               
               
                   
                 GAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGA 
                   
               
               
                   
                 TCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG 
                   
               
               
                   
                 ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGA 
                   
               
               
                   
                 AAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGC 
                   
               
               
                   
                 AGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACT 
                   
               
               
                   
                 TCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCC 
                   
               
               
                   
                 CGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACA 
                   
               
               
                   
                 AGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA 
                   
               
               
                   
                 GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCC 
                   
               
               
                   
                 TGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC 
                   
               
               
                   
                 AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGG 
                   
               
               
                   
                 GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA 
                   
               
               
                   
                 GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACAC 
                   
               
               
                   
                 TGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCG 
                   
               
               
                   
                 ACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAG 
                   
               
               
                   
                 AAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATC 
                   
               
               
                   
                 TCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAT 
                   
               
               
                   
                 AGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACA 
                   
               
               
                   
                 CCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTT 
                   
               
               
                   
                 TCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG 
                   
               
               
                   
               
               
                 Cas9 ORF 
                 ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAAC 
                 52 
               
               
                 with 
                 AGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCC 
                   
               
               
                 minimal 
                 AGCAAGAAGTTCAAGGTGCTGGGCAACACCGACAGACACAGC 
                   
               
               
                 uridine 
                 ATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGC 
                   
               
               
                 codons 
                 GAGACCGCCGAGGCCACCAGACTGAAGAGAACCGCCAGAAGA 
                   
               
               
                 frequently 
                 AGATACACCAGAAGAAAGAACAGAATCTGCTACCTGCAGGAG 
                   
               
               
                 used in 
                 ATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCC 
                   
               
               
                 humans in 
                 ACAGACTGGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGC 
                   
               
               
                 general; 
                 ACGAGAGACACCCCATCTTCGGCAACATCGTGGACGAGGTGG 
                   
               
               
                 12.75% U 
                 CCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGA 
                   
               
               
                 content 
                 AGCTGGTGGACAGCACCGACAAGGCCGACCTGAGACTGATCT 
                   
               
               
                   
                 ACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTCCT 
                   
               
               
                   
                 GATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAA 
                   
               
               
                   
                 GCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAG 
                   
               
               
                   
                 GAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATC 
                   
               
               
                   
                 CTGAGCGCCAGACTGAGCAAGAGCAGAAGACTGGAGAACCTG 
                   
               
               
                   
                 ATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGC 
                   
               
               
                   
                 AACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGA 
                   
               
               
                   
                 GCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCA 
                   
               
               
                   
                 AGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGA 
                   
               
               
                   
                 TCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCT 
                   
               
               
                   
                 GAGCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACAC 
                   
               
               
                   
                 CGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGAG 
                   
               
               
                   
                 ATACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCT 
                   
               
               
                   
                 GGTGAGACAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTT 
                   
               
               
                   
                 CGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGG 
                   
               
               
                   
                 CGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTG 
                   
               
               
                   
                 GAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAAC 
                   
               
               
                   
                 AGAGAGGACCTGCTGAGAAAGCAGAGAACCTTCGACAACGGC 
                   
               
               
                   
                 AGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCC 
                   
               
               
                   
                 TGAGAAGACAGGAGGACTTCTACCCCTTCCTGAAGGACAACA 
                   
               
               
                   
                 GAGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCCTACT 
                   
               
               
                   
                 ACGTGGGCCCCCTGGCCAGAGGCAACAGCAGATTCGCCTGGA 
                   
               
               
                   
                 TGACCAGAAAGAGCGAGGAGACCATCACCCCCTGGAACTTCG 
                   
               
               
                   
                 AGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCG 
                   
               
               
                   
                 AGAGAATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGG 
                   
               
               
                   
                 TGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTA 
                   
               
               
                   
                 CAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGAG 
                   
               
               
                   
                 AAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGT 
                   
               
               
                   
                 GGACCTGCTGTTCAAGACCAACAGAAAGGTGACCGTGAAGCA 
                   
               
               
                   
                 GCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAG 
                   
               
               
                   
                 CGTGGAGATCAGCGGCGTGGAGGACAGATTCAACGCCAGCCT 
                   
               
               
                   
                 GGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGA 
                   
               
               
                   
                 CTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACAT 
                   
               
               
                   
                 CGTGCTGACCCTGACCCTGTTCGAGGACAGAGAGATGATCGA 
                   
               
               
                   
                 GGAGAGACTGAAGACCTACGCCCACCTGTTCGACGACAAGGT 
                   
               
               
                   
                 GATGAAGCAGCTGAAGAGAAGAAGATACACCGGCTGGGGCAG 
                   
               
               
                   
                 ACTGAGCAGAAAGCTGATCAACGGCATCAGAGACAAGCAGAG 
                   
               
               
                   
                 CGGCAAGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCC 
                   
               
               
                   
                 AACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT 
                   
               
               
                   
                 TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCG 
                   
               
               
                   
                 ACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCG 
                   
               
               
                   
                 CCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACG 
                   
               
               
                   
                 AGCTGGTGAAGGTGATGGGCAGACACAAGCCCGAGAACATCG 
                   
               
               
                   
                 TGATCGAGATGGCCAGAGAGAACCAGACCACCCAGAAGGGCC 
                   
               
               
                   
                 AGAAGAACAGCAGAGAGAGAATGAAGAGAATCGAGGAGGGC 
                   
               
               
                   
                 ATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTG 
                   
               
               
                   
                 GAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTAC 
                   
               
               
                   
                 CTGCAGAACGGCAGAGACATGTACGTGGACCAGGAGCTGGAC 
                   
               
               
                   
                 ATCAACAGACTGAGCGACTACGACGTGGACCACATCGTGCCC 
                   
               
               
                   
                 CAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG 
                   
               
               
                   
                 ACCAGAAGCGACAAGAACAGAGGCAAGAGCGACAACGTGCC 
                   
               
               
                   
                 CAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGAGACA 
                   
               
               
                   
                 GCTGCTGAACGCCAAGCTGATCACCCAGAGAAAGTTCGACAA 
                   
               
               
                   
                 CCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAGCTGGACAA 
                   
               
               
                   
                 GGCCGGCTTCATCAAGAGACAGCTGGTGGAGACCAGACAGAT 
                   
               
               
                   
                 CACCAAGCACGTGGCCCAGATCCTGGACAGCAGAATGAACAC 
                   
               
               
                   
                 CAAGTACGACGAGAACGACAAGCTGATCAGAGAGGTGAAGGT 
                   
               
               
                   
                 GATCACCCTGAAGAGCAAGCTGGTGAGCGACTTCAGAAAGGA 
                   
               
               
                   
                 CTTCCAGTTCTACAAGGTGAGAGAGATCAACAACTACCACCAC 
                   
               
               
                   
                 GCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGA 
                   
               
               
                   
                 TCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCG 
                   
               
               
                   
                 ACTACAAGGTGTACGACGTGAGAAAGATGATCGCCAAGAGCG 
                   
               
               
                   
                 AGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACA 
                   
               
               
                   
                 GCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAA 
                   
               
               
                   
                 CGGCGAGATCAGAAAGAGACCCCTGATCGAGACCAACGGCGA 
                   
               
               
                   
                 GACCGGCGAGATCGTGTGGGACAAGGGCAGAGACTTCGCCAC 
                   
               
               
                   
                 CGTGAGAAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAA 
                   
               
               
                   
                 GAAGACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCAT 
                   
               
               
                   
                 CCTGCCCAAGAGAAACAGCGACAAGCTGATCGCCAGAAAGAA 
                   
               
               
                   
                 GGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCAC 
                   
               
               
                   
                 CGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGG 
                   
               
               
                   
                 CAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT 
                   
               
               
                   
                 CACCATCATGGAGAGAAGCAGCTTCGAGAAGAACCCCATCGA 
                   
               
               
                   
                 CTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCT 
                   
               
               
                   
                 GATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAAC 
                   
               
               
                   
                 GGCAGAAAGAGAATGCTGGCCAGCGCCGGCGAGCTGCAGAAG 
                   
               
               
                   
                 GGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGT 
                   
               
               
                   
                 ACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGG 
                   
               
               
                   
                 ACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACT 
                   
               
               
                   
                 ACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGA 
                   
               
               
                   
                 GAGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCG 
                   
               
               
                   
                 CCTACAACAAGCACAGAGACAAGCCCATCAGAGAGCAGGCCG 
                   
               
               
                   
                 AGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCC 
                   
               
               
                   
                 CGCCGCCTTCAAGTACTTCGACACCACCATCGACAGAAAGAG 
                   
               
               
                   
                 ATACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCA 
                   
               
               
                   
                 CCAGAGCATCACCGGCCTGTACGAGACCAGAATCGACCTGAG 
                   
               
               
                   
                 CCAGCTGGGCGGCGACGGCGGCGGCAGCCCCAAGAAGAAGAG 
                   
               
               
                   
                 AAAGGTGTGA 
                   
               
               
                   
               
               
                 Cas9 
                 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT 
                 53 
               
               
                 transcript 
                 GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAGA 
                   
               
               
                 with 5′ UTR 
                 AGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGCT 
                   
               
               
                 of HSD, 
                 GGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGT 
                   
               
               
                 ORF 
                 TCAAGGTGCTGGGCAACACCGACAGACACAGCATCAAGAAGA 
                   
               
               
                 corresponding 
                 ACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGA 
                   
               
               
                 to SEQ 
                 GGCCACCAGACTGAAGAGAACCGCCAGAAGAAGATACACCAG 
                   
               
               
                 ID NO: 52, 
                 AAGAAAGAACAGAATCTGCTACCTGCAGGAGATCTTCAGCAA 
                   
               
               
                 Kozak 
                 CGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGA 
                   
               
               
                 sequence, 
                 GGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGAGACA 
                   
               
               
                 and 3′ UTR 
                 CCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAG 
                   
               
               
                 of ALB 
                 AAGTACCCCACCATCTACCACCTGAGAAAGAAGCTGGTGGAC 
                   
               
               
                   
                 AGCACCGACAAGGCCGACCTGAGACTGATCTACCTGGCCCTG 
                   
               
               
                   
                 GCCCACATGATCAAGTTCAGAGGCCACTTCCTGATCGAGGGCG 
                   
               
               
                   
                 ACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCC 
                   
               
               
                   
                 AGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCA 
                   
               
               
                   
                 TCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCA 
                   
               
               
                   
                 GACTGAGCAAGAGCAGAAGACTGGAGAACCTGATCGCCCAGC 
                   
               
               
                   
                 TGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCG 
                   
               
               
                   
                 CCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGA 
                   
               
               
                   
                 CCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTA 
                   
               
               
                   
                 CGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCA 
                   
               
               
                   
                 GTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCC 
                   
               
               
                   
                 ATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACC 
                   
               
               
                   
                 AAGGCCCCCCTGAGCGCCAGCATGATCAAGAGATACGACGAG 
                   
               
               
                   
                 CACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGAGACAG 
                   
               
               
                   
                 CAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGC 
                   
               
               
                   
                 AAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAG 
                   
               
               
                   
                 GAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATG 
                   
               
               
                   
                 GACGGCACCGAGGAGCTGCTGGTGAAGCTGAACAGAGAGGAC 
                   
               
               
                   
                 CTGCTGAGAAAGCAGAGAACCTTCGACAACGGCAGCATCCCC 
                   
               
               
                   
                 CACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGAGAAGA 
                   
               
               
                   
                 CAGGAGGACTTCTACCCCTTCCTGAAGGACAACAGAGAGAAG 
                   
               
               
                   
                 ATCGAGAAGATCCTGACCTTCAGAATCCCCTACTACGTGGGCC 
                   
               
               
                   
                 CCCTGGCCAGAGGCAACAGCAGATTCGCCTGGATGACCAGAA 
                   
               
               
                   
                 AGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGG 
                   
               
               
                   
                 TGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGAGAATGA 
                   
               
               
                   
                 CCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCA 
                   
               
               
                   
                 AGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCT 
                   
               
               
                   
                 GACCAAGGTGAAGTACGTGACCGAGGGCATGAGAAAGCCCGC 
                   
               
               
                   
                 CTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCT 
                   
               
               
                   
                 GTTCAAGACCAACAGAAAGGTGACCGTGAAGCAGCTGAAGGA 
                   
               
               
                   
                 GGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGAT 
                   
               
               
                   
                 CAGCGGCGTGGAGGACAGATTCAACGCCAGCCTGGGCACCTA 
                   
               
               
                   
                 CCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGA 
                   
               
               
                   
                 CAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGAC 
                   
               
               
                   
                 CCTGACCCTGTTCGAGGACAGAGAGATGATCGAGGAGAGACT 
                   
               
               
                   
                 GAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCA 
                   
               
               
                   
                 GCTGAAGAGAAGAAGATACACCGGCTGGGGCAGACTGAGCAG 
                   
               
               
                   
                 AAAGCTGATCAACGGCATCAGAGACAAGCAGAGCGGCAAGAC 
                   
               
               
                   
                 CATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCAACAGAAAC 
                   
               
               
                   
                 TTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGG 
                   
               
               
                   
                 ACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGC 
                   
               
               
                   
                 ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGA 
                   
               
               
                   
                 AGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGA 
                   
               
               
                   
                 AGGTGATGGGCAGACACAAGCCCGAGAACATCGTGATCGAGA 
                   
               
               
                   
                 TGGCCAGAGAGAACCAGACCACCCAGAAGGGCCAGAAGAAC 
                   
               
               
                   
                 AGCAGAGAGAGAATGAAGAGAATCGAGGAGGGCATCAAGGA 
                   
               
               
                   
                 GCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACAC 
                   
               
               
                   
                 CCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAA 
                   
               
               
                   
                 CGGCAGAGACATGTACGTGGACCAGGAGCTGGACATCAACAG 
                   
               
               
                   
                 ACTGAGCGACTACGACGTGGACCACATCGTGCCCCAGAGCTTC 
                   
               
               
                   
                 CTGAAGGACGACAGCATCGACAACAAGGTGCTGACCAGAAGC 
                   
               
               
                   
                 GACAAGAACAGAGGCAAGAGCGACAACGTGCCCAGCGAGGA 
                   
               
               
                   
                 GGTGGTGAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAA 
                   
               
               
                   
                 CGCCAAGCTGATCACCCAGAGAAAGTTCGACAACCTGACCAA 
                   
               
               
                   
                 GGCCGAGAGAGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTT 
                   
               
               
                   
                 CATCAAGAGACAGCTGGTGGAGACCAGACAGATCACCAAGCA 
                   
               
               
                   
                 CGTGGCCCAGATCCTGGACAGCAGAATGAACACCAAGTACGA 
                   
               
               
                   
                 CGAGAACGACAAGCTGATCAGAGAGGTGAAGGTGATCACCCT 
                   
               
               
                   
                 GAAGAGCAAGCTGGTGAGCGACTTCAGAAAGGACTTCCAGTT 
                   
               
               
                   
                 CTACAAGGTGAGAGAGATCAACAACTACCACCACGCCCACGA 
                   
               
               
                   
                 CGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAA 
                   
               
               
                   
                 GTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAA 
                   
               
               
                   
                 GGTGTACGACGTGAGAAAGATGATCGCCAAGAGCGAGCAGGA 
                   
               
               
                   
                 GATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATC 
                   
               
               
                   
                 ATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAG 
                   
               
               
                   
                 ATCAGAAAGAGACCCCTGATCGAGACCAACGGCGAGACCGGC 
                   
               
               
                   
                 GAGATCGTGTGGGACAAGGGCAGAGACTTCGCCACCGTGAGA 
                   
               
               
                   
                 AAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGACC 
                   
               
               
                   
                 GAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCC 
                   
               
               
                   
                 AAGAGAAACAGCGACAAGCTGATCGCCAGAAAGAAGGACTG 
                   
               
               
                   
                 GGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGC 
                   
               
               
                   
                 CTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAG 
                   
               
               
                   
                 CAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCATCACCAT 
                   
               
               
                   
                 CATGGAGAGAAGCAGCTTCGAGAAGAACCCCATCGACTTCCT 
                   
               
               
                   
                 GGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCAT 
                   
               
               
                   
                 CAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCAG 
                   
               
               
                   
                 AAAGAGAATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAA 
                   
               
               
                   
                 CGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTG 
                   
               
               
                   
                 GCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAAC 
                   
               
               
                   
                 GAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTG 
                   
               
               
                   
                 GACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGAGAGTG 
                   
               
               
                   
                 ATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCGCCTAC 
                   
               
               
                   
                 AACAAGCACAGAGACAAGCCCATCAGAGAGCAGGCCGAGAA 
                   
               
               
                   
                 CATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCC 
                   
               
               
                   
                 GCCTTCAAGTACTTCGACACCACCATCGACAGAAAGAGATAC 
                   
               
               
                   
                 ACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAG 
                   
               
               
                   
                 AGCATCACCGGCCTGTACGAGACCAGAATCGACCTGAGCCAG 
                   
               
               
                   
                 CTGGGCGGCGACGGCGGCGGCAGCCCCAAGAAGAAGAGAAA 
                   
               
               
                   
                 GGTGTGACTAGCCATCACATTTAAAAGCATCTCAGCCTACCAT 
                   
               
               
                   
                 GAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATC 
                   
               
               
                   
                 TCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAA 
                   
               
               
                   
                 AACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCA 
                   
               
               
                   
                 ATTAATAAAAAATGGAAAGAACCTCGAG 
                   
               
               
                   
               
               
                 Cas9 ORF 
                 ATGGACAAAAAATACAGCATAGGGCTAGACATAGGGACGAAC 
                 54 
               
               
                 with 
                 AGCGTAGGGTGGGCGGTAATAACGGACGAATACAAAGTACCG 
                   
               
               
                 minimal 
                 AGCAAAAAATTCAAAGTACTAGGGAACACGGACCGACACAGC 
                   
               
               
                 uridine 
                 ATAAAAAAAAACCTAATAGGGGCGCTACTATTCGACAGCGGG 
                   
               
               
                 codons 
                 GAAACGGCGGAAGCGACGCGACTAAAACGAACGGCGCGACG 
                   
               
               
                 infrequently 
                 ACGATACACGCGACGAAAAAACCGAATATGCTACCTACAAGA 
                   
               
               
                 used in 
                 AATATTCAGCAACGAAATGGCGAAAGTAGACGACAGCTTCTT 
                   
               
               
                 humans in 
                 CCACCGACTAGAAGAAAGCTTCCTAGTAGAAGAAGACAAAAA 
                   
               
               
                 general; 
                 ACACGAACGACACCCGATATTCGGGAACATAGTAGACGAAGT 
                   
               
               
                 12.75% U 
                 AGCGTACCACGAAAAATACCCGACGATATACCACCTACGAAA 
                   
               
               
                 content 
                 AAAACTAGTAGACAGCACGGACAAAGCGGACCTACGACTAAT 
                   
               
               
                   
                 ATACCTAGCGCTAGCGCACATGATAAAATTCCGAGGGCACTTC 
                   
               
               
                   
                 CTAATAGAAGGGGACCTAAACCCGGACAACAGCGACGTAGAC 
                   
               
               
                   
                 AAACTATTCATACAACTAGTACAAACGTACAACCAACTATTCG 
                   
               
               
                   
                 AAGAAAACCCGATAAACGCGAGCGGGGTAGACGCGAAAGCG 
                   
               
               
                   
                 ATACTAAGCGCGCGACTAAGCAAAAGCCGACGACTAGAAAAC 
                   
               
               
                   
                 CTAATAGCGCAACTACCGGGGGAAAAAAAAAACGGGCTATTC 
                   
               
               
                   
                 GGGAACCTAATAGCGCTAAGCCTAGGGCTAACGCCGAACTTC 
                   
               
               
                   
                 AAAAGCAACTTCGACCTAGCGGAAGACGCGAAACTACAACTA 
                   
               
               
                   
                 AGCAAAGACACGTACGACGACGACCTAGACAACCTACTAGCG 
                   
               
               
                   
                 CAAATAGGGGACCAATACGCGGACCTATTCCTAGCGGCGAAA 
                   
               
               
                   
                 AACCTAAGCGACGCGATACTACTAAGCGACATACTACGAGTA 
                   
               
               
                   
                 AACACGGAAATAACGAAAGCGCCGCTAAGCGCGAGCATGATA 
                   
               
               
                   
                 AAACGATACGACGAACACCACCAAGACCTAACGCTACTAAAA 
                   
               
               
                   
                 GCGCTAGTACGACAACAACTACCGGAAAAATACAAAGAAATA 
                   
               
               
                   
                 TTCTTCGACCAAAGCAAAAACGGGTACGCGGGGTACATAGAC 
                   
               
               
                   
                 GGGGGGGCGAGCCAAGAAGAATTCTACAAATTCATAAAACCG 
                   
               
               
                   
                 ATACTAGAAAAAATGGACGGGACGGAAGAACTACTAGTAAAA 
                   
               
               
                   
                 CTAAACCGAGAAGACCTACTACGAAAACAACGAACGTTCGAC 
                   
               
               
                   
                 AACGGGAGCATACCGCACCAAATACACCTAGGGGAACTACAC 
                   
               
               
                   
                 GCGATACTACGACGACAAGAAGACTTCTACCCGTTCCTAAAAG 
                   
               
               
                   
                 ACAACCGAGAAAAAATAGAAAAAATACTAACGTTCCGAATAC 
                   
               
               
                   
                 CGTACTACGTAGGGCCGCTAGCGCGAGGGAACAGCCGATTCG 
                   
               
               
                   
                 CGTGGATGACGCGAAAAAGCGAAGAAACGATAACGCCGTGGA 
                   
               
               
                   
                 ACTTCGAAGAAGTAGTAGACAAAGGGGCGAGCGCGCAAAGCT 
                   
               
               
                   
                 TCATAGAACGAATGACGAACTTCGACAAAAACCTACCGAACG 
                   
               
               
                   
                 AAAAAGTACTACCGAAACACAGCCTACTATACGAATACTTCAC 
                   
               
               
                   
                 GGTATACAACGAACTAACGAAAGTAAAATACGTAACGGAAGG 
                   
               
               
                   
                 GATGCGAAAACCGGCGTTCCTAAGCGGGGAACAAAAAAAAGC 
                   
               
               
                   
                 GATAGTAGACCTACTATTCAAAACGAACCGAAAAGTAACGGT 
                   
               
               
                   
                 AAAACAACTAAAAGAAGACTACTTCAAAAAAATAGAATGCTT 
                   
               
               
                   
                 CGACAGCGTAGAAATAAGCGGGGTAGAAGACCGATTCAACGC 
                   
               
               
                   
                 GAGCCTAGGGACGTACCACGACCTACTAAAAATAATAAAAGA 
                   
               
               
                   
                 CAAAGACTTCCTAGACAACGAAGAAAACGAAGACATACTAGA 
                   
               
               
                   
                 AGACATAGTACTAACGCTAACGCTATTCGAAGACCGAGAAAT 
                   
               
               
                   
                 GATAGAAGAACGACTAAAAACGTACGCGCACCTATTCGACGA 
                   
               
               
                   
                 CAAAGTAATGAAACAACTAAAACGACGACGATACACGGGGTG 
                   
               
               
                   
                 GGGGCGACTAAGCCGAAAACTAATAAACGGGATACGAGACAA 
                   
               
               
                   
                 ACAAAGCGGGAAAACGATACTAGACTTCCTAAAAAGCGACGG 
                   
               
               
                   
                 GTTCGCGAACCGAAACTTCATGCAACTAATACACGACGACAG 
                   
               
               
                   
                 CCTAACGTTCAAAGAAGACATACAAAAAGCGCAAGTAAGCGG 
                   
               
               
                   
                 GCAAGGGGACAGCCTACACGAACACATAGCGAACCTAGCGGG 
                   
               
               
                   
                 GAGCCCGGCGATAAAAAAAGGGATACTACAAACGGTAAAAGT 
                   
               
               
                   
                 AGTAGACGAACTAGTAAAAGTAATGGGGCGACACAAACCGGA 
                   
               
               
                   
                 AAACATAGTAATAGAAATGGCGCGAGAAAACCAAACGACGCA 
                   
               
               
                   
                 AAAAGGGCAAAAAAACAGCCGAGAACGAATGAAACGAATAG 
                   
               
               
                   
                 AAGAAGGGATAAAAGAACTAGGGAGCCAAATACTAAAAGAA 
                   
               
               
                   
                 CACCCGGTAGAAAACACGCAACTACAAAACGAAAAACTATAC 
                   
               
               
                   
                 CTATACTACCTACAAAACGGGCGAGACATGTACGTAGACCAA 
                   
               
               
                   
                 GAACTAGACATAAACCGACTAAGCGACTACGACGTAGACCAC 
                   
               
               
                   
                 ATAGTACCGCAAAGCTTCCTAAAAGACGACAGCATAGACAAC 
                   
               
               
                   
                 AAAGTACTAACGCGAAGCGACAAAAACCGAGGGAAAAGCGA 
                   
               
               
                   
                 CAACGTACCGAGCGAAGAAGTAGTAAAAAAAATGAAAAACTA 
                   
               
               
                   
                 CTGGCGACAACTACTAAACGCGAAACTAATAACGCAACGAAA 
                   
               
               
                   
                 ATTCGACAACCTAACGAAAGCGGAACGAGGGGGGCTAAGCGA 
                   
               
               
                   
                 ACTAGACAAAGCGGGGTTCATAAAACGACAACTAGTAGAAAC 
                   
               
               
                   
                 GCGACAAATAACGAAACACGTAGCGCAAATACTAGACAGCCG 
                   
               
               
                   
                 AATGAACACGAAATACGACGAAAACGACAAACTAATACGAGA 
                   
               
               
                   
                 AGTAAAAGTAATAACGCTAAAAAGCAAACTAGTAAGCGACTT 
                   
               
               
                   
                 CCGAAAAGACTTCCAATTCTACAAAGTACGAGAAATAAACAA 
                   
               
               
                   
                 CTACCACCACGCGCACGACGCGTACCTAAACGCGGTAGTAGG 
                   
               
               
                   
                 GACGGCGCTAATAAAAAAATACCCGAAACTAGAAAGCGAATT 
                   
               
               
                   
                 CGTATACGGGGACTACAAAGTATACGACGTACGAAAAATGAT 
                   
               
               
                   
                 AGCGAAAAGCGAACAAGAAATAGGGAAAGCGACGGCGAAAT 
                   
               
               
                   
                 ACTTCTTCTACAGCAACATAATGAACTTCTTCAAAACGGAAAT 
                   
               
               
                   
                 AACGCTAGCGAACGGGGAAATACGAAAACGACCGCTAATAGA 
                   
               
               
                   
                 AACGAACGGGGAAACGGGGGAAATAGTATGGGACAAAGGGC 
                   
               
               
                   
                 GAGACTTCGCGACGGTACGAAAAGTACTAAGCATGCCGCAAG 
                   
               
               
                   
                 TAAACATAGTAAAAAAAACGGAAGTACAAACGGGGGGGTTCA 
                   
               
               
                   
                 GCAAAGAAAGCATACTACCGAAACGAAACAGCGACAAACTAA 
                   
               
               
                   
                 TAGCGCGAAAAAAAGACTGGGACCCGAAAAAATACGGGGGGT 
                   
               
               
                   
                 TCGACAGCCCGACGGTAGCGTACAGCGTACTAGTAGTAGCGA 
                   
               
               
                   
                 AAGTAGAAAAAGGGAAAAGCAAAAAACTAAAAAGCGTAAAA 
                   
               
               
                   
                 GAACTACTAGGGATAACGATAATGGAACGAAGCAGCTTCGAA 
                   
               
               
                   
                 AAAAACCCGATAGACTTCCTAGAAGCGAAAGGGTACAAAGAA 
                   
               
               
                   
                 GTAAAAAAAGACCTAATAATAAAACTACCGAAATACAGCCTA 
                   
               
               
                   
                 TTCGAACTAGAAAACGGGCGAAAACGAATGCTAGCGAGCGCG 
                   
               
               
                   
                 GGGGAACTACAAAAAGGGAACGAACTAGCGCTACCGAGCAAA 
                   
               
               
                   
                 TACGTAAACTTCCTATACCTAGCGAGCCACTACGAAAAACTAA 
                   
               
               
                   
                 AAGGGAGCCCGGAAGACAACGAACAAAAACAACTATTCGTAG 
                   
               
               
                   
                 AACAACACAAACACTACCTAGACGAAATAATAGAACAAATAA 
                   
               
               
                   
                 GCGAATTCAGCAAACGAGTAATACTAGCGGACGCGAACCTAG 
                   
               
               
                   
                 ACAAAGTACTAAGCGCGTACAACAAACACCGAGACAAACCGA 
                   
               
               
                   
                 TACGAGAACAAGCGGAAAACATAATACACCTATTCACGCTAA 
                   
               
               
                   
                 CGAACCTAGGGGCGCCGGCGGCGTTCAAATACTTCGACACGA 
                   
               
               
                   
                 CGATAGACCGAAAACGATACACGAGCACGAAAGAAGTACTAG 
                   
               
               
                   
                 ACGCGACGCTAATACACCAAAGCATAACGGGGCTATACGAAA 
                   
               
               
                   
                 CGCGAATAGACCTAAGCCAACTAGGGGGGGACGGGGGGGGG 
                   
               
               
                   
                 AGCCCGAAAAAAAAACGAAAAGTATGA 
                   
               
               
                   
               
               
                 Cas9 
                 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT 
                 55 
               
               
                 transcript 
                 GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAAA 
                   
               
               
                 with 5′ UTR 
                 AATACAGCATAGGGCTAGACATAGGGACGAACAGCGTAGGGT 
                   
               
               
                 ofHSD. 
                 GGGCGGTAATAACGGACGAATACAAAGTACCGAGCAAAAAAT 
                   
               
               
                 ORF 
                 TCAAAGTACTAGGGAACACGGACCGACACAGCATAAAAAAAA 
                   
               
               
                 corresponding 
                 ACCTAATAGGGGCGCTACTATTCGACAGCGGGGAAACGGCGG 
                   
               
               
                 to SEQ 
                 AAGCGACGCGACTAAAACGAACGGCGCGACGACGATACACGC 
                   
               
               
                 ID NO: 54, 
                 GACGAAAAAACCGAATATGCTACCTACAAGAAATATTCAGCA 
                   
               
               
                 Kozak 
                 ACGAAATGGCGAAAGTAGACGACAGCTTCTTCCACCGACTAG 
                   
               
               
                 sequence, 
                 AAGAAAGCTTCCTAGTAGAAGAAGACAAAAAACACGAACGAC 
                   
               
               
                 and 3′ UTR 
                 ACCCGATATTCGGGAACATAGTAGACGAAGTAGCGTACCACG 
                   
               
               
                 of ALB 
                 AAAAATACCCGACGATATACCACCTACGAAAAAAACTAGTAG 
                   
               
               
                   
                 ACAGCACGGACAAAGCGGACCTACGACTAATATACCTAGCGC 
                   
               
               
                   
                 TAGCGCACATGATAAAATTCCGAGGGCACTTCCTAATAGAAG 
                   
               
               
                   
                 GGGACCTAAACCCGGACAACAGCGACGTAGACAAACTATTCA 
                   
               
               
                   
                 TACAACTAGTACAAACGTACAACCAACTATTCGAAGAAAACC 
                   
               
               
                   
                 CGATAAACGCGAGCGGGGTAGACGCGAAAGCGATACTAAGCG 
                   
               
               
                   
                 CGCGACTAAGCAAAAGCCGACGACTAGAAAACCTAATAGCGC 
                   
               
               
                   
                 AACTACCGGGGGAAAAAAAAAACGGGCTATTCGGGAACCTAA 
                   
               
               
                   
                 TAGCGCTAAGCCTAGGGCTAACGCCGAACTTCAAAAGCAACTT 
                   
               
               
                   
                 CGACCTAGCGGAAGACGCGAAACTACAACTAAGCAAAGACAC 
                   
               
               
                   
                 GTACGACGACGACCTAGACAACCTACTAGCGCAAATAGGGGA 
                   
               
               
                   
                 CCAATACGCGGACCTATTCCTAGCGGCGAAAAACCTAAGCGA 
                   
               
               
                   
                 CGCGATACTACTAAGCGACATACTACGAGTAAACACGGAAAT 
                   
               
               
                   
                 AACGAAAGCGCCGCTAAGCGCGAGCATGATAAAACGATACGA 
                   
               
               
                   
                 CGAACACCACCAAGACCTAACGCTACTAAAAGCGCTAGTACG 
                   
               
               
                   
                 ACAACAACTACCGGAAAAATACAAAGAAATATTCTTCGACCA 
                   
               
               
                   
                 AAGCAAAAACGGGTACGCGGGGTACATAGACGGGGGGGCGA 
                   
               
               
                   
                 GCCAAGAAGAATTCTACAAATTCATAAAACCGATACTAGAAA 
                   
               
               
                   
                 AAATGGACGGGACGGAAGAACTACTAGTAAAACTAAACCGAG 
                   
               
               
                   
                 AAGACCTACTACGAAAACAACGAACGTTCGACAACGGGAGCA 
                   
               
               
                   
                 TACCGCACCAAATACACCTAGGGGAACTACACGCGATACTAC 
                   
               
               
                   
                 GACGACAAGAAGACTTCTACCCGTTCCTAAAAGACAACCGAG 
                   
               
               
                   
                 AAAAAATAGAAAAAATACTAACGTTCCGAATACCGTACTACG 
                   
               
               
                   
                 TAGGGCCGCTAGCGCGAGGGAACAGCCGATTCGCGTGGATGA 
                   
               
               
                   
                 CGCGAAAAAGCGAAGAAACGATAACGCCGTGGAACTTCGAAG 
                   
               
               
                   
                 AAGTAGTAGACAAAGGGGCGAGCGCGCAAAGCTTCATAGAAC 
                   
               
               
                   
                 GAATGACGAACTTCGACAAAAACCTACCGAACGAAAAAGTAC 
                   
               
               
                   
                 TACCGAAACACAGCCTACTATACGAATACTTCACGGTATACAA 
                   
               
               
                   
                 CGAACTAACGAAAGTAAAATACGTAACGGAAGGGATGCGAAA 
                   
               
               
                   
                 ACCGGCGTTCCTAAGCGGGGAACAAAAAAAAGCGATAGTAGA 
                   
               
               
                   
                 CCTACTATTCAAAACGAACCGAAAAGTAACGGTAAAACAACT 
                   
               
               
                   
                 AAAAGAAGACTACTTCAAAAAAATAGAATGCTTCGACAGCGT 
                   
               
               
                   
                 AGAAATAAGCGGGGTAGAAGACCGATTCAACGCGAGCCTAGG 
                   
               
               
                   
                 GACGTACCACGACCTACTAAAAATAATAAAAGACAAAGACTT 
                   
               
               
                   
                 CCTAGACAACGAAGAAAACGAAGACATACTAGAAGACATAGT 
                   
               
               
                   
                 ACTAACGCTAACGCTATTCGAAGACCGAGAAATGATAGAAGA 
                   
               
               
                   
                 ACGACTAAAAACGTACGCGCACCTATTCGACGACAAAGTAAT 
                   
               
               
                   
                 GAAACAACTAAAACGACGACGATACACGGGGTGGGGGCGACT 
                   
               
               
                   
                 AAGCCGAAAACTAATAAACGGGATACGAGACAAACAAAGCG 
                   
               
               
                   
                 GGAAAACGATACTAGACTTCCTAAAAAGCGACGGGTTCGCGA 
                   
               
               
                   
                 ACCGAAACTTCATGCAACTAATACACGACGACAGCCTAACGTT 
                   
               
               
                   
                 CAAAGAAGACATACAAAAAGCGCAAGTAAGCGGGCAAGGGG 
                   
               
               
                   
                 ACAGCCTACACGAACACATAGCGAACCTAGCGGGGAGCCCGG 
                   
               
               
                   
                 CGATAAAAAAAGGGATACTACAAACGGTAAAAGTAGTAGACG 
                   
               
               
                   
                 AACTAGTAAAAGTAATGGGGCGACACAAACCGGAAAACATAG 
                   
               
               
                   
                 TAATAGAAATGGCGCGAGAAAACCAAACGACGCAAAAAGGG 
                   
               
               
                   
                 CAAAAAAACAGCCGAGAACGAATGAAACGAATAGAAGAAGG 
                   
               
               
                   
                 GATAAAAGAACTAGGGAGCCAAATACTAAAAGAACACCCGGT 
                   
               
               
                   
                 AGAAAACACGCAACTACAAAACGAAAAACTATACCTATACTA 
                   
               
               
                   
                 CCTACAAAACGGGCGAGACATGTACGTAGACCAAGAACTAGA 
                   
               
               
                   
                 CATAAACCGACTAAGCGACTACGACGTAGACCACATAGTACC 
                   
               
               
                   
                 GCAAAGCTTCCTAAAAGACGACAGCATAGACAACAAAGTACT 
                   
               
               
                   
                 AACGCGAAGCGACAAAAACCGAGGGAAAAGCGACAACGTAC 
                   
               
               
                   
                 CGAGCGAAGAAGTAGTAAAAAAAATGAAAAACTACTGGCGAC 
                   
               
               
                   
                 AACTACTAAACGCGAAACTAATAACGCAACGAAAATTCGACA 
                   
               
               
                   
                 ACCTAACGAAAGCGGAACGAGGGGGGCTAAGCGAACTAGACA 
                   
               
               
                   
                 AAGCGGGGTTCATAAAACGACAACTAGTAGAAACGCGACAAA 
                   
               
               
                   
                 TAACGAAACACGTAGCGCAAATACTAGACAGCCGAATGAACA 
                   
               
               
                   
                 CGAAATACGACGAAAACGACAAACTAATACGAGAAGTAAAAG 
                   
               
               
                   
                 TAATAACGCTAAAAAGCAAACTAGTAAGCGACTTCCGAAAAG 
                   
               
               
                   
                 ACTTCCAATTCTACAAAGTACGAGAAATAAACAACTACCACCA 
                   
               
               
                   
                 CGCGCACGACGCGTACCTAAACGCGGTAGTAGGGACGGCGCT 
                   
               
               
                   
                 AATAAAAAAATACCCGAAACTAGAAAGCGAATTCGTATACGG 
                   
               
               
                   
                 GGACTACAAAGTATACGACGTACGAAAAATGATAGCGAAAAG 
                   
               
               
                   
                 CGAACAAGAAATAGGGAAAGCGACGGCGAAATACTTCTTCTA 
                   
               
               
                   
                 CAGCAACATAATGAACTTCTTCAAAACGGAAATAACGCTAGC 
                   
               
               
                   
                 GAACGGGGAAATACGAAAACGACCGCTAATAGAAACGAACG 
                   
               
               
                   
                 GGGAAACGGGGGAAATAGTATGGGACAAAGGGCGAGACTTCG 
                   
               
               
                   
                 CGACGGTACGAAAAGTACTAAGCATGCCGCAAGTAAACATAG 
                   
               
               
                   
                 TAAAAAAAACGGAAGTACAAACGGGGGGGTTCAGCAAAGAA 
                   
               
               
                   
                 AGCATACTACCGAAACGAAACAGCGACAAACTAATAGCGCGA 
                   
               
               
                   
                 AAAAAAGACTGGGACCCGAAAAAATACGGGGGGTTCGACAGC 
                   
               
               
                   
                 CCGACGGTAGCGTACAGCGTACTAGTAGTAGCGAAAGTAGAA 
                   
               
               
                   
                 AAAGGGAAAAGCAAAAAACTAAAAAGCGTAAAAGAACTACT 
                   
               
               
                   
                 AGGGATAACGATAATGGAACGAAGCAGCTTCGAAAAAAACCC 
                   
               
               
                   
                 GATAGACTTCCTAGAAGCGAAAGGGTACAAAGAAGTAAAAAA 
                   
               
               
                   
                 AGACCTAATAATAAAACTACCGAAATACAGCCTATTCGAACTA 
                   
               
               
                   
                 GAAAACGGGCGAAAACGAATGCTAGCGAGCGCGGGGGAACT 
                   
               
               
                   
                 ACAAAAAGGGAACGAACTAGCGCTACCGAGCAAATACGTAAA 
                   
               
               
                   
                 CTTCCTATACCTAGCGAGCCACTACGAAAAACTAAAAGGGAG 
                   
               
               
                   
                 CCCGGAAGACAACGAACAAAAACAACTATTCGTAGAACAACA 
                   
               
               
                   
                 CAAACACTACCTAGACGAAATAATAGAACAAATAAGCGAATT 
                   
               
               
                   
                 CAGCAAACGAGTAATACTAGCGGACGCGAACCTAGACAAAGT 
                   
               
               
                   
                 ACTAAGCGCGTACAACAAACACCGAGACAAACCGATACGAGA 
                   
               
               
                   
                 ACAAGCGGAAAACATAATACACCTATTCACGCTAACGAACCT 
                   
               
               
                   
                 AGGGGCGCCGGCGGCGTTCAAATACTTCGACACGACGATAGA 
                   
               
               
                   
                 CCGAAAACGATACACGAGCACGAAAGAAGTACTAGACGCGAC 
                   
               
               
                   
                 GCTAATACACCAAAGCATAACGGGGCTATACGAAACGCGAAT 
                   
               
               
                   
                 AGACCTAAGCCAACTAGGGGGGGACGGGGGGGGGAGCCCGA 
                   
               
               
                   
                 AAAAAAAACGAAAAGTATGACTAGCCATCACATTTAAAAGCA 
                   
               
               
                   
                 TCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCA 
                   
               
               
                   
                 ATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAA 
                   
               
               
                   
                 CACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCT 
                   
               
               
                   
                 TTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG 
                   
               
               
                   
               
               
                 Cas9 
                 AGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT 
                 56 
               
               
                 transcript 
                 GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAGA 
                   
               
               
                 with AGGas 
                 AGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT 
                   
               
               
                 first three 
                 GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGT 
                   
               
               
                 nucleotides 
                 TCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGA 
                   
               
               
                 for use with 
                 ACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAG 
                   
               
               
                 CleanCap™, 
                 AAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACA 
                   
               
               
                 5′ UTR of 
                 AGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC 
                   
               
               
                 HSD, ORF 
                 AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTG 
                   
               
               
                 corresponding 
                 GAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGA 
                   
               
               
                 to SEQ 
                 CACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACG 
                   
               
               
                 ID NO: 4, 
                 AAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCG 
                   
               
               
                 Kozak 
                 ACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC 
                   
               
               
                 sequence, 
                 TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGG 
                   
               
               
                 and 3′ UTR 
                 AGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCAT 
                   
               
               
                 of ALB 
                 CCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCC 
                   
               
               
                   
                 GATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGC 
                   
               
               
                   
                 AAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACA 
                   
               
               
                   
                 GCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGAT 
                   
               
               
                   
                 CGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTC 
                   
               
               
                   
                 GACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACA 
                   
               
               
                   
                 TACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGAC 
                   
               
               
                   
                 CAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGAC 
                   
               
               
                   
                 GCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATC 
                   
               
               
                   
                 ACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGAC 
                   
               
               
                   
                 GAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGA 
                   
               
               
                   
                 CAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAG 
                   
               
               
                   
                 AGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGC 
                   
               
               
                   
                 CAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAG 
                   
               
               
                   
                 ATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAA 
                   
               
               
                   
                 GACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATC 
                   
               
               
                   
                 CCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGA 
                   
               
               
                   
                 AGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAA 
                   
               
               
                   
                 AAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCG 
                   
               
               
                   
                 GACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAA 
                   
               
               
                   
                 GAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAG 
                   
               
               
                   
                 TCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAA 
                   
               
               
                   
                 TGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGC 
                   
               
               
                   
                 CGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA 
                   
               
               
                   
                 ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCC 
                   
               
               
                   
                 GGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCT 
                   
               
               
                   
                 GCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAA 
                   
               
               
                   
                 GGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGA 
                   
               
               
                   
                 AATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAAC 
                   
               
               
                   
                 ATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTG 
                   
               
               
                   
                 GACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTG 
                   
               
               
                   
                 ACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGA 
                   
               
               
                   
                 CTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAG 
                   
               
               
                   
                 CAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAG 
                   
               
               
                   
                 CAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAA 
                   
               
               
                   
                 AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACA 
                   
               
               
                   
                 GAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAA 
                   
               
               
                   
                 GGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACA 
                   
               
               
                   
                 GCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAA 
                   
               
               
                   
                 TCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAAC 
                   
               
               
                   
                 TGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCA 
                   
               
               
                   
                 TCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAG 
                   
               
               
                   
                 AAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAAT 
                   
               
               
                   
                 CAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGA 
                   
               
               
                   
                 AAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT 
                   
               
               
                   
                 GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACAT 
                   
               
               
                   
                 CAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCA 
                   
               
               
                   
                 GAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGAC 
                   
               
               
                   
                 AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGA 
                   
               
               
                   
                 GCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGC 
                   
               
               
                   
                 TGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACC 
                   
               
               
                   
                 TGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAG 
                   
               
               
                   
                 GCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATC 
                   
               
               
                   
                 ACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACA 
                   
               
               
                   
                 AAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTC 
                   
               
               
                   
                 ATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC 
                   
               
               
                   
                 TICCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACG 
                   
               
               
                   
                 CACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGA 
                   
               
               
                   
                 TCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAG 
                   
               
               
                   
                 ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCG 
                   
               
               
                   
                 AACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA 
                   
               
               
                   
                 GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA 
                   
               
               
                   
                 ACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGA 
                   
               
               
                   
                 GAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCA 
                   
               
               
                   
                 ACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTC 
                   
               
               
                   
                 AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAG 
                   
               
               
                   
                 CATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAA 
                   
               
               
                   
                 GAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCC 
                   
               
               
                   
                 GACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAA 
                   
               
               
                   
                 GGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGG 
                   
               
               
                   
                 GAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGA 
                   
               
               
                   
                 TCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG 
                   
               
               
                   
                 ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGA 
                   
               
               
                   
                 AAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGC 
                   
               
               
                   
                 AGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACT 
                   
               
               
                   
                 TCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCC 
                   
               
               
                   
                 CGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACA 
                   
               
               
                   
                 AGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA 
                   
               
               
                   
                 GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCC 
                   
               
               
                   
                 TGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC 
                   
               
               
                   
                 AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGG 
                   
               
               
                   
                 GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA 
                   
               
               
                   
                 GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACAC 
                   
               
               
                   
                 TGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCG 
                   
               
               
                   
                 ACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAG 
                   
               
               
                   
                 AAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATC 
                   
               
               
                   
                 TCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAT 
                   
               
               
                   
                 AGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACA 
                   
               
               
                   
                 CCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTT 
                   
               
               
                   
                 TCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG 
                   
               
               
                   
               
               
                 Cas9 
                 GGGCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC 
                 57 
               
               
                 transcript 
                 ATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAAC 
                   
               
               
                 with 5′ UTR 
                 GGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACC 
                   
               
               
                 from CMV, 
                 GTCCTTGACACGGCCACCATGGACAAGAAGTACAGCATCGGA 
                   
               
               
                 ORF 
                 CTGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACA 
                   
               
               
                 corresponding 
                 GACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGA 
                   
               
               
                 to SEQ 
                 AACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCA 
                   
               
               
                 ID NO: 4, 
                 CTGCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTG 
                   
               
               
                 Kozak 
                 AAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACAG 
                   
               
               
                 sequence, 
                 AATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAA 
                   
               
               
                 and 3′ UTR 
                 GGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTG 
                   
               
               
                 of ALB 
                 GTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGA 
                   
               
               
                   
                 AACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACA 
                   
               
               
                   
                 ATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAG 
                   
               
               
                   
                 GCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCA 
                   
               
               
                   
                 AGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGG 
                   
               
               
                   
                 ACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGAC 
                   
               
               
                   
                 ATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGG 
                   
               
               
                   
                 AGTCGACGCAAAGGCAATCCTGAGCGCAAGACTGAGCAAGAG 
                   
               
               
                   
                 CAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAA 
                   
               
               
                   
                 GAAGAACGGACTGTTCGGAAACCTGATCGCACTGAGCCTGGG 
                   
               
               
                   
                 ACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGA 
                   
               
               
                   
                 CGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCT 
                   
               
               
                   
                 GGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCT 
                   
               
               
                   
                 GTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAG 
                   
               
               
                   
                 CGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCT 
                   
               
               
                   
                 GAGCGCAAGCATGATCAAGAGATACGACGAACACCACCAGGA 
                   
               
               
                   
                 CCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGA 
                   
               
               
                   
                 AAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATA 
                   
               
               
                   
                 CGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTA 
                   
               
               
                   
                 CAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGA 
                   
               
               
                   
                 AGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAA 
                   
               
               
                   
                 GCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCA 
                   
               
               
                   
                 CCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTT 
                   
               
               
                   
                 CTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGAT 
                   
               
               
                   
                 CCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGA 
                   
               
               
                   
                 GGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGA 
                   
               
               
                   
                 AACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGG 
                   
               
               
                   
                 AGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGA 
                   
               
               
                   
                 CAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCT 
                   
               
               
                   
                 GCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTC 
                   
               
               
                   
                 AAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGC 
                   
               
               
                   
                 GGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACA 
                   
               
               
                   
                 AACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTC 
                   
               
               
                   
                 AAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGCGGAGTC 
                   
               
               
                   
                 GAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTG 
                   
               
               
                   
                 CTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAA 
                   
               
               
                   
                 AACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGT 
                   
               
               
                   
                 TCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACG 
                   
               
               
                   
                 CACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAA 
                   
               
               
                   
                 GAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCA 
                   
               
               
                   
                 ACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACT 
                   
               
               
                   
                 TCCTGAAGAGCGACGGATTCGCAAACAGAAACTTCATGCAGC 
                   
               
               
                   
                 TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGA 
                   
               
               
                   
                 AGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACA 
                   
               
               
                   
                 TCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCC 
                   
               
               
                   
                 TGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGG 
                   
               
               
                   
                 GAAGACACAAGCCGGAAAACATCGTCATCGAAATGGCAAGAG 
                   
               
               
                   
                 AAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAA 
                   
               
               
                   
                 AGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAG 
                   
               
               
                   
                 CCAGATCCTGAAGGAACACCCGGTCGAAAACACACAGCTGCA 
                   
               
               
                   
                 GAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGA 
                   
               
               
                   
                 CATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGA 
                   
               
               
                   
                 CTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGAC 
                   
               
               
                   
                 GACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAAC 
                   
               
               
                   
                 AGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAG 
                   
               
               
                   
                 AAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTG 
                   
               
               
                   
                 ATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGA 
                   
               
               
                   
                 GGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGA 
                   
               
               
                   
                 CAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAG 
                   
               
               
                   
                 ATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGAC 
                   
               
               
                   
                 AAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAG 
                   
               
               
                   
                 CTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCA 
                   
               
               
                   
                 GAGAAATCAACAACTACCACCACGCACACGACGCATACCTGA 
                   
               
               
                   
                 ACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGC 
                   
               
               
                   
                 TGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACG 
                   
               
               
                   
                 TCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAG 
                   
               
               
                   
                 GCAACAGCAAAGTACTTCTTCTACAGCAACATCATGAACTTCT 
                   
               
               
                   
                 TCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGA 
                   
               
               
                   
                 GACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCT 
                   
               
               
                   
                 GGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGA 
                   
               
               
                   
                 GCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGA 
                   
               
               
                   
                 CAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACA 
                   
               
               
                   
                 GCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGA 
                   
               
               
                   
                 AGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCC 
                   
               
               
                   
                 TGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGA 
                   
               
               
                   
                 AGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAA 
                   
               
               
                   
                 GCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGG 
                   
               
               
                   
                 GATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGA 
                   
               
               
                   
                 AGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGC 
                   
               
               
                   
                 TGGCAAGCGCAGGAGAACTGCAGAAGGGAAACGAACTGGCAC 
                   
               
               
                   
                 TGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTA 
                   
               
               
                   
                 CGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGC 
                   
               
               
                   
                 AGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCAT 
                   
               
               
                   
                 CGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGA 
                   
               
               
                   
                 CGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAG 
                   
               
               
                   
                 AGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCT 
                   
               
               
                   
                 GTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTA 
                   
               
               
                   
                 CTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAA 
                   
               
               
                   
                 GGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGG 
                   
               
               
                   
                 ACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGA 
                   
               
               
                   
                 CGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAG 
                   
               
               
                   
                 CCATCACATTFAAAAGCATCTCAGCCTACCATGAGAATAAGAG 
                   
               
               
                   
                 AAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTT 
                   
               
               
                   
                 CGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTC 
                   
               
               
                   
                 TTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAA 
                   
               
               
                   
                 ATGGAAAGAACCTCGAG 
                   
               
               
                   
               
               
                 Cas9 
                 GGGacatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccggatctgccaccAT 
                 58 
               
               
                 transcript 
                 GGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAG 
                   
               
               
                 with 5′ UTR 
                 CGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCGAG 
                   
               
               
                 from HBB, 
                 CAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCAT 
                   
               
               
                 ORF 
                 CAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGA 
                   
               
               
                 corresponding 
                 AACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA 
                   
               
               
                 to SEQ 
                 GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAA 
                   
               
               
                 ID NO: 4, 
                 TCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCA 
                   
               
               
                 Kozak 
                 CAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCA 
                   
               
               
                 sequence, 
                 CGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGC 
                   
               
               
                 and 3′ UTR 
                 ATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAGAA 
                   
               
               
                 of HBB 
                 GCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTA 
                   
               
               
                   
                 CCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTG 
                   
               
               
                   
                 ATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAG 
                   
               
               
                   
                 CTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAG 
                   
               
               
                   
                 AAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCC 
                   
               
               
                   
                 TGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGA 
                   
               
               
                   
                 TCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAA 
                   
               
               
                   
                 ACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAGA 
                   
               
               
                   
                 GCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCA 
                   
               
               
                   
                 AGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGA 
                   
               
               
                   
                 TCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACC 
                   
               
               
                   
                 TGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACA 
                   
               
               
                   
                 CAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGA 
                   
               
               
                   
                 GATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCAC 
                   
               
               
                   
                 TGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCT 
                   
               
               
                   
                 TCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAG 
                   
               
               
                   
                 GAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCC 
                   
               
               
                   
                 TGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGA 
                   
               
               
                   
                 ACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACG 
                   
               
               
                   
                 GAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCAA 
                   
               
               
                   
                 TCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACA 
                   
               
               
                   
                 ACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGT 
                   
               
               
                   
                 ACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCAT 
                   
               
               
                   
                 GGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACT 
                   
               
               
                   
                 TCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCA 
                   
               
               
                   
                 TCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAA 
                   
               
               
                   
                 AGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGT 
                   
               
               
                   
                 CTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAAT 
                   
               
               
                   
                 GAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAAT 
                   
               
               
                   
                 CGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAA 
                   
               
               
                   
                 GCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGA 
                   
               
               
                   
                 CAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAG 
                   
               
               
                   
                 CCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAA 
                   
               
               
                   
                 GGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGA 
                   
               
               
                   
                 CATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATC 
                   
               
               
                   
                 GAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAG 
                   
               
               
                   
                 GTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGA 
                   
               
               
                   
                 AGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAG 
                   
               
               
                   
                 AGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTC 
                   
               
               
                   
                 GCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTG 
                   
               
               
                   
                 ACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAG 
                   
               
               
                   
                 GGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGC 
                   
               
               
                   
                 CCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTC 
                   
               
               
                   
                 GACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAAC 
                   
               
               
                   
                 ATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAAG 
                   
               
               
                   
                 GGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGA 
                   
               
               
                   
                 AGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCC 
                   
               
               
                   
                 GGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTA 
                   
               
               
                   
                 CTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACT 
                   
               
               
                   
                 GGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGT 
                   
               
               
                   
                 CCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGT 
                   
               
               
                   
                 CCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACG 
                   
               
               
                   
                 TCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGA 
                   
               
               
                   
                 GACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCG 
                   
               
               
                   
                 ACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTG 
                   
               
               
                   
                 GACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGA 
                   
               
               
                   
                 CAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATG 
                   
               
               
                   
                 AACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTC 
                   
               
               
                   
                 AAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGA 
                   
               
               
                   
                 AAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTAC 
                   
               
               
                   
                 CACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACA 
                   
               
               
                   
                 GCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC 
                   
               
               
                   
                 TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCA 
                   
               
               
                   
                 AAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTC 
                   
               
               
                   
                 TTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACAC 
                   
               
               
                   
                 TGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAA 
                   
               
               
                   
                 ACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC 
                   
               
               
                   
                 TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAAC 
                   
               
               
                   
                 ATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAG 
                   
               
               
                   
                 GAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCA 
                   
               
               
                   
                 AGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGAC 
                   
               
               
                   
                 AGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCG 
                   
               
               
                   
                 AAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTG 
                   
               
               
                   
                 CTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAAC 
                   
               
               
                   
                 CCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAG 
                   
               
               
                   
                 AAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAAC 
                   
               
               
                   
                 TGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAA 
                   
               
               
                   
                 CTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTC 
                   
               
               
                   
                 AACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGA 
                   
               
               
                   
                 AGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAG 
                   
               
               
                   
                 CACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAA 
                   
               
               
                   
                 TTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAG 
                   
               
               
                   
                 GTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGA 
                   
               
               
                   
                 GAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAAC 
                   
               
               
                   
                 CTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATC 
                   
               
               
                   
                 GACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCA 
                   
               
               
                   
                 ACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGA 
                   
               
               
                   
                 ATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCG 
                   
               
               
                   
                 AAGAAGAAGAGAAAGGTCTAGctagcgctcgctttcttgctgtccaatttctattaaa 
                   
               
               
                   
                 ggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctg 
                   
               
               
                   
                 cctaataaaaaacatttattttcattgcctcgag 
                   
               
               
                   
               
               
                 Cas9 
                 GGGaagctcagaataaacgctcaactttggccggatctgccacCATGGACAAGAAGT 
                 59 
               
               
                 transcript 
                 ACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG 
                   
               
               
                 with 5′ UTR 
                 CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCA 
                   
               
               
                 from XBG, 
                 AGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGAACC 
                   
               
               
                 ORF 
                 TGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAG 
                   
               
               
                 corresponding 
                 CAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGA 
                   
               
               
                 to SEQ 
                 AGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC 
                   
               
               
                 ID NO: 4, 
                 GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAA 
                   
               
               
                 Kozak 
                 GAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACAC 
                   
               
               
                 sequence, 
                 CCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAA 
                   
               
               
                 and 3′ UTR 
                 AAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGAC 
                   
               
               
                 of XBG 
                 AGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTG 
                   
               
               
                   
                 GCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGA 
                   
               
               
                   
                 GACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATC 
                   
               
               
                   
                 CAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCG 
                   
               
               
                   
                 ATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCA 
                   
               
               
                   
                 AGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAG 
                   
               
               
                   
                 CTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATC 
                   
               
               
                   
                 GCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTC 
                   
               
               
                   
                 GACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACA 
                   
               
               
                   
                 TACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGAC 
                   
               
               
                   
                 CAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGAC 
                   
               
               
                   
                 GCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATC 
                   
               
               
                   
                 ACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGAC 
                   
               
               
                   
                 GAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGA 
                   
               
               
                   
                 CAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAG 
                   
               
               
                   
                 AGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGC 
                   
               
               
                   
                 CAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAG 
                   
               
               
                   
                 ATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAA 
                   
               
               
                   
                 GACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATC 
                   
               
               
                   
                 CCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGA 
                   
               
               
                   
                 AGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAA 
                   
               
               
                   
                 AAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCG 
                   
               
               
                   
                 GACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAA 
                   
               
               
                   
                 GAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAG 
                   
               
               
                   
                 TCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAA 
                   
               
               
                   
                 TGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGC 
                   
               
               
                   
                 CGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA 
                   
               
               
                   
                 ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCC 
                   
               
               
                   
                 GGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCT 
                   
               
               
                   
                 GCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAA 
                   
               
               
                   
                 GGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGA 
                   
               
               
                   
                 AATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAAC 
                   
               
               
                   
                 ATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTG 
                   
               
               
                   
                 GACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTG 
                   
               
               
                   
                 ACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGA 
                   
               
               
                   
                 CTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAG 
                   
               
               
                   
                 CAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAG 
                   
               
               
                   
                 CAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAA 
                   
               
               
                   
                 AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACA 
                   
               
               
                   
                 GAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAA 
                   
               
               
                   
                 GGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACA 
                   
               
               
                   
                 GCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAA 
                   
               
               
                   
                 TCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAAC 
                   
               
               
                   
                 TGGTCAAGGTCATOGGAAGACACAAGCCGGAAAACATCGTCA 
                   
               
               
                   
                 TCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAG 
                   
               
               
                   
                 AAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAAT 
                   
               
               
                   
                 CAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGA 
                   
               
               
                   
                 AAACACACAGCTGCAGAACGAAAAGCTGTACCIGTACTACCT 
                   
               
               
                   
                 GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACAT 
                   
               
               
                   
                 CAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCA 
                   
               
               
                   
                 GAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGAC 
                   
               
               
                   
                 AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGA 
                   
               
               
                   
                 GCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGC 
                   
               
               
                   
                 TGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACC 
                   
               
               
                   
                 TGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAG 
                   
               
               
                   
                 GCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATC 
                   
               
               
                   
                 ACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACA 
                   
               
               
                   
                 AAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTC 
                   
               
               
                   
                 ATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC 
                   
               
               
                   
                 TTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACG 
                   
               
               
                   
                 CACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGA 
                   
               
               
                   
                 TCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAG 
                   
               
               
                   
                 ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCG 
                   
               
               
                   
                 AACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA 
                   
               
               
                   
                 GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA 
                   
               
               
                   
                 ACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGA 
                   
               
               
                   
                 GAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCA 
                   
               
               
                   
                 ACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTC 
                   
               
               
                   
                 AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAG 
                   
               
               
                   
                 CATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAA 
                   
               
               
                   
                 GAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCC 
                   
               
               
                   
                 GACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAA 
                   
               
               
                   
                 GGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGG 
                   
               
               
                   
                 GAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGA 
                   
               
               
                   
                 TCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG 
                   
               
               
                   
                 ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGA 
                   
               
               
                   
                 AAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGC 
                   
               
               
                   
                 AGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACT 
                   
               
               
                   
                 TCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAACCC 
                   
               
               
                   
                 CGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACA 
                   
               
               
                   
                 AGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA 
                   
               
               
                   
                 GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCC 
                   
               
               
                   
                 TGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC 
                   
               
               
                   
                 AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGG 
                   
               
               
                   
                 GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA 
                   
               
               
                   
                 GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACAC 
                   
               
               
                   
                 TGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCG 
                   
               
               
                   
                 ACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAG 
                   
               
               
                   
                 AAGAAGAGAAAGGTCTAGctagcaccagcctcaagaacacccgaatggagtctctaa 
                   
               
               
                   
                 gctacataataccaacttacactttacaaaatgttgtcccccaaaatgtagccattcgtatctgctcctaata 
                   
               
               
                   
                 aaaagaaagtttcttcacattctctcgag 
                   
               
               
                   
               
               
                 Cas9 
                 AGGaagctcagaataaacgctcaactttggccggatctgccacCATGGACAAGAAGT 
                 60 
               
               
                 transcript 
                 ACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG 
                   
               
               
                 with AGG as 
                 CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCA 
                   
               
               
                 first three 
                 AGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGAACC 
                   
               
               
                 nucleotides 
                 TGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAG 
                   
               
               
                 for use with 
                 CAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGA 
                   
               
               
                 CleanCap™, 
                 AGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC 
                   
               
               
                 5′ UTR from 
                 GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAA 
                   
               
               
                 XBG, ORF 
                 GAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACAC 
                   
               
               
                 corresponding 
                 CCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAA 
                   
               
               
                 to SEQ 
                 AAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGAC 
                   
               
               
                 ID NO: 4, 
                 AGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTG 
                   
               
               
                 Kozak 
                 GCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGA 
                   
               
               
                 sequence, 
                 GACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATC 
                   
               
               
                 and 3′ UTR 
                 CAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCG 
                   
               
               
                 of XBG 
                 ATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCA 
                   
               
               
                   
                 AGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAG 
                   
               
               
                   
                 CTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATC 
                   
               
               
                   
                 GCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTC 
                   
               
               
                   
                 GACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACA 
                   
               
               
                   
                 TACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGAC 
                   
               
               
                   
                 CAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGAC 
                   
               
               
                   
                 GCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATC 
                   
               
               
                   
                 ACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGAC 
                   
               
               
                   
                 GAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGA 
                   
               
               
                   
                 CAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAG 
                   
               
               
                   
                 AGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGC 
                   
               
               
                   
                 CAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAG 
                   
               
               
                   
                 ATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAA 
                   
               
               
                   
                 GACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATC 
                   
               
               
                   
                 CCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGA 
                   
               
               
                   
                 AGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAA 
                   
               
               
                   
                 AAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCG 
                   
               
               
                   
                 GACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAA 
                   
               
               
                   
                 GAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAG 
                   
               
               
                   
                 TCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAA 
                   
               
               
                   
                 TGACAAACITCGACAAGAACCTGCCGAACGAAAAGGTCCTGC 
                   
               
               
                   
                 CGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA 
                   
               
               
                   
                 ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCC 
                   
               
               
                   
                 GGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCT 
                   
               
               
                   
                 GCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAA 
                   
               
               
                   
                 GGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGA 
                   
               
               
                   
                 AATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAAC 
                   
               
               
                   
                 ATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTG 
                   
               
               
                   
                 GACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTG 
                   
               
               
                   
                 ACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGA 
                   
               
               
                   
                 CTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAG 
                   
               
               
                   
                 CAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAG 
                   
               
               
                   
                 CAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAA 
                   
               
               
                   
                 AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACA 
                   
               
               
                   
                 GAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAA 
                   
               
               
                   
                 GGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACA 
                   
               
               
                   
                 GCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAA 
                   
               
               
                   
                 TCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAAC 
                   
               
               
                   
                 TGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCA 
                   
               
               
                   
                 TCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAG 
                   
               
               
                   
                 AAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAAT 
                   
               
               
                   
                 CAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGA 
                   
               
               
                   
                 AAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT 
                   
               
               
                   
                 GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACAT 
                   
               
               
                   
                 CAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCA 
                   
               
               
                   
                 GAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGAC 
                   
               
               
                   
                 AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGA 
                   
               
               
                   
                 GCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGC 
                   
               
               
                   
                 TGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACC 
                   
               
               
                   
                 TGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAG 
                   
               
               
                   
                 GCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATC 
                   
               
               
                   
                 ACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACA 
                   
               
               
                   
                 AAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTC 
                   
               
               
                   
                 ATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC 
                   
               
               
                   
                 TTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACG 
                   
               
               
                   
                 CACACGACGCATACCTGAACGCAGTCGTCGGAACAGGACTGA 
                   
               
               
                   
                 TCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAG 
                   
               
               
                   
                 ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCG 
                   
               
               
                   
                 AACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA 
                   
               
               
                   
                 GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA 
                   
               
               
                   
                 ACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGA 
                   
               
               
                   
                 GAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCA 
                   
               
               
                   
                 ACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTC 
                   
               
               
                   
                 AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAG 
                   
               
               
                   
                 CATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAA 
                   
               
               
                   
                 GAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCC 
                   
               
               
                   
                 GACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAA 
                   
               
               
                   
                 GGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGG 
                   
               
               
                   
                 GAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGA 
                   
               
               
                   
                 TCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG 
                   
               
               
                   
                 ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGA 
                   
               
               
                   
                 AAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGC 
                   
               
               
                   
                 AGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACT 
                   
               
               
                   
                 TCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCC 
                   
               
               
                   
                 CGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACA 
                   
               
               
                   
                 AGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA 
                   
               
               
                   
                 GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCC 
                   
               
               
                   
                 TGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC 
                   
               
               
                   
                 AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGG 
                   
               
               
                   
                 GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA 
                   
               
               
                   
                 GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACAC 
                   
               
               
                   
                 TGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCG 
                   
               
               
                   
                 ACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAG 
                   
               
               
                   
                 AAGAAGAGAAAGGTCTAGctagcaccagcctcaagaacacccgaatggagtctctaa 
                   
               
               
                   
                 gctacataataccaacttacactttacaaaatgttgtcccccaaaatgtagccattcgtatctgctcctaata 
                   
               
               
                   
                 aaaagaaagtttcttcacattctctcgag 
                   
               
               
                   
               
               
                 Cas9 
                 AGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT 
                 61 
               
               
                 transcript 
                 GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAGA 
                   
               
               
                 with AGGas 
                 AGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT 
                   
               
               
                 first three 
                 GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGT 
                   
               
               
                 nucleotides 
                 TCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGA 
                   
               
               
                 for use with 
                 ACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAG 
                   
               
               
                 CleanCap™, 
                 AAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACA 
                   
               
               
                 5′ UTR from 
                 AGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC 
                   
               
               
                 HSD, ORF 
                 AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTG 
                   
               
               
                 corresponding 
                 GAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGA 
                   
               
               
                 to SEQ 
                 CACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACG 
                   
               
               
                 ID NO: 4, 
                 AAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCG 
                   
               
               
                 Kozak 
                 ACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC 
                   
               
               
                 sequence, 
                 TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGG 
                   
               
               
                 and 3′ UTR 
                 AGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCAT 
                   
               
               
                 of ALB 
                 CCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCC 
                   
               
               
                   
                 GATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGC 
                   
               
               
                   
                 AAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACA 
                   
               
               
                   
                 GCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGAT 
                   
               
               
                   
                 CGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTC 
                   
               
               
                   
                 GACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACA 
                   
               
               
                   
                 TACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGAC 
                   
               
               
                   
                 CAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGAC 
                   
               
               
                   
                 GCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATC 
                   
               
               
                   
                 ACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGAC 
                   
               
               
                   
                 GAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGA 
                   
               
               
                   
                 CAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAG 
                   
               
               
                   
                 AGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGC 
                   
               
               
                   
                 CAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAG 
                   
               
               
                   
                 ATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAA 
                   
               
               
                   
                 GACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATC 
                   
               
               
                   
                 CCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGA 
                   
               
               
                   
                 AGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAA 
                   
               
               
                   
                 AAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCG 
                   
               
               
                   
                 GACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAA 
                   
               
               
                   
                 GAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAG 
                   
               
               
                   
                 TCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAA 
                   
               
               
                   
                 TGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGC 
                   
               
               
                   
                 CGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA 
                   
               
               
                   
                 ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCC 
                   
               
               
                   
                 GGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCT 
                   
               
               
                   
                 GCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAA 
                   
               
               
                   
                 GGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGA 
                   
               
               
                   
                 AATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAAC 
                   
               
               
                   
                 ATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTG 
                   
               
               
                   
                 GACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTG 
                   
               
               
                   
                 ACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGA 
                   
               
               
                   
                 CTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAG 
                   
               
               
                   
                 CAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAG 
                   
               
               
                   
                 CAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAA 
                   
               
               
                   
                 AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACA 
                   
               
               
                   
                 GAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAA 
                   
               
               
                   
                 GGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACA 
                   
               
               
                   
                 GCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAA 
                   
               
               
                   
                 TCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAAC 
                   
               
               
                   
                 TGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCA 
                   
               
               
                   
                 TCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAG 
                   
               
               
                   
                 AAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAAT 
                   
               
               
                   
                 CAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGA 
                   
               
               
                   
                 AAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT 
                   
               
               
                   
                 GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACAT 
                   
               
               
                   
                 CAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCA 
                   
               
               
                   
                 GAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGAC 
                   
               
               
                   
                 AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGA 
                   
               
               
                   
                 GCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGC 
                   
               
               
                   
                 TGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACC 
                   
               
               
                   
                 TGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAG 
                   
               
               
                   
                 GCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATC 
                   
               
               
                   
                 ACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACA 
                   
               
               
                   
                 AAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTC 
                   
               
               
                   
                 ATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC 
                   
               
               
                   
                 TTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACG 
                   
               
               
                   
                 CACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGA 
                   
               
               
                   
                 TCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAG 
                   
               
               
                   
                 ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCG 
                   
               
               
                   
                 AACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA 
                   
               
               
                   
                 GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA 
                   
               
               
                   
                 ACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGA 
                   
               
               
                   
                 GAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCA 
                   
               
               
                   
                 ACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTC 
                   
               
               
                   
                 AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAG 
                   
               
               
                   
                 CATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAA 
                   
               
               
                   
                 GAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCC 
                   
               
               
                   
                 GACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAA 
                   
               
               
                   
                 GGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGG 
                   
               
               
                   
                 GAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGA 
                   
               
               
                   
                 TCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG 
                   
               
               
                   
                 ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGA 
                   
               
               
                   
                 AAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGC 
                   
               
               
                   
                 AGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACT 
                   
               
               
                   
                 TCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCC 
                   
               
               
                   
                 CGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACA 
                   
               
               
                   
                 AGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA 
                   
               
               
                   
                 GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCC 
                   
               
               
                   
                 TGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC 
                   
               
               
                   
                 AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGG 
                   
               
               
                   
                 GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA 
                   
               
               
                   
                 GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACAC 
                   
               
               
                   
                 TGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCG 
                   
               
               
                   
                 ACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAG 
                   
               
               
                   
                 AAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATC 
                   
               
               
                   
                 TCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAT 
                   
               
               
                   
                 AGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACA 
                   
               
               
                   
                 CCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTT 
                   
               
               
                   
                 TCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG 
                   
               
               
                   
               
               
                 30/30/39 
                 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCGAAAAAAA 
                 62 
               
               
                 poly-A 
                 AAAAAAAAAAAAAAAAAAAAAAACCGAAAAAAAAAAAAAAA 
                   
               
               
                 sequence 
                 AAAAAAAAAAAAAAAAAAAAAAAA 
                   
               
               
                   
               
               
                 poly-A100 
                 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 
                 63 
               
               
                 sequence 
                 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 
                   
               
               
                   
                 AAAAAAAAAAAAA 
                   
               
               
                   
               
               
                 G209 guide 
                 mC*mC*mA*GUCCAGCGAGGCAAAGGGUUUUAGAGCUAGAAA 
                 64 
               
               
                 RNA 
                 UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA 
                   
               
               
                   
                 AGUGGCACCGAGUCGGUGCmU*mU*mU*U 
                   
               
               
                   
               
               
                 ORF 
                 ATGGCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGAC 
                 65 
               
               
                 encoding 
                 TGGACATCGGAATCGCATCGGTCGGATGGGCAATGGTCGAAA 
                   
               
               
                 
                   Neisseria 
                 
                 TCGACGAAGAAGAAAACCCGATCAGACTGATCGACCTGGGAG 
                   
               
               
                 
                   meningitidis 
                 
                 TCAGAGTCTTCGAAAGAGCAGAAGTCCCGAAGACAGGAGACT 
                   
               
               
                 Cas9 
                 CGCTGGCAATGGCAAGAAGACTGGCAAGATCGGTCAGAAGAC 
                   
               
               
                   
                 TGACAAGAAGAAGAGCACACAGACTGCTGAGAACAAGAAGA 
                   
               
               
                   
                 CTGCTGAAGAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGAC 
                   
               
               
                   
                 GAAAACGGACTGATCAAGTCGCTGCCGAACACACCGTGGCAG 
                   
               
               
                   
                 CTGAGAGCAGCAGCACTGGACAGAAAGCTGACACCGCTGGAA 
                   
               
               
                   
                 TGGTCGGCAGTCCTGCTGCACCTGATCAAGCACAGAGGATACC 
                   
               
               
                   
                 TGTCGCAGAGAAAGAACGAAGGAGAAACAGCAGACAAGGAA 
                   
               
               
                   
                 CTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCACACGCA 
                   
               
               
                   
                 CTGCAGACAGGAGACTTCAGAACACCGGCAGAACTGGCACTG 
                   
               
               
                   
                 AACAAGTTCGAAAAGGAATCGGGACACATCAGAAACCAGAGA 
                   
               
               
                   
                 TCGGACTACTCGCACACATTCTCGAGAAAGGACCTGCAGGCA 
                   
               
               
                   
                 GAACTGATCCTGCTGTTCGAAAAGCAGAAGGAATTCGGAAAC 
                   
               
               
                   
                 CCGCACGTCTCGGGAGGACTGAAGGAAGGAATCGAAACACTG 
                   
               
               
                   
                 CTGATGACACAGAGACCGGCACTGTCGGGAGACGCAGTCCAG 
                   
               
               
                   
                 AAGATGCTGGGACACTGCACATTCGAACCGGCAGAACCGAAG 
                   
               
               
                   
                 GCAGCAAAGAACACATACACAGCAGAAAGATTCATCTGGCTG 
                   
               
               
                   
                 ACAAAGCTGAACAACCTGAGAATCCTGGAACAGGGATCGGAA 
                   
               
               
                   
                 AGACCGCTGACAGACACAGAAAGAGCAACACTGATGGACGAA 
                   
               
               
                   
                 CCGTACAGAAAGTCGAAGCTGACATACGCACAGGCAAGAAAG 
                   
               
               
                   
                 CTGCTGGGACTGGAAGACACAGCATTCTTCAAGGGACTGAGA 
                   
               
               
                   
                 TACGGAAAGGACAACGCAGAAGCATCGACACTGATGGAAATG 
                   
               
               
                   
                 AAGGCATACCACGCAATCTCGAGAGCACTGGAAAAGGAAGGA 
                   
               
               
                   
                 CTGAAGGACAAGAAGTCGCCGCTGAACCTGTCGCCGGAACTG 
                   
               
               
                   
                 CAGGACGAAATCGGAACAGCATTCTCGCTGTTCAAGACAGAC 
                   
               
               
                   
                 GAAGACATCACAGGAAGACTGAAGGACAGAATCCAGCCGGAA 
                   
               
               
                   
                 ATCCTGGAAGCACTGCTGAAGCACATCTCGTTCGACAAGTTCG 
                   
               
               
                   
                 TCCAGATCTCGCTGAAGGCACTGAGAAGAATCGTCCCGCTGAT 
                   
               
               
                   
                 GGAACAGGGAAAGAGATACGACGAAGCATGCGCAGAAATCTA 
                   
               
               
                   
                 CGGAGACCACTACGGAAAGAAGAACACAGAAGAAAAGATCT 
                   
               
               
                   
                 ACCTGCCGCCGATCCCGGCAGACGAAATCAGAAACCCGGTCG 
                   
               
               
                   
                 TCCTGAGAGCACTGTCGCAGGCAAGAAAGGTCATCAACGCAG 
                   
               
               
                   
                 TCGTCAGAAGATACGGATCGCCGGCAAGAATCCACATCGAAA 
                   
               
               
                   
                 CAGCAAGAGAAGTCGGAAAGTCGTTCAAGGACAGAAAGGAA 
                   
               
               
                   
                 ATCGAAAAGAGACAGGAAGAAAACAGAAAGGACAGAGAAAA 
                   
               
               
                   
                 GGCAGCAGCAAAGTTCAGAGAATACTTCCCGAACTTCGTCGG 
                   
               
               
                   
                 AGAACCGAAGTCGAAGGACATCCTGAAGCTGAGACTGTACGA 
                   
               
               
                   
                 ACAGCAGCACGGAAAGTGCCTGTACTCGGGAAAGGAAATCAA 
                   
               
               
                   
                 CCTGGGAAGACTGAACGAAAAGGGATACGTCGAAATCGACCA 
                   
               
               
                   
                 CGCACTGCCGTTCTCGAGAACATGGGACGACTCGTTCAACAAC 
                   
               
               
                   
                 AAGGTCCTGGTCCTGGGATCGGAAAACCAGAACAAGGGAAAC 
                   
               
               
                   
                 CAGACACCGTACGAATACTTCAACGGAAAGGACAACTCGAGA 
                   
               
               
                   
                 GAATGGCAGGAATTCAAGGCAAGAGTCGAAACATCGAGATTC 
                   
               
               
                   
                 CCGAGATCGAAGAAGCAGAGAATCCTGCTGCAGAAGTTCGAC 
                   
               
               
                   
                 GAAGACGGATTCAAGGAAAGAAACCTGAACGACACAAGATAC 
                   
               
               
                   
                 GTCAACAGATTCCTGTGCCAGTTCGTCGCAGACAGAATGAGAC 
                   
               
               
                   
                 TGACAGGAAAGGGAAAGAAGAGAGTCTTCGCATCGAACGGAC 
                   
               
               
                   
                 AGATCACAAACCTGCTGAGAGGATTCTGGGGACTGAGAAAGG 
                   
               
               
                   
                 TCAGAGCAGAAAACGACAGACACCACGCACTGGACGCAGTCG 
                   
               
               
                   
                 TCGTCGCATGCTCGACAGTCGCAATGCAGCAGAAGATCACAA 
                   
               
               
                   
                 GATTCGTCAGATACAAGGAAATGAACGCATTCGACGGAAAGA 
                   
               
               
                   
                 CAATCGACAAGGAAACAGGAGAAGTCCTGCACCAGAAGACAC 
                   
               
               
                   
                 ACTTCCCGCAGCCGTGGGAATTCTTCGCACAGGAAGTCATGAT 
                   
               
               
                   
                 CAGAGTCTTCGGAAAGCCGGACGGAAAGCCGGAATTCGAAGA 
                   
               
               
                   
                 AGCAGACACACTGGAAAAGCTGAGAACACTGCTGGCAGAAAA 
                   
               
               
                   
                 GCTGTCGTCGAGACCGGAAGCAGTCCACGAATACGTCACACC 
                   
               
               
                   
                 GCTGTTCGTCTCGAGAGCACCGAACAGAAAGATGTCGGGACA 
                   
               
               
                   
                 GGGACACATGGAAACAGTCAAGTCGGCAAAGAGACTGGACGA 
                   
               
               
                   
                 AGGAGTCTCGGTCCTGAGAGTCCCGCTGACACAGCTGAAGCTG 
                   
               
               
                   
                 AAGGACCTGGAAAAGATGGTCAACAGAGAAAGAGAACCGAA 
                   
               
               
                   
                 GCTGTACGAAGCACTGAAGGCAAGACTGGAAGCACACAAGGA 
                   
               
               
                   
                 CGACCCGGCAAAGGCATTCGCAGAACCGTTCTACAAGTACGA 
                   
               
               
                   
                 CAAGGCAGGAAACAGAACACAGCAGGTCAAGGCAGTCAGAGT 
                   
               
               
                   
                 CGAACAGGTCCAGAAGACAGGAGTCTGGGTCAGAAACCACAA 
                   
               
               
                   
                 CGGAATCGCAGACAACGCAACAATGGTCAGAGTAGACGTCTT 
                   
               
               
                   
                 CGAAAAGGGAGACAAGTACTACCTGGTCCCGATCTACTCGTG 
                   
               
               
                   
                 GCAGGTCGCAAAGGGAATCCTGCCGGACAGAGCAGTCGTCCA 
                   
               
               
                   
                 GGGAAAGGACGAAGAAGACTGGCAGCTGATCGACGACTCGTT 
                   
               
               
                   
                 CAACTTCAAGTTCTCGCTGCACCCGAACGACCTGGTCGAAGTC 
                   
               
               
                   
                 ATCACAAAGAAGGCAAGAATGTTCGGATACTTCGCATCGTGCC 
                   
               
               
                   
                 ACAGAGGAACAGGAAACATCAACATCAGAATCCACGACCTGG 
                   
               
               
                   
                 ACCACAAGATCGGAAAGAACGGAATCCTGGAAGGAATCGGAG 
                   
               
               
                   
                 TCAAGACAGCACTGTCGTTCCAGAAGTACCAGATCGACGAACT 
                   
               
               
                   
                 GGGAAAGGAAATCAGACCGTGCAGACTGAAGAAGAGACCGCC 
                   
               
               
                   
                 GGTCAGATCCGGAAAGAGAACAGCAGACGGATCGGAATTCGA 
                   
               
               
                   
                 ATCGCCGAAGAAGAAGAGAAAGGTCGAATGA 
                   
               
               
                   
               
               
                 ORF 
                 GCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGACTGG 
                 66 
               
               
                 encoding 
                 ACATCGGAATCGCATCGGTCGGATGGGCAATGGTCGAAATCG 
                   
               
               
                 
                   Neisseria 
                 
                 ACGAAGAAGAAAACCCGATCAGACTGATCGACCTGGGAGTCA 
                   
               
               
                 
                   meningitidis 
                 
                 GAGTCTTCGAAAGAGCAGAAGTCCCGAAGACAGGAGACTCGC 
                   
               
               
                 Cas9 (no 
                 TGGCAATGGCAAGAAGACTGGCAAGATCGGTCAGAAGACTGA 
                   
               
               
                 start or stop 
                 CAAGAAGAAGAGCACACAGACTGCTGAGAACAAGAAGACTGC 
                   
               
               
                 codons; 
                 TGAAGAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGACGAAA 
                   
               
               
                 suitable for 
                 ACGGACTGATCAAGTCGCTGCCGAACACACCGTGGCAGCTGA 
                   
               
               
                 inclusion in 
                 GAGCAGCAGCACTGGACAGAAAGCTGACACCGCTGGAATGGT 
                   
               
               
                 fusion 
                 CGGCAGTCCTGCTGCACCTGATCAAGCACAGAGGATACCTGTC 
                   
               
               
                 protein 
                 GCAGAGAAAGAACGAAGGAGAAACAGCAGACAAGGAACTGG 
                   
               
               
                 coding 
                 GAGCACTGCTGAAGGGAGTCGCAGGAAACGCACACGCACTGC 
                   
               
               
                 sequence) 
                 AGACAGGAGACTTCAGAACACCGGCAGAACTGGCACTGAACA 
                   
               
               
                   
                 AGTTCGAAAAGGAATCGGGACACATCAGAAACCAGAGATCGG 
                   
               
               
                   
                 ACTACTCGCACACATTCTCGAGAAAGGACCTGCAGGCAGAAC 
                   
               
               
                   
                 TGATCCTGCTGTTCGAAAAGCAGAAGGAATTCGGAAACCCGC 
                   
               
               
                   
                 ACGTCTCGGGAGGACTGAAGGAAGGAATCGAAACACTGCTGA 
                   
               
               
                   
                 TGACACAGAGACCGGCACTGTCGGGAGACGCAGTCCAGAAGA 
                   
               
               
                   
                 TGCTGGGACACTGCACATTCGAACCGGCAGAACCGAAGGCAG 
                   
               
               
                   
                 CAAAGAACACATACACAGCAGAAAGATTCATCTGGCTGACAA 
                   
               
               
                   
                 AGCTGAACAACCTGAGAATCCTGGAACAGGGATCGGAAAGAC 
                   
               
               
                   
                 CGCTGACAGACACAGAAAGAGCAACACTGATGGACGAACCGT 
                   
               
               
                   
                 ACAGAAAGTCGAAGCTGACATACGCACAGGCAAGAAAGCTGC 
                   
               
               
                   
                 TGGGACTGGAAGACACAGCATTCTTCAAGGGACTGAGATACG 
                   
               
               
                   
                 GAAAGGACAACGCAGAAGCATCGACACTGATGGAAATGAAGG 
                   
               
               
                   
                 CATACCACGCAATCTCGAGAGCACTGGAAAAGGAAGGACTGA 
                   
               
               
                   
                 AGGACAAGAAGTCGCCGCTGAACCTGTCGCCGGAACTGCAGG 
                   
               
               
                   
                 ACGAAATCGGAACAGCATTCTCGCTGTTCAAGACAGACGAAG 
                   
               
               
                   
                 ACATCACAGGAAGACTGAAGGACAGAATCCAGCCGGAAATCC 
                   
               
               
                   
                 TGGAAGCACTGCTGAAGCACATCTCGTTCGACAAGTTCGTCCA 
                   
               
               
                   
                 GATCTCGCTGAAGGCACTGAGAAGAATCGTCCCGCTGATGGA 
                   
               
               
                   
                 ACAGGGAAAGAGATACGACGAAGCATGCGCAGAAATCTACGG 
                   
               
               
                   
                 AGACCACTACGGAAAGAAGAACACAGAAGAAAAGATCTACCT 
                   
               
               
                   
                 GCCGCCGATCCCGGCAGACGAAATCAGAAACCCGGTCGTCCT 
                   
               
               
                   
                 GAGAGCACTGTCGCAGGCAAGAAAGGTCATCAACGGAGTCGT 
                   
               
               
                   
                 CAGAAGATACGGATCGCCGGCAAGAATCCACATCGAAACAGC 
                   
               
               
                   
                 AAGAGAAGTCGGAAAGTCGTTCAAGGACAGAAAGGAAATCGA 
                   
               
               
                   
                 AAAGAGACAGGAAGAAAACAGAAAGGACAGAGAAAAGGCAG 
                   
               
               
                   
                 CAGCAAAGTTCAGAGAATACTTCCCGAACTTCGTCGGAGAACC 
                   
               
               
                   
                 GAAGTCGAAGGACATCCTGAAGCTGAGACTGTACGAACAGCA 
                   
               
               
                   
                 GCACGGAAAGTGCCTGTACTCGGGAAAGGAAATCAACCTGGG 
                   
               
               
                   
                 AAGACTGAACGAAAAGGGATACGTCGAAATCGACCACGCACT 
                   
               
               
                   
                 GCCGTTCTCGAGAACATGGGACGACTCGTTCAACAACAAGGTC 
                   
               
               
                   
                 CTGGTCCTGGGATCGGAAAACCAGAACAAGGGAAACCAGACA 
                   
               
               
                   
                 CCGTACGAATACTTCAACGGAAAGGACAACTCGAGAGAATGG 
                   
               
               
                   
                 CAGGAATTCAAGGCAAGAGTCGAAACATCGAGATTCCCGAGA 
                   
               
               
                   
                 TCGAAGAAGCAGAGAATCCTGCTGCAGAAGTTCGACGAAGAC 
                   
               
               
                   
                 GGATTCAAGGAAAGAAACCTGAACGACACAAGATACGTCAAC 
                   
               
               
                   
                 AGATTCCTGTGCCAGTTCGTCGCAGACAGAATGAGACTGACAG 
                   
               
               
                   
                 GAAAGGGAAAGAAGAGAGTCTTCGCATCGAACGGACAGATCA 
                   
               
               
                   
                 CAAACCTGCTGAGAGGATTCTGGGGACTGAGAAAGGTCAGAG 
                   
               
               
                   
                 CAGAAAACGACAGACACCACGCACTGGACGCAGTCGTCGTCG 
                   
               
               
                   
                 CATGCTCGACAGTCGCAATGCAGCAGAAGATCACAAGATTCG 
                   
               
               
                   
                 TCAGATACAAGGAAATGAACGCATTCGACGGAAAGACAATCG 
                   
               
               
                   
                 ACAAGGAAACAGGAGAAGTCCTGCACCAGAAGACACACTTCC 
                   
               
               
                   
                 CGCAGCCGTGGGAATTCTTCGCACAGGAAGTCATGATCAGAGT 
                   
               
               
                   
                 CTTCGGAAAGCCGGACGGAAAGCCGGAATTCGAAGAAGCAGA 
                   
               
               
                   
                 CACACTGGAAAAGCTGAGAACACTGCTGGCAGAAAAGCTGTC 
                   
               
               
                   
                 GTCGAGACCGGAAGCAGTCCACGAATACGTCACACCGCTGTTC 
                   
               
               
                   
                 GTCTCGAGAGCACCGAACAGAAAGATGTCGGGACAGGGACAC 
                   
               
               
                   
                 ATGGAAACAGTCAAGTCGGCAAAGAGACTGGACGAAGGAGTC 
                   
               
               
                   
                 TCGGTCCTGAGAGTCCCGCTGACACAGCTGAAGCTGAAGGAC 
                   
               
               
                   
                 CTGGAAAAGATGGTCAACAGAGAAAGAGAACCGAAGCTGTAC 
                   
               
               
                   
                 GAAGCACTGAAGGCAAGACTGGAAGCACACAAGGACGACCCG 
                   
               
               
                   
                 GCAAAGGCATTCGCAGAACCGTTCTACAAGTACGACAAGGCA 
                   
               
               
                   
                 GGAAACAGAACACAGCAGGTCAAGGCAGTCAGAGTCGAACAG 
                   
               
               
                   
                 GTCCAGAAGACAGGAGTCTGGGTCAGAAACCACAACGGAATC 
                   
               
               
                   
                 GCAGACAACGCAACAATGGTCAGAGTAGACGTCTTCGAAAAG 
                   
               
               
                   
                 GGAGACAAGTACTACCTGGTCCCGATCTACTCGTGGCAGGTCG 
                   
               
               
                   
                 CAAAGGGAATCCTGCCGGACAGAGCAGTCGTCCAGGGAAAGG 
                   
               
               
                   
                 ACGAAGAAGACTGGCAGCTGATCGACGACTCGTTCAACTTCA 
                   
               
               
                   
                 AGTTCTCGCTGCACCCGAACGACCTGGTCGAAGTCATCACAAA 
                   
               
               
                   
                 GAAGGCAAGAATGTTCGGATACTTCGCATCGTGCCACAGAGG 
                   
               
               
                   
                 AACAGGAAACATCAACATCAGAATCCACGACCTGGACCACAA 
                   
               
               
                   
                 GATCGGAAAGAACGGAATCCTGGAAGGAATCGGAGTCAAGAC 
                   
               
               
                   
                 AGCACTGTCGTTCCAGAAGTACCAGATCGACGAACTGGGAAA 
                   
               
               
                   
                 GGAAATCAGACCGTGCAGACTGAAGAAGAGACCGCCGGTCAG 
                   
               
               
                   
                 ATCCGGAAAGAGAACAGCAGACGGATCGGAATTCGAATCGCC 
                   
               
               
                   
                 GAAGAAGAAGAGAAAGGTCGAA 
                   
               
               
                   
               
               
                 Transcript 
                 GGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGATCC 
                 67 
               
               
                 comprising 
                 GCCACCATGGCAGCATTCAAGCCGAACTCGATCAACTACATCC 
                   
               
               
                 SEQ ID NO: 
                 TGGGACTGGACATCGGAATCGCATCGGTCGGATGGGCAATGG 
                   
               
               
                 65 (encoding 
                 TCGAAATCGACGAAGAAGAAAACCCGATCAGACTGATCGACC 
                   
               
               
                 
                   Neisseria 
                 
                 TGGGAGTCAGAGTCTTCGAAAGAGCAGAAGTCCCGAAGACAG 
                   
               
               
                 
                   meningitidis 
                 
                 GAGACTCGCTGGCAATGGCAAGAAGACTGGCAAGATCGGTCA 
                   
               
               
                 Cas9) 
                 GAAGACTGACAAGAAGAAGAGCACACAGACTGCTGAGAACA 
                   
               
               
                   
                 AGAAGACTGCTGAAGAGAGAAGGAGTCCTGCAGGCAGCAAAC 
                   
               
               
                   
                 TTCGACGAAAACGGACTGATCAAGTCGCTGCCGAACACACCG 
                   
               
               
                   
                 TGGCAGCTGAGAGCAGCAGCACTGGACAGAAAGCTGACACCG 
                   
               
               
                   
                 CTGGAATGGTCGGCAGTCCTGCTGCACCTGATCAAGCACAGAG 
                   
               
               
                   
                 GATACCTGTCGCAGAGAAAGAACGAAGGAGAAACAGCAGAC 
                   
               
               
                   
                 AAGGAACTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCA 
                   
               
               
                   
                 CACGCACTGCAGACAGGAGACTTCAGAACACCGGCAGAACTG 
                   
               
               
                   
                 GCACTGAACAAGTTCGAAAAGGAATCGGGACACATCAGAAAC 
                   
               
               
                   
                 CAGAGATCGGACTACTCGCACACATTCTCGAGAAAGGACCTG 
                   
               
               
                   
                 CAGGCAGAACTGATCCTGCTGTTCGAAAAGCAGAAGGAATTC 
                   
               
               
                   
                 GGAAACCCGCACGTCTCGGGAGGACTGAAGGAAGGAATCGAA 
                   
               
               
                   
                 ACACTGCTGATGACACAGAGACCGGCACTGTCGGGAGACGCA 
                   
               
               
                   
                 GTCCAGAAGATGCTGGGACACTGCACATTCGAACCGGCAGAA 
                   
               
               
                   
                 CCGAAGGCAGCAAAGAACACATACACAGCAGAAAGATTCATC 
                   
               
               
                   
                 TGGCTGACAAAGCTGAACAACCTGAGAATCCTGGAACAGGGA 
                   
               
               
                   
                 TCGGAAAGACCGCTGACAGACACAGAAAGAGCAACACTGATG 
                   
               
               
                   
                 GACGAACCGTACAGAAAGTCGAAGCTGACATACGCACAGGCA 
                   
               
               
                   
                 AGAAAGCTGCTGGGACTGGAAGACACAGCATTCTTCAAGGGA 
                   
               
               
                   
                 CTGAGATACGGAAAGGACAACGCAGAAGCATCGACACTGATG 
                   
               
               
                   
                 GAAATGAAGGCATACCACGCAATCTCGAGAGCACTGGAAAAG 
                   
               
               
                   
                 GAAGGACTGAAGGACAAGAAGTCGCCGCTGAACCTGTCGCCG 
                   
               
               
                   
                 GAACTGCAGGACGAAATCGGAACAGCATTCTCGCTGTTCAAG 
                   
               
               
                   
                 ACAGACGAAGACATCACAGGAAGACTGAAGGACAGAATCCAG 
                   
               
               
                   
                 CCGGAAATCCTGGAAGCACTGCTGAAGCACATCTCGTTCGACA 
                   
               
               
                   
                 AGTTCGTCCAGATCTCGCTGAAGGCACTGAGAAGAATCGTCCC 
                   
               
               
                   
                 GCTGATGGAACAGGGAAAGAGATACGACGAAGCATGCGCAGA 
                   
               
               
                   
                 AATCTACGGAGACCACTACGGAAAGAAGAACACAGAAGAAA 
                   
               
               
                   
                 AGATCTACCTGCCGCCGATCCCGGCAGACGAAATCAGAAACC 
                   
               
               
                   
                 CGGTCGTCCTGAGAGCACTGTCGCAGGCAAGAAAGGTCATCA 
                   
               
               
                   
                 ACGGAGTCGTCAGAAGATACGGATCGCCGGCAAGAATCCACA 
                   
               
               
                   
                 TCGAAACAGCAAGAGAAGTCGGAAAGTCGTTCAAGGACAGAA 
                   
               
               
                   
                 AGGAAATCGAAAAGAGACAGGAAGAAAACAGAAAGGACAGA 
                   
               
               
                   
                 GAAAAGGCAGCAGCAAAGTTCAGAGAATACTTCCCGAACTTC 
                   
               
               
                   
                 GTCGGAGAACCGAAGTCGAAGGACATCCTGAAGCTGAGACTG 
                   
               
               
                   
                 TACGAACAGCAGCACGGAAAGTGCCTGTACTCGGGAAAGGAA 
                   
               
               
                   
                 ATCAACCTGGGAAGACTGAACGAAAAGGGATACGTCGAAATC 
                   
               
               
                   
                 GACCACGCACTGCCGTTCTCGAGAACATGGGACGACTCGTTCA 
                   
               
               
                   
                 ACAACAAGGTCCTGGTCCTGGGATCGGAAAACCAGAACAAGG 
                   
               
               
                   
                 GAAACCAGACACCGTACGAATACTTCAACGGAAAGGACAACT 
                   
               
               
                   
                 CGAGAGAATGGCAGGAATTCAAGGCAAGAGTCGAAACATCGA 
                   
               
               
                   
                 GATTCCCGAGATCGAAGAAGCAGAGAATCCTGCTGCAGAAGT 
                   
               
               
                   
                 TCGACGAAGACGGATTCAAGGAAAGAAACCTGAACGACACAA 
                   
               
               
                   
                 GATACGTCAACAGATTCCTGTGCCAGTTCGTCGCAGACAGAAT 
                   
               
               
                   
                 GAGACTGACAGGAAAGGGAAAGAAGAGAGTCTTCGCATCGAA 
                   
               
               
                   
                 CGGACAGATCACAAACCTGCTGAGAGGATTCTGGGGACTGAG 
                   
               
               
                   
                 AAAGGTCAGAGCAGAAAACGACAGACACCACGCACTGGACGC 
                   
               
               
                   
                 AGTCGTCGTCGCATGCTCGACAGTCGCAATGCAGCAGAAGATC 
                   
               
               
                   
                 ACAAGATTCGTCAGATACAAGGAAATGAACGCATTCGACGGA 
                   
               
               
                   
                 AAGACAATCGACAAGGAAACAGGAGAAGTCCTGCACCAGAAG 
                   
               
               
                   
                 ACACACTTCCCGCAGCCGTGGGAATTCTTCGCACAGGAAGTCA 
                   
               
               
                   
                 TGATCAGAGTCTTCGGAAAGCCGGACGGAAAGCCGGAATTCG 
                   
               
               
                   
                 AAGAAGCAGACACACTGGAAAAGCTGAGAACACTGCTGGCAG 
                   
               
               
                   
                 AAAAGCTGTCGTCGAGACCGGAAGCAGTCCACGAATACGTCA 
                   
               
               
                   
                 CACCGCTGTTCGTCTCGAGAGCACCGAACAGAAAGATGTCGG 
                   
               
               
                   
                 GACAGGGACACATGGAAACAGTCAAGTCGGCAAAGAGACTGG 
                   
               
               
                   
                 ACGAAGGAGTCTCGGTCCTGAGAGTCCCGCTGACACAGCTGA 
                   
               
               
                   
                 AGCTGAAGGACCTGGAAAAGATGGTCAACAGAGAAAGAGAA 
                   
               
               
                   
                 CCGAAGCTGTACGAAGCACTGAAGGCAAGACTGGAAGCACAC 
                   
               
               
                   
                 AAGGACGACCCGGCAAAGGCATTCGCAGAACCGTTCTACAAG 
                   
               
               
                   
                 TACGACAAGGCAGGAAACAGAACACAGCAGGTCAAGGCAGTC 
                   
               
               
                   
                 AGAGTCGAACAGGTCCAGAAGACAGGAGTCTGGGTCAGAAAC 
                   
               
               
                   
                 CACAACGGAATCGCAGACAACGCAACAATGGTCAGAGTAGAC 
                   
               
               
                   
                 GTCTTCGAAAAGGGAGACAAGTACTACCTGGTCCCGATCTACT 
                   
               
               
                   
                 CGTGGCAGGTCGCAAAGGGAATCCTGCCGGACAGAGCAGTCG 
                   
               
               
                   
                 TCCAGGGAAAGGACGAAGAAGACTGGCAGCTGATCGACGACT 
                   
               
               
                   
                 CGTTCAACTTCAAGTTCTCGCTGCACCCGAACGACCTGGTCGA 
                   
               
               
                   
                 AGTCATCACAAAGAAGGCAAGAATGTTCGGATACTTCGCATC 
                   
               
               
                   
                 GTGCCACAGAGGAACAGGAAACATCAACATCAGAATCCACGA 
                   
               
               
                   
                 CCTGGACCACAAGATCGGAAAGAACGGAATCCTGGAAGGAAT 
                   
               
               
                   
                 CGGAGTCAAGACAGCACTGTCGTTCCAGAAGTACCAGATCGA 
                   
               
               
                   
                 CGAACTGGGAAAGGAAATCAGACCGTGCAGACTGAAGAAGAG 
                   
               
               
                   
                 ACCGCCGGTCAGATCCGGAAAGAGAACAGCAGACGGATCGGA 
                   
               
               
                   
                 ATTCGAATCGCCGAAGAAGAAGAGAAAGGTCGAATGATAGCT 
                   
               
               
                   
                 AGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTC 
                   
               
               
                   
                 GACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCC 
                   
               
               
                   
                 CCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTT 
                   
               
               
                   
                 TCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGT 
                   
               
               
                   
                 GTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGG 
                   
               
               
                   
                 GGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGG 
                   
               
               
                   
                 TGGGCTCTATGG 
                   
               
               
                   
               
               
                 Amino acid 
                 MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVF 
                 68 
               
               
                 secpence of 
                 ERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREG 
                   
               
               
                 
                   Neisseria 
                 
                 VLQAANFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLI 
                   
               
               
                 
                   meningitidis 
                 
                 KHRGYLSQRKNEGETADKELGALLKGVAGNAHALQTGDFRTPA 
                   
               
               
                 Cas9 
                 ELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEFGN 
                   
               
               
                   
                 PHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAA 
                   
               
               
                   
                 KNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKS 
                   
               
               
                   
                 KLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAI 
                   
               
               
                   
                 SRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLKDR 
                   
               
               
                   
                 IQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIY 
                   
               
               
                   
                 GDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRR 
                   
               
               
                   
                 YGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFRE 
                   
               
               
                   
                 YFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYV 
                   
               
               
                   
                 EIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDN 
                   
               
               
                   
                 SREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYV 
                   
               
               
                   
                 NRFLCQFVADRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVR 
                   
               
               
                   
                 AENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTID 
                   
               
               
                   
                 KETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLE 
                   
               
               
                   
                 KLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVK 
                   
               
               
                   
                 SAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARL 
                   
               
               
                   
                 EAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVW 
                   
               
               
                   
                 VRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDR 
                   
               
               
                   
                 AVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYFA 
                   
               
               
                   
                 SCHRGTGNGNIRIHDLDHKIGKNGILEGIGVKTALSFQKYQIDELG 
                   
               
               
                   
                 KEIRPCRLKKRPPVRSGKRTADGSEPESPKKKRKVE 
                   
               
               
                   
               
               
                 G390 guide 
                 mG*mC*mC*GAGUCUGGAGAGCUGCAGUUUUAGAmGmCmUm 
                 69 
               
               
                 RNA 
                 AmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGU 
                   
               
               
                   
                 UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmC 
                   
               
               
                   
                 mCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G502 guide 
                 mA*mC*mA*CAAAUACCAGUCCAGCGGUUUUAGAmGmCmUm 
                 70 
               
               
                 RNA 
                 AmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGU 
                   
               
               
                   
                 UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmC 
                   
               
               
                   
                 mCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G509 guide 
                 mA*mA*mA*GUUCUAGAUGCCGUCCGGUUUUAGAmGmCmUm 
                 71 
               
               
                 RNA 
                 AmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGU 
                   
               
               
                   
                 UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmC 
                   
               
               
                   
                 mCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 
                   
               
               
                   
               
               
                 G534 guide 
                 mA*mC*mG*CAAAUAUCAGUCCAGCGGUUUUAGAmGmCmUm 
                 72 
               
               
                 RNA 
                 AmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGU 
                   
               
               
                   
                 UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmC 
                   
               
               
                   
                 mCmGmAmGmUmCmGmGmUmCmCmU*mU*mU*mU 
                   
               
            
           
           
               
               
            
               
                 * = PS linkage; ‘m’ = 2′-O-Me nucleotide 
                   
               
               
                   
               
               
                 Mouse G000282 NGS primer sequences 
                   
               
            
           
           
               
            
               
                 Forward primer: 
               
               
                 CACTCTTTCCCTACACGACGCTCTTCCGATCTGTTTTGTTCCAGAGTCTATCACCG 
               
               
                   
               
               
                 Reverse primer: 
               
               
                 GGAGTTCAGACGTGTGCTCTTCCGATCTACACGAATAAGAGCAAATGGGAAC 
               
               
                   
               
               
                 Rat G000390 NGS primer sequences 
               
               
                 Forward Primer: 
               
               
                 CACTCTTTCCCTACACGACGCTCTTCCGATCTTGCATTTCATGAGACCGAAAACA 
               
               
                   
               
               
                 Reverse Primer: 
               
               
                 GGAGTTCAGACGTGTGCTCTTCCGATCTGCTACAGTAGAGCTGTACATAAAACTT 
               
               
                   
               
               
                 GFP sequence: 
               
               
                 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGAC 
               
               
                 GGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGC 
               
               
                 GTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCA 
               
               
                 GATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAG 
               
               
                 GAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGA 
               
               
                 AGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATG 
               
               
                 TGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT 
               
               
                 AAAACGACGGCCAGTGAATTCTAATACGACTCACTATAGGGTCCCGCAGTCGGC 
               
               
                 GTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGCAGGCCTTATTCGGATCCAT 
               
               
                 GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCT 
               
               
                 GGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCG 
               
               
                 ATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCC 
               
               
                 CGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGC 
               
               
                 CGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAA 
               
               
                 GGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACC 
               
               
                 CGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG 
               
               
                 GGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAAC 
               
               
                 TACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAG 
               
               
                 GTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGAC 
               
               
                 CACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC 
               
               
                 CACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGAT 
               
               
                 CACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACG 
               
               
                 AGCTGTACAAGTAATAGGAATTATGCAGTCTAGCCATCACATTTAAAAGCATCTC 
               
               
                 AGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTC 
               
               
                 TTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTT 
               
               
                 TAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTC 
               
               
                 GAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 
               
               
                 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAT 
               
               
                 CTAGACTTAAGCTTGATGAGCTCTAGCTTGGCGTAATCATGGTCATAGCTGTTTC 
               
               
                 CTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCAT 
               
               
                 AAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG 
               
               
                 CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAA 
               
               
                 TCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTC 
               
               
                 GCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCAC 
               
               
                 TCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAAC 
               
               
                 ATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT 
               
               
                 GGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCA 
               
               
                 AGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT 
               
               
                 GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGT 
               
               
                 CCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTAT 
               
               
                 CTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCG 
               
               
                 TTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT 
               
               
                 AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGC 
               
               
                 GAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTAC 
               
               
                 ACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAA 
               
               
                 AAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTT 
               
               
                 TTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCC 
               
               
                 TTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG 
               
               
                 ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAA 
               
               
                 AATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTA 
               
               
                 CCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCA 
               
               
                 TAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATC 
               
               
                 TGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTA 
               
               
                 TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACT 
               
               
                 TTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTT 
               
               
                 CGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTC 
               
               
                 ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGA 
               
               
                 GTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGA 
               
               
                 TCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACT 
               
               
                 GCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGT 
               
               
                 ACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCC 
               
               
                 GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCAT 
               
               
                 CATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGA 
               
               
                 TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTT 
               
               
                 CACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGG 
               
               
                 GAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTA 
               
               
                 TTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTT 
               
               
                 AGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTG 
               
               
                 ACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCAC 
               
               
                 GAGGCCCTTTCGTCG