PROGRAMMABLE DNA BASE EDITING USING NME2CAS9-DESAMINASE FUSION PROTEINS.

Abstract

The present invention relates to the field of gene editing. In particular, gene editing is directed toward the editing of a single nucleotide base. For example, such single-nucleotide base editing results in the conversion of a C•G base pair to a T•A base pair. The high accuracy and precision of the currently disclosed single-nucleotide base gene editor is achieved by fusing an NmeCas9 nuclease with a nucleotide deaminase protein. The compact nature of NmeCas9 coupled with a greater number of compatible protospacer-adjacent motifs provides the Cas9 fusion constructs contemplated herein with a gene-editing window capable of editing sites that are not susceptible targets for other conventional SpyCas9 base-editing platforms.

Description

Cross reference to related request This application claims priority to US Provisional Patent Application No. 62 / 745,666 filed October 15, 2018, incorporated herein by reference in its entirety. field of invention The present invention relates to the field of gene editing. In particular, gene editing is directed toward editing a single nucleotide base. For example, such editing of a single nucleotide base results in a conversion of a C*G base pair to a Τ·Α base pair. The high accuracy and precision of the currently disclosed single nucleotide base gene editor is achieved by a NmeCas9 nuclease that is fused to a protein nucleotide deaminase. The compact nature of NmeCas9 coupled with a greater number of compatible protospacer-adjacent motifs provides the Cas9 fusion constructs contemplated herein to have a gene editing window that can edit sites that are not amenable targets for other base editing platforms. by conventional SpyCas9. Background of the invention Many human diseases arise due to single base mutation. The ability to correct such genetic aberrations is critical in the treatment of these genetic disorders. Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated proteins (Cas) comprise an RNA-guided adaptive immune system in archaea and bacteria. These systems provide immunity by targeting nucleic acids originating from foreign genetic elements and inactivating them. The base editing platforms by SpyCas9 cannot be used to drive all single base mutations due to their limited editing windows. The editing window is limited in part by the requirement of an NGG PAM and by the requirement that the edited base(s) be at a very precise distance from the PAM. SpyCas9 is also intrinsically associated with high non-specific effects in genome editing. What is needed in the art is a high precision Cas9 single base editing platform that has programmable target specificity due to recognition of a diverse population of PAM sites. Brief description of the invention The present invention relates to the field of gene editing. In particular, gene editing is directed toward editing a single nucleotide base. For example, such editing of a single nucleotide base results in a conversion of an OG base pair to a Τ·Α base pair. The high accuracy and precision of the currently disclosed single nucleotide base gene editor is achieved by a NmeCas9 nuclease that is fused to a protein nucleotide deaminase. The compact nature of NmeCas9 coupled with a greater number of compatible protospacer-adjacent motifs provides the Cas9 fusion constructs contemplated herein with a superior gene editing window to other conventional SpyCas9 base editing platforms. In one embodiment, the present invention contemplates a mutated NmeCas9 protein comprising a fused nucleotide deaminase and a binding region for an N4CC nucleotide sequence. In one embodiment, said protein is Nme2Cas9. In one embodiment, said protein further comprises a nuclear localization signal protein. In one embodiment, said nucleotide deaminase is a cytidine deaminase. In one embodiment, said nucleotide deaminase is an adenosine deaminase. In one embodiment, the protein further comprises an inhibitor of uracil glycosylase. In one embodiment, said nuclear localization signal protein includes, but is not limited to, nucleoplasmin NLS and / or SV40 NLS and / or C-myc NLS. In one embodiment, said junction region is an interaction domain with the protospacer accessory motif. In one embodiment, said protospacer accessory motif interaction domain comprises said mutation. In one embodiment, said mutation is a D16A mutation. In one embodiment, said mutated NmeCas9 protein further comprises CBE4. In one embodiment, said mutated NmeCas9 protein further comprises a linker. In one embodiment, said linker is a 73 aa linker. In one embodiment, said linker is a 3xHA tag. In one embodiment, the present invention contemplates a construct, wherein said construct is an optimized nNme2Cas9-ABEmax. In one embodiment, the present invention contemplates a construct, wherein said construct is nNme2Cas9-CBE4. In one embodiment, the present invention contemplates a construct, wherein said construct is a YE1-BE3-nNme2Cas9(D16A)-UGL In one embodiment, the present invention contemplates an adeno-associated virus comprising a mutated NmeCas9 protein, said mutated NmeCas9 protein comprising a fused nucleotide deaminase and a binding region for an N4CC nucleotide sequence. In one embodiment, said virus is an adeno-associated virus 8. In one embodiment, said virus is an adeno-associated virus 6. In one embodiment, said protein is Nme2Cas9. In one embodiment, said protein further comprises a nuclear localization signal protein. In one embodiment, said nucleotide deaminase is a cytidine deaminase. In one embodiment, said nucleotide deaminase is an adenosine deaminase. In one embodiment, the protein further comprises an inhibitor of uracil glycosylase. In one embodiment, the nuclear localization signal protein includes, but is not limited to, nucleoplasmin NLS and / or SV40 NLS and / or C-myc NLS. In one embodiment, said junction region is an interaction domain with the protospacer accessory motif. In one embodiment, said domain of interaction with the motif zQQncn / Lznz / q / Yi accessory to the protospacer comprises said mutation. In one embodiment, said mutation is a D16A mutation. In one embodiment, said mutated NmeCas9 protein further comprises CBE4. In one embodiment, said mutated NmeCas9 protein further comprises a linker. In one embodiment, said linker is a 73 aa linker. In one embodiment, said linker is a 3xHA tag. In one embodiment, the present invention contemplates a construct, wherein said construct is an optimized nNme2Cas9-ABEmax. In one embodiment, the present invention contemplates a construct, wherein said construct is nNme2Cas9-CBE4. In one embodiment, the present invention contemplates a construct, wherein said construct is a YE1-BE3-nNme2Cas9(D16A)-UGL In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a nucleotide sequence comprising a single base mutated gene, wherein said gene is flanked by an N4CC nucleotide sequence; ii) a mutated NmeCas9 protein comprising a fused nucleotide deaminase and a binding region for said N4CC nucleotide sequence; b) contacting said nucleotide sequence with said mutated NmeCas9 protein under conditions such that said binding region binds said N4CC nucleotide sequence; and c) replacing said individual mutated base with a wild-type base with said mutated NmeCas9 protein. In one embodiment, said protein is Nme2Cas9. In one embodiment, said protein further comprises a nuclear localization signal protein. In one embodiment, said nucleotide deaminase is a cytidine deaminase. In one embodiment, said nucleotide deaminase is an adenosine deaminase. In one embodiment, the protein further comprises an inhibitor of uracil glycosylase. In one embodiment, the nuclear localization signal protein includes, but is not limited to, nucleoplasmin NLS and / or SV40 NLS and / or C-myc NLS. In one embodiment, said junction region is an interaction domain with the protospacer accessory motif. In one embodiment, said protospacer accessory motif interaction domain comprises said mutation. In one embodiment, said mutation is a D16A mutation. In one embodiment, said mutated NmeCas9 protein further comprises CBE4. In one embodiment, said mutated NmeCas9 protein further comprises a linker. In one embodiment, said linker is a 73 aa linker. In one embodiment, said linker is a 3xHA tag. In one embodiment, said gene encodes a tyrosinase. In one embodiment, said gene is Fah. In one embodiment, said gene is c-fos. In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a patient comprising a nucleotide sequence comprising a single base mutated gene, wherein said gene is flanked by an N4CC nucleotide sequence, wherein said mutated gene causes a genetically based medical condition; ii) an adeno-associated virus comprising a mutated NmeCas9 protein, said mutated NmeCas9 protein comprising a fused nucleotide deaminase and a binding region for said N4CC nucleotide sequence; b) treating said patient with said adeno-associated virus under conditions such that said mutated NmeCas9 protein replaces said mutated single base with a wild-type single base, such that said medical condition of zQQncn / iznz / u / Yl genetic basis is not I developed. In one embodiment, said gene encodes a protein tyrosinase. In one embodiment, said genetically based medical condition is tyrosinemia. In one embodiment, said virus is an adeno-associated virus 8. In one embodiment, said virus is an adeno-associated virus 6. In one embodiment, said protein is Nme2Cas9. In one embodiment, said protein further comprises a nuclear localization signal protein. In one embodiment, said nucleotide deaminase is a cytidine deaminase. In one embodiment, said nucleotide deaminase is an adenosine deaminase. In one embodiment, the protein further comprises an inhibitor of uracil glycosylase. In one embodiment, the nuclear localization signal protein includes, but is not limited to, nucleoplasmin NLS and / or SV40 NLS and / or Cmyc NLS. In one embodiment, said junction region is an interaction domain with the protospacer accessory motif. In one embodiment, said protospacer accessory motif interaction domain comprises said mutation. In one embodiment, said mutation is a D16A mutation. In one embodiment, said mutated NmeCas9 protein further comprises CBE4. In one embodiment, said mutated NmeCas9 protein further comprises a linker. In one embodiment, said linker is a 73 aa linker. In one embodiment, said linker is a 3xHA tag. In one embodiment, said gene encodes a tyrosinase. In one embodiment, said gene is Fah. In one embodiment, said gene is c-fos. In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a patient comprising a nucleotide sequence comprising a single base mutated gene, wherein said gene is flanked by an N4CC nucleotide sequence, wherein said mutated gene causes a genetically based medical condition; ii) an optimized nNme2Cas9-ABEmax, comprising a mutated NmeCas9 protein, said mutated NmeCas9 protein comprising a fused nucleotide deaminase and a binding region for said N4CC nucleotide sequence; b) treating said patient with said optimized nNme2Cas9-ABEmax under conditions such that said mutated NmeCas9 protein replaces said mutated single base with a wild-type single base such that said genetically based medical condition does not develop. In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a patient comprising a nucleotide sequence comprising a single base mutated gene, wherein said gene is flanked by an N4CC nucleotide sequence, wherein said mutated gene causes a genetically based medical condition; ii) an nNme2Cas9-CBE4, comprising a mutated NmeCas9 protein, said mutated NmeCas9 protein comprising a fused nucleotide deaminase and a binding region for said N4CC nucleotide sequence; b) treating said patient with said nNme2Cas9-CBE4 under conditions such that said mutated NmeCas9 protein replaces said mutated single base with a wild-type single base, such that said genetically based medical condition does not develop. In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a patient comprising a nucleotide sequence comprising a single base mutated gene, wherein said gene is flanked by an N4CC nucleotide sequence, wherein said mutated gene causes a genetically based medical condition; ii) a YE1-BE3-nNme2Cas9(D16A)-UGI, comprising a mutated NmeCas9 protein, said mutated NmeCas9 protein comprising a fused zQQncn / Lznz / q / Yi deaminase nucleotide and a binding region for said N4CC nucleotide sequence; b) treating said patient with said nNme2Cas9-CBE4 under conditions such that said mutated NmeCasS protein replaces said mutated single base with a wild-type single base, such that said genetically based medical condition does not develop. Definitions To facilitate understanding of this invention, various terms are defined below. The terms defined herein have the meanings commonly understood by those skilled in the art in the areas relevant to the present invention. Terms such as a, an, and the are not intended to refer solely to a singular entity, but rather include the general class of which a specific example may be used as an illustration. The terminology herein is used to describe specific embodiments of the invention, but its use does not limit the invention, except as described in the claims. As used herein, the term "editing" refers to a method of altering a nucleic acid sequence of a polynucleotide (eg, a natural wild-type nucleic acid sequence or a mutated natural sequence) by selective deletion. of a specific genomic target. Such specific genomic target includes, but is not limited to, a chromosomal region, gene, promoter, open reading frame, or any nucleic acid sequence. As used herein, the term "single base" refers to one, and only one, nucleotide within a nucleic acid sequence. When used in the context of single base editing, it means that the base at a specific position within the nucleic acid sequence is replaced with a different base. This replacement can occur by many mechanisms, including, but not limited to, substitution or modification. As used herein, the term "target" or "target site" refers to a pre-identified nucleic acid sequence of any composition and / or length. Such target sites include, but are not limited to, a chromosomal region, a gene, a promoter, an open reading frame, or any nucleic acid sequence. In some embodiments, the present invention interrogates these specific genomic target sequences with complementary gRNA sequences. The term "specific (on-target) binding sequence" used herein refers to a specific genomic target subsequence that may be fully complementary to a programmable DNA binding domain or unique guide RNA sequence. The term "off-target binding sequence" used herein refers to a specific genomic target subsequence that may be partially complementary to a programmable DNA binding domain or unique guide RNA sequence. The term "effective amount" used herein refers to a particular amount of a pharmaceutical composition comprising a therapeutic agent, which produces a clinically detrimental result (ie, for example, a reduction in symptoms). The toxicity and therapeutic efficacy of such compositions can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, to determine the LD50 (the lethal dose for 50% of the zQQncn / Lznz / q / Yi population, for its and the EDso (the therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compounds that exhibit broad therapeutic indices are preferred. Data obtained from cell culture assays and additional animal studies can be used in formulating a dosage range for human use. Doses of these compounds are preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The doses vary within this scale depending on the pharmaceutical form used, the sensitivity of the patient and the route of administration. The term "symptom" used herein refers to any subjective or objective evidence of disease or physical disturbance observed by the patient. For example, subjective evidence is usually based on a patient's self-report and may include, without limitation, pain, headache, visual disturbances, nausea, or vomiting. Alternatively, objective evidence is usually the result of medical tests including, without limitation, body temperature, complete blood count, lipid panels, thyroid panels, blood pressure, heart rate, electrocardiogram, tissue or body imaging scans. The term "disease" or "condition" used herein refers to any deterioration of the normal state of the body of a living animal or plant or one of its parts, which interrupts or modifies the performance of vital functions. Typically manifested by distinguishing signs and symptoms, it is usually a response to: i) environmental factors (such as poor nutrition, industrial hazards or weather); ii) specific infectious agents (such as worms, bacteria or viruses); iii) inherent defects of the organism (such as genetic abnormalities); or iv) combinations of these factors. The terms “reduce”, “inhibit”, “decrease”, “suppress3', “decrease”, “prevent” and grammatical equivalents (including “less” or “smaller”, etc.) when referring to the expression of any symptoms in an untreated subject relative to a treated subject, mean that the amount or magnitude of the symptoms in the treated subject is less than in the untreated subject by any amount that is recognized as clinically relevant by any medically trained personnel . In one embodiment, the amount or magnitude of symptoms in the treated subject is at least 10% less, at least 25% less, at least 50% less, at least 75% less, or at least 90% less. than the amount or magnitude of symptoms in the untreated subject. The term "bound" used herein refers to any interaction between a medium (or vehicle) and a drug. The union can be reversible or irreversible. Such binding includes, without limitation, covalent binding, ionic binding, Van der Waals forces or friction, and the like. A drug is bound to a medium (or vehicle) if it is impregnated, incorporated or covered by it, or is in suspension, solution or mixture with it, etc. The term "drug" or "compound" used herein refers to any pharmacologically active substance capable of being administered that achieves a desired effect. Drugs or compounds can be synthetic or natural, non-peptide, protein or peptide, oligonucleotide or nucleotide, polysaccharide or sugar. zQQncn / Lznz / q / Yi The term "administered" or "administer" as used herein refers to any method of providing a composition to a patient in such a way that the composition has its desired effect on the patient. An exemplary method of administration is by a direct mechanism such as local tissue delivery (ie, eg, extravascular placement), oral ingestion, transdermal patch, topical, inhalation, suppository, etc. The term "patient" or "subject" used herein is a human or animal and need not be hospitalized. For example, outpatients, people in nursing homes, are "patients." A patient can comprise any age of a human or non-human animal and thus includes both adults and juveniles (ie, children). The term "patient" is considered to denote a need for medical treatment and therefore a patient, voluntarily or involuntarily, can be part of either clinical experimentation or in support of basic science studies. The term "affinity" used herein refers to any attractive force between substances or particles that causes them to enter and remain in chemical combination. For example, an inhibitory compound that has high affinity for a receptor will provide greater efficacy in preventing the receptor from interacting with its natural ligands than an inhibitor with a low affinity. The term "pharmaceutically" or "pharmacologically acceptable" as used herein refers to molecular entities and compositions that do not cause adverse, allergic, or other adverse reactions when administered to an animal or human. The term "pharmaceutically acceptable carrier," as used herein, includes any and all solvents, or a dispersion medium including, without limitation, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol and the like), suitable mixtures thereof and vegetable oils, coatings, isotonic and absorption delaying agents, liposomes, commercially available cleansers and the like. Complementary bioactive ingredients can also be incorporated into these vehicles. The term "viral vector" encompasses any nucleic acid construct derived from a viral genome capable of incorporating heterologous nucleic acid sequences for expression in a host organism. For example, these viral vectors can include, without limitation, adeno-associated viral vectors, lentiviral vectors, SV40 viral vectors, retroviral vectors, adenoviral vectors. Although viral vectors are occasionally created from pathogenic viruses, they can be modified to minimize their overall health risk. This usually involves the deletion of a part of the viral genome involved in viral replication. Such a virus can efficiently infect cells, but once infection has occurred, the virus may require a helper virus to provide the missing proteins for the production of new virions. Preferably, viral vectors should have minimal effect on the physiology of the cell they infect, and exhibit genetically stable properties (eg, not undergo spontaneous genome rearrangement). Most viral vectors are modified to infect as wide a range of cell types as possible. Even so, a viral receptor can be modified to direct the virus to a specific kind of cell. Viruses modified in this way are said to be pseudotyped. Viral vectors are frequently modified to incorporate certain zQQncn / Lznz / q / Yi genes that help identify which cells incorporated the viral genes. These genes are called marker genes. For example, a common marker gene confers antibiotic resistance for a certain antibiotic. As used herein the "ROSA26 gene" or "Rosa26 gene" refers to a human or mouse locus (respectively) that is widely used to achieve widespread expression in the mouse. Targeting to the ROSA26 locus can be achieved by introducing a desired gene into the first intron of the locus, at a unique Xbal site approximately 248 bp upstream of the original gene trap line. A construct can be constructed using an adenovirus splice acceptor followed by a gene of interest and a polyadenylation site inserted into the unique Xbal site. A neomycin resistance cassette can also be included in the targeting vector. As used herein, the "PCSK9 gene" or "PCSk9 gene" refers to a human or mouse locus (respectively) that encodes a PCSK9 protein. The PCSK9 gene resides on chromosome 1 in band 1p32.3 and includes 13 exons. This gene can produce at least two isoforms through alternative splicing. The terms "proprotein convertase subtilisin / kexin type 9" and "PCSK9" refer to a protein encoded by a gene that modulates low-density lipoprotein levels. Proprotein convertase subtilisin / kexin type 9, also known as PCSK9, is an enzyme that in humans is encoded by the PCSK9 gene. Seidah et al., The secretory proprotein convertase neural apoptosisregulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation Proc. nati. Acad. Sci. USA 100(3): 928-933 (2003). Similar genes (orthologs) are found among many species. Many enzymes, including PSCK9, are inactive when first synthesized because they have a section of peptide chains that blocks their activity; proprotein convertases remove that section to activate the enzyme. PSCK9 is believed to play a regulatory role in cholesterol homeostasis. For example, PCSK9 can bind to the epidermal growth factor-like A (EGF-A) repeat domain of the low-density lipoprotein receptor (LDL-R), giving as Internalization and degradation of LDL-R result. Clearly, reduced LDL-R levels would be expected to reduce LDL-C metabolism, which could lead to hypercholesterolemia. The term "hypercholesterolemia" used herein refers to any condition where blood cholesterol levels are elevated above clinically recommended levels. For example, if cholesterol is measured using low-density lipoproteins (LDL), hypercholesterolemia may exist if the measured LDL levels are above, for example, about 70 mg / dL. Alternatively, if cholesterol is measured using plasma free cholesterol, hypercholesterolemia may exist if the measured free cholesterol levels are above, for example, about 200-220 mg / dL. As used herein, the term "CRISPR" or "clustered regularly interspaced short palindromic repeats" refers to an acronym for a DNA loci containing multiple short direct repeats of base sequences. Each repeat contains a series of bases followed by about 30 base pairs known as spacer DNA. Spacers are short segments of a virus's DNA and can serve as a "memory" of past exposures to facilitate an adaptive defense against future invasions. As used herein, the term "Cas" or "CRISPR-associated (cas)" refers to genes frequently associated with CRISPR repeat-spacer arrangements. As used herein, the term Cas9 refers to a CRISPR Type II nuclease, an enzyme specialized for generating double-strand breaks in DNA, with two active cleavage sites (the HNH and RuvC domains), one for each strand of the double helix. Jinek combined tracrRNA and spacer RNA into a single guide RNA (sgRNA) molecule that, mixed with Cas9, could find and cleave DNA targets through Watson-Crick pairing between the guide sequence within the sgRNA and the target DNA sequence. . The term "protospacer-adjacent motif" (or PAM) as used herein refers to a DNA sequence that may be required for a Cas9 / sgRNA to form an R-loop to interrogate a DNA sequence. Specific DNA by means of the Watson-Crick pairing of its guide RNA with the genome. The specificity for PAM may be a function of the DNA-binding specificity of the Cas9 protein (eg, a "protospacer-adjacent motif recognition domain" at the C-terminus of Cas9). As used herein, the term "sgRNA" refers to unique guide RNA used in conjunction with CRISPR-associated (Cas) systems. sgRNAs are a fusion of crRNA and tracrRNA and contain nucleotides of sequence complementary to the desired target site. Jinek et al., “A programmable dual-RNAguided DNA endonuclease in adaptive bacterial immunity” Science 337(6096):816-821 (2012). Watson-Crick pairing of the sgRNA with the target site allows formation of the R loop, which in conjunction with a functional PAM allows DNA cleavage or, in the case of Cas9 nuclease deficient, allows DNA binding at that locus. As used herein, the term "fluorescent protein" refers to a protein domain comprising at least an organic compound portion that fluoresces light in response to appropriate wavelengths. For example, fluorescent proteins can emit red, blue, and / or green light. Such proteins are commercially available, including, but not limited to: i) mCherry (Clonetech Laboratories): excitation: 556 / 20 nm (wavelength / bandwidth); emission: 630 / 91 nm; ii) sfGFP (Invitrogen): excitation: 470 / 28 nm; emission: 512 / 23nm; iii) TagBFP (Evrogen): excitation 387 / 11 nm; emission 464 / 23 nm. As used herein, the term "sgRNA" refers to unique guide RNA used in conjunction with CRISPR-associated (Cas) systems. sgRNAs contain nucleotides of sequence complementary to the desired target site. Watson-Crick pairing of the sgRNA to the target site recruits nuclease-deficient Cas9 to bind to DNA at that locus. As used herein, the term orthogonal refers to non-overlapping, uncorrelated, or independent targets. For example, if two nuclease-deficient orthogonal Cas9 genes were implemented fused to different effector domains, the sgRNAs encoded for each zQQncn / Lznz / q / Yi would not cross or overlap. Not all nuclease-deficient Cas9 genes function in the same way, allowing the use of the nuclease-deficient orthogonal Cas9 gene fused to different effector domains that provide the appropriate orthogonal gRNAs. As used herein, the term "phenotypic change" or "phenotype" refers to the combination of observable characteristics or traits of an organism, such as its morphology, development, biochemical or physiological properties, phenology, behavior, and behavioral products. Phenotypes result from the expression of an organism's genes, as well as the influence of environmental factors and the interactions between the two. "Nucleic acid sequence" and "nucleotide sequence" as used herein refer to an oligonucleotide or polynucleotide and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin that may be single-stranded. or double and represent the sense or antisense strand. The term "an isolated nucleic acid" used herein refers to any nucleic acid molecule that has been separated from its natural state (eg, separated from a cell and, in a preferred embodiment, is free of other genomic nucleic acids). ). The terms "amino acid sequence" and "polypeptide sequence" used herein are interchangeable and refer to a sequence of amino acids. As used herein, the term "portion" when referring to a protein (as in "a portion of a given protein") refers to fragments of that protein. Fragments can range in size from four amino acid residues to the complete amino acid sequence minus one amino acid. The term "portion", used to refer to a nucleotide sequence, refers to fragments of that nucleotide sequence. Fragments can range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue. As used herein, the terms "complementary" or "complementarity" are used to refer to "polynucleotides" and "oligonucleotides" (which are interchangeable terms referring to a sequence of nucleotides) related by the pairing rules of bases. For example, the sequence C-A-G-T" is complementary to the sequence G-T-C-A". Complementarity can be “partial” or “total”. "Partial" complementarity is where one or more nucleic acid bases do not match according to the base pairing rules. "Total" or "complete" complementarity between nucleic acids is where each and every base in the nucleic acid matches other bases under base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions and also detection methods that depend on binding between nucleic acids. The terms homology and homolog, as used herein in reference to nucleotide sequences, refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (ie, identity). A nucleotide sequence that is zQQncn / Lznz / q / Yi partially complementary, that is, substantially homologous to a nucleic acid sequence is one that inhibits, at least partially, the hybridization of a fully complementary sequence to a target nucleic acid sequence. . Inhibition of hybridization of the fully complementary sequence to the target sequence can be examined using a hybridization assay (Southern or Northern blot, solution hybridization, and the like) under low stringency conditions. A substantially homologous sequence or probe will compete for and inhibit the binding (ie, hybridization) of a fully homologous sequence to a target sequence under low stringency conditions. This is not to say that low stringency conditions are such that non-specific binding is allowed; low stringency conditions require that the binding of two sequences to each other be a specific (ie, selective) interaction. Absence of nonspecific binding can be tested by using a second target sequence that lacks even a partial degree of complementarity (eg, less than about 30% identity); in the absence of non-specific binding, the probe will not hybridize to the second non-complementary target. The terms homology and homolog, as used herein in reference to amino acid sequences, refer to the degree of primary structure identity between two amino acid sequences. Such a degree of identity may be directed to a portion of each amino acid sequence, or to the entire length of the amino acid sequence. Two or more amino acid sequences that are substantially homologous may have at least 50% identity, preferably at least 75% identity, more preferably at least 85% identity, most preferably at least 95% or 100% identity. An oligonucleotide sequence that is a homologue is defined herein as an oligonucleotide sequence that exhibits greater than or equal to 50% identity to a sequence, when comparing sequences that are 100 bp or longer in length. Low stringency conditions comprise conditions equivalent to binding or hybridization at 42 °C in a solution consisting of 5 x SSPE (43.8 g / L NaCI, 6.9 g / L NaH2PO4'H2O, and 1.85 g / L EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, Denhardt's reagent 5X {Denhardt 50X contains per 500 mL: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)}, and 100 pg / mL denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42°C when using a probe approximately 500 nucleotides in length. Numerous equivalent conditions can also be used to encompass conditions of low stringency; factors such as the length and nature (DNA, RNA, composition of bases) of the probe and the nature of the target (DNA, RNA, composition of bases, present in solution or immobilized, etc.) and the concentration of salts and other components (eg, the presence or absence of formamide, dextran sulfate, polyethylene glycol) as well as the components of the hybridization solution can be varied to generate low stringency hybridization conditions different from, but equivalent to, the conditions listed above. In addition, conditions that promote hybridization under high stringency conditions can also be used (eg, increasing the temperature of the hybridization and / or washing steps, the use of formamide in the hybridization solution, etc.). zQQncn / Lznz / q / Yi As used herein, the term "hybridization" is used to refer to the pairing of complementary nucleic acids using any method by which a nucleic acid strand is joined to a complementary strand by means of base pairing, to form a hybridization complex. Hybridization and hybridization strength (i.e., the strength of association between nucleic acids) are affected by factors such as the degree of complementarity between nucleic acids, the stringency of the conditions involved, the Tm of the hybrid formed, and the G ratio. :C within nucleic acids. As used herein, the term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of hydrogen bonding between complementary G and C bases and between complementary A and T bases; these hydrogen bonds can be further stabilized by base-stacking interactions. The two complementary nucleic acid sequences are joined by hydrogen bonding in an antiparallel configuration. A hybridization complex can be formed in solution (for example, Co t or Ro t analysis) or between a nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (for example, a nylon membrane or a nitrocellulose filter as used in Southern and Northern blots, dot blots, or a glass slide as used in in situ hybridization, including FISH (fluorescence in situ hybridization). DNA molecules are said to have 5'" ends and 3'" ends because mononucleotides react to produce oligonucleotides in such a way that the 5' phosphate of one mononucleotide pentose ring binds to the 3' oxygen of its neighbor in a direction through a phosphodiester bond. Therefore, an end of an oligonucleotide is called the 5'" end if its 5' phosphate is not attached to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is called the 3' end if its 3' oxygen is not attached to a 5' phosphate of another mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, can also be said to have 5' and 3' ends. In a linear or circular DNA molecule, the discrete elements are referred to as being upstream or in the 5' direction of the elements downstream" or in the 3' direction. This terminology reflects the fact that transcription proceeds 5' to 3' along the DNA strand. Promoter and enhancer elements that direct transcription of a linked gene are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located downstream of the promoter element and the coding region. The transcription termination and polyadenylation signals are located 3' or downstream of the coding region. The term "transfection" or "transfected" refers to the introduction of foreign DNA into a cell. As used herein, the terms "coding nucleic acid molecule," "coding DNA sequence," and "coding DNA" refer to the order or sequence of deoxyribonucleotides along a deoxyribonucleic acid strand. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Therefore, the DNA sequence codes for the amino acid sequence. zQQncn / Lznz / q / Yi As used herein, the term "gene" means the deoxyribonucleotide sequences that comprise the coding region of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends, by a distance of approximately 1 kb at either end, such that the gene corresponds to the length of the full-length mRNA. Sequences that are located 5' to the coding region and that are present in the mRNA are referred to as 5' non-translated sequences. Sequences that are located 3' or downstream of the coding region and that are present in the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both the cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences called "introns" or "intermediate regions" or "intermediate sequences." Introns are segments of a gene that are transcribed into heterologous nuclear RNA (hnRNA); the introns may contain regulatory elements such as enhancers. Introns are removed or "spliced" from the nuclear or primary transcript; therefore, introns are absent in the messenger RNA (mRNA) transcript. mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. In addition to containing introns, genomic forms of a gene may also include sequences located at both the 5' and 3' ends of the sequences, which are present in the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the untranslated sequences present in the mRNA transcript). The 5' flanking region may contain regulatory sequences, such as promoters and enhancers, that control or alter the transcription of the gene. The 3' flanking region may contain sequences that direct transcription termination, post-transcriptional cleavage, and polyadenylation. The term label or detectable label is used herein to refer to any composition detectable by spectroscopic, photochemical, diochemical, immunochemical, electrical, optical, or chemical means. Such labels include biotin for staining with labeled streptavidin conjugate, magnetic beads (eg, Dynabeads®), fluorescent dyes (eg, fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (eg, 3H, 125l,35S,14C or 32P), enzymes (for example, horseradish peroxidase, alkaline phosphatase, and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic beads (for example, polystyrene, polypropylene, latex, etc.). Patents teaching the use of such tags include, but are not limited to, US Patent No. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference in their entireties). The markers contemplated by the present invention can be detected by many methods. For example, radiolabels can be detected using photographic film or scintillation counters, fluorescent labels can be detected using a photodetector to detect the emitted light. Enzyme labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected simply by viewing the colored label. zQancn / i znz / u / Yl· Brief description of the figures The application or patent file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided to the Office upon request and payment of the necessary fee. Figures 1A through 1E illustrate exemplary schematic embodiments of a NmeCas9 deaminase fusion protein single base editor and exemplary base editor constructed plasmids. Figure 1A shows an exemplary construct YE1 -BE3-nNme2Cas9(D16A)-LJGI. Figure 1B shows an exemplary construct ABE7.10 nNme2Cas9 (D16A). Figure 1C shows an exemplary ABE7.10-nNme2Cas9(D16A) construct comprising two SV40 NLS sequences. Figure 1D shows an exemplary construct nNme2Cas9-CBE4 (also called BE4nNme2Cas9(D16A)-UGI-UGI). Figure 1 E shows an exemplary optimized nNme2Cas9-ABEmax construct. Figures 2A to 2C present exemplary data from electroporation of HEK293T cells with DNA plasmids comprising a YE1-BE3-nNme2Cas9(D16A)-UGI fusion protein that efficiently converts C to T at endogenous target site 25 (TS25) in HEK293T cells by nucleofection. Figure 2A shows exemplary sequences for an endogenous TS25 target site (inside the black box). GN23 sgRNA base pairs with the target DNA strand, leaving the displaced DNA strand for editing by cytidine deaminase (eg, new green nucleotides). Figure 2B shows exemplary sequencing data showing a doublet nucleotide peak (7th apposition from the 5' end; arrow) demonstrating successful single base editing of a cytidine to a thymidine (eg, a conversion of a pair of OG bases in a Τ·Α base pair). Figure 2C shows an exemplary quantization of the data shown in Figure 2B which plots the percentage conversion of the single base edit C T. The percentage of C converted to T is approximately 40% in the sample treated with the editor. base and with sRNA (p-value = 6.88 x 10-6). Control without sgRNA shows source noise due to Sanger sequencing. EditR (Kluesner et al., 2018) was used to perform the analysis. Figures 3A through 3F present exemplary specific UGI target sites that were respectively integrated into YE1-BE3-nNme2Cas9 / D16A mutant fusion proteins and co-expressed with enhanced green fluorescent protein (EGFP) in a cell line derived from K562 stable. Converted bases are highlighted in orange. Source signals were filtered using negative control samples (YE1-BE3-nNme2Cas9 nucleofected K562 cells without sgRNA constructs). The N4CC PAMs are locked up. The right column shows the percentage of total reads that exhibit mutations at sites targeted to the base editor. Figure 3A shows an example of EGFP-Site 1. 7QQOCO / I 7O7 / 3 / YL Figure 3B shows an example of EGFP-Site 2. Figure 3C shows an example of EGFP-Site 3. Figure 3D shows an example of EGFP-Site 4. Figure 3E shows an exemplary deep sequencing analysis indicating where YE1-BE3-nNme2Cas9 converts C residues to T residues in the endogenous c-fos promoter region. The right column shows the percentage of total reads that exhibit mutations at sites targeted to the base editor. Converted bases are highlighted in orange or yellow. Source signals were filtered using negative control samples. The highest editing percentage is 32.50%. Figure 3F shows an exemplary deep sequencing analysis indicating where ABE7.10-nNme2Cas9 or ABEmax (Koblan et al., 2018)-nNme2Cas9 converts A residues to G residues in the endogenous c-fos promoter region. The right column shows the percentage of total reads that exhibit mutations at sites targeted to the base editor. Converted bases are highlighted in orange. Source signals were filtered using negative control samples. The editing percentage is 0.53% by ABE7.10-nNme2Cas9 or 2.33% by ABEmax-nNme2Cas9 (D16A). Figure 4 presents an exemplary alignment of the wild-type Fah gene with the tyrosinemia mutant Fah gene showing a single base A-G gene editing target site (position 9). The respective single PAM site for SpyCas9 and the dual PAM sites for NmeCas9 are indicated to demonstrate the suboptimal targeting window relative to the PAM site for SpyCas9. Figures 5A to 5E illustrate examples of three closely related Neisseria meningitidis Cas9 orthologs that have distinct MAPs. Figure 5A shows an exemplary schematic showing mutated residues (orange spheres) between Nme2Cas9 (left) and Nme3Cas9 (right) mapped to the predicted structure of Nme1Cas9, revealing the cluster of mutations in the PID (black). Figure 5B shows an exemplary experimental workflow of the in vitro PAM discovery assay with a 10 bp randomized PAM region. After in vitro digestion, adapters were ligated to excise products for library construction and sequencing. Figure 5C shows exemplary sequence logos resulting from in vitro PAM discovery revealing the enrichment of a N4GATT PAM for Nme1Cas9, consistent with its previously established specificity. Figure 5D shows exemplary sequence logos indicating that Nme1Cas9 with its PID swapped with that of Nme2Cas9 (left) or Nme3Cas9 (right) requires a C at position 5 of PAM. The remaining nucleotides were not determined with high confidence due to the modest cleavage efficiency of the PID-swapped protein chimeras (see Figure 6C). Figure 5E shows an exemplary sequence logo showing that full-length Nme2Cas9 recognizes an N4CC PAM, based on efficient substrate cleavage of a target group with a fixed C at position 5 of the PAM, and with nt 1-4 and 6-8 of the PAM randomized. Figures 6A to 6D present a characterization of the rapidly evolving Neisseria meningitidis Cas9 orthologs with PID, relative to Figures 5A to 5E. Figure 6A shows an exemplary rootless phylogenetic tree of NmeCas9 orthologs that are >80% identical to Nme1Cas9. Three distinct branches emerge with most of the mutations clustered in the PID. Groups 1 (blue), 2 (orange), and 3 (green) have PIDs with >98%, ~52%, and ~86% identity to Nme1Cas9, respectively. Three representative Cas9 orthologs (one from each group) are indicated (Nme1Cas9, Nme2Cas9 and Nme3Cas9). Figure 6B shows an exemplary schematic showing the CRISPR-cas loci of the strains encoding the three Cas9 orthologs (Nme1Cas9, Nme2Cas9 and Nme3Cas9) of Figure 6A. Percentage identity of each CRISPR-Cas component to N. meningitidis 8013 (encoding Nme1Cas9) is shown. Blue and red arrows indicate the pre-crRNA and tracrRNA transcription start sites, respectively. Figure 6C shows an example of normalized read counts (7th of total reads) of cleaved DNAs from in vitro assays for intact Nme1Cas9 (grey), for chimeras with PIDs of Nme1Cas9 swapped with those of Nme2Cas9 and Nme3Cas9 (mixed colors). , and for full-length Nme2Cas9 (orange), are plotted. The reduction of normalized read counts indicates lower cleavage efficiencies in the chimeras. Figure 6D shows an example of in vitro PAM discovery assay sequence logos in a pool of PAM NNNNCNNN by Nme1Cas9 with their PID swapped with those of Nme2Cas9 (left) or Nme3Cas9 (right). Figures 7A through 7D present exemplary data showing that Nme2Cas9 uses a 22-24 nt spacer to edit sites adjacent to an N4CC PAM. All experiments were performed in triplicate and the error bars represent the standard error of the mean (s.e.m.). Figure 7A shows an exemplary schematic diagram depicting transient transfection and editing of HEK293T TLR2.0 cells, with mCherry+ cells detected by flow cytometry 72 hours post-transfection. Figure 7B shows an exemplary Nme2Cas9 editing of the TLR2.0 reporter. Sites with the N4CC PAMs were targeted with different efficiencies, whereas Nme2Cas9 targeting was not observed in a N4GATT PAM or in the absence of sgRNA. SpyCas9 (targeting a site previously validated with a PAM NGG) and Nme1Cas9 (targeting N4GATT) were used as positive controls. Figure 7C shows an exemplary effect of spacer length on Nme2Cas9 editing efficiency. An sgRNA targeting a single TLR2.0 site, with spacer lengths ranging from 24 to 20 nts (including the 5'-terminal G required by the U6 promoter), indicate that the highest editing efficiencies are obtained with 22 spacers. -24nt. Figure 7D shows an example of a Nme2Cas9 dual nickase that can be used in zQQncn / Lznz / q / Yi tandem to generate NHEJ and HDR-based edits in TLR2.0. Plasmids expressing Nme2Cas9 and sgRNA, together with an 800 bp dsDNA donor for homologous repair, were electroporated into HEK293T TLR2.0 cells, and NHEJ (mCherry+) and HDR (GFP+) results scored by cytometry. flow. HNH nickase, Nme2Cas9D16A; RuvC nickasa, Nme2Cas9H588A. Cleavage sites 32 bp and 64 bp apart were targeted using either nickase. HNH nickase (Nme2Cas9D16A) produced efficient editing, particularly with cleavage sites that were 32 bp apart, whereas RuvC nickase (Nme2Cas9H588A) was not efficient. Wild-type Nme2Cas9 was used as a control. Figures 8A through 8D present exemplary data showing the PAM, spacer, and seed requirements for Nme2Cas9 targeting in mammalian cells, relative to Figures 7A through 7D. All experiments were done in triplicate and error bars represent s.e.m. Figure 8A shows an example Nme2Cas9 targeting N4CD sites in TLR2.0, with estimated editing based on mCherry+ cells. Four sites for each non-C nucleotide at the tested position (N4CA, N4CT, and N4CG) were examined and one N4CC site was used as a positive control. Figure 8B shows an example Nme2Cas9 targeting N4DC sites in TLR2.0 (similar to Figure 8A). Figure 8C shows exemplary leader truncations at a TLR2.0 site (different from Figure 2C) with a N4CCA PAM, revealing similar length requirements to those observed at the other site. Figure 8D shows the targeting efficiency of exemplary Nme2Cas9 which is differentially sensitive to single nucleotide mismatches in the sgRNA seed region. The data show the effects of single nucleotide sgRNA mismatches found along the 23 nt spacer at a TLR2.0 target site. Figures 9A through 9C present exemplary data showing genome editing of Nme2Cas9 at endogenous loci in mammalian cells via multiple delivery methods. All results represent 3 independent biological replicates and error bars represent s.e.m. Figure 9A shows an example of Nme2Cas9 genome editing of endogenous human sites in HEK293T cells after transient transfection of plasmids expressing Nme2Cas9 and sgRNA. Initially, 40 sites were selected (Table 1); the 14 sites shown (selected to include representatives of various editing efficiencies, as measured by TIDE) were reanalyzed in triplicate. A Nme1Cas9 target site (with a N4GATT PAM) was used as a negative control. Figure 9B shows plots of exemplary data: Left panel: Transient transfection of a single plasmid expressing both Nme2Cas9 and sgRNA (targeting the Pcsk9 and Posa26 loci) allows editing in Hepa1-6 mouse cells, as detected by TIDE. Right panel: Electroporation of sgRNA plasmids into K562 cells stably expressing Nme2Cas9 from a lentivector results in efficient indel formation. 7QQncn / L7n7 / q / Yi Figure 9C shows an example of Nme2Cas9 that can be electroporated as an RNP complex to induce genome editing. 40 picomole of Cas9 was electroporated along with 50 pimole of in vitro transcribed sgRNAs targeting three different loci in HEK293T cells. Indels were measured after 72h using TIDE. Figures 10A and 10B present exemplary data showing dose dependence and segmental deletions by Nme2Cas9, as referenced in Figures 9A to 9C. Figure 10A shows an exemplary dose increase of electroporated Nme2Cas9 plasmid (500 ng, vs. 200 ng in Figure 3A) that improves editing efficiency at two sites (TS16 and TS6). Data provided in yellow is reused from Figure 9A. Figure 10B shows an example of Nme2Cas9 that can be used to create precise segmental deletions. Two TLR2.0 targets with cleavage sites 32 bp apart were co-targeted with Nme2Cas9. Most of the lesions created were deletions of exactly 32 bp (blue). Figures 11A to 11C present exemplary data showing that Nme2Cas9 is subject to inhibition by a subset of type ll-C anti-CRISPR families in vitro and in cells. All experiments were done in triplicate and error bars represent s.e.m. Figure 11A shows an exemplary in vitro cleavage assay of Nme1 Cas9 and Nme2Cas9 in the presence of five previously characterized anti-CRISPR proteins (10:1 ratio of Acr:Cas9). Top: Nme1Cas9 efficiently cleaves a fragment containing a protospacer with a N4GATT PAM in the absence of an Acr or in the presence of a negative control Acr (AcrE2). All five previously characterized type ll-C Acr families inhibited Nme1Cas9, as expected. Bottom: Nme2Cas9 inhibition mirrors that of Nme1Cas9, except for the lack of inhibition by AcrllC5smu Figure 11B shows exemplary genome editing in the presence of the five previously described anti-CRISPR families. Plasmids expressing Nme2Cas9 (200 ng), sgRNA (100 ng) and each respective Acr (200 ng) were cotransfected into HEK293T cells, and genome editing was measured using tracking indels by decay (TIDE). English) 72 hours after transfection. Consistent with in vitro analyses, all type ll-C anti-CRISPR except AcrllC5smu inhibited genome editing, albeit with different efficiencies. Figure 11C shows an example of dose dependent Acr inhibition of Nme2Cas9 with different apparent potencies. Nme2Cas9 is fully inhibited by AcHICIn™ and AcrllC4Hpa in 2:1 and 1:1 mass ratios of cotransfected Acr and Nme2Cas9 plasmids, respectively. Figure 12 presents exemplary data showing that a Nme2Cas9 PID swap renders Nme1Cas9 insensitive to AcrllC5s™ inhibition, relative to Figures 11A to 11C. In vitro cleavage by the Nme1Cas9-Nme2Cas9PID chimera in the presence of previously characterized Acr proteins (Cas9-sgRNA 10 pM + Acr 100 μΜ). zQQncn / Lznz / q / Yi Figures 13A to 13E present exemplary data showing the orthogonality and relative accuracy of Nme2Cas9 and SpyCas9 at dual target sites, relative to Figure 12. Figure 13A shows examples of Nme2Cas9 and SpyCas9 guides that are orthogonal. The TIDE results show the frequencies of indels created by both DS2-targeting nucleases with either their cognate sgRNAs or the other orthologous sgRNAs. Figure 13B shows Nme2Cas9 and SpyCas9 samples exhibiting comparable specific editing efficiencies as assessed by GUIDE-seq. Bars indicate counts of specific GUIDE-Seq reads at the three dual sites targeted by each ortholog. The orange bars represent Nme2Cas9 and the black bars represent SpyCas9. Figure 13C shows an example of specific SpyCas9 read counts vs. nonspecific for each site. The orange bars represent the specific readings while the black bars represent the non-specific readings. Figure 13D shows exemplary reads of specific Nme2Cas9 vs. nonspecific for each site. Figure 13E are bar graphs showing exemplary indel efficiencies (as measured by TIDE) at non-specific potential sites predicted by CRISPRSeek. Site-specific and non-specific sequences are shown on the left, with the PAM region underlined and non-consensus PAM sgRNA and nucleotide mismatches in red. Figures 14A to 14E present exemplary data showing that Nme2Cas9 exhibits little or no detectable nonspecific action in mammalian cells. Figure 14A shows an exemplary schematic depicting dual sites (DS) that both SpyCas9 and Nme2Cas9 can target by virtue of their non-overlapping PAMs. The PAM for Nme2Cas9 (orange) and the PAM for SpyCas9 (blue) are highlighted. A 24 nt leader sequence for Nme2Cas9 is indicated in yellow; the corresponding leader sequence for SpyCas9 would be 4 nt shorter at the 5' end. Figure 14B shows an example of Nme2Cas9 and SpyCas9 both inducing indels in DSs. Six DS in VEGFA (with GN3GN19NGGNCC sequences) were selected for direct comparisons of editing of the two orthologs. Plasmids expressing each Cas9 (with the same promoter, linkers, tags and NLS) and its cognate leader were transfected into HEK293T cells. Indel efficiencies were determined by TIDE 72 hours after transfection. Editing by Nme2Cas9 was detectable at all six sites and was marginally or significantly more efficient than SpyCas9 at two sites (DS2 and DS6, respectively). SpyCas9 edited four of the six sites (DS1, DS2, DS4, and DS6), with two sites showing significantly higher editing efficiencies than Nme2Cas9 (DS1 and DS4). DS2, DS4, and DS6 were selected for GUIDE-Seq analysis, as Nme2Cas9 was equally efficient, less efficient, and more efficient than SpyCas9, respectively, at these sites. Figure 14C shows an example of genome editing with Nme2Cas9 that is highly accurate in human cells. The number of non-specific sites detected by GUIDE zQancn / Lznz / q / Yi is shown Seq for each nuclease at individual target sites. In addition to the dual sites, we analyzed the TS6 (due to their high specific editing efficiency) and Pcsk9 and Rosa26 sites in mouse Hepa1-6 cells (to measure accuracy in another cell type). Figure 14D shows exemplary targeted deep sequencing to detect indels in edited cells confirming the high accuracy of Nme2Cas9 indicated by GUIDE-seq. Figure 14E shows an exemplary sequence for the validated non-specific site of the Rosa26 guide, showing the PAM region (underlined), the consensus PAM dinucleotide CC (bold), and three mismatches in the PAM distal portion of the spacer (red). Figures 15A to 15C present exemplary data showing genome editing of Nme2Cas9 in vivo by all-in-one AAV delivery. Figure 15A shows an exemplary workflow for delivery of AAV8.sgRNA.Nme2Cas9 to reduce cholesterol levels in mice by targeting Pcsk9. Top: Schematic of the all-in-one AAV vector expressing Nme2Cas9 and sgRNA (individual genome elements not to scale). BGH, bovine growth hormone poly(A) site; HA, epitope tag; NLS, nuclear localization sequence; h, codon optimized for human. Bottom: Schedule for tail vein injections of AAV8.sgRNA.Nme2Cas9 (4 x 1011GC), followed by cholesterol measurements on day 14 and indel, histology, and cholesterol analysis on day 28 post-injection. Figure 15B shows an exemplary TIDE assay to measure indels in DNA extracted from the livers of mice injected with AAV8.Nme2Cas9+sgRNA targeting the Pcsk9 and Rosa26 loci (control). The efficiency of indel at the only non-specific site identified by GUIDE-seq for these two sgRNAs (Rosa26|OT1) was also evaluated by TIDE. Figure 15C shows an example of reduced serum cholesterol levels in mice injected with Pcsk9 targeting guide compared to Rosa26 targeting controls. P values ​​were calculated using an unpaired two-tailed t-test. Figures 16A and 16B present exemplary data showing PCSK9 deletion and liver histology after AAV Nme2Cas9 delivery and editing, related to Figures 15A to 15C. Figure 16A shows an exemplary Western blot using an anti-PCSK9 antibody revealing strongly reduced levels of PCSK9 in the livers of sgPcsk9-treated mice, compared to sgRosa26-treated mice. 2 ng of recombinant PCSK9 was used as the mobility standard (leftmost lane), and a cross-reactive band in the liver samples is indicated with an asterisk. GAPDH was used as a loading control (bottom panel). Figure 16B shows exemplary H&E staining of livers from mice injected with AAV8.Nme2Cas9+sgfiosa26 (left) or AAV8.Nme2Cas9+sgPcsk9 (right) vectors. Scale bars, 25 pm. zQQncn / Lznz / q / Yi Figures 17A to 17C present exemplary data showing ex vivo Tyr editing in mouse zygotes, related to Figures 16A and 16B. Figure 17A shows an example of two sites on Tyr, each with the N4CC PAMs, tested for editing in Hepa1-6 cells. The sgTyr2 guide showed higher editing efficiency and was selected for further testing. Figure 17B shows an example of seven mice that survived postnatal development, each exhibiting coat color phenotypes as well as specific editing, as assessed by TI DE. Figure 17C shows an example indel spectrum of tail DNA from each mouse in Figure 17B, as well as an unedited C57BL / 6NJ mouse, as indicated by TIDE analysis. The efficiency of insertions (positive) and deletions (negative) of various sizes is indicated. Figures 18A to 18C present exemplary data showing Nme2Cas9 genome editing ex vivo by all-in-one AAV delivery. Figure 18A shows an example workflow for Nme2Cas9 ex vivo editing of single AAV to generate C57BL / 6NJ albino mice targeting the Tyr gene. Zygotes are grown in KSOM containing AAV6.Nme2Cas9:sgTyr for 5-6 hours, rinsed in M2, and grown for one day before transferring into the oviduct of pseudopregnant recipients. Figure 18B shows an example of albino mice (left) and chinchilla or motley mice (middle) generated by 3x109GC, and chinchilla or motley mice (right) generated by 3x108GC of AAV6.Nme2Cas9:sg Tyr zygotes. Figure 18C shows an example summary of ex vivo Tyr editing experiments with single AAV from Nme2Cas9.sg Tyr with two doses of AAV. Figures 19A to 19C show an exemplary mCherry reporter assay for nSpCas9-ABEmax and optimized ABEmax-nNme2Cas9 (D16A) activities. Figure 19A shows an example of ABEmCherry reporter sequence information. There is a TAG stop codon in the mCherry coding region. In the reporter-integrated stable cell line, there is no mCherry signal. The mCherry signal will appear if the optimized nSpCas9ABEmax or ABEmax-nNme2Cas9(D16A) can convert TAG to CAG (which is encoded as Gln). Figure 19B shows an example of mCherry signals lighting as SpCas9ABE or ABEmax-nNme2Cas9 (D16A) is active in the specific region of the mCherry reporter. The upper panel is the negative control, the middle panel shows the illuminated mCherry signals in reporter cells treated with nSpCas9-ABEmax, the lower panel shows the illuminated mCherry signals in reporter cells treated with optimized ABEmax-nNme2Cas9(D16A). Figure 19C shows an example of FAC quantification of base editing events in mCherry reporter cells transfected with SpCas9-ABE or ABEmax-nNme2Cas9 (D16A). Ν = 6; error bars represent S.D. Results are from biological replicates performed on technical duplicates. Figures 20A to 20C show an example of a GFP reporter assay for nSpCas9-CBE4 (Addgene #100802) and CBE4-nNme2Cas9 (D16A)-UGI-UGI activities (CBE4 was cloned from Addgene #100802). Figure 20A shows an example of CBEGFP reporter sequence information. There is a mutation in the fluorophore core region of the GFP reporter line, which converts GYG to GHG. Therefore, there is no GFP signal. The GFP signal will appear if nSpCas9CBE4 or CBE4-nNme2Cas9(D16A)-UGI-UGI can convert CAC to TAC / TAT (Histidine to Tyrosine). Figure 20B shows an example of a GFP signal (green) as nSpCas9-CBE4 or CBE4nNme2Cas9(D16A)-UGI-UGI is active in the specific region of the GFP reporter. The upper panel is the negative control. The middle panel shows that mCherry signals light up in CBE4-nNme2Cas9(D16A)-UGI-UGI-treated reporter cells. The bottom panel shows that GFP signals light up in reporter cells treated with CBE4nNme2Cas9(D16A)-UGI-UGI). Figure 20C shows an example of FAC quantification of base editing events in GFP reporter cells transfected with nSpCas9-CBE4 or CBE4-nNme2Cas9(D16A)UGI-UGI. No.=6; error bars represent S.D. Results are from biological replicates performed on technical duplicates. Figure 21 shows an example of cytokine editing by CBE4-nNme2Cas9(D16A)-UGI-UGI. The upper panel shows information about the KANK3 targeting sequence (PAM sequences are indicated in red) of Nme2Cas9 and base editing in the negative control samples. The lower panel shows the quantification of the substitution rate of each base type in the editing window by CBE4-nNme2Cas9(D16A)-UGI-UGI of the KANK3 target sequences. The sequence tables show nucleotide frequencies at each position. The expected conversion frequencies from C to T are highlighted in red. Figure 22 shows an example of cytosine and adenine editing by optimized CBE4-nNme2Cas9(D16A)UGI-UGI and ABEmax-nNme2Cas9(D16A), respectively. The upper panel shows the information on the PLXNB2 targeting sequence (PAM sequences are indicated in red) of Nme2Cas9 and base editing in the negative control samples. The central panel shows the quantification of the substitution rate of each base type in the optimized ABEmaxnNme2Cas9 (D16A) editing windows of the PLXNB2 target sequences. The sequence tables show nucleotide frequencies at each position. The expected conversion frequencies from A to G are highlighted in red. The lower panel shows the quantification of the substitution rate of each base type in the editing windows by CBE4-nNme2Cas9 (D16A)-UGI-UGI of the PLXNB2 target sequences. The sequence tables show nucleotide frequencies at each position. The expected conversion frequencies from C to T are highlighted in red. Detailed description of the invention The present invention relates to the field of gene editing. In particular, gene editing is directed toward editing a single nucleotide base. For example, such editing of a single nucleotide base results in a conversion of an OG base pair to a Τ·Α base pair. The high accuracy and precision of the currently disclosed single nucleotide base gene editor is achieved by a NmeCas9 nuclease that is fused to a protein nucleotide deaminase. The compact nature of NmeCas9 coupled with a greater number of compatible protospacer-adjacent motifs, provides that the Cas9 fusion constructs contemplated herein can edit sites that are not selectable for conventional SpyCas9 base-editing platforms. A. Single base editing by NmeCas9 Cas9 is a programmable nuclease that uses a guide RNA to create a double-strand break at any desired genomic locus. This programming ability has been harnessed for biomedical and therapeutic approaches. However, Cas9-induced breaks often lead to imprecise repair by the cellular machinery, precluding its therapeutic application for single base corrections as well as uniform and precise gene deletions. Furthermore, it is extremely challenging to combine Cas9-induced DNA double-strand breaks and a repair template for homology-directed repair (HDR) to correct genetic mutations in postmitotic cells (eg, neuronal cells). Single nucleotide base editing is a genome editing approach where an inactive or deficient Cas9 nuclease (for example, inactive Cas9 (dCas9) or Cas9 nickase (nCas9)) is fused with another enzyme capable of editing nucleotide bases without causing breaks double-stranded DNA. To date, two broad classes of Cas9 base editors have been developed: i) cytidine deaminase fusion protein SpyCas9 (edits an OG base pair into a Τ·Α base pair); and ii) SpyCas9 adenosine deaminase. and Luí et al., “Fusions of cas9 domains and nucleic acid-editing domains” US 2015 / 0166980; (both incorporated herein by reference). However, as mentioned above, the SpyCas9 base editing platforms cannot be used to target all single base mutations due to their limited editing windows. The editing window is limited by the requirement of a PAM NGG. SpyCas9 is also intrinsically associated with high non-specific effects in genome editing. In one embodiment, the present invention contemplates a deaminase fusion protein with a compact and hyperexact Nme2Cas9 (Neisseria meningitidis). This Nme2Cas9 has 1,082 amino acids compared to SpyCas9 which has 1,368 amino acids. This Nme2Cas9 ortholog works efficiently in mammalian cells, recognizes a N4CC PAM, and is inherently hyperaccurate. Edraki et al., Mol Cell. (in preparation). Although it is not necessary to understand the mechanism of an invention, it is believed that the compactness and hyperaccuracy of an NmeCas9 base editor targets single base mutations. 7QQncn / L7n7 / q / Yi that could not be previously achieved by other Cas9 platforms currently known in the art. It is further believed that the NmeCas9 base editors contemplated herein target pathogenic mutations that are not feasible through current base editor platforms and with greater base editing accuracy. In one embodiment, the present invention contemplates a fusion protein comprising a Nme2Cas9 and a deaminase protein, exemplary examples include ABE7.10-nNme2Cas9 (D16A); nNme2Cas9-ABEmax optimized; nNme2Cas9-CBE4 (equal to BE4-nNme2Cas9(D16A)-UGI-UGI) as well as ABEmax-nNme2Cas9 (D16A). See, Figure 1A, Figure 1B, Figure 1C, Figure 1D, and Figure 1E. Figure 1 illustrates exemplary schematic embodiments of a NmeCas9 deaminase fusion protein single base editor and exemplary base editor constructed plasmids. Figure 1A shows an exemplary construct YE1-BE3-nNme2Cas9(D16A)-UGI. Figure 1B shows an exemplary construct ABE7.10 nNme2Cas9 (D16A). Figure 1C shows an exemplary construct ABE7.10 nNme2Cas9 (D16A). Figure 1C shows an exemplary ABE7.10-nNme2Cas9(D16A) construct comprising two SV40 NLS sequences. Figure 1D shows an exemplary construct nNme2Cas9CBE4 (also called BE4-nNme2Cas9(D16A)-UGI-UGI). Figure 1E shows an exemplary optimized nNme2Cas9-ABEmax construct. In one embodiment, the protein deaminase is Apobecl (YE1-BE3). It is not intended to limit Apobecl to one organism. In one embodiment, the Apobecl is derived from a species of rat. Kim et al., “Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.” Nature Biotechnology 35 (2017). In one embodiment, the Nme2Cas9 comprises a nNme2Cas9 D16A mutant. In one embodiment, the fusion protein further comprises a uracil glycosylase inhibitor (UGI) protein. In one embodiment, the fusion protein comprises a YE1-BE3nNme2Cas9(D16A)-UGL construct. In one embodiment, the YE1-BE3-nNme2Cas9(D16A)-UGI construct has the sequence of: M^jrETTOPyAyDPTLRRMFE / RHIYFTrV^^ NTRCSITWFLSYSPCGECSRAITEFLSRYPHVTLFIYL ARLYHHADPENRQGLRDLISSGVTIQIMTEQESGYCWRNF VNYSPSNEAHWPRYPHLWVRLYVLELYCIIL GLPPCLNILRRKQPQLTFFTIAL QSCHYQRLPPHILWATGLKSGSET PGTSE SATPE SMAAFKPNPINYIL GLAIGIASVGWAMVEIDEEEENPIRLIDLGVR VFERAEVPKTGDSLAMARRLARS VRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRK NEGETADKELGALLKGVANNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQA ELILLFEKQKEFG NPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTE RATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSSELQ DEIGTAFSLFK TDEDITGRLKDRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKN TEEKIYLPPIPADEIRNPWLRALSQARKVINGWRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAA KFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYS GKEINLVRLNEKGYVEIDAALPFSRTWDDSFNNKVLVLGSENQN KGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLÜKFDEDGFKECNLNDTRYVNRFLCQFVADHILLTGKG KRR VFASNGQITNLLRGFWGLRKVRAENDRHHALDA VWA CS TVAMQQKITRFVR Y KEMNAFDGKTIDKE TGKVLHQK THFPQPWEFFAQE\MIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGAHKDTLR zQQncn / Lznz / q / Yi SAKRF VKHNEKIS VKRVWLTEIKLADLENMVNYKNGREIELYEALKARLEAYGGNAKQAFDPKDNPFYKKGGQLVKAV RVEKTQESGVLLNKKNAYTIADNGDMVRVDVFCKVDKKGKNQYFIVPIYAWQVAENILPDIDCKGYRIDDSYTFCFSL HKYDLIAFQKDEKSK VEFAYYINCDSSNGRFYLAWHDKGSKEQQFRISTQNLVLIQKYQVNELGKEIRPCRLKKRPPV RSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQD SNGENKIKMLSGGSP KKKRKV* YE1-BE3 (underlined); linker (bold), nNme2Cas9 (italics), UGI (bold / underline), SV40 NLS (plain). In one embodiment, the construct YE1 -BE3-nNme2Cas9(D16A)-UGI has the sequence of: MSj5ETGRyAVDPTLRRMEE / RKE / FE^^ NTRCSITWFLSYSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPENRQGLRDLISSGVTIQIMTEQESGYCWRNF WYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSET PGTSE SATPE SMAAFKPNPINYILGLAIGIA SVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGDSLAMARRLARS VRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRK NEGETADKELGALLKGVANNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFE KQKEFG NPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTE RATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSSELQ DEIGTAFSLFKTDEDIT GRLKDRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKN TEEKIYLPPIPADEIRNPWLRALSQARKVINGWRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAA KFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEIN LVRLNEKGYVEIDAALPFSRTWDDSFNNKVLVLGSENQN KGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKECNLNDTRYVNRFLCQFVADHILLTGKG KRR VFASNGQITNLLRGFWGLRKVRAENDRHHALDA VWA CS TVAMQQKITRFVR YKEMNAFD GKTIDKE TGKVLHQK THFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGAHKDTLR SAKRFVKHNEKISVKRVWLTEIKLADLENMVNYKNGREIELYEALKARLEAYGGNAKQAFDPKDNPFYKKGGQLVKAV RVEK TQESGVLLNKKNAYTIADNGDMVRVDVFCKVDKKGKNQYFIVPIYAWQVAENILPDIDCKGYRIDDSYTFCFSL HKYDLIAFQKDEKSKVEFAYYINCDSSNGRFYLAWHDKGSKEQQFRISTQNLVLIQKYQVNELGKEIRPCRLKKRPPV RSGGSTNLSDIIEKETGKQLVI QE SILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQD SNGENKIKMLSGGSP KKKRKV* YE1-BE3 (underlined); linker (bold), nNme2Cas9 (italics), UGI (bold / underline), SV40 NLS (plain). In one embodiment, the present invention contemplates a fusion protein comprising a NmeCas9 / ABE7.10 deaminase protein. In one embodiment, the deaminase protein is TadA. In one embodiment, the protein deaminase is TadA 7.10. In one embodiment, the ABE7.10-nNme2Cas9 (D16A) construct has the following sequence: MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDA TLYVTLEPCVMCAGAMIHSRIGRWFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEI KAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSG GSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVL NNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRWFGVRNAKTGAA GSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSSETFGTSESATPESS G GSSGGSMAAFKPNPINYILGLAIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGDSLAMARRLARSVRRL zQoncn / Lznz / q / Yi TRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGE TADKELGALLKGVANNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHV SGGLKEGIETLLMTQRPALSGDAVQKMLG HCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATL MDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSSELQDEIG TAFSLFKTDEDITGRLKDRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRY DEACAEIYGDHYGKKNTEEK IYLPPIPADEIRNPWLRALSQARKVINGWRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFRE YFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLVRLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQ TP YEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKECNLNDTRYVNRFLCQFVADHILLTGKGKRRV FASNGQITNLLRGFWGLRKVRAENDRHHALDAVWACSTVAMQQKITRFVRYKEMNAFDGKTLDKETGKVLHQKTHFP QPWEFFAQEVMIRVFGKPDGKPE FEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGAHKDTLRSAKR FVKHNEKISVKRVWLTEIKLADLENMVNYKNGREIELYEALKARLEAYGGNAKQAFDPKDNPFYKKGGQLVKAVRVEK TQESGVLLNKKNAYTIADNGDMVRVDVFCKVDKKGKNQYFIVPI YAWQVAENILPDLDCKGYRIDDSYTFCFSLHKYD LIAFQKDEKSKVEFAYYINCDSSNGRFYLAWHDKGSKEQQFRISTQNLVLIQKYQVNELGKEIRPCRLKKRPPVREDE RPAATKKAGQAKKKK* TadA (underlined), TadA 7.10 (underlined / bold), linker (bold), nNme2Cas9 (italics), Nucleoplasmin NLS (plain). In one embodiment, an ABE7.10-nNme2Cas9 (D16A) construct has the following amino acid sequence: MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDA TLYVTLEPCVMCAGAMIHSRIGRWFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEI KAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSG GSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVL NNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRWFGVRNAKTGAA GSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS G GSSGGSMAAFKPNPINYILGLAIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGDSLAMARRLARS VRRL TRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGE TADKELGALLKGVANNAHALQTGDFRTPAELALNKFEKESGHI RNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHV SGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATL MDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLS SELQDEIG TAFSLFKTDEDITGRLKDRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEK IYLPPIPADEIRNPWLRALSQARKVINGWRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFRE YFPNFVGEPKSKDILKLRL YEQQHGKCLYSGKEINLVRLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQ TPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKECNLNDTRYVNRFLCQFVADHILLTGKGKRRV FASNGQITNLLRGFWGLRKVRAENDRHHALDAVWACS TVAMQQKITRFVRYKEMNAFDGKTIDKETGKVLHQKTHFP QPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGAHKDTLRSAKR FVKHNEKISVKRVWLTEIKLADLENMVNYKNGREIELYEALKARLEAYGGNAKQAFDPKDNPF YKKGGQLVKAVRVEK TQESGVLLNKKNAYTIADNGDMVRVDVFCKVDKKGKNQYFIVPIYAWQVAENILPDLDCKGYRIDDSYTFCFSLHKYD LIAFQKDEKSKVEFAYYINCDSSNGRFYLAWHDKGSKEQQFRISTQNLVLIQKYQVNELGKEIRPCRLKKRPPVREDK RPAATKKAGQAKKKK* TadA (underline), TadA 7.10 (underline / bold), linker (bold and italics), nNme2Cas9 zQancn / Lznz / q / Yi (italics), Nucleoplasmin NLS (plain). In one embodiment, an ABEmax-nNme2Cas9 (D16A) construct has the following amino acid sequence: MKRTAPGSEFESPKKKEtKVSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRWFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILA D E CAAL LSD F FRMRRQ EIKAQ KKAQ S S T D SG GSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFS HEYWMRHALTL AKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIH SRIGRWFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQ S STD SGGSSGG SSGSETPGTSESATPESSGGSSGGSMAAFKPNPINYILGLAIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEVPK TGDSLAMARRLARSVRRLLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVL LHLIKHRGYLSQRKNEGETADKELGALLK GVANNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQ AELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLR ILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNA EASTLMEMKAYHAISRALEKEGL KDKKSPLNLSSELQDEIGTAFSLFKTDEDITGRLKDRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDE ACAEIYGDHYGKKRTEEKIYLPPIPADEIRNPWLRALSQARKVINGWRRYGSPARIHIETAREVGKSFKDRKEIEK RQEENRKDRE KAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLVRLNEKGYVEIDHALPFSRTWDDS FNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKECNLNDTRYVNRFL CQFVADHILLTGKGKRRVFASNGQ ITNLLRGFWGLRKVRAENDRHHALDAVWACSTVAMQQKITRFVRYKEMNAFDG KTIDKETGKVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRA PNRKMSGAHKDTLRSAKRFVKHNEKISVKRVWLTEIKLADLENMVNYKNG REIELYEALKARLEAYGGNAKQAFDPKD NPFYKKGGQLVKAVRVEKTQESGVLLNKKNAYTIADNGDMVRVDVFCKVDKKGKNQYFIVPIYAWQVAENILPDIDCK GYRIDDSYTFCFSLHKYDLIAFQKDEKSKVEFAYYINCDSSNGRFYLAWHDKGSKEQQFRISTQNLV LIQKYQVNELG KPPRPCRÍKKRPPVREDKRPAATKKAGQAKKKK.FEPKKKRKV* TadA (underline), TadA* 7.10 (underline / bold), linker (bold and italics), nNme2Cas9 (italics), nucleoplasmin NLS (plain), and SV40 NLS (BOLD). In one embodiment, a CBE4-nNme2Cas9(D16A)-UGI-UGI construct has the following amino acid sequence: PAAKRVKl DGgSGgGSGGGSGPΆAKRVKLDGGSGGGSGGGSGPZEPKKKRKVP¥SSETGPVAVDPTLRRRIEPHEFEV ΓΕΌΡΡΕ®ΡΚΕ7ΓΟΚΡΥΙ«Ν1λΚ|6ΕΡ^^ LSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLE LYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGgSSGgSSgSEIPGTSESArPESSGgSSg GSIDKLAAFKPNPINYILGLAIGIA SVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGDSLAMARRLARSVRRLTR RRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETA DKELGALLKGVANNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFE KQKEFGNPHVSG GLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMD EPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSSELQDEIGTA FSLFKTDEDITG RLKDRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIY LPPIPADEIRNPWLRALSQARKVINGWRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYF PNFVGEPKSKDILKLRLYEQQHGKCLYSGKEIN LVRLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTP zQoncn / Lznz / x / Yi YEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKECNLNDTRYVNRFLCQFVADHILLTGKGKRRVFA SNGQITNLLRGFWGLRKVRAENDRHHALDAVWACSTVAMQÜKITRFVRYKEMNAFDGKTIDKETGKVLHQKTHFPQP WEFFAQEVMIRVFGKPD GKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGAHKDTLRSAKRFV KHNEKISVKRVWLTEIKLADLENMVNYKNGREIELYEALKARLEAYGGNAKQAFDPKDNPFYKKGGQLVKAVRVEKTQ ESGVLLNKKNAYTIADNGDMVRVDVFCKVDKKGKNQYFI VPIYAWQVAENILPDIDCKGYRIDDSYTFCFSLHKYDLI AFQKDEKSKVEFAYYINCDSSNGRFYLAWHDKGSKEQQFRISTQNLVLIQKYQVNELGKEIRPCRLKKRPPVRVYPYD VPDYAGYPYDVPDYAGSYPYDVPDYAGSAAPAAKKKKLDFESGEFLQPGIDLSQLGGDSGGSGGSGGSTNLSDITEKE TGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML SGGSG GSGGSTNLSDIIEKETGKQLVIQESILMLPE EVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQD SNGENKIKMLSGGSPKKKRKVSRGSAaPAAKRVKLDGGSGGGSGGGSGSGPAAKRVKLD rApobed (underline), UGI (underline / bold), linker (bold italic), nNme2Cas9 (D16A) (italic), N LS of Cmyc (raw) and NLS of SV40 (BOLD). In one embodiment, an optimized nNme2Cas9-ABEmax construct refers to an optimized version with an improved promoter, NLS sequences, and linker sequences. In some embodiments, an optimized nNme2Cas9-ABEmax construct comprises, from 5' to 3', a C-myc NLS, 12 aa linker, 15 aa linker, an SV40 NLS, TadA, TadA*7.10, 48 linker aa, nNme2Cas9, a 73 aa linker (3xHA tag), 15 aa linker and a C-myc NLS. In some embodiments, an optimized nNme2Cas9-ABEmax construct further comprises at least two alternating C-myc NLSs and a 12 aa linker at the 3' end. In some embodiments, an optimized nNme2Cas9-ABEmax construct further comprises at least two alternating 15 aa linkers and C-myc NLS at the 5' end. See, Figure 1 E for an example. In one embodiment, an optimized nNme2Cas9-ABEmax construct has the following amino acid sequence: PAAKRVKLDGGSGGGSGGGSGPAAKRVKLDGGSGGGSGGGSGPLEPKKKRKVSEVEFSHEYWMRHALTLAKR AWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCWCAGAMIHSRI GRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD.SGG.S.SGGSSG SETPGTSFSATRESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAH AEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRWFGVRNAKTGAAGSLMDVLEYPGMNHRVEITEG ILADECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGG SSGSETPGTSESATPESSGGSSGGSMAAFKPNPINYDIR KLAAFKPNPINYILGLAIGIASVGWAMVEIDEEEENPIRLIDEGVR VFERAEVPKTGDSLAMARRLARSVRRLTRRRAH RLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKEL GALLKGVANNAHALQTGDFRTPAELALNKFEKESGHIRNQ RGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKE GIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYR KSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSS ELQDEIGTAFSLF KTDEDITGRLKDRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPI PADEIRNPWLRALSQARKVINGWRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFV GEPKSKDILKLRLYE QQHGKCLYSGKEINLVRLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYF NGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKECNLNDTRYVNRFLCQFVADHILLTGKGKRRVFASNGQ ITNLLRGFWGLRKVRAENDRHHALDAVWACSTVAM QQKITRFVRYKEMNAFDGKTIDKETGKVLHQKTHFPQPWEFF zQancn / Lznz / q / Yi AQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGAHKDTLRSAKRFVKHNE KISVKRVWLTEIKLADLENMVNYKNGREIELYEALKARLEAYGGNAKQAFDPKDNPFYKKGGQLVKAVRVEKTQESGV LLNKKNAYTIADNGDMVRVDVF CKVDKKGKNQYFIVPIYAWQVAENILPDIDCKGYRIDDSYTFCFSLHKYDLIAFQK DEKSKVEFAYYINCDSSNGRFYLAWHDKGSKEQQFRISTQNLVLIQKYQVNELGKEIRPCRLKKRPPVRVYPYDVPDY AGYPYDVPDYAGSYPYDVPDYAGSAAPAAAKKKLD FESGEFLQPGGSTSSRGSAAPAAKRVKEOGGSGGGSGGGSGSG PAAKRVKLD hTadA7.10 (underline), hTadA*7.10 (underline / bold), Linker (bold and italic), nNme2Cas9 (italic), Cmyc NLS (raw), SV40 NLS (bold). In some embodiments, an nSpCas9-ABEmax plasmid (Addgene ID:112095) was used for experimental controls and for molecular cloning. In some embodiments, an nSpCas9-CBE4 plasmid (Addgene ID: 100802) was used for experimental controls and for molecular cloning. Electroporation of HEK293T cells with DNA plasmids comprising a YE1-BE3-Nme2Cas9 nucleotide deaminase fusion protein achieved robust single base editing from an OG base pair to a Τ Α base pair at an endogenous target site (TS25 ). See, Figures 2A to 2C. Figures 2A to 2C present exemplary data from electroporation of HEK293T cells with DNA plasmids comprising a YE1-BE3-nNme2Cas9(D16A)-UGI fusion protein that efficiently converts C to T at endogenous target site 25 (TS25) in HEK293T cells by nucleofection. Figure 2A shows exemplary sequences for an endogenous TS25 target site (inside the black box). GN23 sgRNA base pairs with the target DNA strand, leaving the displaced DNA strand for editing by cytidine deaminase (eg, new green nucleotides). Figure 2B shows exemplary sequencing data showing a doublet nucleotide peak (7th apposition from the 5' end; arrow) demonstrating successful single base editing of a cytidine to a thymidine (eg, a conversion of a pair of OG bases in a Τ·Α base pair). Figure 2C shows an exemplary quantization of the data shown in Figure 2B which plots the percentage conversion of the single base edit C T. The percentage of C converted to T is approximately 40% in the sample treated with the editor. base and with sRNA (p-value = 6.88 x 10'6). Control without sgRNA shows source noise due to Sanger sequencing. EditR (Kluesner et al., 2018) was used to perform the analysis. Four other YE1 -BE3-nNme2Cas9 / D16 mutant fusion proteins were co-expressed with enhanced green fluorescent protein (EGFP) in a K562-derived stable cell line expressing enhanced green fluorescent protein (EGFP). Each YE1-BE3nNme2Cas9 / D16A mutant fusion protein has a specific UGI target site. See, Figures 3A to 3D. Deep sequencing analysis indicates that YE1 -BE3-nNme2Cas9 converts C residues to T residues at each of the four EGFP target sites. The editing percentage ranged from 0.24% to 2%. The potential base editing window is nucleotides 2-8 in the displaced DNA strand, counting the nucleotide at the 5' end (PAM-distal) as nucleotide #1. See, Figures 3A to 3D. zQQncn / Lznz / q / Yi Figures 3A through 3F present exemplary specific UGI target sites that were respectively integrated into YE1-BE3-nNme2Cas9 / D16A mutant fusion proteins and co-expressed with enhanced green fluorescent protein (EGFP) in a cell line derived from K562 stable. Converted bases are highlighted in orange. Source signals were filtered using negative control samples (YE1-BE3-nNme2Cas9 nucleofected K562 cells without sgRNA constructs). The N^CC PAMs are locked up. The right column shows the percentage of total reads that exhibit mutations at sites targeted to the base editor. Figure 3A shows an example of EGFP-Site 1. Figure 3B shows an example of EGFP-Site 2. Figure 3C shows an example of EGFP-Site 3. Figure 3D shows an example of EGFP-Site 4. Electroporation of HEK293T cells with DNA plasmids comprising a YE1-BE3-nNme2Cas9 cfos promoter achieved robust single base editing from a C*G base pair to a Τ Α base pair at endogenous target sites in the c promoter. -fos (Figure 3E). Figure 3E shows an exemplary deep sequencing analysis indicating where YE1-BE3-nNme2Cas9 converts C residues to T residues in the endogenous c-fos promoter region. The right column shows the percentage of total reads that exhibit mutations at sites targeted to the base editor. Converted bases are highlighted in orange or yellow. Source signals were filtered using negative control samples. The highest editing percentage is 32.50%. Figure 3F shows an exemplary deep sequencing analysis indicating where ABE7.10-nNme2Cas9 or ABEmax (Koblan et al., 2018)nNme2Cas9 converts A residues to G residues in the endogenous c-fos promoter region. The right column shows the percentage of total reads that exhibit mutations at sites targeted to the base editor. Converted bases are highlighted in orange. Source signals were filtered using negative control samples. The editing percentage is 0.53% by ABE7.10-nNme2Cas9 or 2.33% by ABEmax-nNme2Cas9 (D16A). In one embodiment, the present invention contemplates expression of an ABE7.10-nNme2Cas9 (D16A) fusion protein for base editing. Although it is not necessary to understand the mechanism of an invention, it is believed that Nme2Cas9 base editing may be an effective treatment for tyrosinemia by reversing a G-a-A point mutation in the Fah gene with an ABE7.10nNme2Cas9 (D16A) fusion protein. G-a-A mutation (red) in the last nucleotide of exon 8 in the Fah gene, causing exon skipping. FAH deficiency leads to toxin accumulation and severe liver damage. The position of a PAM for SpyCas9 (black rectangular box) downstream of the mutation is not optimal for designing the sgRNA as the A mutation is outside the efficient base editing window of ABE7.10, which is 4-7 ° nt at the 5' end (PAM-distal) (underlined) (Gaudelli et al., 2017). However, there are two PAMs for Nme2Cas9 (red rectangular box) in the downstream sequences that can potentially correct the mutation and revert the DNA sequence to wild-type via ABE7.10-nNme2Cas9 (D16A). See, Figure 4. Figure 4 presents an exemplary alignment of the wild-type Fah gene with the tyrosinemia mutant Fah gene showing a single base A-G gene editing target site (position 9). The respective single PAM site for SpyCas9 and the dual PAM sites for NmeCas9 are indicated to demonstrate zQQncn / Lznz / q / Yi the suboptimal targeting window relative to the PAM site for SpyCasO. This figure serves as a potential example of a site where Nme2Cas9 could overcome the limitations of existing base editors. In addition, it is believed that the NmeCas9 base editor described herein can perform precise base editing that cannot be achieved with conventional SpyCas9-derived base editors due to a suboptimal base editing window relative to the available PAMs. close. In addition, it is contemplated to extend base editing to a tyrosinemia mouse model to reverse the G-a-A point mutation by viral delivery methods using ABEmax-nNme20as9 (D16A), where the desired editing cannot be achieved with SpyCas9-derived base editors. due to a suboptimal base editing window relative to nearby available PAMs (eg, Figure 4). B. NmeCas9 Constructs: Compact and Hyperaccurate Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated proteins (Cas) constitute adaptive immune pathways of bacteria and archaea against phages and other mobile genetic elements (MGEs). ) (Barrangou et al., 2007; Brouns et al., 2008; Marraffini and Sontheimer, 2008). In type II CRISPR systems, CRISPR RNA (crRNA) is linked to a trans-activating crRNA (tracrRNA) and loaded into a Cas9 effector protein that cleaves MGE nucleic acids complementary to the crRNA (Garneau et al. , 2010; Deltcheva et al., 2011; Sapranauskas et al., 2011; Gasiunas et al., 2012; Jinek et al., 2012). The hybrid crRNA:tracrRNA can be fused into a single guide RNA (sgRNA) (Jinek et al., 2012). The RNA programming ability of Cas9 endonucleases has made them a powerful genome-editing platform in biotechnology and medicine (Cho et al., 2013; Gong et al., 2013; Hwang et al., 2013; Jiang et al. ., 2013; Jinek et al., 2013; Mali et al., 2013b). In addition to sgRNA, Cas9 target recognition is generally associated with a signature 1-5 nucleotides downstream of the complementary DNA sequence, called the protospacer-adjacent motif (PAM) (Deveau et al., 2008; Mojica et al. , 2009). Cas9 orthologs exhibit considerable diversity in PAM length and sequence. Among the Cas9 orthologs that have been characterized, Streptococcus pyogenes Cas9 (SpyCas9) is the most widely used, partly because it recognizes a short PAM NGG (Jinek et al., 2012) (N represents any nucleotide) that provides a high density of target sites. However, the relatively large size of Spy (ie, 1,368 amino acids) makes this Cas9 difficult to package (along with sgRNAs and promoters) into a single recombinant adeno-associated virus (rAAV). This has been shown to be a drawback for therapeutic applications given the promise that AAV vectors show for gene delivery in vivo (Keeler et al., 2017). Furthermore, SpyCas9 and its RNA guides have required extensive characterization and engineering to minimize the tendency to edit non-specific, quasi-cognate sites. (Bolukbasi et al., 2015b; Tsai and Joung, 2016; Tycko et al., 2016; Chen et al., 2017; Casini et al., 2018; Yin et al., 2018). To date, subsequent engineering efforts have not overcome these size limitations. Several Cas9 orthologs less than 1,100 amino acids in length obtained from various species have been validated for mammalian genome editing, including strains of N. meningitidis (NmeCas9, 1,082 aa) (Esvelt et al., 2013; Hou et al., 2013), Staphylococcus aureus (SauCas9, 1,053 aa) (Ran et al., 2015), Campylobacter jejuni (CjeCas9, 984 aa) (Kim et al., 2017), and Geobacillus stearothermophilus (GeoCas9, 1,089 aa) (Harrington et al. ., 2017b). NmeCas9, CjeCasQ, and GeoCas9 are representatives of the Il-C type Cas9s (Mir et al., 2018), most of which are <1,100 aa. With the exception of GeoCas9, each of these shorter-sequence orthologs has been successfully implemented for in vivo editing via all-in-one AAV delivery (in which a single vector expresses both leader and effector) ( Ran et al., 2015; Kim et al., 2017; Ibraheim et al., 2018, submitted). Furthermore, NmeCas9 and CjeCas9 have been shown to be naturally resistant to non-specific editing (Lee et al., 2016; Kim et al., 2017; Amrani et al., 2018, submitted). However, the PAMs that are recognized by compact Cas9 are usually longer than those of SpyCas9, which substantially reduces the number of target sites at or near a given locus; for example, i) N4GAYW / N4GYTT / N4GTCT for NmeCas9 (Esvelt et al., 2013; Hou et al., 2013; Lee et al., 2016; Amrani et al., 2018); ii) N2GRRT for SauCas9 (Ran et al., 2015); iii) N4RYAC for CjeCas9 (Kim et al., 2017); and iv) N4CRAA / N4GMAA for GeoCas9s (Harrington et al., 2017b) (Y = C, T; R = A, G; M = A, C; W = A, T). A smaller subset of target sites is advantageous for high precision and accuracy gene editing tasks including, but not limited to: i) small target (eg miRNA) editing; ii) correction of mutations by base editing that alters a very narrow window of bases in relation to the PAM (Komor et al., 2016; Gaudelli et al., 2017); or iii) precise editing by homology-directed repair (HDR), which is more efficient when the rewritten bases are close to the cleavage site (Gallagher and Haber, 2018). Due to PAM restrictions, many editing sites cannot be targeted using all-in-one AAV vectors for in vivo delivery even with these shorter Cas9 proteins. For example, a SauCas9 mutant (SauCas9KKH) has been developed that has reduced PAM (N3RRT) restrictions, although this increase in targeting interval often comes at the cost of lower specific editing efficiency, and non-specific editing is still observed. . (Kleinstiver et al., 2015). Safe and effective CRISPR-based therapeutic gene editing will be greatly enhanced by Cas9 orthologs and variants that are highly active in human cells, resistant to nonspecific editing, compact enough for all-in-one AAV delivery, and capable of access a high density of genomic sites. In one embodiment, the present invention contemplates a compact and hyperexact Cas9 (Nme2Cas9) from a strain other than N. meningitidis. In one embodiment, the present invention contemplates a method for delivery of Nme2Cas9 unique AAV and its sgRNA to perform efficient genome editing in vivo and / or ex vivo. Although it is not necessary to understand the mechanism of an invention, this ortholog is believed to function efficiently in mammalian cells and recognizes an N4CC PAM that provides a target site density identical to that of wild-type SpyCas9 (eg, every 8 bp on average, when both DNA strands are considered). 1. Domains of interaction with PAM and anti-CRISPR proteins Recognition of PAM by Cas9 orthologs occurs predominantly through 7Qoncn / i 7n7 / 3 / YL protein-DNA interactions between the PAM-interacting domain (PID) and nucleotides adjacent to the protospacer (Jiang and Doudna, 2017). PAM mutations often allow phages to escape type II CRISPR immunity (Paez-Espino et al., 2015), placing these systems under selective pressure not only to acquire new CRISPR spacers, but also to develop new ones. PAM specificities through PID mutations. In addition, some phages and MGE express anti-CRISPR proteins (Acr) that inhibit Cas9 (Pawluk et al., 2016; Hynes et al., 2017; Rauch et al., 2017). PID binding is an effective inhibitory mechanism adopted by some Acr (Dong et al., 2017; Shin et al., 2017; Yang and Patel, 2017), suggesting that PID variation may also be pressure-driven. selective to escape Acr inhibition. Cas9 PIDs can evolve such that closely related orthologs recognize distinct PAMs, as recently illustrated in two Geobacillus species. Cas9 encoded by G. stearothermophilus recognizes a N4CRAA PAM, but when its PID was changed to that of Cas9 from the LC300 strain, its PAM requirement changed to N4GMAA (Harrington et al., 2017b). In one embodiment, the present invention contemplates a plurality of N. meninigitidis Cas9 orthologs with divergent PIDs that recognize different PAMs. In one embodiment, the present invention contemplates a Cas9 protein with high sequence identity (>80% over its entire length) to that of NmeCas9 strain 8013 (Nme1Cas9) (Zhang et al., 2013). Nme1Cas9 also has a small size and naturally high accuracy as discussed above. (Lee et al., 2016; Amrani et al., 2018). Alignments revealed three clades of meningococcal Cas9 orthologs, each with >98% identity at the N-terminal -820 amino acid (aa) residues, including all regions of the protein other than the PID. See, Figure 5A and Figure 6A. All of these Cas9 orthologs are 1,078-1,082 aa in length. The first clade (group 1) includes orthologs in which >98% aa sequence identity to Nme1 Cas9 spans across the PID. In contrast, the other two groups have PIDs that diverged significantly from Nme1Cas9, with group 2 and group 3 orthologs averaging -52% and -86% PID sequence identity to Nme1Cas9, respectively. One meningococcal strain from each group was selected: i) From 1444 from group 2; and ii) 98002 from group 3 for detailed analysis, which are referred to herein as Nme2Cas9 (1.082 aa) and Nme3Cas9 (1.081 aa), respectively. The CRISPR-cas loci from these two strains have identical repeat sequences and spacer lengths as strain 8013. See, Figure 6B. This strongly suggested that their mature crRNAs also have 24 nt guide sequences and 24 nt repeat sequences (Zhang et al., 2013). Similarly, the tracrRNA sequences of Del 1444 and 98002 were 100% identical to those of the tracrRNA 8013. See, Figure 6B. These observations imply that the same sgRNA sequence scaffold can guide DNA cleavage by all three Cas9s. To determine if these Cas9 orthologs have distinct PAMs, the PID of Nme1Cas9 was replaced with that of Nme2Cas9 or Nme3Cas9. To identify the corresponding PAM requirements, these protein chimeras were expressed in Escherichia coli, purified, and used for in vitro PAM identification (Karvelis et al., 2015; Ran et al., 2015; Kim et al., 2017). Briefly, a set of DNA fragments containing a protospacer followed by a 10 nt randomized sequence were excised in vitro using recombinant Cas9 and an in vitro transcribed cognate sgRNA. See, Figure 5B. Only DNAs containing a Cas9 PAM sequence were expected to be cleaved. The cleavage products were then sequenced to identify the PAMs. See, Figures 5C to 5D. The expected N4GATT consensus PAM was validated in the recovered full-length Nme1Cas9. See, Figure 5C. The PID-swapped chimeric derivatives showed a strong preference for a C residue at the fifth position rather than the G recognized by Nme1Cas9. See, Figure 5D. In one embodiment, ABE7.10-nNme2Cas9(D16A) is used for single base editing from a Α·Τ base pair to a G*C base pair. In one embodiment, BEmax-nNme2Cas9 (D16A) is used for single base editing from a Α·Τ base pair to a G*C base pair. (See, Figure 3F). Figures 5A to 5E illustrate examples of three closely related Neisseria meningitidis Cas9 orthologs that have distinct MAPs. Figure 5A shows an exemplary schematic showing mutated residues (orange spheres) between Nme2Cas9 (left) and Nme3Cas9 (right) mapped to the predicted structure of Nme1Cas9, revealing the cluster of mutations in the PID (black). Figure 5B shows an exemplary experimental workflow of the in vitro PAM discovery assay with a 10 bp randomized PAM region. After in vitro digestion, adapters were ligated to excise products for library construction and sequencing. Figure 5C shows exemplary sequence logos resulting from in vitro PAM discovery revealing the enrichment of a N4GATT PAM for Nme1Cas9, consistent with its previously established specificity. Figure 5D shows exemplary sequence logos indicating that Nme1Cas9 with its PID swapped with that of Nme2Cas9 (left) or Nme3Cas9 (right) requires a C at position 5 of PAM. The remaining nucleotides were not determined with high confidence due to the modest cleavage efficiency of the PID-swapped protein chimeras (see Figure 6C). Figure 5E shows an exemplary sequence logo showing that full-length Nme2Cas9 recognizes an N4CC PAM, based on efficient substrate cleavage of a target group with a fixed C at position 5 of the PAM, and with nt 1-4 and 6-8 of the PAM randomized. The remaining PAM nucleotides could not be assigned with certainty due to the low cleavage efficiencies of the chimeric proteins under the conditions used. See, Figure 6C. To further resolve PAMs, in vitro assays were performed on a library with a 7 nt randomized sequence possessing an invariant C at the 58 position of the PAM (eg, 5'-NNNNCNNN-3' on the non-complementary strand of sgRNA). ). This strategy produced much higher cleavage efficiency and the results indicated that the Nme2Cas9 and Nme3Cas9 PIDs recognize the NNNNCC(A) and NNNNCAAA PAMs, respectively. See Figures 6C and 6D. The Nme3Cas9 consensus is similar to that of GeoCas9 (Harrington et al., 2017b). These tests were repeated using a full-length Nme2Cas9 (rather than a PID-swapped chimera) with the NNNNCNNN DNA pool, and again a consensus NNNNCC(A) was recovered. See, Figure 5E. This test was observed to have more efficient cleavage. See, Figure 6C. These data suggest that one or more of the 15 amino acid changes in Nme2Cas9 (relative to Nme1Cas9) outside the PID support efficient DNA cleavage activity. See, Figure 6C. Because the unique 2-3 nt PAM of Nme2Cas9 provides a higher density of potential target sites than previously described compact Cas9 orthologs, it was selected for further analysis. Figures 6A through 6D present a characterization of rapidly evolving Neisseria meningitidis Cas9 orthologs with PIDs, relative to Figure 5. Figure 6A shows an exemplary rootless phylogenetic tree of NmeCas9 orthologs that are >80% identical to Nme1Cas9. Three distinct branches emerge with most of the mutations clustered in the PID. Pools 1 (blue), 2 (orange), and 3 (green) have PIDs with >98%, approximately 52%, and approximately 86% identity with Nme1Cas9, respectively. Three representative Cas9 orthologs (one from each group) are indicated (Nme1Cas9, Nme2Cas9 and Nme3Cas9). Figure 6B shows an exemplary schematic showing the CRISPR-cas loci of the strains encoding the three Cas9 orthologs (Nme1Cas9, Nme2Cas9 and Nme3Cas9) of (A). Percentage identity of each CRISPR-Cas component to N. meningitidis 8013 (encoding Nme1Cas9) is shown. Blue and red arrows indicate the pre-crRNA and tracrRNA transcription start sites, respectively. Figure 6C shows an example of normalized read counts (% of total reads) of excised DNAs from in vitro assays for intact Nme1Cas9 (grey), for chimeras with PIDs of Nme1Cas9 swapped with those of Nme2Cas9 and Nme3Cas9 (mixed colors). , and for full-length Nme2Cas9 (orange), are plotted. The reduction of normalized read counts indicates lower cleavage efficiencies in the chimeras. Figure 6D shows an example of in vitro PAM discovery assay sequence logos in a pool of PAM NNNNCNNN by Nme1Cas9 with their PID swapped with those of Nme2Cas9 (left) or Nme3Cas9 (right). 2. Gene editing targeting the PAM N4CC To test the efficacy of Nme2Cas9 in human genome editing, a full-length human codon-optimized Nme2Cas9 construct (eg, no PID swapped) was cloned into a mammalian expression plasmid with nuclear localization signals (NLS). adjuncts and linkers previously validated for Nme1Cas9 (Amrani et al., 2018). For initial tests, a modified fluorescence-based Traffic Light Reporter (TLR2.0) was used (Certo et al., 2011). Briefly, an interrupted GFP is followed by an out-of-frame T2A peptide and an mCherry cassette. When DNA double-strand breaks (DSBs) are introduced into the broken GFP cassette, a subset of non-homologous end-joining (NHEJ) repair events leave displaced indels +1 from frame, placing mCherry in frame and producing red fluorescence that can be easily quantified by flow cytometry. See, Figure 7A. Homology-directed repair (HDR) results can also be simultaneously qualified by including a DNA donor that restores the functional GFP sequence, producing a green fluorescence (Certo et al., 2011). Because some indels do not introduce a +1 frameshift, fluorescence readout generally provides an underestimate of the true editing efficiency. However, the speed, simplicity, and low cost of the assay make it useful as an initial semiquantitative measure of genome editing in HEK293T cells carrying a single TLR2.0 locus. 7QQnC0 / L7A7 / 3 / YL incorporated by lentivector. For initial tests, the Nme2Cas9 plasmid was transiently co-transfected with one of fifteen sgRNA plasmids carrying spacers targeting TLR2.0 sites with PAM N4CC. No HDR donor was included, so only the NHEJ-based (mCherry) edit was scored. Most sgRNAs were in the G23 format (i.e., a 5' terminal G to facilitate transcription, followed by a 23 nt leader sequence), as is commonly used for Nme1Cas9 (Lee et al., 2016; Pawluk et al., 2016; Amrani et al., 2018; Ibraheim et al., 2018). Neither sgRNA nor a sgRNA targeting a N4GATT PAM were used as negative controls, and SpyCas9+sgRNA and Nme1Cas9+sgRNA co-transfections (targeting NGG and N4GATT protospacers, respectively) were included as positive controls. Editing by SpyCas9 and Nme1Cas9 was easily detectable (~28% and 10% of mCherry, respectively). See, Figure 7B. For Nme2Cas9, all 15 targets with the N4CC PAMs were functional, albeit to varying degrees ranging from 4% to 20% mCherry. These fifteen sites include examples with each of four possible nucleotides at the seventh position of PAM (for example, after dinucleotide CC), indicating that a slight preference for an A residue was observed in vitro (Figure 5E) not reflective. a PAM requirement for editing applications in human cells. The PAM N4GATT control produced a mCherry signal similar to the control without sgRNA. See, Figure 7B. To determine whether both C residues in the N4CC PAM are involved in editing, a series of N4DC (D = A, T, G) and N4CD PAM sites were probed in TLR2.0 reporter cells. See, Figures 8A and 8B. No detectable editing was found at either of these sites, providing an initial indication that both C residues of the N4CC consensus PAM are required for efficient Nme2Cas9 activity. The spacer length in the crRNA differs between Cas9 orthologs and may affect the specific activity vs. unspecific. (Cho et al., 2014; Fu et al., 2014). The optimal spacer length of SpyCas9 is 20 nt, with truncations up to 17 nt tolerated (Fu et al., 2014). In contrast, Nme1Cas9 generally has 24 nt spacers (Hou et al., 2013; Zhang et al., 2013), and tolerates truncations of up to 18-20 nt (Lee et al., 2016; Amrani et al., 2018). . To test the spacer length requirements for Nme2Cas9, guide RNA plasmids were created for each unique target TLR2.0 site, but with different spacer lengths. See, Figure 7C and Figure 8C. Comparable activities were observed with the G23, G22 and G21 leads, but significantly decreased activity upon further truncation to G20 and G19 lengths. See, Figure 7C. These results validate Nme2Cas9 as a genome editing platform, with 22-24 nt guide sequences, at PAM N4CC sites in cultured human cells. Figures 7A through 7D present exemplary data showing that Nme2Cas9 uses a 22-24 nt spacer to edit sites adjacent to an N4CC PAM. All experiments were performed in triplicate and the error bars represent the standard error of the mean (s.e.m.). Figure 7A shows an exemplary schematic diagram depicting transient transfection and editing of HEK293T TLR2.0 cells, with mCherry+ cells detected by flow cytometry 72 hours post-transfection. The Figure 7B shows an exemplary Nme2Cas9 editing of the TLR2.0 reporter. Sites with the N4CC PAMs were targeted with different efficiencies, whereas Nme2Cas9 targeting was not observed in a N4GATT PAM or in the absence of sgRNA. SpyCas9 (targeting a site previously validated with a PAM NGG) and Nme1Cas9 (targeting N4GATT) were used as positive controls. Figure 7C shows an exemplary effect of spacer length on Nme2Cas9 editing efficiency. An sgRNA targeting a single TLR2.0 site, with spacer lengths ranging from 24 to 20 nts (including the 5'-terminal G required by the U6 promoter), indicate that the highest editing efficiencies are obtained with 22 spacers. -24nt. Figure 7D shows an example of a Nme2Cas9 dual nickase that can be used in tandem to generate NHEJ and HDR-based edits in TLR2.0. Plasmids expressing Nme2Cas9 and sgRNA, together with an 800 bp dsDNA donor for homologous repair, were electroporated into HEK293T TLR2.0 cells, and NHEJ (mCherry+) and HDR (GFP+) results scored by cytometry. flow. HNH nickase, Nme2Cas9D16A; RuvC nickasa, Nme2Cas9H588A. Cleavage sites 32 bp and 64 bp apart were targeted using either nickase. HNH nickase (Nme2Cas9D16A) produced efficient editing, particularly with cleavage sites that were 32 bp apart, whereas RuvC nickase (Nme2Cas9H588A) was not efficient. Wild-type Nme2Cas9 was used as a control. 3. Precise editing by HDR and HNH Nickasa Cas9 enzymes use their HNH and RuvC domains to cleave the guide complementary and non-complementary strand of target DNA, respectively. SpyCas9 (nCas9) nickases, in which the HNH or RuvC domain is inactivated by mutations, have been used to induce homology-directed repair (HDR) and to improve the specificity of genome editing by DSB induction by dual nickases (Mali et al., 2013a; Ran et al., 2013). To test the efficacy of Nme2Cas9 as a nickase, Nme2Cas9D16A(HNH nickase) and Nme2Cas9H588A(RuvC nickase) were created that possess alanine mutations in catalytic residues of the RuvC and HNH domains, respectively (Esvelt et al., 2013; Hou et al., 2013; Zhang et al., 2013). TLR2.0 cells, together with a GFP donor double-stranded DNA, were used to determine whether Nme2Cas9-induced incisions can induce precise edits via HDR. Target sites within TLR2.0 were used to test the functionality of each nickase using guides targeting cleavage sites spaced 32 bp and 64 bp apart. See, Figure 7D. Wild-type Nme2Cas9 targeting a single site showed efficient editing, with NHEJ and HDR as the results of repair. For the nickases, cleavage sites 32 bp and 64 bp apart showed editing using the Nme2Cas9D16A(HNH nickase), but none of the target pairs worked with Nme2Cas9H588A. These results suggest that the Nme2Cas9 HNH nickase can be used for efficient genome editing, as long as the sites are in close proximity. Previously characterized studies on Cas9 have identified a specific region close to the PAM where Cas9 activity is highly sensitive to sequence mismatches. This 8 to 12 nt region is known as the seed sequence and has been observed among all Cas9s characterized to date (Gorski et al., 2017). To determine if Nme2Cas9 also possesses a seed sequence, a series of transient transfections were performed, each targeting the same locus in TLR2.0, but with a single nucleotide mismatch at different leader positions. See, Figure 8D. A significant decrease in the number of mCherry-positive cells was observed for mismatches in the first 10-12 nt proximal to the PAM, suggesting that Nme2Cas9 possesses a seed sequence in this region. Figures 8A through 8D present exemplary data showing the PAM, spacer, and seed requirements for Nme2Cas9 targeting in mammalian cells, relative to Figures 7A through 7D. All experiments were done in triplicate and error bars represent s.e.m. Figure 8A shows an example Nme2Cas9 targeting N4CD sites in TLR2.0, with estimated editing based on mCherry+ cells. Four sites for each non-C nucleotide at the tested position (N4CA, N4CT and N4CG) were examined and one N4CC site was used as a positive control. Figure 8B shows an example Nme2Cas9 targeting N4DC sites in TLR2.0 [similar to (A)]. Figure 8C shows exemplary leader truncations at a TLR2.0 site (different from Figure 2C) with a N4CCA PAM, revealing similar length requirements to those observed at the other site. Figure 8D shows the targeting efficiency of exemplary Nme2Cas9 which is differentially sensitive to single nucleotide mismatches in the sgRNA seed region. The data show the effects of single nucleotide sgRNA mismatches found along the 23 nt spacer at a TLR2.0 target site. 4. Methods of Delivery to Mammalian Cell Types The ability of Nme2Cas9 to function in different mammalian cell lines was tested using various delivery methods. As initial testing, forty (40) different sites (29 with a PAM N4CC and 11 sites were tested with a PAM N4CD). Several loci (AAVS1, VEGFA, etc.) were selected and target sites with the N4CC PAMs were randomly chosen for editing with Nme2Cas9. Editing (%) was determined by transiently transfecting 150 ng of Nme2Cas9 together with 150 ng of sgRNA plasmids followed by TIDE analysis 72 hours after transfection. A subset of sites showing a range of editing efficiencies at this initial selection were selected for repeat analyzes in triplicate. See, Figure 9A; and Table 1. Figures 9A to 9C present exemplary data showing genome editing of Nme2Cas9 at endogenous loci in mammalian cells via multiple delivery methods. All results represent 3 independent biological replicates and error bars represent s.e.m. Figure 9A shows an example of Nme2Cas9 genome editing of endogenous human sites in HEK293T cells after transient transfection of plasmids expressing Nme2Cas9 and sgRNA. Initially, 40 sites were selected (Table 1); the 14 sites shown (selected to include representatives of various editing efficiencies, as measured by TIDE) were reanalyzed in triplicate. A Nme1Cas9 target site (with a N4GATT PAM) was used as a negative control. Figure 9B shows plots of exemplary data: Left panel: Transient transfection of a single plasmid expressing both Nme2Cas9 and sgRNA (targeting the Pcsk9 and Rosa26 loci) allows editing in Hepa1-6 mouse cells, as detected by TIDE. Right panel: Electroporation of sgRNA plasmids into K562 cells stably expressing Nme2Cas9 from a lentivector results in efficient indel formation Figure 9C shows a zQQncn / Lznz / q / Yi example of Nme2Cas9 that can be electroporated as an RNP complex to induce genome editing.40 pimole of Cas9 was electroporated along with 50 pimole of in vitro transcribed sgRNAs targeting three different loci in HEK293T cells.Ludels were measured after 72 h using TIDE. ZQQnCn / L7Π7 / Σ1 / Υ Table 1. Exemplary endogenous human genome editing sites directed by Nme2Cas9. No. Site Name Spacer Sequence PAM Locus (%) Edition 1 TS1 GGTTCTGGGTACTTTTATCTGTCC CCTCCACC AAVS1 ND 2 TS4 GTCTGCCTAACAGGAGGTGGGGGT TAGAQGAA AAVS1 11 3 TS5 GAATATCAGGAGACTAGGAAGGAG GAGGCCTA AAVS1 15 4 TS6 GCCTCCCTGCAGGGCTGCTCCC CAGCCCAA UNC01588 20 5 TS10 «AGCTAGTCTTCTTCCTCCAACCC GGGCCCTA AAVS1 3.5 6 TS11 GATCTGTCCCCTCCACCCCACAGT GGGGCCAC AAVS1 9 7 TS12 GG C C C AAAT GAAAG GAG T GAGAG G TGACGCGA AAVS1 10 8 TS13 •G-CATCCTCTTGCTTTCTTTGCCTG GACACC'CCA AVS1 2 9 TS16 GGAGTCGCCAGAGGCCGGTGGTGG ATTTCQTC UNC01588 28 10 TS17 GCCCAGCGGCCGGATATCAGCTGC CACGCQCG LINC01588 NA 11 TS18 GGAAGGGAACATATTACTATTGC TTTCCCTC CYBB 1 12 TS19 GTGGAGTGGCCTGCTATCAGCTAC CTATCCAA CYBB 6 13 TS20 GAGGAAGGGAACATATTACTATTG CTTTCCCT CYBB 11.2 14 TS21 GTGAATTCTCATCAGCTAAAATGC CAAGCCTT CYBB 1 15 TS25 G C T CAC T CAC C CACACAGACACAC ACGTCCTC VEGFA 15.6 16 TS26 GGAAGAATTTCATTCTGTTCTCAG TTT TCCTG CFTR 2 17 TS27 GCTCAGTTTTCCTGGATTATGCCT GGCACCAT CFTR 4 18 TS31 GCGTTGGAGCGGGGAGAAGGCCAG GGGTQACT VEGFA 9 19 TS34 GGGCCGCGGAGATAGCTGCAGGGC GGGGíXCC LINC01588 ND 20 TS35 GCCCA CCCGGCGGCGCCTCCCTGC AGGGC'iOC UNC01588 ND 21 TS36 GCGTGGCAGCTGATATCCGGCCGC TGGGCG-TC LINC01588 ND 22 TS37 GCCGCGGCGCGACGTGGAGCCAGC CCCGGAAA LINC01588 ND 23 TS38 GTGCTCCCCAGCCCAAACCGCCGC GGCGCGAC LINC01 588 2 24 TS41 GTCAGATTGGCTTGCTCGGAATTG CCAGCCAA AGA 3 25 TS44 GCTGGGTGAATGGAGCGAGCAGCG TCTTCGAG VEGFA 3 26 TS45 GTCCTGGAGTGACCCCTGGCCTTC TCCCCGCT VEGFA 7.4 27 TS46 •GATCCTGGAGTGACCCCTG GCCTT CTCCCQGC VEGFA 6 28 TS47 gtgtgtccctctccccacccgtcc CTGTCQGG VEGFA 23.1 29 TS48 GTTGGAGCGGGGAGAAGGCCAGGG GTCACaCC VEGFA 2 30 TS49 GCGTTGGAGCGGGGAGAAGGCCAG GGGTCACT VEGFA 4 31 TS50 GTACCCTCCAATAATTTGGCTGG C AATTCCGA AGA 6 32 TS51 GATAATTTGGCTGGCAATTCCGAG CAAGOCAA AGA 4.5 33 TS58 (DS1) GCAGGGGCCAGGTGTCCTTCTCTG GGGGCCTC VEGFA 5 34 TS59 (DS2) GAATGGCAGGCGGAGGTTGTACTG GGGGCCAG VEGFA 11.5 35TS60 (DS3) GAG T GAGAGAG T GAGAG AGAGAC A CGGGCCAG VEGFA 3 36 TS61 (DS4) GTGAGCAGGCACCTGTGCCAACAT GGGCCCGC VEGFA 3.5 37 TS62 (DS5) GCGTGGGGGCTCCGTGCCCCACGC GGGTCQAT VEGFA 3.4 38 TS63 (DS 6) GCATGGGCAGGGGCTGGGGTGCAC AGGCCCAG VEGFA 16 39 TS64 G AAAAT TGTGATTTC C AG AT C CAC AAGCCCAA FANCJ 7 40 TS65 GAGCAGAAAAAATTGTGAT T T C C AGATíXAC FANCJ ND No. Site name Primer name TIDE Sense primer (FW) TIDE Antisense primer (RV) TIDE 1 TS1 AAVS1 TIDE1 TGGCTTAGCACCTTCCCAT AGAAC T C AG GAC C AAC T T AT T C T G 2 TS4 AAVS1 TIDE1 TGGCTTAGCACCTTCCCAT AGAAC T C AG GAC C AAC T T AT T C T G 3 TS5 AAVS1 TIDE1 TGGCTTAGCACCTTCCCAT AGAAC T C AG GAC C AAC T T AT T C T G 4 TS6 LINC01588 TIDE AGAGGAGCCTTCTGACTGCTGCAGA ATGACAGACACAACCAGAGGGCA 5 TS10 AAVS1 TIDE1 TGGCTTAGCACCTTCCCAT AGAAC T C AG GAC C AAC T T AT T C T G 6 TS11 AAVS1 TIDE1 TGGCTTAGCACCTTCCCAT AGAAC T C AG GAC C AAC T T AT T C T G 7 TS12 AAVS1 TIDE2 TCCGTCTTCTCCCACTCC TAGGAAGGAGGAGGCCTAAG 8 TS13 AAVS1 TIDE2 TCCGTCTTCTCCCACTCC TAGGAAGGAGGAGGCCTAAG 9 TS16 LINC01588 TIDE AGAGGAGCCTTCTGACTGCTGCAGA ATGACAGACACAACCAGAGGGCA 10 TS17 LINC01588 TIDE AGAGGAGCCTTCTGACTGCTGCAGA ATGACAGACACAACCAGAGGGCA 11 TS18 NTS55 TIDE TAGAGAACTGGGTAGTGTG CCAATATTGC ATGGGATGG 12 TS19 NTS55 TIDE TAGAGAACTGGGTAGTGTG CCAATATTGCATGGGATGG 13 TS20 NTS55 TIDE TAGAGAACTGGGTAGTGTG CCAATATTGCATGGGATGG 14 TS21 NTS55 TIDE TAGAGAACTGGGTAGTGTG CCAATATTGCATGG GATGG 15 TS25 VEGF TIDE3 GTACATGAAGCAACTCCAGTCCCA AT C AAAT T C C AG C AC C GAGC G C 16 TS26 hCFTR TIDE1 TGGTGATTATGGGAGAACTGGAGC ACCATTGAGGACGTTTGTCTCAC 17 TS27 hCFTR TIDE1 TGGTGATTATGGGAGAACTGGAGC ACCATTGAGGACGTTTGTCTCAC 18 TS31 VEGF TIDE3 GTACATGAAGCAACTCCAGTCCCA AT CAAAT T C CAG CAC C GAGC GC 19 TS34 LINC01588 TIDE AGAGGAGCCTTCTGACTGCTGCAGA ATGACAGACACAACCAGAGGGCA 20 TS35 LINC01588 TIDE AGAGGAGCCTTCTGACTGCTGCAGA ATGACAGACACAACCAGAGGGCA 21 TS36 LINC01588 TIDE AGAGGAGCCTTCTG ACTGCTGCAGA ATGACAGACACAACCAGAGGGCA 22 TS37 LINC01588 TIDE AGAGGAGCCTTCTGACTGCTGCAGA ATGACAGACACAACCAGAGGGCA 23 TS38 LINC01588 TIDE AGAGGAGCCTTCTGACTGCTGCAGA ATGACAGACACAACCAGAGGGCA 24 TS41 AGA TIDE1 GGCATAAGGAAATCGAAGGTC CAI' Gl' C C TCAAGT CAAGAACAAG 25 TS44 VEGF TIDE3 GTACATGAAGCAACTCCAGTCCCA AT CAAAT T C C AG C AC C G AGC G C 26 TS45 VEGF TIDE3 GTACATGAAGCAACTCCAGTCCCA AT CAAAT T C C AG C AC C GAGC G C 27 TS46 VEGF TIDE3 GTACATGAAGCAACTCCAGTCCCA AT CAAAT T C C AG C AC C GAGC G C 28 TS47 VEGF TIDE3 GTACATGAAGCAACTCCAGTCCCA AT CAAAT T C CAG CAC C GAGC G C 29 TS48 VEGF TIDE3 GTACATGAAGCAACTCCAGTCCCA AT CAAAT T C CAGCAC C GAGC GC 30 TS49 VEGF TIDE3 GTACATGAAGCAACT CCAGTCCCA AT CAAAT T C CAGCAC C GAGC GC 31 TS50 AGA TIDE1 GGCATAAGGAAAT C GAAGGT C C AT G T C C T C AAGT CAAGAACAAG 32 TS51 AGA TIDE1 GGCATAAGGAAATCGAAGGTC C AT G T C C T C AAG T CAAGAACAAG 33 TS58 (DS1) VEGF_TIDE4 ACACGGGCAGCATGGGAATAGTC GCTAGGGGAGAGTCCCACTGTCCA 3 4 TS59 (DS2) VEGF_TIDE5 CCTGTGTGGCTTTGCTTTGGTC GGTAGGGTGTGATGGGAGGCTAAGC 35 TS60 (DS3) VEGF_TIDE5 CCTGTGTGGCTTTGCTTTGGTC GGTAGGGTGTGATGGGAGGCTAAGC 36 TS61 (DS4) VEGF_TIDE 5 CCTGTGTGGCTTTGCTTTGGTC TS62 (DS5) VEGF_TIDE6 TS64 FancJ TIDE5 GTTGGGGGCTCTAAGTTATGTAT CTTCATCTGTATCTTCAGGATCA 40 TS65 FancJ TIDE5 GTTGGGGGCTCTAAGTTATGTAT CTTCATCTGTATCTTCAGGATCA zQoncn / Lznz / q / Yi HEK293T cells were used to support transient transfections and, 72 hours post-transfection, cells were harvested, followed by genomic DNA extraction and selective amplification of the target locus. TIDE analysis was used to measure indel efficiency at each locus (Brinkman et al., 2014). Editing by Nme2Cas9 was detectable at most of these sites, although efficiencies varied depending on the target sequence. Table 1. Interestingly, Nme2Cas9 induced indels at various genomic sites with the N4CD PAMs, albeit less consistently and at lower levels. Table 1. Fourteen (14) sites were tested with the N4CC PAMs in triplicate and consistent editing was observed. See, Figure 9A. Furthermore, editing efficiency could be significantly improved by increasing the amount of Nme2Cas9 plasmid delivered, and this high efficiency could be extended to a precise segmental deletion with two guides. See, Figures 10A and 10B. The ability of Nme2Cas9 to function in mouse Hepal -6 cells (derived from hepatoma) was tested. For Hepal-6 cells, a single plasmid encoding both Nme2Cas9 and a sgRNA (targeting Rosa26 or Pcsk9) was transiently transfected and indels were measured after 72 h. Editing was easily observed at both sites. See, Figure 9B, left. The functionality of Nme2Cas9 when stably expressed in K562 human leukemia cells was also tested. To this end, a lentiviral construct expressing Nme2Cas9 was created and cells transduced to stably express Nme2Cas9 under the control of the SFFV promoter. This stable cell line showed no visible differences with respect to growth and morphology compared to non-transduced cells, suggesting that Nme2Cas9 is non-toxic when stably expressed. These cells were transiently electroporated with sgRNA expressing plasmids and analyzed by TIDE after 72 hours to measure indel efficiencies. Efficient editing (>50%) was observed at all three sites tested, validating the ability of Nme2Cas9 to function upon lentiviral delivery in K562 cells. See, Figure 9B. The ribonucleoprotein (RNP) delivery of Cas9 and its sgRNA is also useful for some genome editing applications, and the increased transience of Cas9 presence may minimize nonspecific editing (Kim et al., 2014; Zuris et al. , 2015). Furthermore, some cell types (for example, certain immune cells) are recalcitrant to transfection-based editing of DNA (Schumann et al., 2015). To test whether Nme2Cas9 is functional by RNP delivery, a 6xHis-tagged Nme2Cas9 (fused to three NLS) was cloned into a bacterial expression construct and the recombinant protein was purified. The recombinant protein was then loaded with sgRNA transcribed by T7 RNA polymerase targeting three previously validated sites. Electroporation of the Nme2Cas9:sgRNA complex induced successful editing at each of the three target sites in HEK293T cells, as detected by TIDE. See, Figure 9C. Taken together, these results indicate that Nme2Cas9 can be efficiently delivered via plasmids or lentiviruses, or as an RNP complex, in multiple cell types. 5. Anti-CRISPR regulation To date, five Acr families from diverse bacterial species have been shown to inhibit Nme1 Cas9 in vitro and in human cells (Pawluk et al., 2016; Lee et al., 2018, submitted). Considering the high sequence identity between Nme1 Cas9 and Nme2Cas9, at least some of these Acr families should inhibit Nme2Cas9. To test this, the five families of the recombinant Acr were expressed, purified and tested for the ability of the Nme2Cas9 to cleave a target in vitro in the presence of a member of each family (10:1 Acr:Cas9 molar ratio). An inhibitor for the type 1-E CRISPR system in E. co / / (AcrE2) was used as a negative control, while Nme1Cas9 was used as a positive control. (Pawluk et al., 2014); (Pawluk et al., 2016). As expected, all 5 families inhibited Nme1 Cas9, whereas AcrE2 was unable to. See, Figure 11A, above. AcrlICI n™, AcrllC2Nme, AcrllC3Nme, and AcrllC4wPa completely inhibited Nme2Cas9. Surprisingly, however, AcrllC5s™, which had previously been reported as the most potent of the Nme1Cas9 inhibitors (Lee et al., 2018), did not inhibit Nme2Cas9 in vitro even with a 10-fold molar excess. This suggests that it probably inhibits Nme1Cas9 by interacting with its PID. Figures 10A and 10B present exemplary data showing dose dependence and segmental deletions by Nme2Cas9, as referenced in Figures 9A to 9C. Figure 10A shows an exemplary dose increase of electroporated Nme2Cas9 plasmid (500 ng, vs. 200 ng in Figure 3A) that improves editing efficiency at two sites (TS16 and TS6). Data provided in yellow is reused from Figure 9A. Figure 10B shows an example of Nme2Cas9 that can be used to create precise segmental deletions. Two TLR2.0 targets with cleavage sites 32 bp apart were co-targeted with Nme2Cas9. Most of the lesions created were deletions of exactly 32 bp (blue). Figures 11A to 11C present exemplary data showing that Nme2Cas9 is subject to inhibition by a subset of type ll-C anti-CRISPR families in vitro and in cells. All experiments were done in triplicate and error bars represent s.e.m. Figure 11A shows an exemplary in vitro cleavage assay of Nme1Cas9 and Nme2Cas9 in the presence of five previously characterized anti-CRISPR proteins (10:1 ratio of Acr:Cas9). Top: Nme1Cas9 efficiently cleaves a fragment containing a protospacer with a N4GATT PAM in the absence of an Acr or in the presence of a negative control Acr (AcrE2). All five previously characterized type ll-C Acr families inhibited Nme1Cas9, as expected. Bottom: Inhibition of Nme2Cas9 mirrors that of Nme1 Cas9, except for the lack of inhibition by AcrllC5smu· Figure 11B shows exemplary genome editing in the presence of the five previously described anti-CRISPR families. Plasmids expressing Nme2Cas9 (200 ng), sgRNA (100 ng), and each respective Acr (200 ng) were cotransfected into HEK293T cells, and genome editing was measured using tracking indels by decay (TIDE). English) 72 hours after transfection. Consistent with in vitro analyses, all type II-C anti-CRISPR except AcrllC5s™ inhibited genome editing, albeit with different efficiencies. Figure 11C shows an example of dose dependent Acr inhibition of Nme2Cas9 with different apparent potencies. Nme2Cas9 is fully inhibited by AcrlICI Nme and AcrllC4Hpa in 2:1 and 1:1 mass ratios of cotransfected Acr and Nme2Cas9 plasmids, respectively. To test this, a Nme1Cas9 / Nme2Cas9 chimera with the PID of Nme2Cas9 was tested. See, Figure 5D and Figure 6D. Due to the reduced activity of this hybrid, a ~30x higher concentration of Cas9 was used to achieve similar cleavage efficiency while maintaining the Cas9:Acr molar ratio of 10:1. No inhibition by AcrllC5smU was observed in this protein chimera. See, Figure 12. These data provide further evidence that AcrllC5smü likely interacts with the Nme1 Cas9 PID. Regardless of the mechanistic basis for the differential inhibition by AcrllC5s™, these results indicate that Nme2Cas9 is subject to inhibition by the other four families of type ll-C Acr. Figure 12 presents exemplary data showing that a Nme2Cas9 PID swap renders Nme1Cas9 insensitive to AcrllC5s™ inhibition, relative to Figures 11A to 11C. In vitro cleavage by the Nme1Cas9-Nme2Cas9PID chimera in the presence of previously characterized Acr proteins (Cas9-sgRNA 10 μΜ + Acr 100 μΜ). Based on the above in vitro data, it was hypothesized that AcrlICI Nme, AcrllC2Nme, AcrllC3wme, and AcrllC4HPa could be used as knockout switches for Nme2Cas9 genome editing. To test this, transfections of Nme2Cas9 / sgRNA plasmids (150 ng of each plasmid) targeting TS16 were performed in HEK293T cells in the presence or absence of Acr expression plasmids, since most Acr have been reported to inhibit Nme1Cas9 in those plasmid ratios (Pawluk et al., 2016). As expected, AcrllICINme, AcrllC2Nme, AcrllC3Nme, and AcrllC4wpa inhibited Nme2Cas9 genome editing, while AcrllC5smu had no effect. See Figure 11Β. Complete inhibition by AcrllC3Nme and AcrllC4Hpa was observed, suggesting that they have high potency against Nme2Cas9 compared to AcrllClNme and AcrllC2Nme. To further compare the potency of AcrlICI n™ and AcrllC4wpa, we repeated the experiments with various ratios of Acr plasmid to Cas9 plasmid. See, Figure 11C. The data show that the AcrllC4Hpa plasmid is especially potent against Nme2Cas9. Taken together, these data suggest that several Acr proteins can be used as kill switches for Nme2Cas9-based applications. 6. Hyperaccuracy Nme1Cas9 demonstrates remarkable fidelity of editing in cells and mouse models (Lee et al., 2016; Amrani et al., 2018; Ibraheim et al., 2018). Furthermore, the similarity of Nme2Cas9 to Nme1 Cas9 over most of its length suggests that it may also be hyperaccurate. However, the greater number of sampled sites in the genome as a result of the dinucleotide PAM might create more opportunities for non-specificity by Nme2Cas9 compared to Nme1Cas9 and its less frequently encountered 4-nucleotide PAM. To assess the non-specific profile of Nme2Cas9, GUIDE-seq (sequencing-enabled unbiased identification of genome-wide double-strand breaks) was used to empirically and unbiasedly identify potential non-specific sites (Tsai et al., 2014). . Even the best nonspecific prediction algorithms are prone to false negatives, so empirical target site profiling methods are needed (Bolukbasi et al., 2015b; Tsai and Joung, 2016; Tycko et al., 2016). . GUIDE-Seq is based on the incorporation of double-stranded oligodeoxynucleotides (dsOD) into double-stranded DNA break sites throughout the genome. These insertion sites are detected by high throughput sequencing and amplification. Because SpyCas9 is a well-characterized Cas9 ortholog, it is useful for applications multiplexed with other Cas9s, and as a benchmark for its editing properties (Jiang and Doudna, 2017; Komor et al., 2017). SpyCas9 and Nme2Cas9 were cloned into identical plasmid backbones, with the same UTRs, linkers, NLS, and promoters, for parallel transient transfections (along with similarly matched sgRNA-expressing plasmids) into HEK293T cells. First, it was confirmed that the RNA guides for SpyCas9 and Nme2Cas9 are orthogonal, that is, that Nme2Cas9 sgRNAs do not perform direct editing by SpyCas9 and vice versa. See, Figure 13A. This is in contrast to previously reported results with Nme1Cas9 (Esvelt et al., 2013; Fonfara et al., 2014). Next, to identify a use of SpyCas9 as a reference point for GUIDE-seq, because SpyCas9 and Nme2Cas9 have non-overlapping PAMs, so they can potentially edit any dual site (DS) flanked by a 5'-sequence. NGGNCC-3', which simultaneously meets the PAM requirements of both Cas9. This allows for side-by-side comparisons of misspecificities with RNA guides that facilitate editing of the exact same specific site. See, Figure 14A. Six (6) DS were targeted in VEGFA, each of which also has a G at the appropriate positions 5' of the PAM, such that the SpyCas9 and Nme2Cas9 guides (driven by the U6 promoter) were 100% complementary to the target site. . Seventy-two (72) hours after transfection, TIDE analysis was performed on these sites targeted by each nuclease. Nme2Cas9 induced indels at all six sites, albeit with low efficiencies at two of them, while SpyCas9 induced indels at four of the six sites. See, Figure 14B. At two of the four sites (DS1 and DS4) where SpyCas9 was effective, it induced ~7-fold more indels than Nme2Cas9, while Nme2Cas9 induced a ~3-fold higher frequency of indels than SpyCas9 in DS6. Both Cas9 orthologs edited DS2 with approximately the same efficiency. For GUIDE-seq, DS2, DS4, and DS6 were selected to sample nonspecific cleavage with Nme2Cas9 guides targeting specific editing efficiently, less efficiently, or more efficiently than corresponding SpyCas9 guides, respectively. In addition to the three dual sites, TS6 was added as it has been observed to be an efficiently edited Nme2Cas9 target site, having an indel efficiency of approximately 30-50% depending on cell type. See, Figures 9A and 10A. Similar data is seen with the mouse Pcsk9 and Rosa26tie Nme2Cas9 sites. See, Figure 9B. Plasmid transfections were performed for each Cas9 along with their cognate sgRNAs and dsODNs. Subsequently, libraries were prepared by GUIDE-seq as previously described (Amrani et al., 2018). A GUIDE-seq analysis revealed specific efficient editing for both Cas9 orthologs, with relative efficiencies (as reflected by GUIDE-seq read counts) that are similar to those observed by TIDE. Figure 13B and Table 2. (Tsai et al., 2014; Zhu et al., 2017). Figures 13A through 13E present exemplary data showing the orthogonality and relative accuracy of Nme2Cas9 and SpyCas9 at dual target sites, relative to Figure 12. Figure 13A shows examples of Nme2Cas9 and SpyCas9 guides that are orthogonal. The TIDE results show the frequencies of indels created by both DS2-targeting nucleases with either their cognate sgRNAs or the other orthologous sgRNAs. Figure 13B shows Nme2Cas9 and SpyCas9 samples exhibiting comparable specific editing efficiencies as assessed by GUIDE-seq. Bars indicate counts of specific GUIDE-Seq reads at the three dual sites targeted by each ortholog. The orange bars represent Nme2Cas9 and the black bars represent SpyCas9. Figure 13C shows an example of specific SpyCas9 read counts vs. nonspecific for each site. The orange bars represent the specific readings while the black bars represent the non-specific readings. Figure 13D shows exemplary reads of specific Nme2Cas9 vs. nonspecific for each site. Figure 13E are bar graphs showing exemplary indel efficiencies (as measured by TIDE) at non-specific potential sites predicted by CRISPRSeek. Site-specific and non-specific sequences are shown on the left, with the PAM region underlined and non-consensus PAM sgRNA and nucleotide mismatches in red. Table 2: GUIDE-seq data SpypSgíSpypSgname^ARNa) Nonspecific Peak Score Predicted Cleavage Score ch r6: - :43748587:43748609 652 100 chr1 :+:82004618:82004640 304 4.1 chr1 :-:31140567:31140589 275 19.6 chr16:+:30357 052:30357074 226 0.6 chr5:-:33453895:33453917 217 4 chr11 :+:116600352:116600374 206 0.4 chr17:-:46938649:46938671 191 0.6 chr9:-: 130859778:130859800 146 5.4 chr15:+:59837681:5983770 3 143 2.6 chr22:-:19135541:19135563 124 0.3 chrX:+:49057600: 49057622 122 0.6 chr7:-:72751388:72751410 117 2.6 chr3:-:51652045:51652067 115 0.3 chr1 :-:9544334:9544356 109 0.7 ch r3: - :478 68006:47868028 99 2.6 chr9:+:140670069:140670091 91 0.4 chr2 :-:149516035:149516057 90 0.3 chr22:-:18245713:18245735 89 0.2 ch r3:+: 154744438:154744460 89 2.6 chr17:-:73320669:73320691 88 0.7 chr1 :-:38479457:38479479 85 2.6 chr7:+:33058792 :33058814 78 0.3 ch r9:+: 108299833:108299855 76 1 chr1 :-:23627429:23627451 74 0.5 ch r2: - :63393272:63393294 74 0.5 chr16:+:71 467786:71467808 70 0.6 chr1:-:111638773:111638795 67 0.3 chr1 :-:213393740:213393762 67 0.5 chr7:+:38284425:38284447 67 0.3 chr7:-:134511606:134511628 66 0.2 chr7:+: 152293366:15 2293388 66 0.7 chr17:+:60243345:60243367 63 0.5 chrX:- :48007735:48007757 60 0.6 chr1 :+:52768707:52768729 58 5.4 chr19:-:38805324:38805346 58 0.3 chrX:-:41283776:41283798 58 2.6 chr 11:-:14539718:14539740 57 2.6 zQancn / Lznz / q / Yi chr6:+:32895093:32895115 57 0.7 chr7:-:138957343:138957365 56 98.6 ch r3: - :63900682:63900704 52 0.4 chr5:-:79624954:79624976 52 2.6 chr7:+:76012229:76012251 52 0.7 chrX:+: 39889198:39889220 52 2.6 chr4:-:99897525:99897547 51 5.4 chr1 :-:25822709:25822731 50 0.7 ch r5:+: 17293204:17293226 50 0.7 ch r13:-:66697991:66698013 49 0.1 chr5:-:80796103:80796125 49 2.6 chr16:+:49239128:49239150 45 1.9 chr3:+:69489884:69489906 43 0.5 chr8:+:113712655:113712677 42 0.3 ch r2: - :24502672:24502 ​​694 39 2.6 chr7:-:65642349:65642371 39 2.6 chrX:- :135700076:135700098 37 2.6 chr1 :-:99795756:99795778 36 6.2 chr19:+:1821377:1821399 36 0.2 chr4:-:75501534:75501556 36 0.3 chr 18:+:74828740:74828762 34 0.3 chrX:+: 133975784:133975806 34 6.2 chr14:+:55717904:55717926 33 98.6 chr13:+:49522615:49522637 32 0.3 chr3:-:77788415:77788437 32 0.7 chr11 :-:48230825:48230 847 31 6.2 chr1 :-:1280441:1280463 30 0.3 chr7:+: 44602379:44602401 30 5.4 chr12:-:108166294:108166316 29 5.4 chr7:-:111929850:111929872 29 4 ch r12: -: 122404237:122404259 27 0.2 chr12:-:79123453:79123475 27 0.7 chr22:-:46412541:46412563 27 6.2 ch r5:+:93889070:93889092 26 0.3 chr10:-:97776548:97776570 25 0.6 ch r2: - :56533335:56533357 24 98.6 chr3:+:149843401:1498 43423 24 0.1 chr1 :-:232769157:232769179 23 2.6 chr15: -:75100050:75100072 21 2.6 chr18:+:37252965:37252987 21 0.6 7QQnen / l 7Π7 / Σ1 / Υ ch r2: - :44506208:44506230 21 2.6 ch r4:+: 182389352:182389374 21 0.6 chr11:+:9360929:9360951 20 98.6 chr12:+:23638452:2363847 4 19 0.4 chr7:-:66498753:66498775 19 1.4 chr13:+ :32055862:32055884 16 6.2 chr15:-:59331986:59332008 16 6.2 chr2:+:126196868:126196890 16 0.7 ch rX:-: 77359566:77359588 16 0 chrX:+:24652788:24652810 16 6.2 chr17:-:17667857:17667879 15 0.4 chr21 :+:34751155:34751177 15 2.6 chr2: - :48734975:48734997 14 5.4 chr1 :-:69755048:69755070 13 2.6 chr16:+:90013282:900 13304 13 1.1 chr18:-:630757:630779 13 5.4 chr3: -: 163905630:163905652 12 0.6 gRNA plus PAM sequence-specific GGCAGGCGGAGGTTGTACTGNGG GGCAGGCGGAGGTTGTACTGGGG GGCAGGCGGAGGTTGTACTGNGG GGAAGGCGGAAGTTGTACTGAGG GGCAGGCGGAGGTTGTACTGNGG GGCA GGCGGAGGTTGTACTGGGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGTGGAGGTTGTACTGAGG GGCAGGCGGAGGTTGTACTGNGG GGCAGGGGGAAGCTGTACTGTGG GGCAGGCGGAGGTTG TACTGNGG AGGAGGCGGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGTAATGAGG GGCAGGCGGAGGTTGTACTGNGG GGCAAGAGGAGGTTGGACTGGGGGGCAGGCG GAGGTTGTACTGNGG AGGAGGCGGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAAGCGGAGGTTGTAATGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGTTGTAATGAGG G GCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG TCCAGGTGGAGGCTGTACTGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCAGAGGTTGCACTGGGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGAT GTAATGAGG GGCAGGCGGAGGTTGTACTGNGG CACAGGCAGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGTAGTGAGG 7QQnen / l 7Π7 / Σ1 / Υ GGCAGGCGGAGGTTGTACTGNGG AGGAGGCAGAGGTTGAACTGAGG GGCAGGCGGAGGTTGTACTGNGG GGCAAGGGGAAGTTGTACTGTGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCAGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG GAGAGGCGGAGGTTGC ACTGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCAGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCAGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG CAGAGGCGGAG A GGAGGCGGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG TGGAGGCGGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG AGGAGGCAGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTG TACTGNGG AGGAGGCAGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG GGGAGGTGGAGGTTGCACTGAGG GGCAG GCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGTTGTA GTGAGG GGCAGGCGGAGGTTGTACTGNGG AGAAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGCTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCAGAGG TTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCAGAGGTTGTAATGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG GGAAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG AGGA GGCAGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG GGGAGGCAGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG GGAAGGTGAAGGCTGTACTGCGG GGCAGGCGGAGGTTGTACTGNGG AGAAGGCAGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG AGTAGGCAGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCAGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG AGGAGGCAGAGGTTGTAATGAGG 7QQnen / l 7Π7 / Σ1 / Υ GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCAGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG GCCAGGCGGGTGCTGTACTGGGGGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAG GTTGTACTGGGC GGCAGGCGGAGGTTGTACTGNGG TGGAGGCGGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG GGGAGGTGGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG AGGAGGTGGAGGTTGTAATGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCAGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG AGGAGGCAGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCAGAGGTTGTAGTGAGG GGCAGGCGGAGG TTGTACTGNGG GGGAGGCGGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG GGTAGGCAAAGGTTGTACCAGGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCAGAGGTTGTACTGAGC GGCA GGCGGAGGTTGTACTGNGG GGGAGGCAGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCAGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAGGCGGAGGTTG TACTGAGC GGCAGGCGGAGGTTGTACTGNGG GGGAGGTGGAGGTTGCACTGAGG GGCAGGCGGAGGTTGTACTGNGG CAGAGGCAGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG GGGAGGCAGAGGTTGTACTGAGT GGCAGGCGGAGGTTGTACTGNGG GGGAGGCA GAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG AGAAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG CGTCTGCGAGGGTACTAGTGAGA GGCAGGCGGAGGTTGTACTGNGG GGGAGGCAGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG GGGAGACGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCAGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGTT GTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG AGAAGGCAGAGGTTGTACTGAGC GGCAGGCGGAGGTTGTACTGNGG GCCAGGCTGAGGATGTACTGTGG GGCAGGCGGAGGTTGTACTGNGG AGGAGGCGGAGGTTGTAGTGAGG GGCAGGCGGAGGTTGTACTGNGG GGGAG GCAGAGGTTGTAGTGAGG guideAlignment2¡unspecific Unspecific string Mismatch, distance2PAM .................... - - . .A.......A............+ 18,10 ............G. . - 3 A.G............C. . . . + 20,18,5 ..G...T............ - 18,14 7QQnen / l 7Π7 / Σ1 / Υ G, , a r + 14,10,8 A. G. . ..... . c - 20,18,5 A. G................. - 20,18 . . G. . ..... TO. . + 18.3 ... .A ..... . G - 16,14,5 A.G. . ..... + 20,18,5 . . G. . .....G... - 18.3 A.G.A ..... A. . - 20,18,16,3 A.G. . ..... TO. . -20,18,3. . G. . .....G... - 18.3 TC. . . T. . c + 20,19,14,8 A.G. . ..... . C-20,18,13,5. . G. . to A. . - 18.8.3 CA. . . , A , ..... + 20,19,13 A.G. . .....G... -20,18,3. . G. . .....G... - 18.3 A.G. . ñ . ..... . A + 20,18,13,5 ... .A G. . At + 16,14,10 . . G. . ..... . C-18,13,5.AG. . ..... -C- - 19,18,5 A.G. . ..... . C + 20,18,5 A.G. . , TO , ..... . C-20,18,13,5. . G. . , TO . ..... . c - 18.13.5 CAG. . .....G... + 20,19,18,3 A. . . . TA. . G - 20,14,13,5 A.G. . .....G... + 20,18,3 . .G. . to ..... + 18,13,5 A. G. . ..... . C - 20,18,5 T.G................. + 20,18 A.G. . , TO . ..... . c-20,18,13,5 . . G. . .....G... - 18.3 A.G. ..... - 20,18,13 A.G. . .....G... + 20,18,3 . . G.................. - 18 . . G. . T ..... - 18,14,5 . . G. . .....G... - 18.3 A.G. .....G... + 20,18,3 . . G. . .....G... + 18.3 A.G................. - 20.18 A.A. . .....G... -20,18,3 zQoncn / Lznz / q / Yi A.G. ............G. . + 20,18,3 A.G. - 20,18,8,5 . . G. ......... G. . -18.3. . G. ......... + 18.5 . . G. . c + 18,13,5 A.G. TO. . + 20,18,13,3 . . G. ......... G. . -18.3. .TO. ......... G. . - 18.3 A.G. - 20.18.13. . G. - 18.13. .TO. T A C + 18,14,12,8 A.A. - 20,18,13,5 Α.Γ. + 20,18,13,5 . . G. A + 18.13 . . G.................. + 18 A.G.A. . + 20,18,13,3 A.G. ......... G. . -20,18,3. . G. - 18.13. C. . ....GT.C. - 19,11,10,8 A.G.................. + 20.18 G.T.................. - 20.18 . . G. - 18.14 A.G. TO. . - 20,18,14,3 A.G. ......... G. . -20,18,3. . G. - 18.13 A.G. + 20,18,13,5 . .G. G. . -18,13,3. . G.................. - 18 . .Γ. . . AAA..... . AC + 18,13,12,2,1 . . G. ......... G. . - 18.3 A.G. - 20,18,13. . G.G.. +18,13,3 . . G. ......... G. . -18.3. . G.G.. +18,13,3 . . G.................. + 18 . . G. + 18,14,5 CAG. to - 20,19,18,13 . . G. + 18.13 . . G. - 18.13 A.A. ......... G. . +20,18,3 zQoncn / iζηζ / π / γ C.TCT.. . AG. . . AC. . G. . -20,18,17,16,12,11,7,6,3. .G. . . . A............ + 18,13 . . G. .A............G. . - 18,15,3 A.G. . ..A............ + 20,18,13 A.G............ - 20,18 A.A. . . .A............ - 20,18,13 .C....T. . . .A....... + 19,13,8 A. G............ - 20,18 . .G. . . . A.........G. . - 18,13,3 no. PAM. mismatches n.guide.mismatches Sequence. PAM 0 0 GGG 0 2 AGG 0 1 GGG 0 3 AGG 0 2 AGG 0 3 TGG 0 3 AGG 1 2 AGC 0 2 AGG 0 3 GGG 0 3 AGG 0 2 AGG 0 4 AGG 0 3 AGG 0 2 AGG 0 4 AGG 0 4 GGG 0 3 AGG 1 3 AGC 0 3 AGG 0 2 AGG 0 4 AGG 0 3 TGG 0 3 AGG 0 3 AGG 0 3 AGG 0 4 AGG 0 3 AGG 0 4 AGG 7QQnCn / L7n7 / q / YIAI 0 4 AGG 0 3 AGG 0 3 AGG 0 3 AGG 1 2 AGC 0 4 AGG 0 2 AGG 1 3 AGC 0 3 AGG 1 1 AGC 0 3 AGG 0 2 AGG 0 3 AGG 0 2 AGG 1 2 AGC 0 3 AGG 0 3 AGG 0 4 AGG 0 2 AGG 0 2 AGG 0 3 AGG 0 4 AGG 0 2 AGG 0 2 AGG 1 3 AGC 1 2 AGC 0 4 CGG 0 4 AGG 0 4 AGG 1 2 AGC 1 1 AGC 0 4 AGG 0 3 AGG 1 2 AGC 0 4 GGG 1 2 GGC 1 2 AGC 1 2 AGC 0 4 AGG 0 3 AGG 7QQnCn / L7n7 / q / YIAI 1 2 AGC 0 4 AGG 0 3 AGG 1 1 AGC 0 5 GGG 0 2 AGG 1 3 AGC 0 3 AGG 0 2 AGG 0 3 AGG 1 1 AGC 0 3 AGG 1 4 AGC 1 2 AGT 1 2 AGC 0 3 AGG 1 9 AGA 1 2 AGC 0 3 AGG 1 3 AGC 1 2 AGC 1 3 AGC 0 3 TGG 1 2 AGC 0 3 AGG lstart_nonspecific End_nonspecific Chromosome 43748587 43748609 chr6 82004618 82004640 chr1 3114 0567 31140589 chr1 30357052 30357074 chr16 33453895 33453917 chr5 116600352 116600374 chr11 46938649 46938671 chr17 130859778 130859800 chr9 59837681 59837703 chr15 19135541 19135563 chr22 49057600 49057622 chrX 72751388 72751410 chr7 51652045 51652067 chr3 7QQnCn / L7n7 / q / YIAI 9544334 9544356 chr1 47868006 47868028 chr3 140670069 140670091 chr9 149516035 149516057 chr2 18245713 18245735 chr22 15474443 8 154744460 chr3 73320669 73320691 chr17 38479457 38479479 chr1 33058792 33058814 chr7 108299833 108299855 chr9 23627429 2362745 1 chr1 63393272 63393294 chr2 71467786 71467808 chr16 111638773 111638795 chr1 213393740 213393762 chr1 38284425 38284447 chr7 13 4511606 134511628 chr7 152293366 152293388 chr7 60243345 60243367 chr17 48007735 48007757 chrX 52768707 52768729 chr1 38805324 38805346 chr19 412 83776 41283798 chrX 14539718 14539740 chr11 32895093 32895115 chr6 138957343 138957365 chr7 63900682 63900704 chr3 79624954 7962 4976 chr5 76012229 76012251 chr7 39889198 39889220 chrX 99897525 99897547 chr4 25822709 25822731 chr1 17293204 17293226 chr5 6669 7991 66698013 chr13 80796103 80796125 chr5 49239128 49239150 chr16 69489884 69489906 chr3 113712655 113712677 chr8 24502672 2450269 4 chr2 65642349 65642371 chr7 7QQnen / l 7Π7 / Σ1 / Υ 135700076 135700098 chrX 99795756 99795778 chr1 1821377 1821399 chr19 75501534 75501556 chr4 74828740 74828762 chr18 133975784 133975806 chrX 55717904 55717926 chr14 49522615 49522637 chr13 77788415 77788437 chr3 48230825 48230847 chr11 1280441 1280463 ch r1 44602379 44602401 chr7 108166294 108166316 chr12 111929850 111929872 chr7 122404237 122404259 chr12 79123453 79123475 chr12 4 6412541 46412563 chr22 93889070 93889092 chr5 97776548 97776570 chr10 56533335 56533357 chr2 149843401 149843423 chr3 232769157 232769179 chr1 7 5100050 75100072 chr15 37252965 37252987 chr18 44506208 44506230 chr2 182389352 182389374 chr4 9360929 9360951 chr11 23638452 23 638474 chr12 66498753 66498775 chr7 32055862 32055884 chr13 59331986 59332008 chr15 126196868 126196890 chr2 77359566 77359588 ch rX 24652788 24652810 chrX 17667857 17667879 chr17 34751155 34751177 chr21 48734975 48734997 chr2 69755048 69755070 chr1 90013282 90013304 chr16 630757 630779 chr18 7QQnen / l 7Π7 / Σ1 / Υ 163905630 163905652 chr3 EnExon EntrezJD Symbol TRUE 7422 VEGFA - 23266 ADGRL2 - - - - - - - 6897 TARS - - - - 10241 CALCOCO2 - 114789 SLC25A25 - - - - - - - - - - 8468 FKBP6 - 23132 RAD54L2 - - - - 22907 DHX30 - 79813 EHMT1 - 26122 EPC2 - 637 BID - 4311 MME - 2885 GRB2 - 51118 UTP11 - 51251 NT5C3A - 83856 FSD1L - - - - 51057 WDPCP - - - - - - - 26750 RPS6KC1 - 445 347 TARP - 800 CALD1 - - - - - - - - - - 9372 ZFYVE9 - 90522 YIF1B - - - - 5682 PSMA1 7QQnen / l 7Π7 / Σ1 / Υ - - - - 254048 UBN2 - 6314 ATXN7 - - - - - - - - - - - - 55219 MACO1 - - - - - - - 23635 SSBP2 - - - - 23150 FRMD4B - 114788 CSMD3 - 50618 ITSN2 - - - - - - - - - - 57455 REXO1 - - - - 4155 MBP - 159091 FAM122C - - - - - - - - - - - - 1855 DVL1 - - - - - - - 11179 ZNF277 - 144406 WDR66 - - - - - - - 285600 KIAA0825 - 728558 ENTPD1-AS1 - 114800 CCDC85A - - - - - - - - - - 647946 MIR924HG ZQQnCn / l 7Π7 / Σ1 / Υ - 6519 SLC3A1 - - - - - - - - - - 55253 TYW1 - - - - 54778 RNF111 - - - - - - - 9468 PCYT1B - 10743 RAI1 - - - - 129285 PPP1R21 - - - - - - - 27098 CLUL1 - - - zoancn / Lznz / q / Yi SpyDS4 (SpyDS4 name.RNAg) gRNA_predicted_cleavage_score plus PAM 100 GCAGGCACCTGTGCCAACATNGG 0.1 GCAGGCACCTGTGCCAACATNGG 0 GCAGGCACCTGTGCCAACATNGG 0 GCAGGCACCTGTGCCAACATNGG 0 GCAGGCACCTGTGCCAACATNGG 0 GCAGGCACCTGTGCCA ACATNGG 0 GCAGGCACCTGTGCCAACATNGG 0 GCAGGCACCTGTGCCAACATNGG 0 GCAGGCACCTGTGCCAACATNGG 0 GCAGGCACCTGTGCCAACATNGG 0 GCAGGCACCTGTGCCAACATNGG sequencenonspecific guideAlignment2nonspecific GCAGGCACCTGTGCCAACATGGG .................... ACAGGCACTGATGCCAACTTTGG A.......TGA.......T. TAATGCCCTGGAGCCTCCCTGGC TA.T..C.TG.A...TC.C. GCAGGGCGCGCCGAGAGCAGCGG .....GCG.GCC.AG.G. . G CCAGCCACCCAGCCCCTCCTCCC C...C....CAGC..CT.C. GTAAGCATATGATAGTCCATTTT . T.A. . . TA. .ATAGTC. . . CCGCGTCCCTGCGCAAACCCAGG C.GC.TC....C..A..,CC GTGCACCCCTGCTCCTACCCCCC .TGCA.C.... CT..T.. CC CCAGGGAGCAATGGCAGCGCGCC C....G.G.AA..G..G.GC GGCGGAAGTTGTACTGAGGTGAG .GC..A.GT...A.TG.GG. GCAGGAACTGGAGTGCACAGGTG .....A. .TG.A.TGC. . .G Unspecific String Mismatch. distance2PAM n.guide.mismatches - - 0 + 20,12,11,10,2 5 + 20,19,17,14,12,11,9,5,4,2 10 + 15,14,13,11, 10,9,7,6,4,1 10 - 20,16,11,10,9,8,5,4,2 9 + 19,17,13,12,9,8,7,6,5, 4 10 - 20,18,17,15,14,9,6,2,1 9 + 19,18,17,16,14,9,8,5,2,1 10 + 20,15,13,11 ,10,7,4,2,1 9 + 19,18,15,13,12,8,6,5,3,2 10 - 15,12,11,9,7,6,5,1 8 Sequence . PAM Start_unspecific End_i unspecific GGG 43748848 43748870 TGG 41551021 41551043 GGC 43748564 43748586 GGG 77359654 77359676 CCC 43741999 43742021 T TT 68132445 68132467 AGG 77359345 77359367 CCC 22774978 22775000 GCC 77359596 77359618 GAG 82004622 82004644 GTG 80003891 80003913 Chromosome EnExon EntrezJD Symbol chr6 NA 7422 VEGFA chr22 TRUE 2033 EP300 chr6 TRUE 7422 VEGFA chrX TRUE 5230 PGK1 chr6 - 7422 VEGFA chr15 - - - chrX - - - chr6 - - - zQoncn / Lznz / q / Yi chrX - - - chr1 - 23266 ADGRL2 chr12 - 5074 PAWR zQoncn / Lznz / q / YiAi SpyDS6 (SpyDS6 name.RNAg) Nonspecific Peak_ScorePredictedcleavagescore chr6:+:80816457:80816479 699 0.2 chr6:-:22774975:22774997 553 1.4 chr6:-:43742023:43742045 458 100 chr7:-:1244981 53:124498175 449 0.2 chr1 :-:79194307:79194329 386 0.2 chr17: +:77835740:77835762 383 5.2 chr19:+:15313634:15313656 382 0.7 chr12:+:96650610:96650632 374 3.7 chr10:-:79681895:79681917 352 1.5 chr6:+:20250488:20250510 338 0.2 chr13:-:49117083:49117105 334 0.1 ch r12: - :80003893:80003915 330 0.1 ch r17: -: 77543039:77543061 302 1.6 chr8:-:65972642:65972664 299 0.1 ch r20: - :3548 8683:35488705 277 2.4 chr11:+:100275645:100275667 271 1.6 ch r22:+:38338356:38338378 268 0.4 ch r13:+:45356854:45356876 255 0.2 chr20:-:31061319:31061341 231 1.9 chr11 :-:66051111:6605 1133 229 0.7 chr17:-:72637693:72637715 225 2.4 chr11:+ :128772408:128772430 198 0.5 ch1 :-:99257317:99257339 172 0.1 ch r15: - :39243269:39243291 171 0.3 ch r14: - :22258408:22258430 1 70 0.2 chr21 :-:42506703:42506725 166 2.1 chr7:-: 150036050 :150036072 163 0.2 chr7:-:1140569:1140591 162 1.5 chr4:+:40239842:40239864 154 0.4 chr22:-:50743552:50743574 151 0.9 chr2:-:24 1904500:241904522 149 3.1 chr9:-:136776149:136776171 146 1 chr8 :+:22487688:22487710 145 0.3 chr1 :-:110032844:110032866 144 4.6 chr1 :-:182626625:182626647 133 0.9 chr5:-:134908150:134908172 127 0.1 chr20:-:61928182:61928204 123 2.1 chr10:-:88042752 :88042774 120 0.4 chr17:-:6131626:6131648 118 0.2 chr4:-:1002743:1002765 117 0.2 chr22:+:19106203:19106225 115 0.2 chr1 :+:4400 3969:44003991 114 1.5ch r1:-: 114792469:114792491 110 0.4 chr19:+:38997988:38998010 110 1.6 chr2:-:46897354:46897376 109 0.1 chr12:+:121011672:121011694 108 0.1 ch r17: -: 75891020:758 91042 105 0.6 chr9:+:139220931:139220953 98 5.7 chr14:+ :24168625:24168647 97 0.1 ch r15: -: 74949775:74949797 92 0.1 ch r19:+:44199443:44199465 86 0.4 chr12:+:75214528:75214550 85 0 .3 chr17:-:46058760:46058782 82 0.2 chr16:-:90077745: 90077767 80 1.3 chr20 :+:62023611:62023633 79 2.1 chr12:+:121013758:121013780 77 0.1 chrX:+:106755923:106755945 75 1 ch r10:+: 44417540:44417562 73 0.3 chrl1:-:118193407:118193429 73 1.4 chr16:-:13411476:13411498 73 0.2 chr4:-:8206405:8206427 73 0.6 chr16:+:1517259:1517281 71 0.1 ch r1:-: 150849202:150849224 69 0 chrl 9:- :2057711:2057733 69 1.1 chr9:-:136075308:136075330 69 0.1 chr12:+:29935821:29935843 67 0.1 chrl 1 :-:70812278:70812300 66 0.2 chr13:-:89703965:89703987 62 2.3 chrl :+:110166721:110166743 60 0.6 chrl 1:-:114079332:114079354 58 0.2 chr10:-:71813737:71813759 57 0.4 chr19:-:17414518:17414540 56 0.3 ch r3:-:18428939 5:184289417 56 0.3 chr14:-:94566714:94566736 55 0.2 chr5 :+:178665449:178665471 55 0.1 chr5:+: 149568491:149568513 54 0.3 chrl 1 :-:70242709:70242731 52 0.1 chr21 :-:45132035:45132057 52 0.1 chrl 7:-:827977:827999 47 0.2 chr18:-: 35056448:35056470 44 0 chr6:+:12990616:12990638 44 0.2 chr8:-:17955569:17955591 44 0.1 ch r1:-: 148932239:148932261 43 0.3 ch r19:-:32734619:32734641 42 0.2 chrl :+:228330887:228330909 41 0.1 chr3:+:140221489:140221511 41 3.3 chr5:-:139938346:139938368 40 0.2 chr22:+:23744878:23744900 39 1.6 chr10:-:16388635:16 388657 38 0.1 chr17:+:34824953:34824975 35 0.1 chr3: -:129656921:129656943 35 0.4 chr14:-:93351573:93351595 34 0 chrl:+ :33169255:33169277 33 0.1 chr18:+:29253123:29253145 33 0 chr 6:+:20984444:20984466 33 0 ch r10: -: 77256682: 77256704 32 2.5 chr15:+:89196634:89196656 32 1.7 chr18: -: 73391522:73391544 32 0 chr10:-:72512814:72512836 31 0 chr8:+:80980 935:80980957 31 0.1 ch r11:+:37704008:37704030 30 0.1 chr12:+:52539310:52539332 29 1.7 chr14:-:56431001:56431023 27 0.3 ch r15: - :66949803:66949825 26 1.6 chr7:+:100879116:10087913 8 26 98.6 chrl 1:-:94784149:94784171 25 0.2 chrl 2: +:111548733:111548755 25 0.8 chr19:+:2212199:2212221 25 0.2 chr13:-:22824517:22824539 22 0.1 chr13:-:84196623:84196645 19 0.2 chr15:+:96534271:96534293 19 0.1 chr21 :-:21304105:21304127 17 0.2 chrl 7:-:39705337:39705359 16 0.2 chr20:-:56582544:56582566 15 0.9 chr20:+:49479068:49479090 15 0.1 chrl:+ :89258185:8 9258207 14 0.1 chrl 5:-:51386687:51386709 13 0.1 ch r19:+:38724286:38724308 13 0.3 chrl 6:-:2286384:2286406 11 0.2 gRNA plus PAM non-specific sequence GGGCAGGGGCTGGGGTGCACNGG CGGCAGGGGCTGAGGGGCACTGG GGGCAGGGGCTGG GGTGCACNGG GGGTAGGAGCAGGGGTGCACTGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGGGGCTGGGGTGCACAGG zQQncn / Lznz / q / γι GGGCAGGGGCTGGGGTGCACNGG GGGCAGGAACTGGAGTGCACAGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGGAACTGGAGTGCACAGG GGGCAGGGGCTGGGGTGCACNGG CAGCAGGGGCTGGGGTGCACAGG GGGCAGGGGCTGGGGTG CACNGG GGGAAGGGCCTGGGGTACACGGGGGCAGGGGCTGGGGTGCACNGG GGGCCGGGGCAGGGGTGCACAGG GGGCAGGGGCTGGGGTGCACNGG AGACAGGGGCCGGGGTGCACAGGGGCAGGGGCTGGGGTGCACNGG GGGCAGGAACTG GAGTGCACCGG GGGCAGGGGCTGGGGTGCACNGG AGGCAGGAACTGGAGTGCACGGG GGGCAGGGGCTGGGGTGCACNGG AGGCAGGAACTGGAGTGCACAGG GGGCAGGGGCTGGGGTGCACNGG AGGAAGGGACTGGGGTGCACTGGGGGCAGGGGCT GGGGTGCACNGG AGGCAGGAACTGGAGTGCACAGG GGGCAGGGGCTGGGGTGCACNGG AGGTGGGGGCTGGGGTGCACAGG GGGCAGGGGCTGGGGTGCACNGG AGGCAGGAACTGGGGTGCACGGGGGCAGGGGCTGGGGTGCACNGG TGG CAGGGGCAGGGGTGAACTGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGGAACTGGAGTGCACGGG GGGCAGGGGCTGGGGTGCACNGG GGCCAGGGGCTGGGGAGCACAGGGGCAGGGGCTGGGGTGCACNGG GGGCAGGGCTGGGGGTGCACAG G. GTGCACNGG GAGAAGGAGCTGGGGAGCACTGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGGAACTGGAGTGCACCAG GGGCAGGGGCTGGGGTGCACNGG GGGCAAGGGCAGGGGTGCACCAG GGGCAGGGGCTGGGGTGCACNGG AAGAAGGGGCAAGGG TGCACAGGGGGCAGGGGCTGGGGTGCACNGGGGCCAGGAGCAGGGGTGCACGGG GGGCAGGGGCTGGGGTGCACNGG TGGCAGCGGCTGGGGAGCACTGG GGGCAGGGGCTGGGGTGCACNGG GGGCGTGGGCAGGGGTGCACTGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGTGGCTGGGGTGCATTGG GGGCAGGGGCTG GGGTGCACNGG GGCCAGGAGCTGGGGTGCTCAGG GGGCAGGGGCTGGGGTGCACNGG CCTCAGGGGCTGGGGTGAACAGG GGGCAGGGGCTGGGGTGCACNGG TGGCAGGGTCTGGGGTGCACAGA GGGCAGGGGCTGGGGTGCACNGG GAGCAGGG TCTGGGGTGCATGGG GGGCAGGGGCTGGGGTGCACNGG GAGCAGGGACTGAGGGGCACAGG GGGCAGGGGCTGGGGTGCACNGG GAGCAGGGGCTGGGGGGCACTGG GGGCAGGGGCTGGGGTGCACNGG TGGCAGGGGTAAGGGTGCACTGG G GGCAGGGGCTGGGGTGCACNGG AGACAGAGGCTGGAGTGCACTGG GGGCAGGGGCTGGGGTGCACNGG AGGCAGGGGCTGGAGTTCACAGG GGGCAGGGGCTGGGGTGCACNGG AGGAAGGGACCAGGGTGCACCAG GGGCAGGGGCTGGGGTGCACNGG GGCCAGGAGCAGGGGTGCACAGGGGGCAGGGGCTGGGGTGCACNGGGGGCCGGGGCTGGGGTGCCAGGG 7QQnen / l 7Π7 / Σ1 / Υ GGGCAGGGGCTGGGGTGCACNGG GGGCGGGGGCTGGGGAGCACAGG GGGCAGGGGCTGGGGTGCACNGG AGGCAGGAGCCAGGGTGCAGAGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGAGGCTGGAGTGCCCAGGGGGCAGGGGCTGGGG TGCACNGG GGACAGGGGCAGGGGTGCCCGGG GGGCAGGGGCTGGGGTGCACNGG AGGGAGGGGCTGGGGTGCACGGA GGGCAGGGGCTGGGGTGCACNGG GGGCAGGAACTGGAGTGCATAGG GGGCAGGGGCTGGGGTGCACNGG AGGCAGGAACTG GAGTGCACAAG GGGCAGGGGCTGGGGTGCACNGG GGGCAGAGGCTAGGGTGCAGTGG GGGCAGGGGCTGGGGTGCACNGG AGGTAGGGGTTGGGGGGCACAGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGAAGCAGGGTGCTCAGG GGGCAG GGGCTGGGGTGCACNGG GGGGAGGGGTGGGGGTGCACCGG GGGCAGGGGCTGGGGTGCACNGG GAGCAGGGGCTGGGGGGCACTGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGAGGCTGGAGTGCCCAGGGGCAGGGGCTGGGGTGCA CNGG GGGTGGGGGCTGGGGTGCCCAGG GGGCAGGGGCTGGGGTGCACNGG GGGCAAGGGCAGGGGTGCCCTGG GGGCAGGGGCTGGGGTGCACNGG GAGAGGGAGCTGGGGTGCACGGGGGCAGGGGCTGGGGTGCACNGG AGGCAGGACT GAGGTGCATAGG GGGCAGGGGCTGGGGTGCACNGG GGGCCAGGGCTGAGGTGCACAGG GGGCAGGGGCTGGGGTGCACNGG TGGGAGGGGCTAGAGTGCACAGG GGGCAGGGGCTGGGTGCACNGG CCGCAGGGGCTGGGATGCTGGGGGGGG GGGCTGGGGTGCACNGG GAGGAGGGGCTGGGGTGCCCTGG GGGCAGGGGCTGGGGTGCACNGG GGGCAAAGGCCGGGGTGCCCAGG GGGCAGGGGCTGGGGTGCACNGG AGGCGGGGGCTGGGGGCTCGGGGGCAGGGGCTGGGGTGCA CNGG AGGCAGGGGCCAGGGTCCACAGGGGGCAGGGGCTGGGGTGCACNGGGGGTTGGGGTTGGGGTGCACAGG GGGCAGGGGCTGGGGTGCACNGG AGGCAGGGGCCGGGGTGCGCAGG GGGCAGGGGCTGGGGTGCACNGG GGGCACAGACTGGGGTGCATTGG GGGCAGGGGCTGGGGTGCACNGG GGGCTGGGGCTGAGGTGCGCCGGGGGCAGGGGCTGG GGTGCACNGG AGGCAGGGGCTGGGGGGCAAGGG GGGCAGGGGCTGGGGTGCACNGG GAGCGGGAGCTGGGGGGCACAGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGGGACTGGGGTGCTTAGG GGGCAGGGGCTGGGGTGCACNGG GGGAAG GGGCTGGAGGGCACAGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGGGGAAGGGGTGGACTGG GGGCAGGGGCTGGGGTGCACNGG AGACAGGGGCTGGAGTGCAGTGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGAGGCTGGAGTGCAATGG GGGCAGGGGCTGGGGTGCACNGG GGGCTGGGGCTGGGGAGCAGGGG GGGCAGGGGCTGGGGTGCACNGG AGGAAAGGGCTGGAGTGCAGGGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGGAACTGGAGTGCACCAG GGGCAGGGGCTGGGGT GCACNGG AGGCAGGAACTGGAGTGCACAAG GGGCAGGGGCTGGGGTGCACNGG AGGCAGAGCCTGGGGTGCAGGGG 7QQnen / l 7Π7 / Σ1 / Υ GGGCAGGGGCTGGGGTGCACNGG GGGCAGGGCCAGGGGAGCACAGG GGGCAGGGGCTGGGGTGCACNGG AGCCAGGGGCTGGGGGGAACAGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGGGGATGGGGTGCAGTGG GGGCAGGGGCTGG GGTGCACNGG AGGCAAGGCCTGGGGTGCCCAGG GGGCAGGGGCTGGGGTGCACNGG GGGCTGGGGCTGGGGAGCACGGGGGCAGGGGCTGGGGTGCACNGG GAGAAGGGGCTGGGAAGCACAGGGGCAGGGGCTGGGGTGCACNGG AGGCAGGA ACTGGAGTGCACAAG GGGCAGGGGCTGGGGTGCACNGG GGGGAGGGGCTGGGGTGCCAGGG GGGCAGGGGCTGGGGTGCACNGG AGGAAGGGGCTGGGGAAAACAGG GGGCAGGGGCTGGGGTGCACNGG CCCCAGGGGCTGGGTGCCTGGGGGGG GGGGCTGGGGTGCACNGG AAGCAGAGGCTGAAGTGCACAGG GGGCAGGGGCTGGGGTGCACNGG AGGCAGGAACTAGAGTGCACAGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGGGGGTGGGGTCCACAGGGGGCAGGGGCTGGGGTGCACNGG GG GGAGGGGCTGGGGAGCACGGA GGGCAGGGGCTGGGGTGCACNGG AGGCAGAGGCTGGAGTGGACCGG GGGCAGGGGCTGGGGTGCACNGG GGGTAGGGGCTGGGGGATACCGG GGGCAGGGGCTGGGGTGCACNGG GGGAAGGGTCTGGAGTCCACTGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGGAACTAGAGTGCACGGG GGGCAGGGGCTGGGGTGCACNGG GGGCAGGGACTGGGGTGCTCTGG GGGCAGGGGCTGGGGTGCACNGG GAGTAGGGGCAGGGGTGCTCTGG GGGCAGGGGCTGGGGTG CACNGG AGGAAGGGCCTGGGGTGCACAGA GGGCAGGGGCTGGGGTGCACNGG GGCCAGGGGCTGGGGTGCACGGT GGGCAGGGGCTGGGGTGCACNGG AGGCAGGGGCCAGGGTGCATGGGGGGCAGGGGCTGGGGTGCACNGG GGGCAGAGGAT GGGGTGCAGGGGGGGCAGGGGCTGGGGTGCACNGGCGGCAGGGGCTGGAGTGCAGTGG GGGCAGGGGCTGGGGTGCACNGG AGGCAGGATCTGGAGTGCACAGG GGGCAGGGGCTGGGGTGCACNGG AGACAGGAGCTGGAGTGCACAAG GGGCAGGGGCTGGGGTGCACNGG TGGCAGGGGCAGGGATGCTCTGGGGCAGGGGCTGGGGTG CACNGG CCTCAGGGGTTGGGATGCACTGG GGGCAGGGGCTGGGGTGCACNGG GAGCAGGGTCAGGGGTGCAGAGG GGGCAGGGGCTGGGGTGCACNGG CAGGAGTGGCTGGGGTGCACAGGGGGCAGGGGCTGGGGTGCACNGGGGGCCTGGGCTGAG ATGCACGGG GGGCAGGGGCTGGGGTGCACNGG ACGCAGGGGCTAGGGAGCACAAG GGGCAGGGGCTGGGGTGCACNGG GGGCTGGGGCTGGGGAGCACGGGGGCAGGGGCTGGGGTGCACNGG GGGCAGGGATTGGGGGGGGGCT GGGGTGCACNGG AGGGAGGGGCCGGGCTGCACTGG guideAlignment2nonspecific Nonspecific String Mismatch. distance2PAM c. .......TO. . G. . . . + 20,8,5,T. ..A..A......... - 17,13,10 ZQQnCn / l 7Π7 / Σ1 / Υ .................... - - ...... .AA . . .A...... - 13,12,7 ...... .AA . . .A...... - 13,12,7 AC.................. + 20,19 . . .TO. . r- ...... A. . . +17,12,4 . . . .C. A......... + 16.10 A.A. . . C......... - 20,18,10 ...... .AA . . . A...... + 13,12,7 A..... .AA . . .A...... - 20,13,12,7 A..... .AA . . .A...... - 20,13,12,7 A. .A. . _ .......... - 20,17,12 A..... .AA . . .A...... - 20,13,12,7 A. .TG............ - 20,17,16 A..... .AA . ......... + 20,13,12 T..... A...... A. . + 20,10,3 ...... .AA . . .A...... + 13,12,7 . .C. . . .....TO. . . . - 18,5 ........CTG......... - 12,11,10 T. .GT............ - 20, 17.16...AG. .....TO. . . . + 17,16,5 .AA . . .TO. .TO. . . - 13,12,7,4 .A.A. . , A , ..... A. . . . - 19,17,13,5 ...... .AA . . .A...... - 13,12,7.....? A......... - 15.10 YY.Y. . AA........ - 20,19,17,10,9 ..C... A......... - 18,13,10 T..... C___ .. ...TO. . . . + 20,14,5 . . . .GT A......... - 16,15,10 T, , .........T - 14,1 . .C. . . , TO . ........T. - 18,13,2 CCT. . . .......TO. . + 20,19,18,3 T..... , . T .......... - 20.12 .A. . . . . . T .........T - 19,12,1 -A.... —A, . .TO. .G. . . . - 19,12,8,5 .A. . . . .....G. . . . - 19.5T..... . . . Ί ΆΑ........ - 20,11,10,9 A.A. . . TO . . . . .A...... - 20,18,14,7 A..... ...A..T... - 20,7,4 A. .A. . . .A AC........ + 20,17,12,10,9 . .C. . . AA A......... + 18,13,10 zQoncn / iζηζ / π / γ ... .c......... . . .CA-16,2,1 . . . . G......... A. . . . + 16.5A......A. .AC. . . . . . G - 20,13,10,9,1 ......A......A . . .C. + 14,7,2 . .A.......A. . . . . .C. - 18,10,2 A. .G................. + 20,17 .......AA....A . . . . T + 13,12,7,1 A......AA. . . . A...... - 20,13,12,7 ......A. . ..TO. . . . . . G + 14,9,1 A. .T....T. . . . G. . . . + 20,17,11,5 ......AA. .TO. . . . . .T. -14,13,10,2. . . G.....TG......... - 17,11,10 .A............ G. . . . + 19.5 ......A......A . . .C. + 14,7,2...TG......... . . .C. + 17,16,2.....A. . . .TO. . . . . .C. + 15,10,2 .A.A.G. .A............ - 19,17,16,13 A.......A. . .TO. . . . . T - 20,12,8,1 .... CA......A....... - 16,15,8 T. .G.......A.A..... + 20,17,9,7 CC............A. . .TG - 20,19,6,2,1 .A.G.......... . . .C. - 19,17,2 .....AA...C... . . .C. - 15,14,10,2 A...G......... G. .T. + 20,16,5,2 A.........CA. . .C. . . -20,10,9,4. . .TT. . . .T.......... - 17,16,11 BC.........C. . . . . .G. + 20,10,2.....CA.A..... . . . . T-15,14,12,1 . . . .T.......A. . . .G. - 16,8,2 A............ G. . .A - 20,5,1 .A. . G. .A...... G. . . . - 19,16,13,5 ........A..... . . .TT - 12,2,1 . . .A.........A G. . . . + 17,7,5 .........AA... . . G. . + 11,10,3 A. A..........A . . . . G - 20,18,7,1 ......A......A ... .A - 14,7,1 . . . .T......... A. . . G - 16,5,1 A. .A.A.......A . . . . G - 20,17,15,7,1 .......AA.. . .A...... + 13,12,7 A......AA. . . . A...... - 20,13,12,7 7QQnen / l 7Π7 / Σ1 / Υ TO. . . . A O ......G - 20,14,12,1 C · A . . A.___ 12,10,5 B.C. . ...... . . G.A. . + 20,18,5,3 to ......G + 11,1 A. . . .j .....C. - 20,15,12,2 ____T . . A .___ 16.5 .A.A. .AA.... - 19,17,6,5 A. . . . .AA... A...... + 20,13,12,7 . . .G. ...... .....CA - 17,2,1 A. .A. ...... . .AAA. . - 20,17,5,4,3 CCC. . ...... .....CT + 20,19,18,2,1 YY. . . A..... XA...... + 20,19,14,8,7 A. . . . .AA. .A A...... + 20,13,12,9,7 cz . . . r. . . 11.4 ___G. . . A .___ 17.5 A. . . . A A. ​​. .G. . -20,14,7,3. . .T. ...... . . GAT . . - 17,5,4,3 ___A . you . c___ 17,12,7,4 .AA. .A A...... + 13,12,9,7 to .....T. + 12.2 .A.T. to.....T. - 19,17,10,2 A. .A. r-20,17,12. . c................. 18 A. . . . ....CA ......T - 20,10,9,1 Λ. .TO. . ......G + 14,11,1 C. . . . ...... A.....G + 20,7,1 A. . . . .AT... A...... - 20,13,12,7 A.A. . to A...... - 20,18,13,7 T. . . . to .A...T. + 20,10,6,2 CCT. . .A..... - 20,19,18,11,6 .A. . . T A ......G - 19,12,10,1 CA.G. Τ' - 20,19,17,14 . . . .CT......A. A..... + 16,15,8,6 BC. . . . . TO. . . . + 20,19,9,5 ____T . . TO . G. . 16,5,3 ..... ..AT.. . .G. . . . + 12,11,5 A. .G. .C..... - 20,17,10,6 n. PAM.mismatches n.guide.mismatches Sequence. PAM 0 3 TGG ZQQnCn / l 7Π7 / Σ1 / Υ 0 3 TGG 0 0 AGG 0 3 AGG 0 3 AGG 0 2 AGG 0 3 GGG 0 2 AGG 0 3 AGG 0 3 CGG 0 4 GGG 0 4 AGG 0 3 TGG 0 4 AGG 0 3 AGG 0 3 GGG 0 3 TGG 0 3 GGG 0 2 AGG 0 3 AGG 0 3 TGG 0 3 AGG 0 4 GGG 0 4 TGG 1 3 CAG 1 2 CAG 0 5 AGG 0 3 GGG 0 3 TGG 0 3 TGG 0 2 TGG 0 3 AGG 0 4 AGG 1 2 AGA 0 3 GGG 0 4 AGG 0 2 TGG 0 4 TGG 0 4 TGG 0 3 AGG 1 5 CAG 7QQnCn / L7n7 / q / YIAI 0 3 AGG 0 3 GGG 0 2 AGG 0 5 AGG 0 3 AGG 0 3 GGG 1 2 GGA 0 4 AGG 1 4 AAG 0 3 TGG 0 4 AGG 0 4 AGG 0 3 CGG 0 2 TGG 0 3 AGG 0 3 AGG 0 3 TGG 0 4 GGG 0 4 AGG 0 3 AGG 0 4 AGG 0 5 GGG 0 3 TGG 0 4 AGG 0 4 GGG 0 4 AGG 0 3 AGG 0 3 AGG 0 4 TGG 0 3 CGG 0 3 GGG 0 4 AGG 0 3 AGG 0 3 AGG 0 3 TGG 0 4 TGG 0 3 TGG 0 3 GGG 0 5 GGG 1 3 CAG 7QQnCn / L7n7 / q / YIAI 1 4 AAG 0 4 GGG 0 3 AGG 0 4 AGG 0 2 TGG 0 4 AGG 0 2 GGG 0 4 AGG 1 4 AAG 0 3 GGG 0 5 AGG 0 5 GGG 0 5 AGG 0 5 AGG 0 2 AGG 1 2 GGA 0 4 CGG 0 4 CGG 0 4 TGG 0 4 GGG 0 2 TGG 0 4 TGG 1 3 AGA 1 1 GGT 0 4 GGG 0 3 GGG 0 3 TGG 0 4 AGG 1 4 AAG 0 4 TGG 0 5 TGG 0 4 AGG 0 4 AGG 0 4 GGG 1 4 AAG 0 3 GGG 0 3 AGG 0 4 TGG Non-specific start Non-specific end Chromosome 80816457 80816479 chr6 zQancn / Lznz / q / YiAi 22774975 22774997 chr6 43742023 43742045 chr6 124498153 124498175 chr7 79194307 79194329 chr1 77835740 77835762 chr17 15313634 15313656 chr19 96650610 96650632 chr12 79681895 79681917 chr10 20250488 20250510 chr6 49117083 49117105 chr13 80003893 80003915 chr12 77543039 77543061 chr17 65972642 65972664 chr8 35488683 35488705 chr20 100275645 100275667 chr11 38338356 38338378 chr22 4 5356854 45356876 chr13 31061319 31061341 chr20 66051111 66051133 chr11 72637693 72637715 chr17 128772408 128772430 chr11 99257317 99257339 chr1 39243269 39243291 chr15 22258408 22258430 chr14 42506703 42506725 chr21 150036050 150036072 chr7 1140569 1140591 chr7 40239842 4 0239864 chr4 50743552 50743574 chr22 241904500 241904522 chr2 136776149 136776171 chr9 22487688 22487710 chr8 110032844 11003286 6 chr1 182626625 182626647 chr1 134908150 134908172 chr5 61928182 61928204 chr20 88042752 88042774 chr10 6131626 6131648 chr17 1002743 1002765 chr4 19106203 19106225 chr22 7QQnen / l 7Π7 / Σ1 / Υ 44003969 44003991 chr1 114792469 114792491 chr1 38997988 38998010 chr19 46897354 46897376 chr2 121011672 121011694 chr12 75891 020 75891042 chr17 139220931 139220953 chr9 24168625 24168647 chr14 74949775 74949797 chr15 44199443 44199465 chr19 75214528 752 14550 chr12 46058760 46058782 chr17 90077745 90077767 chr16 62023611 62023633 chr20 121013758 121013780 chr12 106755923 10675594 5 chr X 44417540 44417562 chr10 118193407 118193429 chr11 13411476 13411498 chr16 8206405 8206427 chr4 1517259 1517281 chr16 150849202 150849224 chr1 205 7711 2057733 chr19 136075308 136075330 chr9 29935821 29935843 chr12 70812278 70812300 chr11 89703965 89703987 chr13 110166721 11 0166743 chr1 114079332 114079354 chr11 71813737 71813759 chr10 17414518 17414540 chr19 184289395 184289417 chr3 94566714 9456673 6 chr14 178665449 178665471 chr5 149568491 149568513 chr5 70242709 70242731 chr11 45132035 45132057 chr21 827977 827999 chr17 35056448 35056470 chr18 12990616 12990638 chr6 7QQnen / l 7Π7 / Σ1 / Υ 17955569 17955591 chr8 148932239 148932261 chr1 32734619 32734641 chr19 228330887 228330909 chr1 140221489 140221511 chr3 1399 38346 139938368 chr5 23744878 23744900 chr22 16388635 16388657 chr10 34824953 34824975 chr17 129656921 129656943 chr3 93351573 9 3351595 chr14 33169255 33169277 chr1 29253123 29253145 chr18 20984444 20984466 chr6 77256682 77256704 chr10 89196634 89196656 chr 15 73391522 73391544 chr18 72512814 72512836 chr10 80980935 80980957 chr8 37704008 37704030 chr11 52539310 52539332 chr12 56431001 56431023 chr14 66 949803 66949825 chr15 100879116 100879138 chr7 94784149 94784171 chr11 111548733 111548755 chr12 2212199 2212221 chr19 22824517 22824539 chr13 84196623 84196645 chr13 96534271 96534293 chr15 21304105 21304127 chr21 39705337 39705359 chr17 56582544 56582566 chr20 49479068 49479090 chr20 89258185 89258207 chr1 51386687 51386709 chr15 38724286 38724308 chr19 2286384 2286406 chr16 TRUE 594 BCKDHB 7QQnen / l 7Π7 / Σ1 / Υ - - - - 7422 VEGFA - 25913 POT1 - - - - - - - - - - 2004 ELK3 - 9231 DLG5 - - - - - - - 5074 PAWR - - - - - - - 140710 SOGA1 - - - TRUE 85377 MICALL1 - - - - 140688 N0L4L TRUE 254263 CNIH2 - - - TRUE 3762 KCNJ5 - - - - - - - - - - - - 5919 RARRES2 - 84310 C7orf50 - 399 RHOH - 23654 PLXNB2 - 200772 LOC200772 - 7 410VAV2 - 55909 BIN3 - 127002 ATXN7L2 - 85397 RGS8 - 9547 CXCL14 - 57642 COL20A1 - 2894 GRID1 - - - - - - - 9993 DGCR2 zQoncn / Lznz / q / Yi - 5792 PTPRF - - - - 6261 RYR1 - - - - 9921 RNF10 - - - TRUE 26102 DKFZP434A062 - - - - 80153 EDC3 - - - - - - - 80279 CDK5RAP3 - 79007 DBNDD1 - - - - 9921 RNF10 - - - - 283033 LINC00841 - - - - - - - 54436 SH3TC1 - 1186 CLCN7 TRUE 405 ARNT - - - - - - TRUE 83857 TMTC1 - 22941 SHANK2 - - - - 271 AMPD2 - 7704 ΖΒΤΒ16 - 55506 H2AFY2 - - - - 2049 EPHB3 - 122509 IFI27L1 - 9509 ADAMTS2 - - - - - - - - - - 64359 NXN - 56853 CELF4 - 221692 PHACTR1 7QQnC0 / l 7Π7 / Σ1 / Υ - - - - 645166 LOC645166 - - - - 2987 GUK1 - 64084 CLSTN2 TRUE 10307 APBB3 - - - - - - - - - - - - - - - - - 9331 B4GALT6 - 54901 CDKAL1 - - - - 3669 ISG20 - - - - 140766 ADAMTS14 - 7163 TPD52 - - - - - - - - - - - - - 24146 CLDN15 - - - - 23316 CUX2 TRUE 84444 DOT1L - - - - - - - - - - - - - 3728 JUP - - - - 55653 BCAS4 - 5586 PKN2 - 388121 TNFAIP8L3 - - - - - - 7QQnen / l 7Π7 / Σ1 / Υ Nme2DS2 Nonspecific Peak score Predicted cleavage score chr6:-:43748582:43748613 547 100 chrX:+:77359550:77359581 44 0 name. qRNA gRNA plus PAM nonspecific sequence Nme2DS2 GAATGGCAGGCGGAGGTTGTACTGNNNNNC CNN GAATGGCAGGCGGAGGTTGTACTGGGGGCCA G Nme2DS2 GAATGGCAGGCGGAGGTTGTACTGNNNNNC CNN GAATGGCAGGCGGAGGTTGTACTGGGGGCCA G leaderAlignment2nonspecific Chain unspecific Discordance. distance2PAM - - A..C..A..C..C.C..CTC...A + 24, 21, 18, 15, 12, 10, 7, 6, 5, 1 n.PAM.mismatches n.lead .mismatches Sequence.PAM 0 0 GGGGCCAG 0 10 GTACCCTC Nonspecific Start Nonspecific End Chromosome 43748582 43748613 chr6 77359550 77359581 chrX EnExon Entrez ID Symbol TRUE 7422 VEGFA - - - 7QQnen / l 7Π7 / Σ1 / Υ Nme2DS4 Nonspecific Peak_Score name.gRNA ch r6 :-:43748843:43748874 66 c Human DeCas9 TS14 gRNA plus PAM GTGAGCAGGCACCTGTGCCAACATNNNNCCNN nonspecific leader sequenceAlignment2nonspecific GTGAGCAGGCACCTGTGCCAACATGGGLLGL ............................ Unspecific Strand Cleavage Score Predicted Mismatch. distance2PAM - 100 - n.PAM.mismatches n.quía.mismatches Sequence.PAM 0 0 GGGCCCGC Nonspecific start Nonspecific end Chromosome 43748843 43748874 chr6 EnExon Entrez ID Symbol - 7422 VEGFA Nme2DS6 Nonspecific Peak score Predicted cleavage score chr6:-:43742018:43742049 483 100 chrX:-:77359465:77359496 12 0 name.gRNA qRNA plus PAM d DeCas9 human TS16 GCATGGGCAGGGGCTGGGGTGCACNNNNCCNN d DeCas9 human TS16 GCATGGGCAGGGGCTGGGGTGCACNNNNCCNN unspecific sequence leaderAlignment2unspecific Unspecific string GCATGGGCAGGGGCTGGGGTGCACAGGC CCAG ... .................... - GCAGGAAGCGTCGCCGGGGGGCCCACAA GGGT ...G.AAGC.TC..C....G.. C. 21,19,18, 17,16,14,13,10,5,2 n.PAM.mismatches Sequence.PAM Sequence.PAM 0 AGGCCCAG AATCCCTT 10 ACAAGGGT ACTCCCTC Beginning unspecific Ending unspecific Chromosome 43742018 43742049 chr6 77359465 77359496 chrX EnExon Entrez ID Symbol - 7422 VEGFA - - V EGFA ZQQnCn / l 7Π7 / Σ1 / Υ rose26 Nonspecific Peak score Predicted cleavage score chr6:-:113076072:113076103 1175 100 chr11 :-:73171296:73171327 24 1.4 name.gRNAgRNA plus PAM Nme2Pink TGAGGACCGCCCTGGGCCTGGGAGNNNNCCNN Nme2Pink TGAG GACCGCCCTGGGCCTGGGAGNNNNCCNN unspecific sequence guide Al in eam i in to2i n specif i c Unspecific chain TGAGGACCGCCCTGGGCCTGGGAGAAT CCCTT .............. - GAAGGAC CAC C C TAGGC C T GGGAGAC T CCCT GA....A. . . .A.......... - Discordance. distance2PAM n.PAM.mismatches n.lead.mismatches - 0 0 24, 23, 16, 11 0 4 Sequence.PAM Start unspecific End unspecific AATCCCTT 113076072 113076103 ACTCCCTC 73171296 73171327 Chromosome EnExon Entrez ID chr6 - 14910 chr11 - 94045 PCSK9 In specific Score peak name.gRNA ch r4: -: 106463720:106463751 266 Nme2PCSK9 gRNA plus PAM nonspecific sequence GGCCTGGCTGATGAGGCCGCACATNNNNNCN N GGCCTGGCTGATGAGGCCGCACATGTGGCCAC guideAlignment2nonspecific Nonspecific chain Scorepredictedcleavage .................... .. ... - 100 Mismatch.distance2PAM n.PAM.mismatches n.guide.mismatches - 0 0 Sequence. PAM Startinspecific Endjnspecific GTGGCCAC 106463720 106463751 Chromosome EnExon EntrezJD chr4 TRUE 100102 7QQOCO / I 7O7 / 3 / YL For identification of nonspecificities, analysis revealed that SpyCas9 DS2, DS4, and DS6 sRNAs appeared to directly edit at 93, 10, and 118 nonspecific candidate sites, respectively, in the normal range of nonspecificities when plasmid-based editing by SpyCas9 it is analyzed by GUIDE-seq (Fu et al., 2014; Tsai et al., 2014). In striking contrast, the Nme2Cas9 DS2, DS4, and DS6 sgRNAs appeared to direct editing at 1.0 and 1 nonspecific sites, respectively. Figure 14C and Table 2. Compared to the GUIDE-seq read counts for SpyCas9 nonspecificities, those for Nme2Cas9 were very low, further suggesting that Nme2Cas9 is highly specific. Figure 13C cf. Figure 13D. GUIDE-seq analyzes of Nme2Cas9 with TS6, Pcsk9, and Rosa26 yielded similar results (0, 0, and 1 nonspecific sites, respectively, with a modest read count for the F?osa26-OT1 nonspecific site). Figure 13C, Figure 14D, and Table 2. Figures 14A to 14E present exemplary data showing that Nme2Cas9 exhibits little or no detectable nonspecific action in mammalian cells. Figure 14A shows an exemplary schematic depicting dual sites (DS) that both SpyCas9 and Nme2Cas9 can target by virtue of their non-overlapping PAMs. The PAM for Nme2Cas9 (orange) and the PAM for SpyCas9 (blue) are highlighted. A 24 nt leader sequence for Nme2Cas9 is indicated in yellow; the corresponding leader sequence for SpyCas9 would be 4 nt shorter at the 5' end. Figure 14B shows an example of Nme2Cas9 and SpyCas9 both inducing indels in DSs. Six DS in VEGFA (with GN3GN19NGGNCC sequences) were selected for direct comparisons of editing of the two orthologs. Plasmids expressing each Cas9 (with the same promoter, linkers, tags and NLSs) and its cognate leader were transfected into HEK293T cells. Indel efficiencies were determined by TIDE 72 hours after transfection. Editing by Nme2Cas9 was detectable at all six sites and was marginally or significantly more efficient than SpyCas9 at two sites (DS2 and DS6, respectively). SpyCas9 edited four of the six sites (DS1, DS2, DS4, and DS6), with two sites showing significantly higher editing efficiencies than Nme2Cas9 (DS1 and DS4). DS2, DS4, and DS6 were selected for GUIDE-Seq analysis, as Nme2Cas9 was equally efficient, less efficient, and more efficient than SpyCas9, respectively, at these sites. Figure 14C shows an example of genome editing with Nme2Cas9 that is highly accurate in human cells. The number of non-specific sites detected by GUIDE-Seq for each nuclease at individual target sites is shown. In addition to the dual sites, we analyzed the TS6 sites (due to their high specific editing efficiency) and Pcsk9 and Rosa26 sites in mouse Hepal -6 cells (to measure accuracy in another cell type). Figure 14D shows exemplary targeted deep sequencing to detect indels in edited cells, confirming the high accuracy of Nme2Cas9 indicated by GUIDE-seq. Figure 14E shows an exemplary sequence for the validated Rosa26 guide nonspecific site, showing the PAM region (underlined), the consensus PAM dinucleotide CC (bold), and three mismatches in the PAM distal portion of the spacer (red). To validate the nonspecific sites detected by GUIDE-Seq, targeted deep sequencing was performed to measure indel formation at major nonspecific loci after independent editing of GUIDE-Seq (ie, without dsODN cotransfection). While SpyCas9 showed considerable editing at most nonspecific sites tested, and in some cases was more efficient than on the corresponding specific site, Nme2Cas9 showed no detectable indels at the only nonspecific candidate sites DS2 and DS6. See, Figure 14D. With Rosa26 sRNA, Nme2Cas9 induced ~1% editing at the Rosa26-OT1 site in Hepal -6 cells, compared to ~30% specific editing. See, Figure 14D. Of note, this non-specific site has a consensus PAM of Nme2Cas9 (ACTCCCT) with only 3 mismatches at the distal end of the guide complementary region PAM (ie, outside the seed region). See, Figure 14E. These data support and reinforce our GUIDESeq results, indicating a high degree of accuracy in Nme2Cas9 genomic editing in mammalian cells. To further corroborate previous GUIDE-Seq results, CRISPRseek was used to computationally predict potential non-specific sites for two active Nme2Cas9 gRNAs targeting TS25 and TS47, both of which are also in VEGFA See, Figure 9A; (Zhu et al., 2014). Three (TS25) or four (TS47) of the predicted sites with the highest agreement, five with the N4CC PAMs and two with the N4CA PAMs; each had 2-5 mismatches, mainly in their distal PAM regions, not seed. See, Figure 13E. The specific edition vs. nonspecific was compared after transfections of the Nme2Cas9+sgRNA plasmid into HEK293T cells by targeted amplification of each locus, followed by TIDE analysis. Consistently, no indels could be detected at non-specific sites for any sgRNA by TIDE, while specific efficient editing was easily detected in the DNA of the same cell populations. Taken together, our data indicate that Nme2Cas9 is a naturally hyperaccurate genome-editing platform in mammalian cells. 7. Supply of associated adenoviruses The compact size, small PAM, and high fidelity of Nme2Cas9 offer important advantages for in vivo genome editing via adeno-associated virus (AAV) delivery. To test whether efficient genome editing by Nme2Cas9 can be achieved by single AAV delivery, Nme2Cas9 with its sgRNA and promoters (U1a and U6, respectively) was cloned into an AAV vector backbone. See, Figure 15A. An all-in-one AAV with a .Nme2Cas9 sRNA packaged in a hepatotropic AAV8 capsid was prepared to target two genes in mouse liver: i) Rosa26 (a safe harbor locus commonly used for transgene insertion) (Friedrich and Soriano, 1991) as a negative control; and i¡) Pcsk9, an important regulator of circulating cholesterol homeostasis (Rashid et al., 2005), as a phenotypic target. Indels induced by SauCas9 or Nme1Cas9 in Pcsk9 in mouse liver outcomes and reduced cholesterol levels provide a useful and easy-to-qualify in vivo benchmark for new editing platforms (Ran et al., 2015; Ibraheim et al., 2018). Nme2Cas9 RNA guides were the same as used above. See, Figure 9B, Figure 13D, and Figures 14A to 14E. Since Rosa26-OT1 was the only Nme2Cas9 nonspecific site that has been validated in cultured mammalian cells, the Rosa26 guide also provided us with the opportunity to assess specific vs. zQQncn / Lznz / q / Yi non-specific in vivo. See, Figures 14D to 14E). The tail veins of two groups of mice (n=5) were injected with 4 x 1011 genome copies (GC) AAV8.sgRNA.Nme2Cas9 targeting Pcsk9o Rosa26. Serum was collected at 0, 14 and 28 days after injection to measure cholesterol level. Mice were sacrificed 28 days after injection and liver tissues were harvested. See, Figure 15A. Targeted deep sequencing of each locus revealed ~38% and -46% indel induction at the Pcsk9 and Rosa26 editing sites, respectively, in the liver. See, Figure 15B. Because hepatocytes constitute only 65-70% of the total cell content in the adult liver, the AAV-induced hepatocyte editing efficiencies for Nme2Cas9 with sgPcsk9 and sgRosa were approximately 5458% and 66-71%, respectively. (Racanelli and Rehermann, 2006). Only 2.25% of overall liver indels (-3-3.5% in hepatocytes) were detected at the Rosa26-OT'\ non-specific site, comparable to the 1% edit we observed at this site in transfected Hepal 6 cells. Figure 15B cf. Figure 14D. At both 14 and 28 days post-injection, editing of Pcsk9 was associated with a -44% reduction in serum cholesterol levels, whereas mice treated with sgfiose26-expressing AAV maintained normal cholesterol for the whole study. See, Figure 15C. The -44% reduction in serum cholesterol in mice treated with AAV Nme2Cas9 / sgPcs / í9 compares well with the -40% reduction reported with the all-in-one AAV for SauCas9 when targeting the same gene (Ran et al. , 2015). Figures 15A to 15C present exemplary data showing Nme2Cas9 genome editing in vivo by all-in-one AAV delivery. Figure 15A shows an exemplary workflow for delivery of AAV8.sgRNA.Nme2Cas9 to reduce cholesterol levels in mice by targeting Pcsk9. Top: Schematic of the all-in-one AAV vector expressing Nme2Cas9 and sgRNA (individual genome elements not to scale). BGH, bovine growth hormone poly(A) site; HA, epitope tag; NLS, nuclear localization sequence; h, codon optimized for human. Bottom: Schedule for tail vein injections of AAV8.sgRNA.Nme2Cas9 (4 x 1011GC), followed by cholesterol measurements on day 14 and indel, histology, and cholesterol analysis on day 28 post-injection. Figure 15B shows an exemplary TIDE assay to measure indels in DNA extracted from the livers of mice injected with AAV8.Nme2Cas9+sgRNA targeting the Pcsk9 and Rosa26 loci (control). The efficiency of indel at the only non-specific site identified by GUIDE-seq for these two sgRNAs (Rosa26|OT1) was also evaluated by TIDE. Figure 15C shows an example of reduced serum cholesterol levels in mice injected with Pcsk9 targeting guide compared to Rosa26 targeting controls. P values ​​were calculated using an unpaired two-tailed t-test. Figures 16A and 16B present exemplary data showing PCSK9 deletion and liver histology after AAV Nme2Cas9 delivery and editing, related to Figures 15A to 15C. Figure 16A shows an exemplary Western blot using an anti-PCSK9 antibody revealing strongly reduced levels of PCSK9 in the livers of sg Pcsk9-treated mice, compared to sgRosa26-treated mice. 2 ng of recombinant PCSK9 was used as the mobility standard (leftmost lane), and a cross-reactive band in the liver samples is indicated with an asterisk. GAPDH was used as a loading control (bottom panel). Figure 16B shows exemplary H&E staining of livers from mice injected with AAV8.Nme2Cas9+sgRosa26 (left) or AAV8.Nme2Cas9+sgPcsk9 (right) vectors. Scale bars, 25 pm. Western blotting was performed using an anti-PCSK9 antibody to estimate PCSK9 protein levels in the livers of sgPcsk9 and sgRosa26-treated mice. Liver PCSK9 was below the detection limit in sg Pcsk9-treated mice, whereas sgRosa26-treated mice had normal PCSK9 levels. See, Figure 16A. Hematoxylin and eosin (H&E) staining and histology revealed no signs of toxicity or tissue damage in either group after Nme2Cas9 expression. See, Figure 16B. These data validate Nme2Cas9 as a highly efficient genome editing system in vivo, even when delivered via single AAV vectors. Recently, AAV vectors have been used for the generation of genome-edited mice, without the need for microinjection or electroporation, simply by submerging the zygotes in culture medium containing AAV vector(s), followed by reimplantation into pseudopregnant females (Yoon et al. , 2018). Editing was previously achieved with a dual AAV system, in which SpyCas9 and its sgRNA were delivered on separate vectors (Yoon et al., 2018). To test whether Nme2Cas9 could perform precise and efficient editing in mouse zygotes with an all-in-one AAV delivery system, we focused on Tyrosinase (Tyr). A bi-allelic inactivation of Tyr disrupts melanin production resulting in an albino phenotype (Yokoyama et al., 1990). An efficient Tyr sRNA that cleaves the Tyr locus just seventeen (17) bp from the site of the classical albino mutation was validated in Hepal -6 cells by transient transfections. See, Figure 17A. Next, C57BL / 6NJ zygotes were incubated for 5-6 hours in culture medium containing 3x109 or 3x108GC of an all-in-one AAV6 vector expressing Nme2Cas9 together with Tyr gRNA. After overnight culture in fresh medium, zygotes that progressed to the two-cell stage were transferred to the oviduct of pseudopregnant recipients and allowed to grow to term. See, Figure 18A. Analysis of the coat color of the pups revealed that the mice were either albino, chinchilla (indicating a hypomorphic Tyrosinase allele), or had motley coat color composed of albino and chinchilla spots, but no black pigmentation. See, Figures 18B and 18C. These results suggest a high frequency of biallelic mutations since the presence of a wild-type Tyrosinase allele should produce black pigmentation. A total of five pups (10%) were born from the 3x109GC experiment. All of them were bearers of indels; phenotypically, two were albino, one was chinchilla, and two had variegated pigmentation, indicating mosaicism. From the 3x108GC experiment, four (4) pups (14%) were obtained, two of which died at birth, precluding a coat color or genome analysis. Analysis of the coat color of the two remaining pups revealed a chinchilla pup and a mosaic pup. These results indicate that single AAV delivery of Nme2Cas9 and its guidance can be used to generate mutations in mouse zygotes without microinjection or electroporation. To measure specific indel formation in the Tyr gene, DNA was isolated from the tails of each mouse, the locus amplified, and TIDE analysis performed. All mice had high levels of specific editing by Nme2Cas9, ranging from 84% to 100%. See, Figures 17B and 17C. Most of the zQQncn / Lznz / q / Yi lesions in the 9-1 albino mouse were a 1- or 4-bp deletion, suggesting mosaicism or transheterozygosity, but the 9-2 albino mouse exhibited a uniform 2-bp deletion. See, Figure 17C. Figures 17A to 17C present exemplary data showing ex vivo Tyr editing in mouse zygotes, related to Figures 16A and 16B. Figure 17A shows an example of two sites on Tyr, each with the N^CC PAMs, tested for editing in Hepa1-6 cells. The sg Tyr2 guide showed higher editing efficiency and was selected for further testing. Figure 17B shows an example of seven mice that survived post-natal development, each exhibiting coat color phenotypes as well as specific editing, as assessed by TIDE. Figure 17C shows an example indel spectrum of tail DNA from each mouse in (B), as well as a raw C57BL / 6NJ mouse, as indicated by TIDE analysis. The efficiency of insertions (positive) and deletions (negative) of various sizes is indicated. Figures 18A to 18C present exemplary data showing ex vivo Nme2Cas9 genome editing by all-in-one AAV delivery. Figure 18A shows an example workflow for Nme2Cas9 ex vivo editing of single AAV to generate C57BL / 6NJ albino mice targeting the Tyr gene. Zygotes are grown in KSOM containing AAV6.Nme2Cas9:sg Tyr for 5-6 hours, rinsed in M2 and grown for one day before transferring into the oviduct of pseudo-pregnant recipients. Figure 18B shows an example of albino mice (left) and chinchilla or motley mice (middle) generated by 3x109GC, and chinchilla or motley mice (right) generated by 3x108GC of AAV6.Nme2Cas9:sg Tyr zygotes. Figure 18C shows an example summary of ex vivo Tyr editing experiments with single AAV from Nme2Cas9.sg Tyr with two doses of AAV. The data is inconclusive as to the absence of mosaicism in the 9-2 mouse, or the absence of additional alleles in the 9-1 mouse, because only tail samples were sequenced and other tissues might have distinct lesions. Analysis of DNA from the tail of chinchilla mice revealed the presence of in-frame mutations that are potentially the cause of chinchilla coat color. The limited mutational complexity suggests that editing occurred early during embryonic development in these mice. These results provide a simplified route to mammalian mutagenesis through the application of a single AAV vector, in this case delivering both Nme2Cas9 and its sgRNA. Figures 19A to 19C show an example of mCherry reporter assay for nSpCas9ABEmax and optimized nNme2Cas9-ABEmax activities. Figure 19A shows exemplary sequence information for the ABE-mCherry reporter. There is a TAG stop codon in the mCherry coding region. In the reporter-integrated stable cell line, there is no mCherry signal due to this stop codon. The mCherry signal will be activated if nSpCas9-ABEmax or optimized nNme2Cas9-ABEmax can convert TAG to CAG, which encodes a glutamine residue. Figure 19B shows an exemplary mCherry signal that is activated due to SpCas9-ABE or Nme2Cas9-ABE activity. Upper panel: negative control (no editing); middle panel: activation of mCherry by nSpCas9-ABEmax; bottom panel: activation of mCherry by optimized nNme2Cas9-ABEmax. Figure 19C shows an example of FACS quantification of base editing events in mCherry reporter cells transfected with SpCas9-ABE or Nme2Cas9-ABE. No.=6; error bars represent S.D. The results are from three biological replicates performed in technical duplicates. Figures 20A to 20C show an example of a GFP reporter assay for the activities of nSpCas9-CBE4 (Addgene #100802) and nNme2Cas9-CBE4 (same plasmid backbone as Addgene #100802). Figure 20A shows exemplary sequence information for the CBEGFP reporter. There is a mutation that converts GYG to GHG in the central region of the fluorophore of the GFP reporter line. There is no GFP signal due to this mutation. The GFP signal will be activated if nSpCas9CBE4 or nNme2Cas9-CBE4 can convert CAO (encoding histidine) to TAC / TAT (encoding tyrosine). Figure 20B shows an exemplary GFP signal that is activated due to nSpCas9-CBE4 or nNme2Cas9-CBE4 activity. Upper panel: negative control (no editing); middle panel: GFP activation by nSpCas9-CBE4; lower panel: GFP activation by nNme2Cas9-CBE4). Figure 20C shows an example of FACS quantification of base editing events in GFP reporter cells transfected with nSpCas9-CBE4 or nNme2Cas9-CBE4. No.=6; error bars represent S.D. Results are from biological replicates performed on technical duplicates. Figure 21 shows an example of cytokine editing by nNme2Cas9-CBE4. The upper panel shows the information on the KANK3 targeting sequence (PAM sequences are indicated in red) of Nme2Cas9 and base editing in the negative control samples. The lower panel shows the quantification of the substitution efficiency of each base type in the nNmeCas9CBE4 editing window of the KANK3 target sequences. The sequence tables show nucleotide frequencies at each position. The expected conversion frequencies from C to T are indicated in red. Figure 22 shows an example of cytosine and adenine editing by nNme2Cas9-CBE4 and nNme2Cas9-ABEmax, respectively. The upper panel shows the information on the PLXNB2 targeting sequence (PAM sequences are indicated in red) of Nme2Cas9 and base editing in the negative control samples. The central panel shows the quantification of the substitution rate of each base type in the editing windows of nNmeCas9-ABEmax of the target sequence PLXNB2. The sequence tables show nucleotide frequencies at each position. The expected conversion frequencies from A to G are highlighted in red. The lower panel shows the quantification of the substitution efficiency of each base type in the nNmeCas9-CBE4 editing windows of the PLXNB2 target sequence. The sequence tables show nucleotide frequencies at each position. The expected conversion frequencies from C to T are highlighted in red. 8. Sequences Alignment of Nme1Cas9 and Nme2Cas9 No aa differences with the PID (underlined in teal); differences of aa with the PID (yellow - bold underlined); active site residues (red - bold). Nme1Cas9 (1-60) MAAFKPNSINYILGLDIGIASVGWAMVEIDEEEENPIRLIDLGVRVFERAEVPKTGDSLAM Nme2Cas9 (1 -60) MAAFKPNPINYILGLDIGIASVGWAMVEIDEEEENPIRLIDLGVRVFERAEVPKTGDSLAM Nme1Cas9 (61-120) ARRLARSVRRLLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIKSLPNTPWQLRAAALDR Nme2Cas9 (61-120) ARRLARSVRRLLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDR Nme1Cas9 (121-180) KLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVAGNAHALQTGDFRTPAEL Nme2Cas9 (121-180) KLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVANNAHALQTGDFRTPAEL Nme1Cas9 (181-240) ALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLM Nme2Cas9 (181-240) ALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLM Nme1Cas9 (241-300) TQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDT Nme2Cas9 (241-300) TQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDT Nme1Cas9 (301-360) ERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRAL Nme2Cas9 (301-360) ERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRAL Nme1Cas9 (361-420) EKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLKDR^QPEILEALLKHISFDKF Nme2Cas9 (361-420) EKEGLKDKKSPLNLSSELQDEIGTAFSLFKTDEDITGRLKDRVQPEILEALLKHISFDKF Nme1Cas9 (421-480) VQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPWLRA Nme2Cas9 (421-480) VQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPWLRA Nme1Cas9 (481-540) LSQARKVINGWRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREY Nme2Cas9 (481-540) LSQARKVINGWRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREY Nme1Cas9 (541-600) FPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSF Nme2Cas9 (541-600) FPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLVRLNEKGYVEIDHALPFSRTWDDSF Nme1Cas9 (601-660) NNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDED zQancn / Lznz / q / Yi Nme2Cas9 (601-660) NNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDED Nme1Cas9 (661-720) GFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVRAEND Nme2Cas9 (661-720) GFKECNLNDTRYVNRFLCQFVADHILLTGKGKRRVFASNGQITNLLRGFWGLRKVRAEND Nme1Cas9 (721-780) RHHALDAVWACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGEVLHQKTHFPQPWEFFA Nme2Cas9 (721-780) RHHALDAVWACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGKVLHQKTHFPQPWEFFA Nme1Cas9 (781-840) QEVMIRVFGKPDGKPEFEEADTLEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSG Nme2Cas9 (781-840) QEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSG Nme1Cas9 (841-895) QGHMETVKSAK---RLDEGVSVLRVPLTQLKLKDLEKMVNR—EREPKLYEALKARLEAH Nme2Cas9 (841-899) AHK-DTLRSAKRFVKHNEKISVKRVWLTEIKLADLENMVNYKNGREIELYEALKARLEAY Nme1Cas9 (896-950) KDDPAKAFAE---P FYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNH —NGIADNATMVRV Nme2Cas9 (900 -954) GGNAKQAFDPKDNPFYKK---G—GQLVKAVRVEKTQESGVLLNKKNAYTIADNGDMVRV Nme1Cas9 (951-1005) DVFEKG-----DKYYLVPIYSWQVAKGIL P DRAWQGKDEEDWQLIDDSFNFKFSLHPND Nme2Cas9 (955-1007) DVFCKVDKKGKNQYFIVPIYAWQVAENILPDIDCKG-------YRIDDSYTFCFSLHKYD Nme1Cas9 (1006-1063) LVEVIT—KKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGIGVKTALSFQKYQI Nme2Cas9 (1008-1063) LIAFQKDEKSKVEFAYYINCDSSNGRFYLAWHDKGSKEQ----QFRISTQNLVLIQKYQV Nme1Cas9 (1064-1082) DELGKEIRPCRLKKRPPVR Nme2Cas9 (1064-1082) NELGKEIRPCRLKKRPPVR Alignment of Nme1Cas9 and Nme3Cas9 No aa differences with the PID (underlined - teal); differences of aa with the PID (yellow - bold underlined); active site residues (red - bold). Nme1Cas9 1 maafkpnsinyilgldigiasvgwamveideeenpirlidlgvrvferae 50 zQancn / Lznz / q / Yi Nme3Cas9 1 maafkpnpinyilgldigiasvgwamveideeenpirlidlgvrvferae 50 Nme1Cas9 51 vpktgdslamarrlarsvrrltrrrahrllrtrrllkregvlqaanfden 100 Nme3Cas9 51 Vpktgdslamarrlarsvrrltrrrahrllrarrllkregvlqaadfden 100 Nme1Cas9101 glikslpntpwqlraaaldrkltplewsavllhlikhrgylsqrkneget 150 Nme3Cas9101 glikslpntpwqlraaaldrkltplewsavllhlikhrgylsqrkneget 150 Nme1Cas9 151 adkelgallkgvagnahalqtgdfrtpaelalnkfekesghirnqrsdys 200 Nme3Cas9 151 adkelgallkgvadnahalqtgdfrtpaelalnkfekecghirnqrgdys 200 Nme1Cas9 201 htfsrkdlqaelillfekqkefgnphvsgglkegietllmtqrpalsgda 250 Nme3Cas9 201 htfsrkdlqaelnllfekqkefgnphvsgglkegietllmtqrpalsgda 250 Nme1Cas9 251 vqkmlghctfepaepkaakntytaerfiwltklnnlrileqgserpltdt 300 Nme3Cas9 251 vqkmlghctfepaepkaakntytaerfiwltklnnlrileqgserpltdt 300 Nme1 Cas9 301 eratlmdepyrkskltyaqarkllgledtaffkglrygkdnaeastlmem 350 Nme3Cas9 301 eratlmdepyrkskltyaqarkllsledtaffkglrygkdnaeastlmem 350 Nme1Cas9 351 kayhaisralekeglkdkksplnlspelqdeigtafslfktdeditgrlk 400 Nme3Cas9 351 kayhtisralekeglkdkksplnlspelqdeigtafslfktdeditgrlk 400 Nme1Cas9 401 driqpeileallkhisfdkfvqislkalrrivplmeqgkrydeacaeiyg 450 Nme3Cas9 401 driqpeileallkhisfdkfvqislkalrrivplmeqgkrydeacaeiyg 450 Nme1Cas9 451 dhygkknteekiylppipadeirnpwlralsqarkvingwrrygspar 500 Nme3Cas9 451 dhygkknteekiylppipadeirnpwlralsqarkvingwrrygspar 500 Nme1Cas9 501 ihietarevgksfkdrkeiekrqeenrkdrekaaakfreyfpnfvgepks 550 Nme3Cas9 501 ihietarevgksfkdrkeiekrqeenrkdrekaaakfreyfpnfvgepks 550 Nme1Cas9 551 kdilklrlyeqqhgkclysgkeinlgrlnekgyveidhalpfsrtwddsf 600 Nme3Cas9 551 kdilklrlyeqqhgkclysgkeinlgrlnekgyveidhalpfsrtwddsf 600 Nme1Cas9 601 nnkvlvlgsenqnkgnqtpyeyfngkdnsrewqefkarvetsrfprskkq 650 Nme3Cas9 601 nnkvlvlgsenqnkgnqtpyeyfngkdnsrewqefkarvetsrfprskkq 650 Nme1Cas9 651 rillqkfdedgfkernlndtrywrflcqfvadrmrltgkgkkrvfasng 700 Nme3Cas9 651 rillqkfdedgfkernlndtrywrflcqfvadrmrltgkgkkrvfasng 700 Nme1Cas9 701 qitnllrgfwglrkvraendrhhaldavvvacstvamqqkitrfvrykem 750 Nme3Cas9 701 qitnllrgfwglrkvraendrhhaldavvvacstvamqqkitrfvrykem 750 Nme1Cas9 751 nafdgktidketgevlhqkthfpqpweffaqevmirvfgkpdgkpefeea 800 Nme3Cas9 751 nafdgktidketgevlhqkthfpqpweffaqevmirvfgkpdgkpefeea 800 Nme1Cas9 801 dtleklrtllaeklssrpeavheyvtplfvsrapnrkmsgqghmetvksa 850 Nme3Cas9 801 dtpeklrtllaeklssrpeavheyvtplfvsrapnrkmsgqghmetvksa 850 Nme1Cas9851 krldegvsvlrvpltqlklkdlekmvnrerepklyealkarleahkddpa 900 Nme3Cas9 851 krldegvsvlrvpltqlklkdlekmvnrerepklyealkarleahkddpa 900 Nme1Cas9 901 KAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRV 950 Nme3Cas9 901 KAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRV 950 Nme1Cas9 951 dvfekgdkyylvpiyswqvakgilpdravvqgkdeedwqliddsfnfkfs 1000 Nme3Cas9 951 dvfekgdkyylvpiyswqvakgilpdrawayadeedwtvidesfrfkfv 1000 Nme1Cas91001 lhpndlvevitkkarmfgyfaschrgtgninirihdldhkigkngilegi 1050 Nme3Cas9 1001 lysndlikvqlkkdsflgyfsgldratgaislrehdlekskgkdg^íRI 1049 Nme1Cas91051 gvktalsfqkyqidelgkeirpcrlkkrppvr1082 Nme3Cas9 1050 gvktalsfqkyqidemgkeirpcrlkkrppvr 1081 Nme2Cas9 expressed on plasmid NLS of SV40 (yellow- BOLD); Label 3X-HA (green-(underlined / black); cMyc-like NLS (teal- plain); Linker (magenta-bold italic) and Nme2Cas9 (italic). MAAFKPNPINYILGLDIGIASVGWAMVEIDEEEENPIRLIDLGVRVFERAEVPKTGDSLAMARRLARSVRRLLTRRRAHR LLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELG ALLKGVANNAHALQTGDFRTPAELALNKFEKESGHIRNQ RGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEG IETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRK SKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSSEL QDEIGTAFSLFK TDEDITGRLKDRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIP ADEIRNPWLRALSQARKVINGWRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFVG EPKSKDILKLRLY EQQHGKCLYSGKEINLVRLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFN GKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKECNLNDTRYVNRFLCQFVADHILLTGKGKRRVFASNGQI TNLLRGFWGLRKVRAENDRHHALDAVWACS TVAMQQKITRFVRYKEMNAFDGKTIDKETGKVLHQKTHFPQPWEFFA QEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGAHKDTLRSAKRFVKHNEK ISVKRVWLTEIKLADLENMVNYKNGREIELYEALKARLEAYGGNAKQAFDPKDNPF YKKGGQLVKAVRVEKTQESGVL LNKKNAYTIADNGDMVRVDVFCKVDKKGKNQYFIVPIYAWQVAENILPDIDCKGYRIDDSYTFCFSLHKYDLIAFQKD EKSKVEFAYYINCDSSNGRFYLAWHDKGSKEQQFRISTQNLVLIQKYQVNELGKEIRPCRLKKRPPVRG TGGP'KKKRK VYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAGSAAPAAKKKKLD FE S G* Nme2Cas9 expressed by AA V NLS of SV40 (yellow- BOLD); 3X-HA tag (green-funderlining / bold): nucleoplasmin-like NLS (red-underlining); NLS of c-myc (teal-raw); Linker (magenta bold italics) and Nme2Cas9 (italics). MVP'KKKRKVEDYRPAAEKKAGQA'KYYYMAAFKPNPINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEV PKTGDSLAMARRLARSVRRLLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSA VLLHLIKHRGYLSQRKNEGETADKELGALLK GVANNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKD LQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNN LRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD NAEASTLMEMKAYHAISRALEKE GLKDKKSPLNLSSELQDEIGTAFSLFKTDEDITGRLKDRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRY DEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPWLRALSQARKVINGWRRYGSPARIHIETAREVGKSFKDRKEI zoancn / i znz / n / Y EKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLVRLNEKGYVEIDHALPFSRTWD DSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKECNLNDTRYVNR FLCQFVADHIL LTGKGKRRVFASNGQITNLLRGFWGLRKVRAENDRHHALDAVWACSTVAMQQKITRFVRYKEMNAF DGKTIDKETGKVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVS RAPNRKMSGAHKDTLRSAKRFVKHNEKISVKRVW LTEIKLADLENMVNYKNGREIELYEALKARLEAYGGNAKQAFDP KDNPFYKKGGQLVKAVRVEKTQESGVLLNKKNAYTIADNGDMVRVDVFCKVDKKGKNQYFIVPIYAWQVAENILPDID CKGYRIDDSYTFCFSLHKYDLIAFQKDEKSKVEFAYYINCDSSNGRFYLAWHDK GSKEQQFRISTQNLVLIQKYQVNE PGKPTPPCRRKKRPPVREDKRPAATKKAGQAKKKKYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAAPAAKKKKLD* Recombinant Nme2Cas9 NLS of SV40 (yellow- BOLD); Nucleoplasmin-like NLS (red-underlined): Linker (magenta - bold italics) and Nme2Cas9 {italics). P'KKKRKVNAMAAFKPNPINYILGLDIGIASVGWAMVEIDEEEENPIRLIDLGVRVFERAEVPKTGDSLAMARRLARSVR RLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNE GETADKELGALLKGVANNAHALQTGDFRTPAELALNK FEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEFGNP HVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERA TLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKD KKSPLNLSSELQDE IGTAFSLFKTDEDITGRLKDRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTE EKIYLPPIPADEIRNPWLRALSQARKVINGWRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKF REYFPNFVGEPK SKDILKLRLYEQQHGKCLYSGKEINLVRLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKG NQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKECNLNDTRYVNRFLCQFVADHILLTGKGKR RVFASNGQITNLLRGFWGLRKVRAEND RHHALDAWVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGKVLHQKTH FPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGAHKDTLRSA KRFVKHNEKISVKRVWLTEIKLADLENMVNYKNGREIELYEALKARLEAYGG NAKQAFDPKDNPFYKKGGQLVKAVRV EKTQESGVLLNKKNAYTIADNGDMVRVDVFCKVDKKGKNQYFIVPIYAWQVAENILPDIDCKGYRIDDSYTFCFSLHK YDLIAFQKDEKSKVEFAYYINCDSSNGRFYLAWHDKGSKEQQFRISTQNLVLIQKYQVNELGKEIR PCRLKKRPPVRG GGGSGGGGSGGGGSPAAKKKKLDGGGSKRPAATKKAGQAKKKK* Recombinant Nme2Cas9 for use in RNP delivery in mammalian cells: NLS of SV40 (yellow- BOLD); Nucleoplasmin-like NLS (red-underlined): Linker (magenta - bold italics) and Nme2Cas9 {italics). PKKKBKVNAMAAFKPNPINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGDSLAMARRLARSVR RLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNE GETADKELGALLKGVANNAHALQTGDFRTPAELALNKFEK ESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEFGNP HVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERA TLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKK SPLNLSSELQDE IGTAFSLFKTDEDITGRLKDRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTE EKIYLPPIPADEIRNPWLRALSQARKVINGWRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKF REYFPNFVGEPKSK DILKLRLYEQQHGKCLYSGKEINLVRLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKG NQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKECNLNDTRYVNRFLCQFVADHILLTGKGKR RVFASNGQITNLLRGFWGLRKVRAENDRH HALDAVWACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGKVLHQKTH zQancn / Lznz / q / Yi FPQPWEFFAQEVMLRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGAHKDTLRSA KRFVKHNEKISVKRVWLTEIKLADLENMVNYKNGREIELYEALKARLEAYGGNAKQAFDPKDNPFYKKGGQLVKAVRV EKTQESGVLLNKKNAYTIADNG DMVRVDVFCKVDKKGKNQYFIVPIYAWQVAENILPDIDCKGYRIDDSYTFCFSLHK YDLIAFQKDEKSKVEFAYYINCDSSNGRFYLAWHDKGSKEQQFRISTQNLVLIQKYQVNELGKEIRPCRLKKRPPVRG GGGSGGGGSGGGGSPAMAKKKLDGGGSKRPAATKFAGQ AKKKK* 9. Therapeutic Applications Although compact Cas9 orthologs have previously been validated for genome editing, even through single AAV delivery, their longer MAPs have restricted therapeutic development due to lower target site frequencies than that of the compact Cas9 orthologs. SpyCas9 most widely adopted. Furthermore, SauCas9 and its KKH variant with relaxed PAM requirements (Kleinstiver et al., 2015) are prone to non-specific editing with some sgRNAs (Friedland et al., 2015; Kleinstiver et al., 2015). These limitations are compounded with target loci that require editing within a narrow sequence window, or that require a precise segmental deletion. We have identified Nme2Cas9 as a compact, high-accuracy Cas9 with a less restrictive dinucleotide PAM for genome editing by AAV delivery in vivo. The development of Nme2Cas9 greatly expands the genomic scope of in vivo editing, especially through viral vector transfer. The all-in-one AAV delivery platform for Nme2Cas9 established in this study can, in principle, be used to target a wide range of sites like SpyCas9 (due to the identical densities of the optimal N4CC and NGG PAMs), but without the need for delivering two separate vectors to the same target cells. The availability of a catalytically inactive version of Nme2Cas9 (dNme2Cas9) also promises to expand the scope of applications such as CRISPRI, CRISPRa, base editing, and related approaches (Domínguez et al., 2016; Komor et al., 2017). Furthermore, the hyperaccuracy of Nme2Cas9 allows for precise editing of target genes, potentially ameliorating safety concerns resulting from non-specific activities. Perhaps counterintuitively, the higher target site density of Nme2Cas9 (compared to that of Nme1Cas9) does not lead to a relative increase in non-specific editing for the former. Similar results have recently been reported with SpyCas9 variants evolved to have shorter MAPs (Hu et al., 2018). Type II-C Cas9 orthologs are generally slower nucleases in vitro than SpyCas9 (Ma et al., 2015; Mir et al, 2018); interestingly, enzymological principles indicate that a reduced apparent feat (within limits) can improve specificity in vs. off-target for RNA-guided nucleases (Bisaría et al., 2017). The discovery of Nme2Cas9 and Nme3Cas9 depended on unexplored Cas9s that are highly related (outside the PID) to an ortholog that was previously validated for human genome editing (Esvelt et al., 2013; Hou et al., 2013; Lee et al. ., 2016; Amrani et al., 2018). The relationship of Nme2Cas9 and Nme3Cas9 with Nme1Cas9 brought an additional benefit, that is, that they use exactly the same sRNA scaffold, avoiding the need to identify and validate functional tracrRNA sequences for each. In the context of natural CRISPR immunity, the accelerated evolution of novel PAM specificities might reflect selective pressure to restore zQQncn / iznz / u / Yl targeting of phage and MGEs that have escaped interference via mutations. of PAMs (Deveau et al., 2008; Paez-Espino et al., 2015). Our observation that AcrllC5s™ inhibits Nme1Cas9 but not Nme2Cas9 suggests a second, non-mutually exclusive basis for accelerated PID variation, specifically, evasion of anti-CRISPR inhibition. We also speculate that the accelerated variability may not be restricted to PIDs, perhaps as a result of selective pressures to evade anti-CRISPR binding to other Cas9 domains. Cas9 inhibitors such as AcrlICI that bind to more conserved regions of Cas9 likely present fewer pathways to mutational escape and therefore exhibit a broader inhibitory spectrum (Harrington et al., 2017a). Regardless of the sources of selective pressure driving the co-evolution of Acr and Cas9, the availability of validated inhibitors of Nme2Cas9 (eg, AcrlICI -4) provides opportunities for additional levels of control over their activities. The approach used in this study (ie, the search for rapidly evolving domains within Cas9) can be implemented elsewhere, especially with bacterial species that are well sampled at the genome sequence level. This approach could also be applied to other CRISPR-Cas effector proteins such as Casi 2 and Casi 3 that have also been developed for genome or transcriptome engineering and other applications. This strategy could be especially convincing with Cas proteins that are closely related to orthologs with proven efficacy in heterologous contexts (eg, in eukaryotic cells), as was the case with Nme1Cas9. Application of this approach to meningococcal orthologs of Cas9 yielded a novel genome editing platform, Nme2Cas9, with a unique combination of features (compact size, PAM dinucleotide, hyperaccuracy, single-AAV delivery capability, and Acr susceptibility) that they promise to accelerate the development of genome-editing tools for general and therapeutic applications. 7Qoncn / i 7n7 / 3 / YL Table 3. Below are examples of sequences for plasmids and oligos as disclosed herein. Example Plasmids Plasmid No. Name Insert Description Main Structure Purpose Insertion Sequence 1 pAE70 Nme3Cas9 PID into Nme1Cas9 pMCSG7 Bacterial Expression of Nme1Cas9 with Nme3Cas9 PID See examples here 2 pAE71 Nme2Cas9 PID into Nme1Cas9 pMCSG7 Bacterial Expression of Nme1Cas9 with Nme2Cas9 PID See examples here 3 pAE113 Nme2TLR1 pLKO Targeting TLR2.0 with Nme2Cas9 GTCACCTGCCTCGTGGAATAC GG 4 pAE114 Nme2TLR2 pLKO Targeting TLR2.0 with Nme2Cas9 GCACCTGCCTCGTGGAATACG GT 5 pAE115 Nme2TLR5 pLKO Targeting TLR2.0 with Nme2Cas9 GTTCAGCGTGTCCGGCTTTGG C 6 pAE116 Nme2TLR11 pLKO Targeting TLR2.0 with Nme2Cas9 GTGGTGAGCAAGGGCGAGGAG CTG 7 pAE117 Nme2TLR12 pLKO Targeting TLR2.0 with Nme2Cas9 GGGCGAGGAGCTGTTCACCGG GGT 8 pAE118 Nme2TLR13 pLKO Targeting TLR2.0 with GTGAACTTGTGGCCG TTTACG Nme2Cas9 TCG 9 pAE119 Nme2TLR14 pLKO Targeting TLR2.0 with Nme2Cas9 GCGTCCAGCTCGACCAGGATG GGC 10 pAE120 Nme2TLR15 pLKO Targeting TLR2.0 with Nme2Cas9 GCGGTGAACAGCTCCTCGCCC TTG 11 pAE121 Nme2TL R16 pLKO Targeting TLR2.0 with Nme2Cas9 GGGCACCACCCCGGTGAACAG CTC 12 pAE122 Nme2TLR17 pLKO Targeting TLR2.0 with Nme2Cas9 GGCACCACCCCGGTGAACAGC TCC 13 pAE123 Nme2TLR18 pLKO Targeting TLR2.0 with Nme2Cas9 GGGATGGGCACCACCCGGTG AAC 14 pAE124 Nme2TLR19 pLKO Targeting TLR2.0 with Nme2Cas9 GCGTGTCCGGCTTTGGCGA GA CAA 15 pAE125 Nme2TLR20 pLKO Targeting TLR2.0 with Nme2Cas9 GTCCGGCTTTGGCGAGACAAA TCA 16 pAE126 Nme2TLR21 pLKO Targeting TLR2 .0 with Nme2Cas9 GATCACCTGCCTCGTGGAATA CGG 17 pAE149 Nme2TLR22 pLKO Targeting TLR2.0 with Nme2Cas9 GACGCTGAACTTGTGGCCGTT TAC 18 pAE150 Nme2TLR23 pLKO Targeting TLR2.0 with Nme2Cas9 GCCAAAGCCGGACACGCT GAA CTT 19 pAE193 Nme2TLR13 with 23 nt spacer pLKO Targeting TLR2.0 with Nme2Cas9 GGAACTTGTGGCCGTTTACGT CG 20 pAE194 Nme2TLR13 with 22 nt spacer pLKO Targeting TLR2.0 with Nme2Cas9 GAACTTGTGGCCGTTTACGTC G 21 pAE195 Nme2TLR13 with 21 nt spacer pLKO Targeting TLR2.0 with Nme2Cas9 GACTTGTGGCCGTTTACGTCG 22 pAE196 Nme2TLR13 with 20 nt spacer pLKO Targeted TLR2.0 with Nme2Cas9 GCTTGTGGCCGTTTACGTCG 23 pAE197 Nme2TLR13 with 19 nt spacer pLKO Targeting TLR2.0 with Nme2Cas9 GTTGTGGCCGTTTACGTCG 24 pAE213 Nme2TLR21 with G22 spacer pLKO Targeting TLR2.0 with Nme2Cas 9 GTCACCTGCCTCGTGGAATAC GG 25 pAE214 Nme2TLR21 with G21 spacer pLKO Targeted at TLR2.0 with Nme2Cas9 GCACCTGCCTCGTGGAATACG G 26 pAE215 Nme2TLR21 with G20 spacer pLKO Targeting TLR2.0 with Nme2Cas9 GACCTGCCTCGTGGAATACGG 27 pAE216 Nme2TLR21 with G19 spacer pLKO Targeting TLR2.0 with Nme2Cas9 G CCTGCCTCGTGGAATACGG 28 pAE90 Nme2TS1 pLKO Targeting AAVS1 with Nme2Cas9 GGTTCTGGGTACTTTTATCTG TCC 29 pAE93 Nme2TS4 pLKO Targeting AAVS1 with Nme2Cas9 GTCTGCCTAACAGGAGGTGGG GGT 30 pAE94 Nme2TS5 pLKO Targeting AAVS1 with Nme2Cas9 GAATATCAGGAGACTAGGAAG GAG 31 pAE129 Nme2TS6 pLKO Targeting LINC01588 with Nme2Cas9 GCCTCCCTGCAGG GCTGCTCC C 32 pAE130 Nme2TS10 pLKO Targeting AAl / Yes with Nme2Cas9 GAGCTAGTCTTCTTCCTCCAA CCC 33 pAE131 Nme2TS11 pLKO Targeting AAVS1 with Nme2Cas9 GATCTGTCCCCTCCCACCCCAC AGT zQQncn / Lznz / q / γι 34 pAE132 Nme2TS12 pLKO Targeted AAVS1 with Nme2Cas9 GGCCCAAATGAAAGGAGTGAG AGG 35 pAE133 Nme2TS13 pLKO Targeted AAVS1 with Nme2Cas9 GCATCCTCTTGCTTTCTTTGC CTG 36 pAE136 Nme2TS16 pLKO Targeted to LINC01588 with Nme2Cas9 GGAGTCGCCAGAGGCCGGTGG TGG 37 pAE137 Nme2TS17 pLKO Targeted at LINC01588 with Nme2Cas9 GCCCAGCGGCCGGATATCAGC TGC 38 pAE138 Nme2TS18 pLKO Targeted at CYBBcon Nme2Cas9 GGAAGGGAACATATTACTATT GC 39 pAE139 Nme2TS19 pLKO Directed at CYBBcon Nme2Cas9 GTGGAGTGGCCTGCTATCAGC TAC 40 pAE140 Nme2TS20 pLKO Directed at CYBBcon Nme2Cas9 GAGGAAGGGAACATATTACTA TTG 41 p AE141 Nme2TS21 pLKO Targeted at CYBB with Nme2Cas9 GTGAATTCTCATCAGCTAAAA TGC 42 pAE144 Nme2TS25 pLKO Targeted at VEGFA with Nme2Cas9 GCTCACTCACCCACACAGACA CAC 43 pAE145 Nme2TS26 pLKO Targeted to CFTR with Nme2Cas9 GGAAGAATTTCATTCTGTTCT CAG 44 pAE146 Nme2TS27 pLKO Targeting CFTR with Nme2Cas9 GCTCAGTTTTCCTGGATTATG CCT 45 pAE152 Nme2TS31 pLKO Targeting VEGFA with Nme2Cas9 GCGTTGGAGCGGGGAGAAGGC CAG 4 6 pAE153 Nme2TS34 pLKO Targeting LINC01588 with Nme2Cas9 GGGCCGCGGAGATAGCTGCAG GGC 47 pAE154 Nme2TS35 pLKO Targeting LINC01588 with Nme2Cas9 GCCCACCCGGCGGCGCCTCCC TGC 48 pAE155 Nme2TS36 pLKO Targeting LINC01588 with Nme2Cas9 GCGTGGCAGCTGATATCCGGC CGC 49 pAE156 Nme2TS37 pLKO Targeting LINC01588 with Nme2Cas9 GCCGCGGCGCGACGTGGAGCC AGC 50 pAE157 Nme2TS38 p LKO Targeted LINC01588 with Nme2Cas9 GTGCTCCCCAGCCCAAACCGC CGC 51 pAE159 Nme2TS41 pLKO Targeted AGA with Nme2Cas9 GTCAGATTGGCTTGCTCGGAA TTG 52 pAE185 Nme2TS44 pLKO Targeted VEGFA with Nme2Cas9 GCTGGGTGAATGGAGCGAGCA GCG 53 pAE186 Nme2TS45 pLKO Targeting VEGFA with Nme2Cas9 GTCCTGGAGTGACCCCTGGCC TTC 54 pAE187 Nme2TS46 pLKO Targeting VEGFA with Nme2Cas9 GATCCTGGAGTGACCCCTGGC C TT 55 pAE188 Nme2TS47 pLKO Targeting VEGFA with Nme2Cas9 GTGTGTCCCTTCCCCCACCCG TCC 56 pAE189 Nme2TS48 pLKO Targeting VEGFA with Nme2Cas9 GTTGGAGCGGGGAGAAGGCCA GGG 57 pAE190 Nme2TS49 pLKO Targeting VEGFA with Nme2Cas9 GCGTTGGAGCGGGGAGAAGGC CAG 58 pAE191 Nme2TS50 pLKO Targeting AGA with Nme2Cas9 GTACCCTCCAATAATTTGGCT GGC 59 pAE192 Nme2TS51 pLKO Targeting AGA with Nme2Cas9 G ATAATTTGGCTGGCAATTCC GAG 60 pAE232 TS64_FancJ1 pLKO Targeted at FANCJ with Nme2Cas9 GAAAATTGTGATTTCCAGATC CAC 61 pAE233 TS65_FancJ2 pLKO Targeted at FANCJ with Nme2Cas9 GAGCAGAAAAAATT GT GATTT CC 62 pAE200 Nme2TS58 (Nme2DS1) pLKO Targeting DS in VEGFA with Nme2Cas9 GCAGGGGCCAGGTGTCCTTCT CTG 63 pAE201 Nme2TS59 (Nme2DS2) pLKO Targeting DS in VEGFA with Nme2Cas9 GAATGGCAGGCGGAGGTTGTA CTG ZQQnCn / l 7Π7 / Σ1 / Υ 64 pAE202 Nme2TS60 (Nme2DS3) pLKO Targeted DS in VEGFA with Nme2Cas9 GAGT GAGAGGT GAGAGAGAG ACA 65 pAE203 Nme2TS61 (Nme2DS4) pLKO Targeted DS in VEGFA with Nme2Cas9 GTGAGCAGGCACCTGTGCCAA CAT 66 pAE2 04 Nme2TS62 (Nme2DS5) pLKO Targeted DS in VEGFA with Nme2Cas9 GCGTGGGGGCTCCGTGCCCCA CGC 67 pAE205 Nme2TS63 (Nme2DS6) pLKO Targeted DS in VEGFA with Nme2Cas9 GCATGGGCAGGGGCTGGGGTG CAC 68 pAE207 SpyDSI pLKO Targeted DS in VEGFA with SpyCas9 GGGCCAGGTGTCCTTCTCTG 69 pAE208 SpyDS2 p LKO Directed to DS in VEGFA with SpyCas9 GGCAGGCGGAGGTTGTACTG 70 pAE209 SpyDS3 pLKO Directed to DS in VEGFA with SpyCas9 GAGAGAGT GAGAGAGAGACA 71 pAE210 SpyDS4 pLKO Targeted DS in VEGFA with SpyCas9 GCAGGCACCTGTGCCAACAT 72 pAE211 SpyDS5 pLKO Targeted DS in VEGFA with SpyCas9 GGGGGCTCCGTGCCCCACGC 73 pAE212 SpyDS 6 pLKO Targeting DS in VEGFA with SpyCas9 GGGCAGGGGCTGGGGTGCAC 74 pAE169 hDeCas9 Wt on AAV AAV backbone Nme2Cas9 all-in-one AAV expression with sgRNA cassette See examples herein. pAE217 hDeCas9 wt in pMSCG7 backbone pMCSG7 Nme2Cas9 wild type for expression in bacteria See examples herein. 76 pAE107 2xNLS Nme2Cas9 with HA pCdest CMV-driven Nme2Cas9 expression plasmid See examples herein. 77 pAE127 hDemonCas9 3X NLS in pMSCG7 pMSCG7 Targeted Endogenous Loci with Nme2Cas9 See examples herein. 78 pAM172 hNme2Cas9 4X NLS with 3XHA pCVL Lentivector containing UCOE, SFFV-driven Nme2Cas9 and Puro See examples herein. 79 pAM174 hNme2Cas9 D16A4XNLS nickase with 3XHA pCVL Lentivector containing UCOE, SFFV driven Nme2Cas9 and Puro See examples herein. 80 pAM175 hNme2Cas9 H588A 4X NLS nickase with 3XHA pCVL Lentivector containing UCOE, SFFV driven Nme2Cas9 and Puro See examples herein. 81 pAM177 hNme2Cas9 4X NLS inactivates with 3XHA pCVL Lentivector containing UCOE, SFFV-driven Nme2Cas9 and Puro See examples herein. 7QQnen / l 7Π7 / Σ1 / Υ Example Oligonucleotides Number Name Sequence Purpose 1 AAVS1TIDE1FW TGGCTTAGCACCTTCCCAT TIDE Analysis 2 LINC01588_TIDE_FW AGAGGAGCCTTCTGACTGCTGCAGA TIDE Analysis 3 AAVS1TIDE2FW TCCGTCTTCTCCCACTCC TIDE Analysis 4 NTS55_TIDE_FW TAGAGAACTGGGTAG TGTG TIDE Analysis 5 VEGF_TIDE3_FW GTACATGAAGCAACTCCAGTCCCA TIDE Analysis 6 hCFTR_TIDE1_FW TGGTGATTATGGGAGAACTGGAGC TIDE Analysis 7 AGATIDE1FW GGCATAAGGAAATCGAAGGTC TIDE Analysis 8 VEGFTIDE4FW ACAC GG GCAG CAT G GGAATAGT C TIDE Analysis 9 VEGF_TIDE5_FW CCTGTGTGGCTTTGCTTTGGTCG TIDE Analysis 10 VEGF_TIDE6_FW GGAGGAAGAGTAGCTCGCCGAGG TIDE Analysis 11 VEGF_T IDE7_FW AGGGAGAGGGAAGTGTGGGGAAGG TIDE Analysis 12 AAVS1_TIDE1_RV AGAACTCAGGACCAACTTATTCTG TIDE Analysis 13 LINC01588_TIDE_RV AT GACAGACACAAC CAGAGGGCA TIDE Analysis 14 AAVS1_TIDE2_RV TAGGAAGGAGGAGGCCTAAG TIDE Analysis 15 NTS 55_TIDE_RV CCAATATTGCATGGGATGG TIDE Analysis 16 VEGF_TIDE3_RV ATCAAATTCCAGCACCGAGCGC TIDE Analysis 17 hCFTR_TIDE1_RV ACCATTGAGGACGTTTGTCTCAC TIDE Analysis 18 AGA_TIDE1_RV CATGTCCTCAAGTCAAGAACAAG TIDE Analysis 19 VEGFTIDE4RV GCTAGGGGAGAGTCCCACTGTC CA TIDE Analysis 20 VEGF_TIDE5_RV GTAGGGTGTGATGGGAGGCTAAGC TIDE Analysis 21 VEGF_TIDE6_RV AGACCGAGTGGCAGTGACAGCAAG TIDE Analysis 22 VEGFTIDE7RV GTCTTCCTGCTCTGTGCGCACGAC TIDE Analysis 23 PAMAIeatorio_FW TAGCGGCCGCTCATGCGCGGCGCATTACC TTTACNNNNNNNNNNGGA TCCTCTAGAGTCG Protospacer with randomized PAM 24 PAMAIrandom_RV ACAGGAAACAGCTATGACCATGAAAGCTTGCATGCCTGCAGGTCGAC TCTAGAGGATC Protospacer with Randomized PAM 25 DS2_ON_FW1 ctacacgacgctcttccgatctCCTGGAGCGTGTACGTTGG Targeted Deep Seum 26 SpyDS2_OT1_FW1 ctacacgacgctcttccgatctCCTGTGGTCCCAGCTACTTG Targeted Deep Seum 27 SpyDS2_OT2_FW1 ctacacgacg ctcttccgatctATCTGCGATGTCCTCGAGG Directed Deep Sec 28 SpyDS2_OT3_FW1 ctacacgacgctcttccgatctTGGTGTGCGCCTCTAACG Directed Deep Sec 29 SpyDS2_OT4_FW1 ctacacgacgctcttccgatctGGAGTCTTGCTTTGTCACTCAGA Directed Deep Sec 30 SpyDS2_OT5_ FW1 ctacacgacgctcttccgatctAGCCTAGACCCAGTCCCAT Directed Deep Sec 31 SpyDS2_OT6_FW1 ctacacgacgctcttccgatctGCTGGGCATAGTAGTGGACT Directed Deep Sec 32 SpyDS2_OT7_FW1 ctacacgacgctcttccgatctTGGGGAGGCTGAGACACGA Sec Directed Deep Sec 33 SpyDS2_OT8_FW1 ctacacgacgctcttccgatctCTTGGGAGGCTGAGGCAAG Directed Deep Sec 34 DS2_ON_RV1 agacgtgtgctcttccgatctCAGGAGGATGAGAGCCAGG Directed Deep Sec 35 SpyDS2_OT1_RV1 agacgtgtgctcttccgatctCAGGGTC TCACTCTATCACCCA Directed Deep Sec 36 SpyDS2_OT2_RV1 agacgtgtgctcttccgatctACTGAATGGGTTGAACTTGGC Directed Deep Sec 37 SpyDS2_OT3_RV1 agacgtgtgctcttccgatctGAGACAGAATCTTGCTCTGTCTCC Directed Deep Sec 7QQnen / l 7Π7 / Σ1 / Υ 38 SpyDS2_OT4_RV1 agacgtgtgctcttccgatctTCCCAGCTACTTGGGAGGC Directed Deep Sec 39 SpyDS2_OT5_RV1 agacgtgtgctcttccgatctCCTGCCCAAATAGGGAAGCAG Directed Deep Sec 40 SpyDS2_OT6_RV1 agacgtgtgctcttccgatctTGGCGCC TTAGTCTCTGCTAC Directed Deep Sec 41 SpyDS2_OT7_RV1 agacgtgtgctcttccgatctGCATGAGACACAGTTTCACTCTG Directed Deep Sec 42 SpyDS2_OT8_RV1 agacgtgtgctcttccgatctGAGAGAGTCTCACTGCGTTGC Directed Deep Sec 43 DS4_ON_FW3 ctacacgacgctctt ccgatctTCTCTCACCCACTGGGCAC Directed Deep Sec 44 DS4_ON_RV3 agacgtgtgctcttccgatctGCTTCCAGACGAGTGCAGA Directed Deep Sec 45 SpyDS4_OT1_FW1 ctacacgacgctcttccgatctAAGTTTTCAAACCAGAAGAACTACG AC Directed Deep Sec 46 SpyDS4_OT2_FW1 ctacacgacgctcttccgatctCCGGTATAAGTCCTGGAGCG Targeted Deep Sec 47 SpyDS4_OT3_FW1 ctacacgacgctcttccgatctGCCAGGGAGCAATGGCAG Targeted Deep Sec 48 SpyDS4_OT6_FW1 ctacacgacgctcttccgatctCCTCGAATTCCACGGG GTT Directed Deep Sec 49 DS16_ON_FW1 ctacacgacgctcttccgatctGTTGGTGGGAGGGAAGTGAG Directed Deep Sec 50 SpyDS6_OT1_FW1 ctacacgacgctcttccgatctGATGGCGGTTGTAGCGGC Directed Deep Sec 51 SpyDS6_OT2_FW1 ctacacgacgctcttccga tctCACATAAACCTATGTTTCAGCAGA Targeted Deep Sec 52 SpyDS6_OT3_FW1 ctacacgacgctcttccgatctGCTAGTTGGATTGAAGCAGGGT Targeted Deep Sec 53 SpyDS6_OT4_FW1 ctacacgacgctcttccgatctTTGAGTGCGGCAGCTTCC Targeted Deep Sec 54 SpyDS6_OT6_FW1 ctacacgacgctcttccgatctATAACCCTCCCAGGCAAAGTC Directed Deep Sec 55 SpyDS6_OT7_FW1 ctacacgacgctcttccgatctAGCCTGCACATCTGAGCTC Directed Deep Sec 56 SpyDS6_OT8_FW1 ctacacgacgctcttccgatctGGAGCATTGAAGTGCCTGG Directed Deep Sec 57 DeDS6_ON_RV1 agacgtgtgctcttccgatctCAGC CTGGGACCACTGA Directed Deep Sec 58 SpyDS6_OT1_RV1 agacgtgtgctcttccgatctCATCCTCGACAGTCGCGG Directed Deep Sec 59 SpyDS6_OT2_RV1 agacgtgtgctcttccgatctGACTGATCAAGTAGAATACTCATGGG Directed Deep Sec 60 SpyDS6_OT3_RV1 agacgtgtgctct tccgatctCCCTGCCAGCACTGAAGC Directed Deep Sec 61 SpyDS6_OT4_Rv1 agacgtgtgctcttccgatctGGTTCCTATTCTTTCTAGACCAGGAGT Directed Deep Sec 62 SpyDS6_OT6_RV1 agacgtgtgctcttccgatctAGTGTGGAGGGCTCAGGG Deep Sec Targeted 63 SpyDS6_OT7_RV1 agacgtgtgctcttccgatctGATGGGCAGAGGAAGGCAA Targeted Deep Sec 64 SpyDS6_OT8_RV1 agacgtgtgctcttccgatctTCACTCTCATGAGCGTCCCA Targeted Deep Sec 65 Nme2DS2_OT1_FW1 ctacacgacgctcttccgatctAAGGTT CCTTGCGGTTCGC Directed Deep Sec 66 Nme2DS2_OT1_RV1 agacgtgtgcttccgatctCGCTGCCATTGCTCCCT Directed Deep Sec 67 Nme2DS6_OT1_FW1 ctacacgacgctcttccgatctTCTCGCACATTCTTCACGTCC Directed Deep Sec 7QQnen / l 7Π7 / Σ1 / Υ 68 Nme2DS6_OT1_RV1 agacgtgtgctcttccgatctAGGAACCTTCCCGACTTAGGG Directed Deep Sec 69 Rosa26_ON_FW1 ctacacgacgctcttccgatctCCCGCCCATCTTCTAGAAAGAC Directed Deep Sec 70 Rosa26_OT1_FW1 ctacacgacgct cttccgat ctTGCCAGGTGAG GGACTGG Directed Deep Sec 71 Rosa26_ON_RV1 agacgtgtgctcttccgatctTCTGGGAGTTCTCTGCTGCC Directed Deep Sec 72 Rosa26_OT1_RV1 agacgtgtgctcttccgatctTGCCCAACCTTAGCAAGGAG Directed Deep Sec 73 pCSK9_ON_FW2 ctacacgacgctcttccgatctta ccttggagcaacggcg Directed Deep Sec 74 PCSK9ONRV2 agacgtgtgctcttccgatctcccaggacgaggatggag Directed Deep Sec 75 Tyr_500_FW3 GATAGTCACTCCAGGGGTTG TIDE Analysis 76 Tyr_500_RV3 GTGGTGAACCAATCAGTCCT TIDE Analysis zQQncn / Lznz / q / Yi RNP supply for mammalian genomic editing For RNP experiments, the Neon electroporation system was used exactly as described (Amrani et al., 2018). Briefly, 40 pimole of 3xNLSNme2Cas9 together with 50 pimole of T7-transcribed sgRNA were assembled in buffer R and electroporated using 10 pL Neon tips. After electroporation, cells were seeded in pre-warmed 24-well plates containing the appropriate culture medium without antibiotics. Electroporation parameters (voltage, width, number of pulses) were 1150 V, 20 ms, 2 pulses for HEK293T cells; 1000 V, 50 ms, 1 pulse for K562 cells. Delivery of AAV8.Nme2Cas9+sgRNA in vivo and processing of liver tissue For AAV8 vector injections, 8-week-old female C57BL / 6NJ mice were injected with 4 x 1011 genomic copies per mouse via the tail vein, with sgRNA targeting a validated site in Pcsk9o Rosa26. Mice were sacrificed 28 days after vector administration and liver tissues were collected for analysis. Liver tissues were fixed in 4% formalin overnight, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Blood was drawn from the facial vein on days 0, 14 and 28 post injection and serum isolated using a serum separator (BD, cat. no. 365967) and stored at -80°C until assay. Serum cholesterol level was measured using the Infinity™ Colorimetric Endpoint Assay (Thermo-Scientific) following the manufacturer's protocol, and as described above. (Ibraheim et al., 2018). For a Western blot of anti-PCSK9, 40 pg of tissue protein or 2 ng of mouse recombinant PCSK9 protein (R&D Systems, 9258-SE-020) were loaded onto a MiniProtean® TGX™ precast gel (Bio-Rad). Separated bands were transferred to a PVDF membrane and blocked with 5% Blocking-Grade (Bio-Rad) blocking solution for 2 h at room temperature. The membrane was then incubated with rabbit anti-GAPDH (Abcam ab9485, 1:2,000) or goat anti-PCSK9 (R&D Systems AF3985, 1:400) antibodies overnight. Membranes were washed in TBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Bio-Rad 1706515, 1:4,000), and donkey anti-goat (R&D Systems HAF109, 1:2,000) secondary antibodies. ) for 2 hours, at room temperature. Membranes were washed again in TBST and visualized using ECL Clarity™ Western Substrate (BioRad) using an M35A XOMAT Processor (Kodak). AAV6.Nme2Cas9 delivery ex vivo in mouse zygotes Zygotes were incubated for 5-6 hours in 15 pL drops of KSOM (Potassium-Supplemented Simple Optimized Medium, Millipore, Cat. No. MR-106-D) containing 3x109o 3x108GC of the vector AAV6.Nme2Cas9.sg Tyr(4 zygotes in each drop). After incubation, zygotes were rinsed in M2 and transferred to fresh KSOM for overnight growth. The next day, embryos that progressed to the 2-cell stage were transferred to the pseudopregnant recipient oviduct and allowed to develop to term. examples Experimental Example I Discovery of Cas9 orthologs with differentially divergent PIDs The Nme1Cas9 peptide sequence was used as a query in BLAST searches to find all Cas9 orthologs in Neisseria meningitidis species. Orthologs with >80% identity to Nme1 Cas9 were selected for the remainder of this study. PIDs were aligned to that of Nme1Cas9 (residues 820-1082) using ClustalW2 and those with clusters of PID mutations were selected for further analyses. A rootless phylogenetic tree of the NmeCas9 orthologs was constructed using FigTree (http: / / tree.bio.ed.ac.uk / software / figtree / ). Example II Cloning, expression and purification of Cas9 and Acr orthologs Examples of plasmids and oligonucleotides used in this study are listed in Table 3. The Nme2Cas9 and Nme3Cas9 PIDs were ordered as gBlocks (IDT) to replace the Nme1Cas9 PID using Gibson Assembly (NEB) on the bacterial expression plasmid pMSCG7 (Zhang et al., 2015), which encodes Nme1Cas9 with a 6xHis tag. The construct was transformed into E. col!, expressed and purified as previously described (Pawluk et al., 2016). Briefly, Rosetta (DE3) cells containing the respective Cas9 plasmids were grown at 37°C to an ODeoo of 0.6 and protein expression was induced with 1 mM IPTG for 16 hours at 18°C. Cells were harvested and used by sonication in lysis buffer [50 mM Tris-HCI (pH 7.5), 500 mM NaCI, 5 mM imidazole, 1 mM DTT] supplemented with 1 mg / mL Lysozyme and a protease inhibitor cocktail ( Sigma). The used was then run through a Ni2+-NTA agarose column (Qiagen), and the bound protein was eluted with 300 mM imidazole and dialyzed into storage buffer (20 mM HEPES-NaOH (pH 7.5). , 250 mM NaCI, 1 mM DTT], For Acr proteins, 6xHis-tagged proteins were expressed in E. coli strain BL21 Rosetta (DE3).Cells were grown at 37 °C to an optical density (ODeoo) of 0.6 in a shaking incubator. Bacterial cultures were cooled to 18 °C and protein expression was induced by adding 1 mM IPTG for overnight expression. The next day, cells were harvested and resuspended in lysis buffer supplemented with 1 mg / mL lysozyme and the zQQncn / Lznz / q / Yi cocktail 100 protease inhibitor (Sigma), and the protein was purified using the same protocol as for Cas9. The 6xHis tag was removed by incubating the resin-bound protein with tobacco etch virus (TEV) protease overnight at 4°C to isolate untagged Acr. Example III In vitro PAM discovery assay A target double-stranded DNA library with random PAM sequences was generated by overlapping PCR, with the sense (FW) primer containing the 10 nt randomized PAM region. The library was gel-purified and subjected to in vitro cleavage reaction by purified Cas9, together with T7-transcribed sgRNAs. A 300 nM Cas9:sgRNA complex was used to cleave 300 nM of the target fragment in 1X NEBuffer 3.1 (NEB) at 37 °C for 1 h. The reaction was then treated with proteinase K at 50 °C for 10 min and run on a 4% agarose / 1xTAE gel. The cleavage product was cut, eluted, and cloned using a previously described protocol (Zhang et al., 2012), with modifications. Briefly, the DNA was end-repaired, template-free 2'deoxyadenosine tails were added, and Y-shaped adapters were ligated. After PCR, the product was quantified with the ΚΑΡΑ Library Quantification Kit and sequenced using a NextSeq. 500 (lllumina) to obtain 75 nt paired-end reads. The sequences were analyzed with custom scripts and R. Example IV Mammalian genome editing and transfections Nme2Cas9 was codon-optimized for human by Gibson Assembly cloned into the backbone of plasmid pCDest2 previously used for expression of Nme1Cas9 and SpyCas9 (Pawluk et al., 2016; Amrani et al., 2018). Transfection of HEK293T and HEK293TTLR2.0 cells was performed as previously described (Amrani et al., 2018). For Hepa1-6 transfections, Lipofectamine LTX was used to transfect 500ng of AAV.sRNAg.Nme2Cas9 plasmid in 24-well plates (~105 cells / well), using cells that had been cultured 24 hours prior to transfection. For lentivector-delivered K562 cells stably expressing Nme2Cas9 (see below), they were electroporated with 500 ng of sgRNA plasmid using 10 µL Neon tips. To measure indels in all cells 72 hours after transfections, cells were harvested and genomic DNA extracted using the DNaesy Blood and Tissue kit (Qiagen). The target locus was amplified by PCR, sequenced by Sanger (Genewiz) and analyzed by TIDE (Brinkman et al., 2014) using the Desktop Genetics web-based interface (http: / / tide.deskgen.com). Example V Lentiviral transduction of K562 cells to stably express Nme2Cas9 K562 cells stably expressing Nme2Cas9 were generated as previously described for Nme1 Cas9 (Amrani et al., 2018). For lentivirus production, the lentiviral vector was co-transfected into HEK293T cells together with the packaging plasmids (Addgene 12260 and 12259) in 6-well plates using TranslT-LT1 transfection reagent (Mirus Bio). After 24 hours, the culture medium was aspirated from the transfected cells and replaced with 1 mL of fresh DMEM. Up to date zQQncn / Lznz / q / Yi 101 following, the virus-containing supernatant was collected and filtered through a 0.45 pm filter. 10 pL of the undiluted supernatant was used together with 2.5 pg of Polybrene to transduce ~106 K562 cells in 6-well plates. Transduced cells were selected using medium supplemented with 2.5 pg / mL puromycin. Example VI RNP supply for mammalian genomic editing For RNP experiments a Neon electroporation system was used exactly as described (Amrani et al., 2018). Briefly, 40 pimole of 3xNLSNme2Cas9 together with 50 pimole of T7-transcribed sgRNA were assembled in buffer R and electroporated using 10 pL Neon tips. After electroporation, cells were seeded in pre-warmed 24-well plates containing the appropriate culture medium without antibiotics. Electroporation parameters (voltage, width, number of pulses) were 1150 V, 20 ms, 2 pulses for HEK293T cells; 1000 V, 50 ms, 1 pulse for K562 cells. vile example GUIDE-seq GUIDE-Seq experiments were done as previously described (Tsai et al., 2014), with minor modifications (Bolukbasi et al., 2015a). Briefly, HEK293T cells were transfected with 200 ng Cas9 plasmid, 200 ng sgRNA plasmid and 7.5 pmol paired GUIDE-Seq oligonucleotides using Polyfect (Qiagen). Alternatively, Hepa1-6 cells were transfected as described above. Genomic DNA was extracted with a DNeasy Blood and Tissue kit (Qiagen) 72 hours after transfection, according to the manufacturer's protocol. Library preparation and sequencing was performed exactly as previously described (Bolukbasi et al., 2015a). For analysis, all sequences with up to ten mismatches with the target site, as well as a C at the fifth position of the PAM (N+CN), were considered potential non-specific sites. Data were analyzed using the GUIDEseq r Biopipeline package, version 1.1.17 (Zhu et al., 2017). Example VIII Targeted Deep Sequencing and Analysis Targeted deep sequencing was used to confirm the GUIDE-seq results and measure indel rates with maximum accuracy. Two-step PCR amplification was used to produce DNA fragments for each specific site vs. unspecific. For editing by SpyCas9 on DS2 and DS6, major non-specific sites were selected based on GUIDE-seq read counts. For editing by SpyCas9 in DS4, fewer non-specific candidate sites were identified by GUIDEseq, and only those with PAM NGG (DS4|OT1, DS4|OT3, DS4|OT6) or NGC (DS4|OT2) were examined by sequencing. In the first step, locus-specific primers with universal overhangs with ends complementary to the adapters were used. In the first step, the 2x PCR master mix (NEB) was used to generate fragments with the overhangs. In the second step, the purified PCR products were amplified with a universal sense primer and indexed antisense (RV) primers. Full-length products (-250 bp) were gel-purified and sequenced in a 102 lllumina MÍSeq in paired-end mode. MÍSeq data analysis was performed as previously described (Pinello et al., 2016; Ibraheim et al., 2018). Example IX Non-specificity analysis using CRISPRseek Global predictions of non-specificities for TS25 and TS47 were made using the Bioconductor CRISPRseek package. Minor changes were made to accommodate features of Nme2Cas9 not shared with SpyCas9. Specifically, the following changes were used for: gRNA.size = 24, PAM = NNNNCC, PAM.size = 6, RNA.PAM.pattern = NNNNCN, and non-specific candidate sites with less than 6 mismatches were collected. Major non-specific potential sites were selected based on the number and position of mismatches. Genomic DNA from cells targeted by each respective sgRNA was used to amplify each non-specific candidate locus and then analyzed by TIDE. Example X Mouse strains and embryo harvesting All animal experiments were performed in accordance with University of Massachusetts Medical School Institutional Animal Care and Use Committee (IACUC) guidance. C57BL / 6NJ (Stock No. 005304). Mice were obtained from The Jackson Laboratory. All animals were maintained on a 12-h light cycle. The mid-day light cycle when a mating plug was observed was considered embryonic day 0.5 (E0.5) of gestation. Zygotes were harvested at E0.5 by tearing the bleb with forceps and incubation in M2 medium containing hyaluronidase to remove cumulus cells. Example XI Delivery of AAV8.Nme2Cas9+sRNA in vivo and processing of liver tissue For AAV8 vector injections, 8-week-old female C57BL / 6NJ mice were injected with 4 x 1011 genomic copies per mouse via the tail vein, with sgRNA targeting a validated site in Pcsk9 or Rosa26. Mice were sacrificed 28 days after vector administration and liver tissues were collected for analysis. Liver tissues were fixed in 4% formalin overnight, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Blood was drawn from the facial vein on days 0, 14 and 28 post injection and serum isolated using a serum separator (BD, cat. no. 365967) and stored at -80°C until assay. Serum cholesterol level was measured using the Infinity™ Colorimetric Endpoint Assay (ThermoScientific) following the manufacturer's protocol, and as described above. (Ibraheim et al., 2018). For a Western blot of anti-PCSK9, 40 pg of tissue protein or 2 ng of mouse recombinant PCSK9 protein (R&D Systems, 9258-SE-020) were loaded onto a MiniProteanSTGX™ precast gel (Bio-Rad). Separated bands were transferred to a PVDF membrane and blocked with 5% Blocking-Grade (Bio-Rad) blocking solution for 2 h at room temperature. The membrane was then incubated with rabbit anti-GAPDH (Abcam ab9485, 1:2,000) or goat anti-PCSK9 (R&D Systems AF3985, 1:400) antibodies overnight. Membranes were washed in TBST and incubated with 103 horseradish peroxidase (HRP) conjugated goat anti-rabbit (Bio-Rad 1706515, 1:4,000), and donkey anti-goat (R&D Systems HAF109, 1:2,000) secondary antibodies for 2 hours, at room temperature. Membranes were washed again in TBST and visualized using ECL Clarity™ Western Substrate (Bio-Rad) using an M35A XOMAT Processor (Kodak). 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Zuris, J.A., Thompson, D.B., Shu, Y., Guilinger, J.P., Bessen, J.L., Hu, J.H., Maeder, M.L., Joung, J.K., Chen, Z.-Y., and Liu, D.R. (2015). Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73-80. All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it is to be understood that the invention as claimed should not be unduly limited to such specific embodiments. In fact, various modifications of the described modes of carrying out the invention that are obvious to those skilled in biological control, biochemistry, molecular biology, entomology, plankton, fishery systems, and freshwater ecology, or related fields, are envisioned within the scope of the following claims.

Claims

1. A mutated NmeCas9 protein comprising a fused nucleotide deaminase and a binding region for an N4CC nucleotide sequence.

2. The protein according to claim 1, wherein said protein is Nme2Cas9.

3. The protein according to nuclear localization signal of claim 1, further comprising a protein of 4. The protein according to claim 1, wherein said nucleotide deaminase is a cytidine deaminase.

5. The protein according to claim 1, wherein said nucleotide deaminase is an adenosine deaminase.

6. The protein according to uracil glycosylase, as described in claim 1, further comprising an inhibitor of the 7. The protein according to claim 1, wherein said nuclear localization signal protein is selected from a nucleoplasmin and an SV40.

8. The protein according to claim 1, wherein said binding region is an interaction domain with the accessory motif to the protospacer.

9. The protein according to claim 8, wherein said interaction domain with the protospacer accessory motif comprises said mutation.

10. The protein according to claim 9, wherein said mutation is a D16A mutation.

11. An adeno-associated virus comprising a mutated NmeCas9 protein, said mutated NmeCas9 protein comprising a fused N4CC nucleotide sequence deaminase and a binding region for a 12. The virus according to claim 11, wherein said virus is an adeno-associated virus 8.

13. The virus according to claim 11, wherein said virus is an adeno-associated virus 6.

14. The virus according to claim 11, wherein said protein is Nme2Cas9.

15. The virus according to claim 11, wherein said protein further comprises a nuclear localization signal protein.

16. The virus according to claim 11, wherein said nucleotide deaminase is a cytidine deaminase.

17. The virus according to claim 11, wherein said nucleotide deaminase is an adenosine deaminase.

18. The virus according to claim 11, wherein said protein further comprises a uracil glycosylase inhibitor.

19. The virus according to claim 11, wherein said nuclear localization signal protein 110 is selected from a nucleoplasmin and SV40.

20. The virus according to claim 11, wherein said binding region is an interaction domain with the accessory motif to the protospacer.

21. The virus according to claim 20, wherein said interaction domain with the protospacer accessory motif comprises said mutation.

22. The virus according to claim 21, wherein said mutation is a D16A mutation 23. A method comprising: a) providing; i) a nucleotide sequence comprising a gene with a mutated single base, wherein said gene is flanked by an N4CC nucleotide sequence; i) a mutated NmeCas9 protein comprising a fused nucleotide deaminase and a binding region for said N4CC nucleotide sequence; b) contacting said nucleotide sequence with said mutated NmeCas9 protein under conditions such that said binding region binds to said N4CC nucleotide sequence; and c) replacing said mutated single base with a wild-type base in said mutated NmeCas9 protein.

24. The method according to claim 23, wherein said protein is Nme2Cas9.

25. The method according to claim 23, wherein said protein further comprises a nuclear localization signal protein.

26. The method according to claim 23, wherein said nucleotide deaminase is a cytidine deaminase.

27. The method according to claim 23, wherein said nucleotide deaminase is an adenosine deaminase.

28. The method according to claim 23, wherein said protein further comprises a uracil glycosylase inhibitor.

29. The method according to claim 23, wherein said nuclear localization signal protein is selected from the group consisting of nucleoplasmin and SV40.

30. The method according to claim 23, wherein said joining region is an interaction domain with the accessory motif to the protospacer.

31. The method according to claim 30, wherein said interaction domain with the protospacer accessory motif comprises said mutation of the Cas9 protein.

32. The method according to claim 31, wherein said mutation in the Cas9 protein is a D16A mutation.

33. A method comprising: (a) providing; (i) a patient comprising a nucleotide sequence comprising a gene with a mutated single base, wherein said gene is flanked by an N4CC nucleotide sequence, wherein said mutated gene causes a genetically based medical condition; (ii) an adeno-associated virus comprising a mutated NmeCas9 protein, said mutated NmeCas9 protein comprising a fused nucleotide deaminase and a binding region for said N4CC nucleotide sequence; (b) treating said patient with said adeno-associated virus under conditions such that said mutated NmeCas9 protein replaces said mutated single base with a wild-type single base, so that said genetically based medical condition does not develop.

34. The method according to claim 33, wherein said gene encodes a tyrosinase protein.

35. The method according to claim 33, wherein said genetically based medical condition is tyrosinemia.

36. The method according to claim 33, wherein said virus is an adeno-associated virus 8.

37. The method according to claim 33, wherein said virus is an adeno-associated virus 6.

38. The method according to claim 33, wherein said protein is Nme2Cas9.

39. The method according to claim 33, wherein said protein further comprises a nuclear localization signal protein.

40. The method according to claim 33, wherein said nucleotide deaminase is a cytidine deaminase.

41. The method according to claim 33, wherein said nucleotide deaminase is an adenosine deaminase.

42. The method according to claim 33, wherein said protein further comprises a uracil glycosylase inhibitor.

43. The method according to claim 33, wherein said nuclear localization signal protein is selected from the group consisting of nucleoplasmin and SV40.

44. The method according to claim 33, wherein said joining region is an interaction domain with the accessory motif to the protospacer.

45. The method according to claim 44, wherein said interaction domain with the accessory motif to the protospacer comprises said mutation.

46. ​​The method according to claim 45, wherein said mutation is a mutation