Targeted crispr delivery platforms

Abstract

To provide targeted CRISPR delivery platforms.SOLUTION: The present invention is related to compositions and methods for gene therapy. Several approaches described herein utilize the Neisseria meningitidis Cas9 system that provides a hyperaccurate CRISPR gene editing platform. Furthermore, the invention incorporates full-length and truncated single guide RNA sequences that permit a complete sgRNA-Nme1Cas9 vector to be inserted into an adeno-associated viral plasmid that is compatible with in vivo administration. Furthermore, Type II-C Cas9 orthologs have been identified that target protospacer adjacent motif sequences limited to one to four required nucleotides.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to compositions and methods for gene therapy. Some techniques described herein utilize a hyperaccurate CRISPR gene editing platform. The present invention utilizes the Neisseria meningitidis Cas9 system, which provides a novel method for targeting the genomic DNA of the present invention. Additionally, the present invention incorporates improvements to this Cas9 system, such as shortening the single guide RNA sequence and packaging Nme1Cas9 or Nme2Ca9 with its guide RNA into an adeno-associated viral vector compatible with in vivo administration. Additionally, type II-C Cas9 orthologs have been identified that target protospacer adjacent motif sequences restricted to between 1 and 4 required nucleotides. [Background technology]

[0002] Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) are unique, RNA-guided adaptive immune systems found in archaea and bacteria. These systems confer immunity by targeting and inactivating nucleic acids originating from foreign genetic elements. To date, many different types of CRISPR-Cas systems have been identified and are categorized into two classes.

[0003] Among the class II CRISPR systems, type II CRISPR-Cas systems are It is characterized by a single effector protein called Cas9, which forms a ribonucleoprotein (RNP) complex with RNA (crRNA) and transactivating RNA (tracrRNA) to target and cleave DNA. The crRNA contains a programmable guide sequence that can direct Cas9 to almost any DNA sequence in a living organism.

[0004] The programmability of the Cas9 RNP complex has been exploited by many researchers for genome editing in eukaryotic systems. It has been used to edit the genomes of mammalian cells, human embryos, plants, rodents, and other living organisms. RNPs have been used for precise (donor template) and non-precise genome editing, both of which are applied in gene therapy, agriculture, and other cases. In addition, nuclease-deactivated versions of Cas9 orthologs have been used for transcriptional modulation, site-specific DNA labeling, and proteome profiling at specific genomic loci. Several different Cas9s have been used for these applications. Central to the programmability of Cas9, and therefore its application, is the ability to introduce any guide sequence into the crRNA. The crRNA and tracrRNA can be fused together to form a single guide RNA (sgRNA), which is more stable and leads to enhanced genome editing.

[0005] There is a need in the art for improved Cas9 and sgRNA sequences that can result in specific and precise editing of a broader range of target sites, particularly when combined with a reliable nucleic acid delivery platform. Summary of the Invention [Means for solving the problem]

[0006] The present invention relates to compositions and methods for gene therapy. Some of the approaches described herein utilize the Neisseria meningitidis Cas9 system, which provides an ultra-high precision CRISPR gene editing platform. Furthermore, the present invention incorporates improvements to this Cas9 system, such as shortening the single guide RNA sequence and packaging Nme1Cas9 or Nme2Cas9 with its guide RNA into an adeno-associated viral vector compatible with in vivo administration. Furthermore, type II-C Cas9 orthologs have been identified that target protospacer adjacent motif sequences restricted to between 1 and 4 required nucleotides.

[0007] In one embodiment, the present invention contemplates a single guide ribonucleic acid (sgRNA) sequence comprising a shortened repeat:anti-repeat region. In one embodiment, the sgRNA sequence further comprises a shortened Stem 2 region. In one embodiment, the sgRNA sequence further comprises a shortened spacer region. In one embodiment, the sgRNA sequence is 121 nucleotides in length. In one embodiment, the sgRNA sequence is selected from the group consisting of 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 101 nucleotides, and 99 nucleotides in length. In one embodiment, the sgRNA sequence is 100 nucleotides in length. In one embodiment, the sgRNA sequence is an Nme1Cas9 single guide ribonucleic acid sequence. In one embodiment, the sgRNA sequence is an Nme2Cas9 single guide ribonucleic acid sequence. In one embodiment, the sgRNA sequence is an Nme1Cas9 single guide ribonucleic acid sequence or an Nme2Cas9 single guide ribonucleic acid sequence.

[0008] In one embodiment, the present invention contemplates a single guide ribonucleic acid (sgRNA) sequence comprising a shortened Stem 2 region. In one embodiment, the sgRNA sequence further comprises a shortened repeat:anti-repeat region. In one embodiment, the sgRNA sequence further comprises a shortened spacer region. In one embodiment, the length of the sgRNA sequence is selected from the group consisting of 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 101 nucleotides, and 99 nucleotides. In one embodiment, the length of the sgRNA sequence is 100 nucleotides.

[0009] In one embodiment, the present invention contemplates an adeno-associated virus (AAV) vector comprising a single guide ribonucleic acid-Neisseria meningitidis Cas9 (sgRNA-Nme1Cas9 or sgRNA-Nme2Cas9) nucleic acid vector. In one embodiment, the single guide ribonucleic acid-Neisseria meningitidis Cas9 nucleic acid vector comprises at least one promoter. In one embodiment, the at least one promoter is selected from the group consisting of a U6 promoter and a U1a promoter. In one embodiment, the single guide ribonucleic acid-Neisseria The Meningitidis Cas9 nucleic acid vector comprises a Kozak sequence. In one embodiment, the sgRNA comprises a nucleic acid sequence complementary to a sequence of a gene of interest. In one embodiment, the sequence of the gene of interest is selected from the group consisting of a PCSK9 sequence and a ROSA26 sequence. In one embodiment, the sgRNA comprises an untruncated sequence that is 145 nucleotides in length. In one embodiment, the sgRNA comprises a truncated repeat-anti-repeat sequence. In one embodiment, the sgRNA further comprises a truncated Stem 2 region. In one embodiment, the sgRNA further comprises a truncated spacer region. In one embodiment, the sgRNA sequence is 121 nucleotides in length. In one embodiment, the sgRNA sequence is selected from the group consisting of 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 101 nucleotides, and 99 nucleotides in length. In one embodiment, the sgRNA sequence is 100 nucleotides in length. In one embodiment, the sgRNA comprises a truncated Stem 2 region. In one embodiment, the sgRNA further comprises a truncated repeat:anti-repeat region. In one embodiment, the sgRNA further comprises a truncated spacer region. In one embodiment, the sgRNA sequence is 100 nucleotides in length. In one embodiment, the sgRNA comprises an untruncated sequence that is 145 nucleotides in length.

[0010] In one embodiment, the present invention contemplates a method comprising: a) providing i) a patient exhibiting at least one symptom of a medical condition, the patient comprising a plurality of genes associated with the medical condition; and ii) a delivery platform comprising a single guide ribonucleic acid-Neisseria meningitidis Cas9 (sgRNA-Nme1Cas9 or sgRNA-Nme2Cas9) nucleic acid vector, the sgRNA comprising a nucleic acid sequence complementary to a portion of at least one of the plurality of genes; and b) administering the AAV plasmid to the patient under conditions in which the at least one symptom of the medical condition is alleviated. In one embodiment, the delivery platform comprises an adeno-associated virus (AAV) vector. In one embodiment, the delivery platform comprises a microparticle. In one embodiment, the medical condition comprises hypercholesterolemia. In one embodiment, the medical condition comprises tyrosinemia. In one embodiment, the at least one of the plurality of genes is a PCSK9 gene. In one embodiment, the sgRNA nucleic acid is complementary to a portion of the PCSK9 gene. In one embodiment, at least one of the plurality of genes is a FAH gene. In one embodiment, the sgRNA nucleic acid is complementary to a portion of the FAH gene. In one embodiment, the sgRNA comprises a shortened repeat-anti-repeat sequence. In one embodiment, the sgRNA further comprises a shortened Stem 2 region. In one embodiment, the sgRNA further comprises a shortened spacer region. In one embodiment, the sgRNA sequence is 121 nucleotides in length. In one embodiment, the sgRNA sequence is selected from the group consisting of 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 101 nucleotides, and 99 nucleotides in length. In one embodiment, the sgRNA sequence is 100 nucleotides in length. In one embodiment, the sgRNA comprises a shortened Stem 2 region. In one embodiment, the sgRNA further comprises a shortened repeat:anti-repeat region. In one embodiment, the sgRNA further comprises a shortened spacer region.In one embodiment, the length of the sgRNA sequence is selected from the group consisting of 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 101 nucleotides, and 99 nucleotides. In one embodiment, the length of the sgRNA sequence is 100 nucleotides. In one embodiment, the sgRNA comprises an untruncated sequence that is 145 nucleotides in length.

[0011] In one embodiment, the invention contemplates an adeno-associated virus (AAV) plasmid encoding a Type II-C Cas9 nuclease protein, said protein comprising a protospacer adjacent motif recognition domain configured with a binding site for a protospacer adjacent motif sequence comprising between 1 and 4 required nucleotides. In one embodiment, said Type II-C Cas9 nuclease protein is selected from the group consisting of Neisseria meningitidis strain De10444 Nme2Cas9 nuclease protein, Haemophilus parainfluenzae HpaCas9 nuclease protein, and Simonsiella muelleri SmuCas9 nuclease protein. In one embodiment, said protospacer adjacent motif sequence comprising between 1 and 4 required nucleotides is selected from the group consisting of Nme2Cas9 nuclease protein, Haemophilus parainfluenzae HpaCas9 nuclease protein, and Simonsiella muelleri SmuCas9 nuclease protein. 4 CN 3 , N 4 C.T., N. 4 C.C.N. 4 CCA, and N 4 GNT 3 In one embodiment, the 1 to 4 required nucleotides are selected from the group consisting of C, CT, CCN, CCA, CN 3 and G.N.T. 2In one embodiment, the type II-C Cas9 nuclease protein is associated with a truncated sgRNA. In one embodiment, the adeno-associated virus plasmid encodes two sgRNA sequences. In one embodiment, the adeno-associated virus plasmid encodes a poly-adenosine sequence. In one embodiment, the adeno-associated virus plasmid encodes a homologous recombination repair donor nucleotide template. In one embodiment, the adeno-associated virus plasmid is an integral adeno-associated virus plasmid.

[0012] In one embodiment, the invention contemplates a method comprising: a) providing i) a patient exhibiting at least one symptom of a medical condition, the patient comprising a plurality of genes associated with the medical condition, the plurality of genes comprising a protospacer adjacent motif comprising between 1 and 4 required nucleotides; and ii) a delivery platform comprising at least one nucleic acid encoding a Cas9 Type II-C nuclease protein, the protein comprising a protospacer adjacent motif recognition domain configured with a binding site for the protospacer adjacent motif sequence comprising between 2 and 4 required nucleotides; and b) administering the delivery platform to the patient under conditions in which the at least one symptom of the medical condition is alleviated. In one embodiment, the medical condition comprises hypercholesterolemia. In one embodiment, the medical condition comprises tyrosinemia. In one embodiment, the at least one of the plurality of genes is a PCSK9 gene. In one embodiment, the sgRNA nucleic acid is complementary to a portion of the PCSK9 gene. In one embodiment, the at least one of the plurality of genes is a FAH gene. In one embodiment, the sgRNA nucleic acid is complementary to a portion of the FAH gene. In one embodiment, the delivery platform comprises an adeno-associated virus plasmid. In one embodiment, the delivery platform comprises a microparticle. In one embodiment, the Type II-C Cas9 nuclease protein is selected from the group consisting of Neisseria meningitidis strain De10444 Nme2Cas9 nuclease protein, Haemophilus parainfluenzae HpaCas9 nuclease protein, and Simonsiella muelleri SmuCas9 nuclease protein. In one embodiment, the protospacer adjacent motif sequence comprising 1 to 4 required nucleotides is selected from the group consisting of Nme2Cas9 nuclease protein, ... 4 CN 3 , N 4 C.T., N. 4 C.C.N. 4 CCA, and N 4 GNT 3In one embodiment, the 1 to 4 required nucleotides are selected from the group consisting of C, CT, CCN, CCA, CN 3 and G.N.T. 2 In one embodiment, the type II-C Cas9 nuclease protein is associated with a truncated sgRNA. In one embodiment, the adeno-associated virus plasmid encodes two sgRNA sequences. In one embodiment, the adeno-associated virus plasmid encodes a poly-adenosine sequence. In one embodiment, the adeno-associated virus plasmid encodes a homologous recombination repair donor nucleotide template. In one embodiment, the adeno-associated virus plasmid is an integral adeno-associated virus plasmid.

[0013] In one embodiment, the present invention contemplates an adeno-associated virus (AAV) plasmid encoding a type II-C Cas9 nuclease protein, said protein comprising a protospacer adjacent motif recognition domain (e.g., a PAM interaction domain; PID) configured to bind to a protospacer adjacent motif (PAM) sequence, said PAM sequence comprising adjacent cytosine dinucleotide pairs. In one embodiment, the adjacent cytosine dinucleotide pairs are at positions 5 and 6 of the PAM. In one embodiment, said type II-C Cas9 nuclease protein is derived from a Neisseria meningitidis strain. In one embodiment, the Neisseria meningitidis strain is De10444. In one embodiment, the type II-C Cas9 nuclease protein is an Nme2Cas9 nuclease protein. In one embodiment, the Neisseria meningitidis strain is 98002. In one embodiment, the type II-C Cas9 nuclease protein is an Nme3Cas9 nuclease protein. In one embodiment, the PAM sequence is 4 C.C., N. 4 CCN 3 , N 4 C.C.A.,N. 4 CC(X), N 4 CA 3 and N 10In one embodiment, the PAM sequence is selected from the group consisting of N 3 In one embodiment, the type II-C Cas9 nuclease protein further comprises an sgRNA sequence. In one embodiment, the sgRNA sequence comprises a spacer ranging in length from about 17 to 24 nucleotides.

[0014] In one embodiment, the present invention contemplates a method comprising: a) providing i) a patient exhibiting at least one symptom of a medical condition, the patient comprising a plurality of genes associated with the medical condition, the plurality of genes comprising a protospacer adjacent motif comprising adjacent cytosine dinucleotide pairs; and ii) a delivery platform comprising at least one nucleic acid encoding a Cas9 type II-C nuclease protein, the protein comprising a protospacer adjacent motif recognition domain (e.g., a PAM-interacting domain; PID) configured to bind to the protospacer adjacent motif sequence comprising adjacent cytosine dinucleotide pairs; and b) administering the delivery platform to the patient under conditions in which the at least one symptom of the medical condition is alleviated. In one embodiment, the delivery platform comprises an adeno-associated viral vector. In one embodiment, the adeno-associated viral vector is an adeno-associated viral vector 8 (AAV8). In one embodiment, the medical condition comprises hypercholesterolemia. In one embodiment, the medical condition comprises tyrosinemia. In one embodiment, the medical condition is X-linked chronic granulomatous disease. In one embodiment, the medical condition is aspartylglycosaminuria. In one embodiment, the at least one of the plurality of genes is a PCSK9 gene. In one embodiment, the sgRNA nucleic acid is complementary to a portion of the PCSK9 gene. In one embodiment, at least one of the plurality of genes is a FAH gene. In one embodiment, the sgRNA nucleic acid is complementary to a portion of the FAH gene. In one embodiment, the adeno-associated virus plasmid encodes at least one sgRNA sequence. In one embodiment, the adeno-associated virus plasmid encodes two sgRNA sequences. In one embodiment, the adeno-associated virus plasmid encodes a poly-adenosine sequence. In one embodiment, the adeno-associated virus plasmid encodes a homologous recombination repair donor nucleotide template. In one embodiment, the adeno-associated virus plasmid is an integral adeno-associated virus plasmid. In one embodiment, the delivery platform comprises a microparticle.In one embodiment, the adjacent cytosine dinucleotide pair is at positions 5 and 6 of the PAM. In one embodiment, the type II-C Cas9 nuclease protein is from a Neisseria meningitidis strain. In one embodiment, the Neisseria meningitidis strain is De10444. In one embodiment, the type II-C Cas9 nuclease protein is an Nme2Cas9 nuclease protein. In one embodiment, the Neisseria meningitidis strain is 98002. In one embodiment, the type II-C Cas9 nuclease protein is an Nme3Cas9 nuclease protein. In one embodiment, the PAM sequence is N. 4 C.C., N. 4 CCN 3 , N 4 C.C.A.,N. 4 CC(X), N 4 CA 3 and N 10 In one embodiment, the PAM sequence is selected from the group consisting of N 3 In one embodiment, the type II-C Cas9 nuclease protein further comprises an sgRNA sequence. In one embodiment, the sgRNA sequence comprises a spacer ranging in length from about 17 to 24 nucleotides.

[0015] definition In order to facilitate understanding of the present invention, certain terms are defined below. Terms defined herein have the meanings commonly understood by those skilled in the art to which the present invention pertains. Terms such as "a", "an" and "the" refer to a singular entity as well as a plurality of entities, and also include general classes for which specific examples may be used for illustration. While the terminology used herein is intended to describe specific embodiments of the present invention, their use does not limit the invention except as outlined in the claims.

[0016] The terms "about" or "approximately" as used herein with respect to any assay measurement, refer to + / - 5% of a given measurement.

[0017] As used herein, "ROSA26 gene" or "Rosa26 gene" refers to the human or mouse locus (respectively) that is widely used to achieve systemic expression in mice. Targeting of the ROSA26 locus can be achieved by introducing the desired gene into the unique XbaI site approximately 248 bp upstream of the original gene trap line, in the first intron of the locus. Constructs can be constructed using an adenovirus splice acceptor, followed by the gene of interest inserted into the unique XbaI site and a polyadenylation site. A neomycin resistance cassette can also be included in the targeting vector.

[0018] As used herein, "PCSK9 gene" or "Pcsk9 gene" refers to the human or mouse (respectively) genetic locus that encodes the PCSK9 protein. The PCSK9 gene is present on chromosome 1 in band 1p32.3 and contains 13 exons. At least two isoforms can be produced from this gene by alternative splicing.

[0019] 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 encoded by the PCSK9 gene in humans. Seidah et al., "The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation" Proc. Natl. Acad. Sci. USA 100 (3): 928-933 (2003). Similar genetic Orthologs are found across many species. Many enzymes, including PSCK9, are inactive when first synthesized because they have a portion of the peptide chain that blocks their activity. The portion is removed by proprotein convertases to activate the enzyme. PSCK9 is thought to play a role in regulating cholesterol homeostasis. For example, PCSK9 binds to the epidermal growth factor-like repeat A (EGF-A) domain of the low-density lipoprotein receptor (LDL-R), which can result in internalization and degradation of LDL-R. Clearly, a reduction in LDL-R levels would be predicted to decrease metabolism of LDL-C, which could lead to hypercholesterolemia.

[0020] The term "hypercholesterolemia" as used herein refers to any medical condition in which blood cholesterol levels are elevated above clinically recommended levels. For example, if cholesterol is measured using low density lipoprotein (LDL), hypercholesterolemia may be present if the measured LDL level is, for example, above approximately 70 mg / dl. Alternatively, if cholesterol is measured using free plasma cholesterol, hypercholesterolemia may be present if the measured free cholesterol level is, for example, above approximately 200-220 mg / dl.

[0021] As used herein, the term "CRISPR" or "clustered regularly interspaced short palindromic repeats" refers to an acronym for DNA loci that contain multiple, short, direct repeats of a base sequence. Each repeat contains a series of bases followed by 30 or so base pairs known as "spacer" sequences. Spacers are short segments of virus-derived DNA that may act as a "memory" of past exposure to facilitate adaptive defense against future invasions. Doudna et al. Genome editing. The new frontier of genome engineering with CRISPR-Cas9" Science 346(6213): 1258096 (2014).

[0022] As used herein, the term "Cas" or "CRISPR-associated (cas)" refers to genes often associated with CRISPR repeat-spacer arrays.

[0023] As used herein, the term "Cas9" refers to a nuclease from the type II CRISPR system, an enzyme specialized for the generation of double-stranded breaks in DNA, with two active cutting sites (HNH and RuvC domains), one for each strand of the double helix. The tracrRNA and spacer RNA can be combined into a "single guide RNA" (sgRNA) molecule, which can be mixed with Cas9 to find and cleave a DNA target through Watson-Crick pairing between the guide sequence in the sgRNA and the target DNA sequence. Jinek et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity" Science 337(6096):816-821 (2012).

[0024] As used herein, the term "catalytically active Cas9" refers to an unmodified Cas9 nuclease that contains full nuclease activity.

[0025] The term "nickases" as used herein refers to nucleases that cleave only a single DNA strand, either due to their natural function or because they have been engineered to cleave only a single DNA strand. Cas9 nickase variants mutated in either the RuvC or HNH domains provide control over which DNA strand is cleaved and which strand remains intact. Jinek et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity" Science 337(6096):816-821 (2012) and Cong et al. Multiplex genome engineering using CRISPR / Cas systems" Science 339(6121):819-823 (2013).

[0026] The term "trans-activating crRNA", "tracrRNA" as used herein refers to a small RNA encoded in trans. For example, CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins) constitutes an RNA-mediated defense system that protects against viruses and plasmids. This defense pathway has three steps. First, a copy of the invading nucleic acid is integrated into the CRISPR locus. Next, CRISPR RNA (crRNA) is transcribed from this CRISPR locus. Then, the crRNA is integrated into an effector complex, where the crRNA guides the complex to the invading nucleic acid and the Cas protein degrades the nucleic acid. There are several pathways for CRISPR activation, one of which requires tracrRNA, which plays a role in the maturation of crRNA. The tracrRNA is complementary to the repeat sequence of the pre-crRNA and forms an RNA duplex. This RNA duplex is cleaved by the RNA-specific ribonuclease RNase III to form a crRNA / tracrRNA hybrid. This hybrid acts as a guide for the endonuclease Cas9, which cleaves the invading nucleic acid.

[0027] The term "protospacer adjacent motif" (or PAM), as used herein, refers to a DNA sequence that may be required for Cas9 / sgRNA to form an R-loop to interrogate a specific DNA sequence through Watson-Crick pairing of its guide RNA with the genome. PAM specificity may be a function of the DNA binding specificity of the Cas9 protein (e.g., the "protospacer adjacent motif recognition domain" at the C-terminus of Cas9).

[0028] The term "protospacer adjacent motif recognition domain," "PAM interaction domain," or "PID," as used herein, refers to a Cas9 amino acid sequence that contains the binding site for the DNA target PAM sequence.

[0029] The term "binding site" as used herein refers to any molecular configuration having a specific tertiary and / or quaternary structure that undergoes physical attachment or close association with a binding component. For example, the molecular configuration may include a sequence of amino acids. Alternatively, the molecular configuration may include a sequence of nucleic acids. Additionally, the molecular configuration may include a lipid bilayer or other biological material.

[0030] As used herein, the term "sgRNA" refers to a single guide RNA used in conjunction with a CRISPR-associated system (Cas). The sgRNA is a fusion of the crRNA and tracrRNA and contains nucleotides of a sequence complementary to a desired target site. Jinek et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity" Science 337(6096):816-821 (2012). Watson-Crick pairing at the target site allows for R-loop formation which, in conjunction with a functional PAM, allows DNA cleavage or, in the case of nuclease-deficient Cas9, binding to DNA at the locus.

[0031] As used herein, the term "orthogonal" refers to non-overlapping, uncorrelated, or independent targets. For example, when utilizing two orthogonal Cas9 isoforms, they will use an orthogonal sgRNA that programs only one of the Cas9 isoforms for DNA recognition and cleavage. Esvelt et al., "Orthogonal Cas9 proteins for RNA-guided gene regulation and editing" Nat Methods 10(11):1116-1121 (2013). For example, this allows one Cas9 isoform to be used as a target. (e.g., S. pyogenes Cas9 or SpyCas9) can function as a nuclease programmed by an sgRNA that can be specific for it, while another Cas9 isoform (e.g., N. meningitidis Cas9 or NmeCas9) can operate as a nuclease-deactivated Cas9 that provides its PAM specificity and DNA targeting to the binding site through an orthogonal sgRNA. Other Cas9s include S. aureus Cas9 or SauCas9 and A. naeslundii Cas9 or AnaCas9.

[0032] The term "truncated" as used herein, when used in reference to a polynucleotide or amino acid sequence, means that at least a portion of the wild-type sequence may be absent. In some cases, a truncated guide sequence in the sgRNA or crRNA may improve the editing accuracy of Cas9. Fu, et al. "Improving CRISPR-Cas nuclease specificity using truncated guide RNAs" Nat Biotechnol. 2014 Mar;32(3):279-284 (2014).

[0033] The term "base pair" as used herein refers to a specific nucleic acid base (also called nitrogenous base) that is a component of the nucleotide sequence that forms the primary structure of both DNA and RNA. Double-stranded DNA can be characterized by a specific hydrogen bond pattern. Base pairs can include, but are not limited to, guanine-cytosine base pairs and adenine-thymine base pairs.

[0034] The term "specific genomic target" as used herein refers to any predefined nucleotide sequence that can bind to the Cas9 protein as intended herein. Targets may include, but are not limited to, nucleotide sequences complementary to a programmable DNA-binding domain or an orthogonal Cas9 protein programmed with a unique guide RNA, nucleotide sequences complementary to a single guide RNA, protospacer adjacent motif recognition sequences, on-target binding sequences, and off-target binding sequences.

[0035] The term "on-target binding sequence," as used herein, refers to a subsequence of a specific genomic target that may be perfectly complementary to a programmable DNA-binding domain and / or a single guide RNA sequence.

[0036] The term "off-target binding sequence," as used herein, refers to a subsequence of a particular genomic target that may be partially complementary to a programmable DNA-binding domain and / or a single guide RNA sequence.

[0037] The term "unable to bind" as used herein refers to any nucleotide-nucleotide or nucleotide-amino acid interaction that exhibits partial complementarity but has insufficient complementarity for recognition to trigger cleavage of the target site by the Cas9 nuclease. Such an inability to bind may result in weak or partial binding of the two molecules, thus abolishing the expected biological function (e.g., nuclease activity).

[0038] The term "cleavage," as used herein, can be defined as the production of a break in DNA, which can be a single-stranded break or a double-stranded break depending on the type of nuclease that can be used.

[0039] As used herein, the terms "edit," "editing," or "edited" refer to a method of altering the nucleic acid sequence of a polynucleotide (e.g., a wild-type naturally occurring nucleic acid sequence or a mutated naturally occurring sequence) by selectively deleting or specifically including a new sequence at a particular genomic target by using an exogenously supplied DNA template. Such particular genomic targets include, but are not limited to, a chromosomal region, mitochondrial DNA, a gene, a promoter, an open reading frame, or any nucleic acid sequence.

[0040] The terms "delete," "deleted," "deleting," or "deletion," as used herein, may be defined as a change in either a nucleotide or amino acid sequence such that one or more nucleotides or amino acid residues, respectively, are absent or become nonexistent.

[0041] The term "gene of interest" as used herein refers to any given gene which may be desirable to delete.

[0042] The term "allele," as used herein, refers to any one of several alternative forms of the same gene or the same genetic locus.

[0043] The term "effective amount," as used herein, refers to a particular amount of a pharmaceutical composition containing a therapeutic agent in which a clinically beneficial result (i.e., reduction in symptoms, for example) is achieved. The toxicity and therapeutic efficacy of such a composition can be determined, for example, by the LD 50 (a dose lethal to 50% of the population) and ED 50 The LD (the dose that is therapeutically effective in 50% of the population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 Therapeutic indices can be expressed as a ratio. Compounds that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and additional animal studies can be used in formulating a range of dosages for human use. The dosage of such compounds can be adjusted to achieve the ED 50 The dosage will vary within this range depending on the dosage form used, sensitivity of the patient, and the route of administration.

[0044] The term "symptoms" as used herein refers to any subjective or objective evidence of disease or physical disorder present in a patient. For example, subjective evidence is usually based on the patient's self-report and may include, but is not limited to, pain, headache, visual disturbances, nausea and / or vomiting. Alternatively, objective evidence is usually the results of medical tests, including, but not limited to, temperature, complete blood count, lipid panel, thyroid panel, blood pressure, heart rate, electrocardiogram, tissue and / or body imaging scans.

[0045] The term "disease" or "medical condition", as used herein, refers to any defect that interferes with or alters the normal performance of vital functions of a living animal or plant organism or part thereof. Generally, this is manifested by noticeable signs and symptoms and is usually a response to i) environmental factors (as malnutrition, industrial accidents, or weather); ii) specific infectious agents (as insects, bacteria, or viruses); iii) an inherent defect of the organism (as a genetic abnormality); and / or iv) a combination of these factors.

[0046] The terms "reduce," "inhibit," "diminish," "suppress," "decrease," "prevent," and grammatical equivalents (including "lower," "smaller," etc.), when referring to any symptom relative to an untreated subject, mean that the number and / or magnitude of the symptom in the treated subject is less than the number and / or magnitude of the symptom in the untreated subject by any amount that would be recognized as clinically significant by any medically trained personnel. In one embodiment, the number and / or magnitude of the symptom in the treated subject is at least 10% less, at least 25% less, at least 50% less, at least 75% less, and / or at least 90% less than the number and / or magnitude of the symptom in the untreated subject.

[0047] The term "attached" as used herein refers to any interaction between a vehicle (or carrier) and a drug. Attachment can be reversible or irreversible. Such attachments include, but are not limited to, covalent bonds, ionic bonds, van der Waals forces, or friction. A drug is attached to a vehicle (or carrier) if it permeates, is incorporated into, is coated with, is in suspension with, is in solution with, is mixed with, or the like.

[0048] The term "drug" or "compound" as used herein refers to any pharmacologically active substance that can be administered to achieve a desired effect. A drug or compound can be synthetic or naturally occurring, a non-peptide, a protein or peptide, an oligonucleotide or nucleotide, a polysaccharide or sugar.

[0049] The terms "administered" or "administering," as used herein, refer to any method of providing a composition to a patient such that the composition has its intended effect on the patient. Exemplary methods of administration are by direct mechanisms such as local tissue administration (i.e., for example, extravascular placement), oral ingestion, transdermal patch, topical, inhalation, suppository, and the like.

[0050] The term "patient" or "subject" as used herein may be a human or animal, and need not be hospitalized. For example, an outpatient, nursing facility resident is a "patient". Patients may include humans or non-human animals of any age, and thus include both adults and young individuals (i.e., children). The term "patient" does not imply a need for medical treatment, and thus patients may willingly or involuntarily participate in experiments, whether clinical or in support of basic scientific research.

[0051] The term "affinity," as used herein, refers to any force of attraction between substances or particles that causes them to chemically combine and remain so. For example, an inhibitory compound that has a high affinity for a receptor will be more effective at preventing the interaction of the receptor with its natural ligand than an inhibitor with a low affinity.

[0052] The term "derived from," as used herein, refers to the source of a compound or sequence. In one respect, the compound or sequence may be derived from an organism or a particular species. In another respect, the compound or sequence may be derived from a larger complex or sequence.

[0053] The term "protein," as used herein, refers to any of a large number of naturally occurring, highly complex substances (such as enzymes or antibodies) made up of amino acid residues joined by peptide bonds, containing the elements carbon, hydrogen, nitrogen, oxygen, and usually sulfur. Generally, proteins contain on the order of several hundred amino acids.

[0054] The term "peptide" as used herein refers to any of a variety of amides derived from two or more amino acids by combination of the amino group of one amino acid with the carboxyl group of another amino acid, usually obtained by partial hydrolysis of proteins. Generally, peptides contain on the order of several dozen amino acids.

[0055] The term "polypeptide" refers to any of a variety of amides derived from two or more amino acids by combination of the amino group of one amino acid with the carboxyl group of another amino acid, usually obtained by partial hydrolysis of proteins. Generally, peptides contain roughly several dozen or more amino acids.

[0056] The terms "pharmacologically" or "pharmacologically acceptable" as used herein refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans.

[0057] The term "pharmaceutically acceptable carrier" as used herein includes, but is not limited to, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, as well as vegetable oils, coatings, isotonic and absorption delaying agents, liposomes, commercially available The carrier may include any and all solvents or dispersion media, including detergents, etc. Supplementary bioactive ingredients may also be incorporated into such carriers.

[0058] "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, which may be single-stranded or double-stranded and represent the sense or antisense strand.

[0059] The term "isolated nucleic acid," as used herein, refers to any nucleic acid molecule that has been removed from its natural state (e.g., removed from a cell and, in preferred embodiments, free of other genomic nucleic acid).

[0060] The terms "amino acid sequence" and "polypeptide sequence" as used herein are interchangeable and refer to a sequence of amino acids.

[0061] As used herein, the term "portion" when in reference to a protein (such 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 entire amino acid sequence minus one amino acid.

[0062] The term "portion" when used in reference to a nucleotide sequence refers to a fragment of that nucleotide sequence. Fragments can range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue.

[0063] The term "biologically active" refers to any molecule that has a structural, regulatory or biochemical function. For example, biological activity can be determined by, for example, restoration of wild-type growth in cells lacking the activity of the protein. Cells lacking the activity of the protein can be created in many ways (i.e., point mutations and frameshift mutations, for example). Complementation is achieved by transfecting cells lacking the activity of the protein with an expression vector that expresses the protein, its derivatives, or parts thereof.

[0064] As used herein, the term "complementary" or "complementarity" is used in reference to base pairing rules with respect to "polynucleotides" and "oligonucleotides" (terms that refer to sequences of nucleotides). For example, the sequence "CAGT" is complementary to the sequence "GTCA". Complementarity can be "partial" or "complete". "Partial" complementarity is when one or more nucleic acid bases do not match according to the base pairing rules. "Complete" or "complete" complementarity between nucleic acids is when each and every nucleic acid base matches another base under the base pairing rules. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions, as well as detection methods that rely on binding between nucleic acids.

[0065] As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids using any process in which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) depend on the degree of complementarity between the nucleic acids, the stringency of the conditions required, the T of the hybrid formed, and the degree of complementarity between the nucleic acids. m It is influenced by factors such as the G:C ratio within the nucleic acid.

[0066] As used herein, the term "hybridization complex" refers to a complex formed between two nucleic acid sequences by the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases, which may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. Hybridization complexes can also be formed in solution (e.g., C 0 t or R 0Interleukin-1 (IL-1) and IL-2 (IL-3) binding assays), or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., nylon membranes or nitrocellulose filters used in Southern and Northern blotting, dot blots or glass slides used in in situ hybridization, including FISH (fluorescence in situ hybridization)).

[0067] Transcriptional control signals in eukaryotes include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription. Maniatis, T. et al., Science 236:1237 (1987). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes from plants, yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on which cell type is used to express the protein of interest.

[0068] The term "poly A site" or "poly A sequence" as used herein refers to a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of recombinant transcripts is desirable, since transcripts lacking a poly A tail are unstable and rapidly degraded. The poly A signal utilized in an expression vector may be "heterologous" or "endogenous." An endogenous poly A signal is one that is naturally found at the 3' end of the coding region of a given gene in the genome. A heterologous poly A signal is one that is isolated from one gene and placed 3' of another gene. Efficient expression of recombinant DNA sequences in eukaryotic cells involves the expression of signals that direct efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are several hundred nucleotides in length.

[0069] The terms "transfection" or "transfected" refer to the introduction of foreign DNA into a cell.

[0070] As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Thus, a DNA sequence encodes an amino acid sequence.

[0071] As used herein, the term "coding region," when used in reference to a structural gene, refers to a nucleotide sequence that encodes the amino acids found in a nascent polypeptide as a result of translation of an mRNA molecule. In eukaryotes, a coding region is bounded at the 5' by the nucleotide triplet "ATG," which encodes an initiator methionine, and at the 3' by one of three triplets that specify a stop codon (i.e., TAA, TAG, TGA).

[0072] As used herein, the term "structural gene" refers to a DNA sequence that codes for an RNA or protein. In contrast, a "regulatory gene" is a structural gene that codes for a product (e.g., a transcription factor) that controls the expression of other genes.

[0073] As used herein, the term "gene" refers to a deoxyribonucleotide sequence comprising the coding region of a structural gene, including sequences located adjacent to the coding region at the 5' and 3' ends for a distance of about 1 kb on either end, and thus a gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of genes. Genomic forms or clones of genes contain coding regions interrupted with non-coding sequences referred to as "introns" or "intervening regions" or "intervening sequences". Introns are segments of a gene that are transcribed into heterologous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced ​​out" from the nuclear or primary transcript; therefore, introns are absent from messenger RNA (mRNA) transcripts. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0074] In addition to containing introns, genomic forms of genes may also contain sequences located at both the 5' and 3' ends of the sequences present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or affect the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation.

[0075] The term "viral vector" encompasses any nucleic acid construct derived from a viral genome that can incorporate a heterologous nucleic acid sequence for expression in a host organism. For example, such viral vectors can include, but are not limited to, adeno-associated viral vectors, lentiviral vectors, SV40 viral vectors, retroviral vectors, and adenoviral vectors. Viral vectors are sometimes created from pathogenic viruses, which can be modified to minimize their overall health risks. This usually involves deleting the part of the viral genome involved in viral replication. Such viruses can efficiently infect cells, but once infection has occurred, the virus may require a helper virus to provide the missing proteins to produce new virions. Preferably, the viral vector should have minimal impact on the physiology of the cells it infects and also exhibit genetically stable properties (e.g., not undergo spontaneous genome rearrangement). Most viral vectors are engineered to infect as wide a range of cell types as possible. Even so, the viral receptor can be modified to target the virus to a specific type of cell. Viruses modified in this way are said to be pseudotyped. Viral vectors are often engineered to incorporate specific genes that help identify which cells have incorporated the viral genes. These genes are called marker genes. For example, a common marker gene confers antibiotic resistance to a particular antibiotic.

[0076] The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. In certain embodiments, for example, the following items are provided: (Item 1) Truncated repeat: A single guide ribonucleic acid (sgRNA) sequence that contains an anti-repeat region. (Item 2) The sgRNA sequence of item 1, further comprising a shortened Stem 2 region. (Item 3) The sgRNA sequence of item 2, further comprising a shortened spacer region. (Item 4) 2. The sgRNA sequence of item 1, which is 121 nucleotides in length. (Item 5) 3. The sgRNA sequence of item 2, wherein the length of the sgRNA sequence is selected from the group consisting of 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 101 nucleotides, and 99 nucleotides. (Item 6) 4. The sgRNA sequence of item 3, which is 100 nucleotides in length. (Item 7) The sgRNA sequence of item 1, which is an Nme1Cas9 single guide ribonucleic acid sequence or an Nme2Cas9 single guide ribonucleic acid sequence. (Item 8) A single guide ribonucleic acid (sgRNA) sequence containing a shortened Stem 2 region. (Item 9) The sgRNA sequence of item 8, further comprising a truncated repeat:anti-repeat region. (Item 10) The sgRNA sequence of item 9, further comprising a shortened spacer region. (Item 11) 10. The sgRNA sequence of claim 9, wherein the length of the sgRNA sequence is selected from the group consisting of 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 101 nucleotides, and 99 nucleotides. (Item 12) 11. The sgRNA sequence of item 10, which is 100 nucleotides in length. (Item 13) Adeno-associated virus (AAV) plasmid containing a single guide ribonucleic acid - Neisseria meningitidis Cas9 nucleic acid vector. (Item 14) 14. The AAV plasmid of item 13, wherein the single guide ribonucleic acid-Neisseria meningitidis Cas9 nucleic acid vector comprises at least one promoter. (Item 15) 15. The AAV plasmid of item 14, wherein the at least one promoter is selected from the group consisting of a U6 promoter and a U1a promoter. (Item 16) 14. The AAV plasmid of item 13, wherein the single guide ribonucleic acid-Neisseria meningitidis Cas9 nucleic acid vector comprises a Kozak sequence. (Item 17) 14. The AAV plasmid of item 13, wherein the sgRNA comprises a nucleic acid sequence complementary to a sequence of a gene of interest. (Item 18) 18. The AAV plasmid of item 17, wherein the sequence of the gene of interest is selected from the group consisting of a PCSK9 sequence and a ROSA26 sequence. (Item 19) 14. The AAV plasmid of claim 13, wherein the sgRNA comprises a truncated repeat-anti-repeat sequence. (Item 20) 20. The AAV plasmid of claim 19, wherein the sgRNA further comprises a shortened Stem 2 region. (Item 21) 21. The AAV plasmid of claim 20, wherein the sgRNA further comprises a shortened spacer region. (Item 22) 20. The AAV plasmid of claim 19, wherein the sgRNA sequence is 121 nucleotides in length. (Item 23) 21. The AAV plasmid of claim 20, wherein the length of the sgRNA sequence is selected from the group consisting of 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 101 nucleotides, and 99 nucleotides. (Item 24) 22. The AAV plasmid of claim 21, wherein the sgRNA sequence is 100 nucleotides in length. (Item 25) 14. The AAV plasmid of item 13, wherein the sgRNA comprises a shortened Stem 2 region. (Item 26) 26. The AAV plasmid of claim 25, wherein the sgRNA further comprises a truncated repeat:anti-repeat region. (Item 27) 27. The AAV plasmid of claim 26, wherein the sgRNA further comprises a shortened spacer region. (Item 28) 27. The AAV plasmid of claim 26, wherein the length of the sgRNA sequence is selected from the group consisting of 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 101 nucleotides, and 99 nucleotides. (Item 29) 28. The AAV plasmid of claim 27, wherein the sgRNA sequence is 100 nucleotides in length. (Item 30) a) i) a patient exhibiting at least one symptom of a medical condition, the patient comprising a plurality of genes associated with said medical condition; ii) an adeno-associated virus (AAV) plasmid comprising a single guide ribonucleic acid-Neisseria meningitidis Cas9 nucleic acid vector, wherein the sgRNA comprises a nucleic acid sequence complementary to a portion of at least one of the plurality of genes; and providing a b) administering said AAV plasmid to said patient under conditions such that said at least one symptom of said medical condition is alleviated. The method includes: (Item 31) 31. The method of claim 30, wherein the medical condition comprises hypercholesterolemia. (Item 32) 31. The method of claim 30, wherein the at least one of the plurality of genes is a PCSK9 gene. (Item 33) 33. The method of claim 32, wherein the sgRNA nucleic acid is complementary to a portion of the PCSK9 gene. (Item 34) 31. The method of claim 30, wherein the sgRNA comprises a truncated repeat-anti-repeat sequence. (Item 35) 35. The method of claim 34, wherein the sgRNA further comprises a shortened Stem 2 region. (Item 36) 36. The method of claim 35, wherein the sgRNA further comprises a shortened spacer region. (Item 37) 35. The method of claim 34, wherein the sgRNA sequence is 121 nucleotides in length. (Item 38) 36. The method of claim 35, wherein the length of the sgRNA sequence is selected from the group consisting of 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 101 nucleotides, and 99 nucleotides. (Item 39) 22. The method of claim 21, wherein the sgRNA sequence is 100 nucleotides in length. (Item 40) 31. The method of claim 30, wherein the sgRNA comprises a shortened Stem 2 region. (Item 41) 41. The method of claim 40, wherein the sgRNA further comprises a truncated repeat:anti-repeat region. (Item 42) 42. The method of claim 41, wherein the sgRNA further comprises a shortened spacer region. (Item 43) 42. The method of claim 41, wherein the length of the sgRNA sequence is selected from the group consisting of 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 101 nucleotides, and 99 nucleotides. (Item 44) 43. The method of claim 42, wherein the sgRNA sequence is 100 nucleotides in length. (Item 45) 1. An adeno-associated virus (AAV) plasmid encoding a Cas9 Type II-C nuclease protein, the protein comprising a protospacer adjacent motif recognition domain configured with a binding site for a protospacer adjacent motif sequence comprising between 1 and 4 required nucleotides. (Item 46) 46. ​​The adeno-associated virus plasmid of item 45, wherein the type II-C Cas9 nuclease protein is selected from the group consisting of Neisseria meningitidis strain De10444 Nme2Cas9 nuclease protein, Haemophilus parainfluenzae HpaCas9 nuclease protein, and Simonsiella muelleri SmuCas9 nuclease protein. (Item 47) The protospacer adjacent motif sequence containing between 1 and 4 required nucleotides is 4 CN 3 , N 4 C.T., N. 4 C.C.N. 4 CCA, and N 4 GNT 3 47. The adeno-associated virus plasmid of item 46, selected from the group consisting of: (Item 48) The 1 to 4 required nucleotides are C, CC, CT, CCN, CCA, CN 3 and G.N.T. 2 46. ​​The adeno-associated virus plasmid of item 45, selected from the group consisting of: (Item 49) 46. ​​The adeno-associated virus plasmid of claim 45, wherein the Type II-C Cas9 nuclease protein is linked to a truncated sgRNA. (Item 50) a) i) a patient exhibiting at least one symptom of a medical condition, the patient comprising a plurality of genes associated with the medical condition, the plurality of genes comprising a protospacer adjacent motif comprising between 2 and 4 required nucleotides; ii) a delivery platform comprising at least one nucleic acid encoding a Cas9 type II-C nuclease protein, said protein comprising a protospacer adjacent motif recognition domain configured with a binding site for said protospacer adjacent motif sequence comprising between 2 and 4 required nucleotides; providing a b) administering said delivery platform to said patient under conditions such that said at least one symptom of said medical condition is alleviated; The method includes: (Item 51) 51. The method of claim 50, wherein the delivery platform comprises an adeno-associated virus plasmid. (Item 52) 51. The method of claim 50, wherein the delivery platform comprises a microparticle. (Item 53) 51. The method of claim 50, wherein the type II-C Cas9 nuclease protein is selected from the group consisting of Neisseria meningitidis strain De10444 Nme2Cas9 nuclease protein, Haemophilus parainfluenzae HpaCas9 nuclease protein, and Simonsiella muelleri SmuCas9 nuclease protein. (Item 54) The protospacer adjacent motif sequence containing 1 to 4 required nucleotides is 4 C 3 , N 4 C.T., N. 4 C.C.N. 4 CCA, and N 4 GNT 3 51. The method of claim 50, selected from the group consisting of: (Item 55) The 1 to 4 required nucleotides are C, CC, CT, CCN, CCA, CN 3 and G.N.T. 2 51. The adeno-associated virus plasmid of item 50, selected from the group consisting of: (Item 56) 51. The method of claim 50, wherein the Type II-C Cas9 nuclease protein is bound to a truncated sgRNA. (Item 57) 51. The method of claim 50, wherein the medical condition is selected from the group consisting of hyperlipidemia and tyrosinemia. [Brief description of the drawings]

[0077] [Figure 1] FIG. 1 provides representative sequences of conventional, full-length, 145 nt Nme1Cas9 sgRNAs and Nme2Cas9 sgRNAs. [Diagram 2] Figure 2 presents exemplary Nme1Cas9 sgRNA sequences with truncated repeat:anti-repeat regions or truncated Stem 2 regions and associated gene editing activity. Nme1Cas9 sgRNA deletion / truncation series. Top: Aligned sequences, color coded as in Figure 1. Bottom: T7E1 assay for editing at Nme1Cas9 target site 7 (NTS7) using the indicated sgRNAs as guides. [Diagram 3] Figure 3 presents exemplary Nme1Cas9 sgRNA sequences with truncated repeat:anti-repeat regions or truncated Stem 2 regions and associated gene editing activity. The shortest Nme1Cas9 sgRNAs (#10-101nt; 24nt guide sequence; and #11-100nt; 23nt guide sequence) efficiently edit three distinct target sites (NTS7, NTS27, and NTS55) in the human genome. Top: Sequences of wild-type and minimized sgRNAs, using the same color scheme as in previous figures. Bottom: T7E1 assay for editing efficiency at three target sites in HEK293T cells. [Figure 4-1]Figure 4 presents exemplary sequences (as secondary structures) of Nme1Cas9 wt sgRNA, and truncated sgRNAs 11 and 12, and associated gene editing by RNP delivery of Nme1Cas9 and sgRNA. Three genomic sites (N-TS72, N-TS55, and N-TS40), and one traffic light reporter site, were targeted in the human genome using HEK293T cells. Top: Sequences are shown as secondary structures of wild-type and minimized sgRNAs. Bottom: Editing efficiency measured by T7E1 assay or flow cytometry is shown as a bar graph. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Diagram 5] Figure 5 is a graph showing gene editing using minimized sgRNA 11 and in vitro transcribed wt sgRNA in PLB985 cells. Cells were transfected with Nme1Cas9 and sgRNA RNP complexes and gene editing at genomic site N-TS72 was measured by TIDE. [Figure 6] 6 is a schematic diagram of one embodiment of an AAV vector containing an intact CRISPR / Cas9 gene editing complex. Representative sequences of various AAV vector regions are color-coded in Appendix 1. [Figure 7] Figure 7 presents one embodiment of a color-coded sequence of the Nme single guide RNA and promoter shown in Figure 4. The backbone has been linearized using SapI to insert a 24 nt target spacer. U6 promoter: turquoise Nme single guide RNA: purple SapI restriction site: bold [Figure 8]Figure 8 presents one embodiment of a color-coded sequence of the Nme1Cas9 and promoter shown in Figure 4. Start and stop codons are bold and underlined. U1a promoter: blue Kozak sequence: grey Humanized Nme1Cas9: red SV40 NLS: green Nucleoplasmin (NP) NLS: yellow HA tag (3x): bold orange Synthetic NLS: cyan Beta globin polyadenylation signal: dark cyan [Figure 9] FIG. 9 presents exemplary data showing the editing efficiency of various target sites using AAV plasmids carrying sgRNA-Nme1Cas9 constructs directed to either the Pcsk9 gene or the Rosa26 gene (control). [Figure 10] Figure 10 presents one embodiment of color-coded target site sequences for sgRNA-Nme1Cas9 constructs guided to either the Pcsk9 gene or the Rosa26 gene (control). 24 nt Nme1Cas9 target spacer, bold blue Nme1Cas9 PAM, underlined [NNNNGATT) T7E1 primer binding site: italics in green TIDE primer binding site: italics in purple [Figure 11] FIG. 11 presents exemplary data showing gene editing efficiency following in vivo hydrodynamic injection of 30 μg of endotoxin-free sgRNA-Nme1Cas9-AAV plasmid targeting Pcsk9 in the tail vein of mice. [Figure 12]Figure 12A is a graph showing exemplary data showing gene editing efficiency in liver Pcsk9 gene and Rosa26 gene 14 days after vector administration by Nme1-Cas9 vector packaged in hepatocyte-specific AAV8 serotype at a dose of 4 x 10 genome copies (gc) per mouse. Figure 12B is a graph showing exemplary data showing gene editing efficiency in liver Pcsk9 gene and Rosa26 gene 50 days after vector administration by Nme1-Cas9 vector packaged in hepatocyte-specific AAV8 serotype at a dose of 4 x 10 genome copies (gc) per mouse. [Figure 13] FIG. 13 is a graph presenting exemplary data showing reduction in mouse cholesterol levels at days 0, 25, and 50 after injection of sgRNA-Cas9-AAV vectors targeting the Pcsk9 gene, Rosa26 gene, and a PBS control group. [Figure 14] Figures 14A and 14B are graphs presenting exemplary data showing a genome-wide unbiased identification of double-stranded breaks (DSBs) enabled by sequencing (e.g., GUIDE-Seq) assay searching for off-target editing sites for both Pcsk9-sgRNA-Cas9-AAV (A) and Rosa26-sgRNA-Cas9-AAV (B). [Figure 15] Figure 15 is a graph presenting exemplary data showing targeted TIDE analysis in mice 14 days after injection with both Pcsk9-sgRNA-Cas9-AAV and Rosa26-sgRNA-Cas9-AAV, revealing minimal cleavage. OnT, on-target site; OT1, OT2, etc.: off-target site. [Figure 16] Figure 16 presents exemplary data showing hematoxylin and eosin staining assays in liver sections from mice sacrificed 14 days after injection of Pcsk9 gene-targeting vector and Rosa26 gene-targeting vector, with no evidence of a host immune response. [Figure 17]Figure 17 illustrates one embodiment of an in vitro PAM library identification workflow. NGS, next generation sequencing. [Figure 18] Figure 18 presents the predicted sequences from the in vitro PAM discovery assay shown in Figure 17. Recombinantly purified Cas9 from each bacterium was incubated with sgRNA and targets with randomized PAMs. Nme1Cas9 was used as a control. [Figure 19] Figure 19 is a graph presenting exemplary data showing percent genome editing at a single site in the human genome in HEK293T cells (top panel). The percentages indicate indel formation estimated using a T7E1 endonuclease assay (Nme2Cas9, HpaCas9) or a fluorescent assay (for SmuCas9) based on a "traffic light" reporter integrated into the genome of HEK293T cells. [Figure 20] Figure 20 is a graph showing exemplary data showing genome editing of integrated traffic light reporters using Nme2Cas9 (X-axis) targeting different protospacers with different PAMs in HEK293T cells. The results suggest that NNNNCC PAM is preferred for Nme2Cas9 in human cells. [Figure 21] Figure 21 presents exemplary data showing genome editing in HEK293T cells in the presence of various anti-CRISPR (Acr) proteins. T7E1 digestion shows genome editing after plasmid transfection (to express Nme2Cas9 and its sgRNA) or RNA / protein delivery (HpaCas9 and its sgRNA). Nme2Cas9 is robustly inhibited by two Acr proteins (AcrIIC3Nme and AcrIIC4Hpa), while HpaCas9 is inhibited by four of the previously reported II-C type Acr proteins. These results indicate that these two Cas9 proteins are subject to anti-CRISPR off-switch control. [Figure 22]Figure 22 is a graph presenting exemplary data of traffic light reporter (TLR) gene editing using Nme2Cas9-sgRNA complexes with a "CC" dinucleotide PAM. Figure 22A. Blue bars are % of cells showing fluorescence, and red bars indicate % editing based on sequencing accuracy ("TIDE analysis"). [Figure 23] FIG. 23 presents exemplary data of gene editing with Nme2Cas9 using T7E1 assays at the AAVS1, chromosome 14 NTS4, VEGF and CFTR loci. [Figure 24] FIG. 24 presents one embodiment of a wild-type Nme2Cas9 bacterial open reading frame DNA sequence. [Diagram 25] FIG. 25 presents one embodiment of a wild-type Nme2Cas9 bacterial protein sequence. [Figure 26] Figure 26 presents one embodiment of the Nme2Cas9 human codon-optimized open reading frame DNA sequence. Yellow - SV40 NLS; Green - 3x-HA tag; Blue: cMyc-like NLS. [Figure 27] Figure 27 presents one embodiment of an Nme2Cas9 humanized protein sequence. Yellow - SV40 NLS; Green - 3x-HA tag; Blue: cMyc-like NLS. [Figure 28] FIG. 28 presents one embodiment of an HpaCas9 bacterial protein sequence. [Figure 29] FIG. 29 presents one embodiment of a SmuCas9 native bacterial open reading frame DNA sequence. [Diagram 30] FIG. 30 presents one embodiment of a SmuCas9 bacterial protein sequence. [Diagram 31] Figure 31 presents one embodiment of a SmuCas9 human codon-optimized open reading frame DNA sequence. Yellow - SV40 NLS; Green - 3x-HA tag; Blue: cMyc-like NLS. [Diagram 32]Figure 32 presents one embodiment of a SmuCas9 humanized protein sequence. Yellow - SV40 NLS; Green - 3x-HA tag; Blue: cMyc-like NLS. [Diagram 33] Figure 33 presents exemplary Type IIC Cas9 ortholog single guide RNA sequences compatible with short C-rich PAMs. Yellow - crRNA; Grey - linker; Purple - tracrRNA. [Figure 34-1] Figure 34 illustrates three closely related Neisseria meningitidis Cas9 orthologues with distinct PAMs. Figure 34A: Schematic showing mutated residues (orange spheres) in Nme2Cas9 (left) and Nme3Cas9 (right) mapped onto the predicted structure of Nme1Cas9. A cluster of mutations in the PID (black) is evident. Figure 34B: Experimental workflow for an in vitro PAM discovery assay with a 10 nt randomized PAM sequence downstream of the protospacer. Adapters were ligated to the cleavage products and sequenced. [Figure 34-2] Figure 34C: Sequence logo of an in vitro PAM discovery assay demonstrating the N4GATT PAM for Nme1Cas9 as previously shown in cells. Figure 34D: Sequence logo showing that Nme1Cas9, with its PID replaced by that of Nme2Cas9 (left) and Nme3Cas9 (right), recognizes a C at position 5. The remaining nucleotides were determined with low confidence due to the modest cleavage efficiency of the protein chimera (Figure 35C). [Figure 34-3] Figure 34E: Sequence logo illustrating that full-length Nme2Cas9 recognizes the N4CC PAM based on PAM discovery assays with a fixed C at position 5 and randomized 1-4 nt and 6-8 nt PAMs. [Figure 35-1]Figure 35 shows the characterization of Neisseria meningitidis Cas9 orthologues with rapidly evolving PIDs according to Figure 34. Figure 35A: Unrooted phylogenetic tree of NmeCas9 orthologues >80% identical to Nme1Cas9. Three distinct branches emerged with the majority of mutations clustered in PIDs. Group 1 (blue) PIDs with >98% identity to Nme1Cas9, group 2 (orange) with PIDs ~52% identical to Nme1Cas9, and group 3 (green) with PIDs ~86% identical to Nme1Cas9. Three representative Cas9 orthologues (Nme1Cas9, Nme2Cas9, and Nme3Cas9) from each group are marked. Figure 35B: Schematic showing the CRISPR loci of strains encoding the three Cas9 orthologues (Nme1Cas9, Nme2Cas9, and Nme3Cas9) from (A). The percent identity of each CRISPR-Cas component to N. meningitidis 8013 (encoding Nme1Cas9) is shown. [Figure 35-2] Figure 35C: Number of reads from cleaved DNA from in vitro assays for intact Nme1Cas9 and for chimeras in which the PID of Nme1Cas9 was replaced with the PIDs of Nme2Cas9 and Nme3Cas9. The reduced read count indicates lower cleavage efficiency in the chimeras. [Figure 35-3] Figure 35D: Sequence logos from an in vitro PAM discovery assay for NNNNCNNN randomized PAM by Nme1Cas9, where the PID was replaced by the PID of Nme2Cas9 (left) or Nme3Cas9 (right). [Figure 36-1]Figure 36: Nme2Cas9 recognizes and edits sites adjacent to the N4CC PAM using a 22-24 nucleotide spacer. All experiments were performed in triplicate. Error bars represent standard error of the mean (sem). Figure 36A: Schematic showing the transient transfection workflow for HEK293T TLR2.0 cells. Nme2Cas9 and sgRNA plasmids were transfected and mCherry+ cells were detected 72 hours post-transfection. Figure 36B: Targeting an array of PAMs using Nme2Cas9 in TLR2.0. All sites with N4CC PAMs were targeted with varying degrees of efficiency, but no targeting by Nme2Cas9 was observed with the N4GATT PAM or in the absence of sgRNA. SpyCas9 (targeting NGG) and Nme1Cas9 (targeting N4GATT) were used as positive controls. [Figure 36-2] Figure 36C: Effect of spacer length on the efficiency of editing by Nme2Cas9. sgRNAs targeting TLR2.0 sites (with N4CCA PAM) with spacer lengths varying from 24nt to 20nt (including a G at the 5' end), showing highest editing efficiency with a spacer of 22-24 nucleotides. Figure 36D: Nme2Cas9 nickases (HNH nickase = Nme2Cas9D16A; RuvC nickase = Nme2Cas9H588A) can be used in tandem to generate indels within TLR2.0. Targets with cut sites 32 and 64 base pairs apart were targeted using either nickase to generate indels. HNH nickase shows efficient editing, especially when the cut sites are close (32bp). Wild-type Nme2Cas9 was used as a control. Green is GFP (HDR) and red is mCherry (NHEJ). [Figure 37-1]Figure 37 is a graph presenting exemplary data for PAM, spacer, and seed elements for targeting by Nme2Cas9 in mammalian cells according to Figure 36. All experiments were performed in triplicate. Error bars represent sem. Figure 37A: Targeting by Nme2Cas9 to the N4CD site in TLR2.0. Four sites were investigated for each non-C nucleotide at the tested position (N4CA, N4CT, and N4CG), and the N4CC site was used as a positive control. [Figure 37-2] Figure 37B: Nme2Cas9 targeting of TLR2.0 to N4 DC sites [same as (A)]. [Figure 37-3] Figure 37C: Guide shortening for another TLR2.0 site. A similar length to that observed in Figure 36C was found to be required. Figure 37D: Targeting efficiency with Nme2Cas9 is differentially sensitive to a single nucleotide mismatch in the seed sequence. Data show the impact of moving a single nucleotide mismatch in the sgRNA along the 23 nt spacer at the TLR target site. [Figure 38]Figure 38 is a graph presenting exemplary data showing genome editing efficiency by Nme2Cas9 at genomic loci in mammalian cells by multiple delivery methods. All results represent three independent biological repeats, and error bars represent sem. Figure 38A: Genome editing by Nme2Cas9 using transient transfection with sgRNA targeting loci throughout the human genome in HEK293T cells. To demonstrate the range of indels induced by Nme2Cas9 at different loci (as detected by TIDE), 14 sites were selected based on an initial screening of 38 sites. Nme1Cas9 target site (with N4GATT PAM) was used as a negative control. Figure 38B: Left panel: Transient transfection of integrated plasmid (Nme2Cas9+sgRNA) targeting Pcsk9 and Rosa26 loci in Hepa1-6 mouse cells as detected by TIDE. Right panel: Electroporation of sgRNA plasmid into K562 cells stably expressing Nme2Cas9 from a lentivector results in efficient indel formation at the intended locus. Figure 38C: Nme2Cas9 can be electroporated as an RNP complex for efficient genome editing. 40 pmoles of Cas9 were electroporated into HEK293T cells along with 50 pmoles of in vitro transcribed sgRNAs targeting three different loci. Indels were measured after 72 hours using TIDE. [Figure 39-1] Figure 39 presents exemplary data showing dose-dependence and block deletion by Nme2Cas9 according to Figure 38. Figure 39A: Increasing the dose of Nme2Cas9 plasmid electroporated (500 ng vs. 200 ng in Figure 3A) improves editing efficiency at two sites (TS16 and TS6). [Figure 39-2] Figure 39B: Nme2Cas9 can be used to create block deletions. Two TLR2.0 targets with cut sites 32 bp apart were targeted simultaneously with Nme2Cas9. The majority of lesions created were precise 32 bp deletions (green). [Figure 40-1] Figure 40 presents exemplary data showing that type II-C anti-CRISPR proteins can be used to inhibit Nme2Cas9 gene editing activity in vitro and in vivo (e.g., as an off switch). All experiments were performed in triplicate. Error bars represent sem. Figure 40A: In vitro cleavage assays of Nme1Cas9 and Nme2Cas9 in the presence of five previously characterized anti-CRISPR proteins (Acr:Cas9 at a ratio of 10:1). Top: In the absence of Acr or in the presence of a control Acr (AcrE2), Nme1Cas9 efficiently cleaves a fragment containing a protospacer with an N4GATT PAM. As expected, all other previously characterized Acrs inhibit Nme1Cas9. Bottom: In the presence of AcrE2 and AcrIIC5Smu, Nme2Cas9 efficiently cleaves targets containing a protospacer with an N4CC PAM, suggesting that AcrIIC5Smu cannot inhibit Nme2Cas9 at a 10:1 molar ratio. [Figure 40-2] Figure 40B: Genome editing in the presence of five previously described anti-CRISPR proteins. HEK293T cells were co-transfected with plasmids expressing Nme2Cas9, sgRNA and each Acr (Cas9 200ng, sgRNA 100ng, Acr 200ng), and genome editing was measured using TIDE 72 hours after transfection. Except for AcrE2 and AcrIIC5Smu, all other Acrs inhibited genome editing, although with different efficiencies. [Figure 40-3] Figure 40C: Acr inhibition of Nme2Cas9 is dose-dependent with distinct potencies. AcrIIC1Nme and AcrIIC4Hpa completely inhibited Nme2Cas9 at co-transfected plasmid ratios of 2:1 and 1:1, respectively. [Diagram 41]Figure 41 presents exemplary data showing that Nme2Cas9 PID replacement renders Nme1Cas9 insensitive to inhibition by AcrIIC5Smu in accordance with Figure 40. In vitro cleavage with the Nme1Cas9-Nme2Cas9PID chimera was performed in the presence of the previously characterized Acr protein (Cas9-sgRNA 10 μM + Acr 100 μM). [Figure 42-1] Figure 42 presents exemplary data showing that Nme2Cas9 has no detectable off-targets in mammalian cells. Figure 42A: Schematic showing a duplex site (DS) that can be targeted by both SpyCas9 and Nme2Cas9 due to non-overlapping PAMs. Nme2Cas9 PAM (orange) and SpyCas9 PAM (blue) are highlighted. Figure 42B: Nme2Cas9 and SpyCas9 induce indels at duplex sites. Six duplex sites in VEGFA with GN3GN19NGGNCC sequence were selected for direct comparison between the two orthologs. Plasmids expressing each Cas9 (with the same promoter and NLS) were transfected into HEK293T cells together with the cognate guide of each ortholog. Indel rates were determined by TIDE 72 hours after transfection. Editing by Nme2Cas9 was detectable at all six sites and was more efficient than SpyCas9 at two sites (DS2 and 6). SpyCas9 edited four of the six sites (DS1, 2, 4, and 6) and showed significantly higher editing rates than Nme2Cas9 at two sites (DS1 and 4). Nme2Cas9 was as efficient as SpyCas9, less efficient than SpyCas9, and more efficient than SpyCas9 at DS2, 4, and 6, respectively, so these sites were selected for GUIDE-Seq analysis. [Figure 42-2]Figure 42C: Nme2Cas9 has a clean off-target profile in human cells. The number of off-target sites detected by GUIDE-Seq for each nuclease at individual target sites is shown. SpyCas9 off-target counts are shown in black. In addition to the dual sites, TS6 (due to its high efficiency and potential for off-targets) and the two mouse sites (to test accuracy in another cell type) also showed zero or one off-target site per guide. [Figure 42-3] Figure 42D: Targeted deep sequencing confirms the high accuracy of Nme2Cas9 as shown by GUIDE-seq. The top off-target loci detected by GUIDE-seq were amplified and deep sequenced. SpyCas9 showed off-targeting at the majority of loci, while Nme2Cas9 showed relatively low levels of editing at off-target loci (-40% on-target vs -1% off-target) at only one (Rosa26 site). Note the logarithmic scale of the y-axis. [Figure 42-4]Figure 42E: Efficiency of Nme2Cas9 and SpyCas9 varies based on locus and target site. Sites throughout the genome (with GN3GN19NGGNCC sequence) were selected for direct comparison of editing by the two orthologs. Plasmids expressing each Cas9 (with the same promoter, linker, tag and NLS) and its cognate guide were transfected into HEK293T cells. Indel efficiency was determined by TIDE 72 hours after transfection. Box plots show the editing efficiency at 28 double sites by Nme2Cas9 and SpyCas9 (left). Sites that showed no editing were excluded from the analysis. The relative efficiency of Nme2Cas9 and SpyCas9 shows that, on average, Nme2Cas9 is less efficient than SpyCas9 (right). Editing efficiency by both Cas9 orthologs at all 28 sites was included in the analysis of relative efficiency in the right panel. Figure 42F presents the nucleic acid sequence of the confirmed off-target site of the Rosa26 guide, showing the PAM region (underlined), the consensus CC PAM dinucleotide (bold), and three mismatches in the PAM-distal portion of the spacer (red). [Figure 43-1] Figure 43 presents exemplary data showing the orthogonality and relative accuracy of Nme2Cas9 and SpyCas9 at dual target sites according to Figure 42. Figure 43A: Nme2Cas9 and SpyCas9 guides are orthogonal. TIDE results show the frequency of indels created by both nucleases targeting DS12, either with their cognate sgRNAs or with other orthologous sgRNAs. [Figure 43-2] Figure 43B: Nme2Cas9 and SpyCas9 show comparable on-target editing efficiency during GUIDE-seq. Bars show on-target read counts at three duplex sites targeted by each ortholog from GUIDE-Seq. Orange bars represent Nme2Cas9 and black bars represent SpyCas9. [Figure 43-3]Figure 43C: SpyCas9 on-target versus off-target reads for each site. Orange bars represent on-target reads and black bars represent off-target. [Figure 43-4] Figure 43D: Nme2Cas9 on-target versus off-target reads for each site. [Figure 43-5] Figure 43E: Bar graph showing TIDEs at off-target sites predicted based on CRISPRseek. No indels were detected at off-target loci. [Figure 44-1] Figure 44 presents exemplary data showing genome editing by Nme2Cas9 via integrated AAV delivery in vivo. Figure 44A: Workflow of delivery of AAV8.Nme2Cas9+sgRNA to reduce cholesterol levels in mice by targeting Pcsk9. Top: Schematic of integrated AAV vector expressing Nme2Cas9 and sgRNA. Bottom: Timeline of AAV8.Nme2Cas9+sgRNA tail vein injection followed by cholesterol measurement on day 14 and indel, histology and cholesterol analysis on day 28. Figure 44B: Deep sequencing analysis to measure indels in DNA extracted from liver of mice injected with AAV8.Nme2Cas9+sgRNA targeting Pcsk9 locus and Rosa26 locus (control). [Figure 44-2] (C) Reduced serum cholesterol levels in mice injected with Pcsk9-targeting guides compared to Rosa26-targeting controls. P-values ​​are calculated by unpaired T-test. (D) H&E staining from livers of mice injected with AAV8.Nme2Cas9+sgRosa26 vector (left) or AAV8.Nme2Cas9+sgPcsk9 vector (right). Scale bar, 25 μm. [Diagram 45] FIG. 45 presents one embodiment of exemplary comparative TLR2.0 data for minimized and conventionally sized AAV scaffolds. [Figure 46]FIG. 46 provides a comparison of the Nme2Cas9 structures of truncated sgRNA 11 and truncated sgRNA 12. [Figure 47] FIG. 47 illustrates one embodiment of a minimized integrated AAV with a short polyA signal. [Figure 48-1] Figure 48 illustrates two embodiments of a minimized integrated AAV backbone: Tandem duplex sgRNA (top); Donor template for homology directed repair (bottom). [Figure 48-2] Same as above. [Figure 49] Figure 49 provides validation of the integrated AAV-sgRNA-hNme1Cas9 construct. Figure 49A: Schematic of a single rAAV vector expressing human codon-optimized Nme1Cas9 and its sgRNA. The backbone is flanked by AAV inverted terminal repeats (ITRs). The poly(a) signal is from rabbit beta globin (BGH). Figure 49B: Schematic of Pcsk9 (top) and Rosa26 (bottom) mouse genes. Red bars represent exons. A close-up shows the protospacer sequence (red) while the Nme1Cas9 PAM sequence is highlighted in green. The site where the double-stranded break is located is indicated (black arrowhead). Figure 49C: Stacked histograms showing representative percentage distribution of insertion-deletions (indels) obtained by TIDE after AAV-sgRNA-hNme1Cas9 plasmid transfection into Hepa1-6 cells targeting Pcsk9 (sgPcsk9) and Rosa26 (sgRosa26) genes. Data are presented as mean ± SD from three biological replicates. Figure 49D: Stacked histograms showing representative percentage distribution of indels in Pcsk9 in the liver of C57Bl / 6 mice obtained by TIDE after hydrodynamic injection of AAV-sgRNA-hNme1Cas9 plasmid. [Figure 50] FIG. 50 is a graph presenting exemplary data showing that many N4GN3 PAMs were inactive in the mouse genome and did not reveal off-target sites with fewer than four mismatches. [Figure 51] Figure 51 presents exemplary data showing that Nme1Cas9-mediated knockout of Hpd rescues the lethal phenotype in hereditary tyrosinemia type I mice. Figure 51A: Schematic of the Hpd mouse gene. Red bars represent exons. Zoom-in shows protospacer sequences (red) for targeting exon 8 (sgHpd1) and exon 11 (sgHpd2). The Nme1Cas9 PAM sequence is in green and the double-stranded break location is indicated (black arrowhead). Figure 51B: Experimental design. Three groups of hereditary tyrosinemia type I Fah- / - mice are injected with PBS or the integrated AAV-sgRNA-hNme1Cas9 plasmid sgHpd1 or sgHpd2. Figure 51C: Body weights of mice hydrodynamically injected with PBS (green), AAV-sgRNA-hNme1Cas9 plasmids sgHpd1 targeting Hpd exon 8 (red) or sgHpd2 targeting Hpd exon 11 (blue) were monitored after NTBC withdrawal. Error bars represent 3 mice in the PBS and sgHpd1 groups and 2 mice in the sgHpd2 group. Data are presented as mean ± SD. Figure 51D: Stacked histograms showing representative percentage distribution of indels in Hpd in livers of Fah- / - mice obtained by TIDE after hydrodynamic injection of PBS or sgHpd1 and sgHpd2 plasmids. Livers were collected at the end of NTBC withdrawal (day 43). [Figure 52] FIG. 52 is a graph presenting exemplary data showing the average indel efficiency of the guides presented in FIG. [Figure 53] FIG. 53 shows exemplary histological micrographs showing that sgHpd1 and sgHpd2 treated mice had substantially less severe liver injury compared to PBS injected Fahmut / mut mice, as indicated by a lower number of multinucleated hepatocytes compared to PBS injected mice. [Figure 54]Figure 54 presents AAV-mediated delivery of Nme1Cas9 for genome editing in vivo. Figure 54A: Experimental outline of AAV8-sgRNA-hNme1Cas9 vector tail vein injection to target Pcsk9 (sgPcsk9) and Rosa26 (sgRosa26) in C57Bl / 6 mice. Mice were sacrificed 4 days (n=1) or 50 days (n=5) after injection, and liver tissue was collected. Serum was collected for cholesterol level measurement at 0, 25, and 50 days after injection. Figure 54B: Serum cholesterol levels. p-values ​​are calculated by unpaired t-test. Figure 54C: Stacked histograms showing representative percentage distribution of indels in Pcsk9 or Rosa26 in mouse livers as measured by targeted deep sequencing analysis. Data are presented as mean ± SD from 5 mice per cohort. Figure 54D: Representative anti-PCSK9 Western blot using total protein harvested from mouse liver homogenates at day 50. 2 ng total of recombinant mouse PCSK9 (r-PCSK9) was included as a mobility standard. Asterisks indicate cross-reactive proteins greater than the control recombinant protein. [Figure 55] Figure 55 is a graph presenting exemplary data showing that mice injected with AAV8-sgRNA-hNme1Cas9 produce anti-Nme1Cas9 antibodies. [Figure 56]Figure 56 presents exemplary data showing GUIDE-seq genome-wide specificity of Nme1Cas9. Data are presented as mean ± SD. Figure 56A: Number of GUIDE-seq reads for on-target (OnT) and off-target (OT) sites. Figure 56B: Targeted deep sequencing to measure the rate of damage at each of the OT sites in Hepa1-6 cells. Mismatches with the OnT protospacer of each OT site are highlighted (blue). Data are presented as mean ± SD from three biological replicates. Figure 56C: Targeted deep sequencing to measure the rate of damage at each of the OT sites using genomic DNA obtained from mice injected with integrated AAV8-sgRNA-hNme1Cas9 sgPcsk9 and sgRosa26 and sacrificed at day 14 (D14) or day 50 (D50) post-injection. [Figure 57] Figure 57 presents exemplary data for ex vivo tyrosinase (Tyr) gene editing by Nme2Cas9 in mouse zygotes, related to Figure 58. Figure 57A: Two sites in the Tyr gene, each with an N4CC PAM, were tested for editing in Hepa1-6 cells. The sgTyr2 guide showed higher editing efficiency and was selected for further testing. Figure 57B: Seven mice survived postnatal development, each showing coat color phenotype as well as on-target editing assayed by TIDE. Figure 57C: Indel spectra from tail DNA of each mouse in Figure 57B, as well as an unedited C57BL / 6NJ mouse, as shown by TIDE analysis. The efficiency of various sizes of insertions (positive) and deletions (negative) is shown. [Figure 58]Figure 58 presents exemplary data of ex vivo Nme2Cas9 genome editing using monolithic AAV delivery. Figure 58A: Workflow of ex vivo single AAV Nme2Cas9 editing to generate albino C57BL / 6NJ mice by targeting the Tyr gene. Zygotes are cultured in KSOM containing AAV6.Nme2Cas9:sgTyr for 5-6 hours, rinsed in M2, cultured for 1 day, and then transferred into the oviducts of pseudopregnant recipients. Figure 58B: Albino (left) and chinchilla or spotted (middle) mice generated by zygotes carrying 3x109 GC AAV6.Nme2Cas9:sgTyr, and chinchilla or spotted mice (right) generated by zygotes carrying 3x108 GC AAV6.Nme2Cas9:sgTyr. Figure 58C: Summary of ex vivo Tyr editing experiments of Nme2Cas9.sgTyr single AAV at two AAV doses. [Figure 59] Figure 59 shows an alignment of the Nme1Cas9 and Nme2Cas9 nucleotide sequences. Legend: Non-PID aa differences (shaded in turquoise); PID aa differences (shaded in yellow); Active site residues (red text). [Figure 60] Figure 60 shows an alignment of Nme1Cas9 and Nme3Cas9 nucleotide sequences. Legend: Non-PID aa differences (shaded in turquoise); PID aa differences (shaded in yellow); Active site residues (red text). [Figure 61] Figure 61 depicts one embodiment of an Nme2Cas9 amino acid sequence. Legend: SV40 NLS (yellow shading); 3x-HA tag (green shading); cMyc-like NLS (cyan shading); Linker (purple shading). [Figure 62] Figure 62 depicts one embodiment of an Nme2Cas9 amino acid sequence. Legend: SV40 NLS (yellow shading); 3x-HA tag (green shading); nucleoplasmin-like NLS (red shading); c-myc NLS (cyan shading); linker (purple shading). [Figure 63]Figure 63 depicts one embodiment of a recombinant Nme2Cas9 (rNme2Cas9) amino acid sequence. Legend: SV40 NLS (yellow shading); Nucleoplasmin-like NLS (red shading); Linker (purple shading). [Figure 64] Figure 64 depicts one embodiment of an integrated AAV-sgRNA-hNmeCas9 plasmid nucleotide sequence. Legend: sgRNA scaffold (brown text); guide sequence (black text); U6 promoter (blue text); U1a promoter (purple text): NLS NLS (green text); hNmeCas9 (red text); NLS 3x-HA and NLS BGH-pA (alternating green / black text). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] The present invention relates to compositions and methods for gene therapy. Some techniques described herein utilize the Neisseria meningitidis Cas9 system, which provides an ultra-high precision CRISPR gene editing platform. Furthermore, the present invention incorporates improvements to this Cas9 system, such as shortening the single guide RNA sequence and packaging -Nme1Cas9 or Nme2Cas9 with its guide RNA into an adeno-associated viral vector compatible with in vivo administration. Furthermore, type II-C Cas9 orthologs have been identified that target protospacer adjacent motif sequences restricted to between 1 and 4 required nucleotides.

[0079] I. Accuracy of Neisseria meningitidis Cas9 (Nme1Cas9) / CRISPR gene editing Previously, an ultra-high accuracy version of the type II-C CRISPR-Cas9 system, called Neisseria meningitidis Cas9 (Nme1Cas9), was reported. In addition to being ultra-high accuracy, Nme1Cas9 is also smaller than the widely used Streptococcus pyogenes Cas9 (SpyCas9), which allows Nme1Cas9 to be more easily delivered by virus- and messenger RNA (mRNA)-based methods. Genome editing with Nme1Cas9 is generally achieved using plasmid transfection. Zhang et al., "Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis" Mol Cell 50:488-503 (2013);Hou et al., "Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis" Procd Natl Acad Sci USA 110:15644-15649 (2013);Esvelt et al., "Orthogonal Cas9 proteins for RNA-guided gene regulation and editing" Nature Methods 10:1116-1121 (2013);Zhang et al. al., "DNase H activity of Neisseria meningitidis Cas9" Mol Cell 60:242-255 (2015);Lee et al., "The Neisseria meningitidis CRISPR-Cas9 system enables specific genome editing in mammalian cells" Molecular Therapy 24:645-654 (2016);Pawluk et al., “Naturally occurring off-switches for CRISPR-Cas9" Cell 167:1829-1838 (2016); and Amrani et al., "Nme1Cas9 is an intrinsically high-fidelity genome editing platform" biorxiv.org / content / early / 2017 / 08 / 04 / 172650 (2017).

[0080] However, virus-based, RNA-based and ribonucleoprotein (RNP)-based Nme1Cas9 delivery have not been extensively explored. RNA-based and RNP-based delivery of Cas9 orthologs to manipulate genomes have several advantages over other delivery methods. RNA-based and RNP-based delivery not only circumvents the expression issues associated with DNA-based delivery of Cas9 and its sgRNA, resulting in faster editing, but also reduces the off-target effects associated with Cas9-based editing. The reduction in off-target activity is due to finer control of Cas9 RNA and RNP concentrations, as well as the relatively rapid degradation of Cas9 RNA and RNP in cells. It has been shown that the long-term presence of active Cas9 in cells is associated with higher off-target effects. Because Cas9 RNA and RNP are degraded more rapidly in cells, Cas9 delivered as RNA or RNP does not persist for long periods, thus reducing off-target effects.

[0081] The conventionally used Nme1Cas9 sgRNA of 145 nt in length contains a 48 nucleotide (nt) crRNA, a 4 nt linker, and a 93 nt tracrRNA. The sgRNA crRNA region consists of a first 24 nt spacer sequence and a second 24 nt repeat sequence that pairs with a 24 nt tracrRNA anti-repeat 5' region, thereby forming a repeat:anti-repeat region. The remaining 69 nt tracrRNA region contains the Stem 1 and Stem 2 regions. Figure 1.

[0082] This full-length Nme1Cas9 sgRNA has been successfully used for genome editing using a plasmid-based method. Furthermore, the in vitro transcribed Nme1Cas9 sgRNA can be complexed with purified Nme1Cas9 and used for genome editing in human cells. Although genome editing in human cells has been successful using in vitro transcribed sgRNA, the editing efficiency of the Nme1Cas9 RNP is reduced in difficult-to-transfect human cell lines such as PLB985.

[0083] It has been previously shown that the editing efficiency of Cas9 RNPs is proportional to the chemical stability of their sgRNAs. Although it is not necessary to understand the mechanism of the invention, it is believed that some cellular mechanism is used to rapidly degrade RNA. For this reason, Cas9 sgRNAs are routinely modified by chemical means. Some chemical modifications that confer increased stability to sgRNAs include, but are not limited to, ribose 2'-O-methylation and / or phosphorothioate linkages. Chemically modified RNAs are an option for improving genome editing with Cas9 RNPs, but their effectiveness is limited by the fact that chemical synthesis of RNA becomes increasingly difficult and expensive as the length of RNA increases. At 145nt, Nme1Cas9 sgRNA synthesis is not accessible to routine genome editing applications using chemically synthesized sgRNAs.

[0084] II. Truncated Nme1Cas9 sgRNA sequence Due to the above identified limitations that the full length 145 nt Nme1Cas9 sgRNA is too large for routine chemical synthesis of sgRNA for genome editing, in one embodiment of the present invention, a truncated Nme1Cas9 sgRNA is contemplated. Although it is not necessary to understand the mechanism of an invention, it is believed that the truncated Nme1Cas-sgRNA does not impair the function of the Nme1Cas9 RNP. Furthermore, the sgRNA for Nme1Cas9 and the sgRNA for Nme2Cas9 are identical and can be exchanged (Figure 35B), and thus sgRNA truncation is equally applicable to both Nme1Cas9 and Nme2Cas9. Exemplary sequences of truncated sgRNAs and associated target sites are disclosed below, where the nt number of the varying sgRNA within the guide region is indicated as "N" residues. Within the target sequence, the 24 nt recognized by the sgRNA guide region is underlined, and the protospacer adjacent motif (PAM) region is indicated in bold. Table 1. [Table 1]

[0085] As intended herein, shortened Nme1Cas9 sgRNA not only allows synthesis at reasonable cost, but also facilitates use in virus-based delivery methods (e.g., adeno-associated virus delivery platform) that limit DNA length.In one embodiment, shortened sgRNA reduces off-target Nme1Cas9 editing effect.In one embodiment, shortened Nme1Cas9 sgRNA comprises at least one chemical modification that increases Nme1Cas9 editing efficiency.

[0086] As discussed above, the full-length 145 nt sgRNA of Nme1Cas9 contains a guide region, a repeat:anti-repeat duplex region, a Stem 1 region, and a Stem 2 region. Figure 1. However, because the length of the sgRNA is problematic for routine genomic editing, it was highly desirable to develop a shortened sgRNA for Nme1Cas9. Currently, commercially available RNA synthesis methods require that the final RNA product not exceed approximately 100 nt.

[0087] In one embodiment, the present invention contemplates an Nme1Cas9 sgRNA that comprises a shortened repeat:anti-repeat duplex. In one embodiment, the present invention contemplates an Nme1Cas9 sgRNA that comprises a shortened stem 2. FIG. 2. Additionally, it has previously been shown that the 5' flexible guide crRNA region (e.g., spacer region) of Nme1Cas9 can also be shortened by several nucleotides without loss of function. Amrani et al., "Nme1Cas9 is an intrinsically high-fidelity genome editing platform" biorxiv.org / content / early / 2017 / 08 / 04 / 172650 (2017); and Lee et al., "The Neisseria meningitidis CRISPR-Cas9 system enables specific genome editing in mammalian cells" Molecular Therapy 24:645-654 (2016).

[0088] In one embodiment, the present invention contemplates a 100nt Nme1Cas9 truncated sgRNA. Figure 3, construct #11. This 100nt Nme1Cas9 truncated sgRNA construct #11 was tested in HEK293T cells by transient transfection at three different human genomic sites, and all three sites support Nme1Cas9 function at the same level, if not better, than the full-length Nme1Cas9 sgRNA. Figure 3, lower panel. Additionally, sgRNA 11 and sgRNA 13 were also tested using RNP-mediated delivery at several genomic target sites, with editing efficiencies similar to or higher than wt sgRNA. Figure 4. A synthetic version of construct #11 was also tested in PLB985 cells, resulting in higher editing efficiencies compared to in vitro transcribed wt sgRNA. Figure 5.

[0089] III. Related Adenoviral CRISPR Delivery Platforms Compared to transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs), Cas9 is distinguished by its flexibility and versatility. Komor et al., "CRISPR-based technologies for the manipulation of eukaryotic genomes" Cell 2017;168:20-36. Such properties make Cas9 ideal for advancing the field of genome engineering. Over the past few years, CRISPR-Cas9 has been used to enhance production in agriculture, food, and industry, in addition to promising applications in gene therapy and personalized medicine. Barrangou et al., "Applications of CRISPR technologies in research and beyond" Nat Biotechnol. 2016;34:933-41. Despite the diversity of class 2 CRISPR systems described, only a few of them have been developed and validated for genome editing in vivo. As shown herein, NmeCas9 is a small, high-fidelity Cas9 that can be explored for future integrated rAAV-based in vivo genome editing applications. The unique PAM of NmeCas9 enables editing at additional targets that are difficult to reach with the other two small, integrated rAAV-enabled orthologues (SauCas9 and CjeCas9).

[0090] Genome editing using the bacterial CRISPR system has opened new avenues for human gene therapy. Derived from the clustered regularly interspaced short palindromic repeats that capture snippets of invasive nucleic acids in bacteria, the CRISPR complex contains a guide RNA (e.g., sgRNA) that directs the nuclease Cas9 (CRISPR-associated protein 9) to cleave complementary double-stranded DNA. Non-homologous repair of Cas9-induced DNA breaks leads to small insertions or deletions (indels) that inactivate the target gene, but the breaks can also be repaired by homologous DNA templates, thereby resulting in gene replacement. Nelson et al., "In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy" Science 351:403-407 (2016); and Ran et al., "In vivo genome editing using Staphylococcus aureus Cas9" Nature 520:186-191 (2015); and Yin et al., "Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype" Nature Biotechnology 32:551-553 (2014).

[0091] Type II-A Streptococcus pyogenes (Spy)Cas9, currently widely used as a flexible genome editing tool, exhibits several disadvantages: i) inefficient delivery; ii) off-target cleavage; and iii) unregulated activity. These disadvantages severely limit SpyCas9 as a potential gene therapy tool. As discussed herein, the highly accurate and precise Nme1Cas9 or Nme2Cas9 complexes can overcome these limitations of SpyCas9.

[0092] Nme1Cas9 and Nme2Cas9 are shown herein to be efficient genome editing platforms in mammalian cells, and are smaller proteins than SpyCas9, making them easier to engineer viral vectors for in vivo delivery.In addition, Nme1Cas9 and Nme2Cas9 have significantly lower off-target editing than SpyCas9, and anti-CRISPR proteins have been identified that allow Nme1Cas9 and Nme2Cas9 activity to be controlled. Esvelt et al., "Orthogonal Cas9 proteins for RNA-guided gene regulation and editing" Nature Methods 10:1116-1121 (2013);Amrani et al., "Nme1Cas9 is an intrinsically high-fidelity genome editing platform" biorxiv.org / content / early / 2017 / 08 / 04 / 172650 (2017);Lee et al., 'The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells" Molecular Therapy 24:645-654 (2016);Hou et al., "'Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis" Procd Natl Acad Sci USA 110:15644-15649 (2013); and Pawluk et al., "Naturally Occurring Off-Switches for CRISPR-Cas9" Cell 167:1829-38 e9 (2016); and Figure 21.

[0093] Adeno-associated viruses (AAVs) have been demonstrated in preclinical and clinical settings to be minimally pathogenic delivery shuttles, but they have limited packaging capacity. Nme1Cas9 and its guide RNAs, encoded by an open reading frame of approximately 3.3 kb, are within the packaging limits of AAV. Nme2Cas9 has similar advantages. Unlike SpyCas9, which requires the sgRNA and Cas9 to be delivered by separate vectors, Nme1Cas9, Nme2Cas9 and their sgRNAs are small enough to be delivered with a single AAV vector.

[0094] Other Cas9 orthologs, such as Campylobacter jejuni Cas9 (CjeCas9) and Staphylococcus aureus (SauCas9), have also been successfully delivered by AAV in vivo. Kim et al., "In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni" Nat Commun 8:14500 (2017); and Ran et al., "In vivo genome editing using Staphylococcus aureus Cas9" Nature 520:186-191 (2015). Nme1Cas9 is normally 4 This involves the GATT PAM, which is similar to the CjeCas9 PAM (e.g., N 4 RYAC), or SauCas9 PAM (e.g., NNGRRT) (R = purine (A or G), Y = pyrimidine (C or T)).

[0095] Nme1Cas9 has been successfully delivered as a ribonucleoprotein (RNP) complex in human cells (Figures 2 and 3). Furthermore, the data presented herein demonstrate that the Nme1Cas9 nucleic acid sequence can be expressed and gene targeted in vivo in mice using an integrated sgRNA-Nme1Cas9-AAV vector following tail vein injection.

[0096] The data presented herein demonstrates the targeting of mouse proprotein convertase subtilisin / kexin type 9 (Pcsk9) gene. PCSK9 functions as an antagonist to low density lipoprotein (LDL) receptors, limiting LDL cholesterol uptake. Detection of reduced cholesterol levels in serum can provide a direct functional readout of efficient Nme1Cas9 editing using PCSK9-directed Cas9 platform.

[0097] In one embodiment, this invention contemplates an adeno-associated viral vector comprising a Nme1Cas9-sgRNA complex or a Nme2Cas9-sgRNA complex. Although it is not necessary to understand the mechanism of an invention, it is believed that the AAV / Nme1Cas9-sgRNA complex or the AAV / Nme2Cas9-sgRNA complex is compatible with an in vivo delivery route to effect gene editing.

[0098] In one embodiment, the present invention contemplates an sgRNA-Nme1Cas9-AAV vector comprising an sgRNA sequence, an RNA polymerase III U6 promoter sequence, a human codon-optimized Nme1Cas9 sequence, and an RNA polymerase II U1a promoter sequence. Figure 6. U1a is a ubiquitous promoter that allows versatile expression of Cas9 in various tissues of interest. A specific gene to be edited can be targeted by inserting a spacer sequence that matches the target gene into the sgRNA cassette using a conventional restriction site (e.g., Sap1). Representative sequences of various elements of sgRNA-Nme1Cas9-AAV are shown by colored annotation. Figures 7 and 8.

[0099] The editing efficiency of several target sites using Pcsk9-sgRNA-Nme1Cas9-AAV and Rosa26-sgRNA-Nme1Cas9-AAV plasmids was estimated by T7E1 assay after transient transfection into mouse Hepa1-6 hepatoma cells (Figure 9). Representative target site sequences within the Pcsk9 and Rosa26 genes complementary to the Pcsk9-sgRNA-Nme1Cas9-AAV and Rosa26-sgRNA-Nme1Cas9-AAV plasmids are shown by colored annotations (Figure 10).

[0100] The plasmid design was validated in vivo in mice by hydrodynamic tail vein injection of 30 μg of endotoxin-free sgRNA-Nme1Cas9-AAV plasmid targeting Pcsk9. Significant gene editing was detected in mouse liver 10 days after injection as measured by Tracking of Indels by DEcomposition (TIDE), a sequencing-based method to assess indel efficiency. FIG. 11.

[0101] Plasmid backbones targeting the Pcsk9 and Rosa26 genes were packaged into hepatocyte-specific AAV8 serotypes and delivered to 4 × 10 cells per mouse. 10A dose of 100 genomic copies (gc) was injected via the tail vein. Preliminary data shows significant indel levels in the liver Pcsk9 and Rosa26 genes from mice sacrificed 14 days after injection. Figure 12A. Deep sequencing data was also collected 50 days after injection.

[0102] At 50 days post-injection, the three groups of mice were sacrificed and liver gDNA was used to measure indel levels in Pcsk9 and Rosa26 using TIDE (Figure 12B). Deep sequencing analysis was also performed to record precise measurements of indel levels.

[0103] PCSK9 protein "knockdown" can lead to a significant reduction in cholesterol levels in mice. Serum cholesterol levels were measured by Infinity™ colorimetric endpoint assay (Thermo-Scientific) in three groups of mice injected with vectors targeting the Pcsk9 gene, vectors targeting the Rosa26 gene, and a PBS control group. The results suggest that Nme1Cas9-induced indel formation led to disruption of the normal reading frame of the Pcsk9 gene, as shown by the significant reduction in serum cholesterol levels 25 and 50 days after injection. Figure 13. Western blot assays were also performed to measure the levels of PCSK9 protein in mouse liver at day 50.

[0104] A genome-wide unbiased identification assay of double-strand breaks (DSBs) enabled by sequencing (e.g., GUIDE-Seq®, Illumina) was used to search for off-target editing sites after injection of vectors targeting the Pcsk9 gene and vectors targeting the Rosa26 gene. The data revealed four potential off-target sites for Pcsk9 and six potential off-target sites for Rosa26. Figures 14A and 14B.

[0105] Targeted TIDE analysis revealed on-target genome editing in cells and mice 14 days after injection of AAV vectors targeting the Pcsk9 gene and AAV vectors targeting the Rosa26 gene, Figure 15. Deep sequencing analysis for off-target cleavage at these sites was also performed 50 days after injection.

[0106] Hematoxylin-eosin staining assay showed no signs of massive immune cell infiltration in liver sections from mice sacrificed 14 days after injection of Pcsk9 gene-targeting vector and Rosa26 gene-targeting vector. Figure 16. Specific immune response assay performed 50 days after injection.

[0107] In one embodiment, the present invention contemplates a method for therapeutic in vivo genome editing by integrated AAV delivery of Nme2Cas9. Although it is not necessary to understand the mechanism of an invention, it is believed that Nme2Cas9, due to its small size, small PAM and high fidelity, makes it an ideal tool for in vivo genome editing using AAV. To this end, Nme2Cas9 was cloned into a single AAV vector backbone along with its cognate sgRNA and their respective promoters. Figure 44A; top. This integrated AAV.sgRNA.Nme2Cas9 was packaged into a hepatocyte-selective AAV8 capsid. Two genes were targeted: i) Rosa26, a locus commonly used as a negative control; and ii) proprotein convertase subtilisin / kexin type 9 (Pcsk9), a key regulator of circulating cholesterol homeostasis. Studies have shown that knocking out Pcsk9 using Cas9 results in reduced cholesterol levels (Ran et al.).

[0108] Two groups of mice (n=5) were injected with packaged AAV8.sgNA.Nme2Cas9 targeting either Pcsk9 or Rosa26. Serum was collected for cholesterol level measurement at 0, 14, and 28 days after vector injection. Mice were sacrificed 28 days after injection and liver tissue was collected (Figure 44A, bottom). Deep sequencing analysis showed significantly higher levels of indels in Pcsk9 and Rosa26. Figure 44B. These indel values ​​were accompanied by a significant reduction in blood cholesterol levels after 14 and 28 days in mice injected with sgPcsk9; mice injected with sgRosa26 maintained normal levels of cholesterol throughout the study. Figure 44C. H&E analysis showed no signs of toxicity or tissue damage in both groups after Nme2Cas9 expression. Figure 44D. These data establish that Nme2Cas9 is highly functional in vivo and can be readily delivered by a convenient integrated AAV platform.

[0109] In one embodiment, the present invention contemplates a minimized AAV.hNmeCas9 construct. See Figure 44A. As discussed above, the present invention contemplates an engineered integrated AAV.sgRNA.hNme1Cas9 construct packaged into AAV8 virions that successfully edited the Pcsk9 and Rosa26 genes in mouse liver.

[0110] In one embodiment, the present invention contemplates an AAV8 backbone that contains an Nme2Cas9 cassette. Similar to Nme1Cas9, Nme2Cas9 has also demonstrated robust editing in Pcsk9 and Rosa26 in mice (see below). The data presented herein show that in vivo administration of AAV8-NmeCas9 in mice is associated with a significant reduction in the level of circulating cholesterol 28 days after vector injection.

[0111] To increase the utility of this integrated AAV platform, various truncations were introduced to minimize the size of the cargo and create space for additional features within the AAV capsid, such as duplex sgRNAs or donor DNA segments.

[0112] To minimize the cargo of the integrated AAV backbone, redundant features (3x HA tags and 2x NLS sequences) were systematically removed without compromising the nuclease activity of Cas9. Using the Traffic Light Reporter (TLR) system, Nme1Cas9 shows that this minimized integrated AAV.sgRNA.hNme1Cas9 (4.468kb) is as potent as the previous longer version with four NLS sequences. See Figure 45. Similar to the sgRNA11 version but with a 3' end. UA A new sgRNA12 with the addition of the following was used to construct a shortened sgRNA to allow for more space. See Figure 46.

[0113] It has previously been reported that short polyA sequences can be useful in Cas9 constructs. Platt et al. (2015). In one embodiment, the present invention provides an AAV-Nm construct containing BGH polyA. e2Cas9 constructs are contemplated. See Figure 47. Although it is not necessary to understand the mechanism of an invention, it is believed that this polyA sequence further reduces the size of the integrated AAV scaffold.

[0114] It is further contemplated that the utility of Nme1Cas9 and Nme2Cas9 will be increased by including separate sgRNAs for double gene knockout or DNA fragment deletion in this minimized (4.4kb) integrated AAV backbone. See Figure 48, top. This configuration also provides free space within the AAV capsid to include a donor template (~600 base pairs) for homology directed repair applications. See Figure 48, bottom. In some embodiments, the dual sgRNA AAV construct is packaged into a single AAV vector.

[0115] The relatively small Nme1Cas9 is active in genome editing in a variety of cell types. To take advantage of the small size of this Cas9 ortholog, an integrated AAV construct was generated using human codon-optimized Nme1Cas9 under the expression of the mouse U1a promoter and its sgRNA driven by the U6 promoter. See Figure 49A. Two sites in the mouse genome were first selected to test the in vivo nuclease activity of Nme1Cas9: the Rosa26 "safe harbor" gene (targeted by sgRosa26); and the proprotein convertase subtilisin / kexin type 9 (Pcsk9) gene (targeted by sgPcsk9), a common therapeutic target for lowering circulating cholesterol and reducing the risk of cardiovascular disease. Figure 49B. Genome-wide off-target predictions for these guides were analyzed using Bioconductor Using package CRISPRseek 1.9.1, 4 GN 3 PAM and up to six mismatches were used to determine the sequence of the target-specific guide RNAs. Zhu et al., "CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome editing systems" PLoS One 2014;9:e108424. 4 GN 3PAM is inert, so these search parameters almost certainly cast a wider net than true off-target profiles. Despite the broad nature of the search, the analysis did not reveal any off-target sites with fewer than four mismatches in the mouse genome. See Figure 50. On-target editing efficiency at these target sites was assessed in mouse Hepa1-6 hepatoma cells by plasmid transfection, and indel quantification was performed by sequence trace decomposition using the Tracking of Indels by Decomposition (TIDE) web tool. Brinkman et al., "Easy quantitative assessment of genome editing by sequence trace decomposition" Nucleic Acids Res. 2014;42:e168. Data are based on >25% indices for selected guides. The results show that the majority of the clones were deletions. See Figure 49C.

[0116] To evaluate the preliminary efficacy of the constructed integrated AAV-sgRNA-hNme1Cas9 vector, endotoxin-free sgPcsk9 plasmid was hydrodynamically administered by tail vein injection into C57Bl / 6 mice. In this method, plasmid DNA can be delivered to approximately 40% of liver cells for transient expression. Liu et al., "Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA" Gene Ther. 1999;6:1258-66. Indel analysis by TIDE using DNA extracted from liver tissue revealed 5-9% indels 10 days after vector administration, which was comparable to the editing efficiency obtained in a similar study of SpyCas9. Figure 49D; and Xue et al., "CRISPR-mediated direct mutation of cancer genes in the liver" Gene Ther. 1999;6:1258-66. See " mouse liver" Nature 2014;514:380-4. These results suggest that Nme These results suggest that it is possible to edit liver cells in vivo using 1Cas9.

[0117] Hereditary tyrosinemia type I (HT-I) is a fatal genetic disease caused by autosomal recessive mutations in the Fah gene, which encodes the fumarylacetoacetate hydroxylase (FAH) enzyme. In patients with attenuated FAH, the tyrosine catabolic pathway is disrupted, leading to the accumulation of toxic fumarylacetoacetate and succinylacetoacetate, causing liver and kidney damage. Grompe M., "The pathophysiology and treatment of hereditary tyrosinemia type 1" Semin Liver Dis. 2001;21:563-71 For the past two decades, this disease has been controlled by 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which inhibits 4-hydroxyphenylpyruvate dioxygenase upstream of the tyrosine degradation pathway, thus preventing the accumulation of toxic metabolites. Lindstedt et al., "Treatment of hereditary tyrosinaemia type I by inhibition of 4-hydroxyphenylpyruvate dioxygenase" Lancet 1992;340:813-7. However, this treatment requires lifelong dietary and drug management and may ultimately require liver transplantation. Das, AM., "Clinical utility of nitisinone for the treatment of hereditary tyrosinemia type-1 (HT-1)" Appl Clin Genet. 2017;10:43-8.

[0118] Several gene therapy strategies have been tested to correct the defective Fah gene using site-directed mutagenesis or CRISPR-Cas9 homologous recombination repair. Paulk et al., "Adenoassociated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo" Hepatology 2010;51:1200-8;Yin et al., "Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo" Nat Biotechnol. 2016;34:328-33; and Yin et al., "Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype" Nat Biotechnol. 2014;32:551-3. Fah m ut / mutIt has been reported that successful modification of only 1 / 10,000 hepatocytes in the liver is sufficient to rescue the mouse phenotype. Recently, a metabolic pathway reprogramming approach was suggested, disrupting the function of the hydroxyphenylpyruvate dioxygenase (HPD) enzyme by deletion of exons 3 and 4 of the Hpd gene in the liver. Pankowicz et al., "Reprogramming metabolic pathways in vivo with CRISPR / Cas9 genome editing to treat hereditary tyrosinaemia" Nat Commun. 2016;7:12642. This provides a situation to test the efficacy of editing with Nme1Cas9, for example, by targeting Hpd and assessing the rescue of the disease phenotype in Fah mutant mice. Grompe et al., "Loss of fumarylacetoacetate hydrolase is responsible for the neonatal hepatic dysfunction phenotype of lethal albino mice" Genes Dev. 1993;7:2298-307. To this end, two target sites were screened and identified within the open reading frame of Hpd, one within exon 8 [sgHpd1] and one within exon 11 [sgHpd2], respectively. See Figure 51A. With these guides (e.g., sgRNAs), plasmid transfection in Hepa1-6 cells promoted an average Nme1Cas9-induced indel efficiency of 10.8% and 9.1%, respectively. Figure 52.

[0119] Three groups of mice were treated with phosphate-buffered saline (PBS) or one of two sgHpd1 and sgHpd2 integrated AAV-sgRNA-hNme1Cas9 plasmids by hydrodynamic injection. One mouse in the sgHpd1 group and two in the sgHpd2 group failed tail vein injection and were excluded from follow-up studies. Seven days after injection, mice were weaned off water containing NTBC and body weight was monitored for 43 days after injection. See Figure 51B. Mice injected with PBS suffered severe weight loss (a hallmark of HT-I) and were sacrificed after losing 20% ​​of their body weight. Overall, all sgHpd1 and sgHpd2 mice successfully maintained their body weight for a total of 43 days and at least 21 days without NTBC. See Figure 51C.

[0120] For two mice receiving sgHpd1 and one receiving sgHpd2, NTBC treatment had to be resumed for 2-3 days during the third week after plasmid injection to restore body weight, likely due to low initial editing efficiency, liver injury caused by hydrodynamic injection, or both. Conversely, indels were achieved at frequencies ranging from 35-60% in all other sgHpd1- and sgHpd2-treated mice. See Figure 51D. This level of gene inactivation may reflect not only the initial editing event but also a competitive expansion (after NTBC withdrawal) of edited cell lineages at the expense of their non-edited counterparts. Liver histology revealed that PBS-injected Fah1- and sgHpd2-treated mice had significantly higher numbers of multinucleated hepatocytes than PBS-injected mice, as indicated by lower numbers of multinucleated hepatocytes. mut / mut The severity of liver injury was found to be substantially less in mice compared to control mice. See Figure 53.

[0121] AAV vectors have recently been used to generate genome-edited mice without the need for microinjection or electroporation by simply immersing zygotes in culture medium containing the AAV vector and then reimplanting them into pseudopregnant females. Editing was previously obtained using a dual AAV system in which SpyCas9 and its sgRNA are delivered on separate vectors. Yoon et al., "Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses" Nat. Commun. 9:412 (2018). To test whether Nme2Cas9 could enable precise and efficient editing in mouse zygotes using an integrated AAV delivery system, we targeted the tyrosinase gene (Tyr), whose biallelic inactivation disrupts melanin production and results in albino offspring. Yokoyama et al., "Conserved cysteine ​​to serine mutation in tyrosinase is responsible for the classical albino mutation in laboratory mice" Nucleic Acids Res. 18:7293-7298 (1990).

[0122] An efficient Tyr sgRNA (cleaving the Tyr locus only 17 bp from the site of the classical albino mutation) was validated by transient transfection in Hepa1-6 cells. See Figure 57. C57BL / 6NJ zygotes were then transfected with 3x10 9 or 3×10 8GCs were incubated for 5-6 hours in culture medium containing an integrated AAV6 vector expressing Nme2Cas9 with Tyr sgRNA. After overnight culture in fresh medium, zygotes that had progressed to the two-cell stage were transferred into the oviducts of pseudopregnant recipients and allowed to develop to term. See Figure 58A. Coat color analysis of the pups revealed mice that were albino, light gray (suggesting a hypomorphic allele of Tyr), or had albino and a patchy coat color composed of light gray spots but lacking black pigmentation. See Figures 58B and 58C. These results suggest that biallelic mutations were frequent, as the presence of a single wild-type Tyr allele should result in black pigmentation. 3×10 9 A total of five pups (10%) were born from the GC experiment. All of them had indels; phenotypically, two were albino, one was light gray, and two had patchy pigmentation indicative of mosaicism. 8 The GC experiment yielded four pups (14%), two of which died at birth, preventing coat color or genomic analysis. Coat color analysis of the remaining two pups revealed one to be light gray and one to be a mosaic pup. These results indicate that single AAV delivery of Nme2Cas9 and its sgRNA can be used to generate mutations in mouse zygotes without microinjection or electroporation.

[0123] To measure on-target indel formation in the Tyr gene, DNA was isolated from the tail of each mouse, the locus was amplified, and TIDE analysis was performed. The data showed that all mice had high levels of on-target editing by Nme2Cas9, varying from 84% to 100%. See Figures 57B and 5C. The majority of lesions in albino mouse 9-1 were 1-bp or 4-bp deletions, suggesting either mosaicism or trans-heterozygosity. Albino mouse 9-2 showed a uniform 2-bp deletion. See Figure 58C. Analysis of tail DNA from light gray mice revealed the presence of an in-frame mutation, potentially responsible for the light gray coat color. The limited complexity of the mutations suggests that editing occurred early in embryonic development in these mice. One female (mouse 9-2) was mated with a classical albino male and all six resulting pups were albino, demonstrating that mutations generated by zygotic AAV delivery of Nme2Cas9+sgRNA can be transmitted through the germline. These results provide a streamlined route to mammalian mutagenesis, in this case by application of a single AAV vector delivering both Nme2Cas9 and its sgRNA.

[0124] Patients with mutations in the Hpd gene are considered to have type III tyrosinemia and exhibit high levels of tyrosine in the blood, but are otherwise largely asymptomatic. Szymanska et al., "Tyrosinemia type III in an asymptomatic girl. Mol Genet Metab Rep. 2015;5:48-50; and Nakamura et al., "Animal models of tyrosinemia" J Nutr. 2007;137:1556S-60S. HPD acts upstream of FAH in the tyrosine catabolic pathway, and disrupting Hpd ameliorates HT-I symptoms by preventing the accumulation of toxic metabolites resulting from loss of FAH. Structural analysis of HPD reveals that the catalytic domain of the HPD enzyme is located at the C-terminus of the enzyme and is encoded by exons 13 and 14. Huang et al., "The different catalytic roles of the metal-binding ligands in human 4-hydroxyphenylpyruvate dioxygenase" Biochem J. 2016;473:1179-89. Therefore, a frameshift upstream of exon 13 Inducible indels should render the enzyme inactive. Using this context, we demonstrate that Hpd inactivation by hydrodynamic injection of Nme1Cas9 plasmid is a viable approach to rescue HT-I mice. Nme1Cas9 can inactivate several different PAMs (N 4 GATT [Consensus], N 4 G.C.T.T., N. 4 G.T.T.T., N 4 GACT, N 4 GATA, N. 4 GTCT, and N 4 Hpd editing experiments have demonstrated that one of the variant PAMs in vivo is N 4 Verified with sgHpd2 guide targeting a site with a GACT PAM.

[0125] Although indels can be generated by plasmid hydrodynamic injection, therapeutic development may require less invasive delivery strategies, such as those using rAAV. To this end, an integrated AAV-sgRNA-hNme1Cas9 plasmid was packaged into a hepatocyte-tropic AAV8 capsid to target Pcsk9 (sgPcsk9) and Rosa26 (sgRosa26). See Figure 49B; Gao et al., "Novel adenoassociated viruses from rhesus monkeys as vectors for human gene therapy" Proc Natl Acad Sci USA 2002;99:11854-9; and Nakai et al., "Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice" J Virol. 2005;79:214-24. Pcsk9 and Rosa26 was used in part to allow AAV delivery of Nme1Cas9 to serve as a benchmark for AAV delivery of other Cas9 orthologs similarly delivered and targeting the same locus. Ran et al., "In vivo genome editing using Staphylococcus aureus Cas9" Nature 2015;520:186-91. Vectors were administered via the tail vein to C57BL / 6 mice. See Figure 54A. Serum cholesterol levels were monitored and PCSK9 protein and indel frequencies were measured in liver tissue 25 and 50 days after injection.

[0126] Using a colorimetric endpoint assay, it was determined that circulating serum cholesterol levels in mice administered Nme1Cas9 / sgPcsk9 were significantly (p<0.001) reduced compared to PBS and Nme1Cas9 / sgRosa26 mice 25 and 50 days after injection. See Figure 54B. Targeted deep sequencing analysis at the Pcsk9 and Rosa26 target sites revealed highly efficient indels of 35% and 55%, respectively, 50 days after vector administration. Figure 54C. Additionally, one mouse from each group was euthanized 14 days after injection, revealing on-target indel efficiencies of 37% and 46% in Pcsk9 and Rosa26, respectively. As expected, PCSK9 protein levels in the livers of mice treated with Nme1Cas9 / sgPcsk9 were substantially reduced compared to mice injected with PBS and mice injected with Nme1Cas9 / sgRosa26. See Figure 54D. Efficient editing, PCSK9 reduction, and attenuation of serum cholesterol demonstrate successful delivery and activity of Nme1Cas9 at the Pcsk9 locus.

[0127] SpyCas9 delivered by viral vectors is known to elicit host immune responses. Chew et al., "A multifunctional AAV-CRISPR-Cas9 and its host response" Nat Methods 2016;13:868-74; and Wang et al., "Adenovirus-mediated somatic genome editing of Pten by CRISPR / Cas9 in mouse liver in spite of Cas9-specific immune responses" Hum Gene Ther. 2015;26:432-42. To investigate whether anti-Nme1Cas9 antibodies were produced in mice injected with AAV8-sgRNA-hNme1Cas9, IgG1 ELISA was performed using serum from treated animals. These results indicate that Nme1Cas9 elicits a humoral response in these animals. See Figure 55. Despite the presence of an immune response, rAAV-delivered Nme1Cas9 is highly functional in vivo with no obvious signs of abnormality or liver damage. See Figure 16.

[0128] A key concern with therapeutic CRISPR / Cas9 genome editing is the potential for off-target editing activity. For example, wild-type Nme1Cas9 has been found to be a naturally accurate genome editing platform in cultured mammalian cells. Lee et al., "The Neisseria meningitidis CRISPR-Cas9 system enables specific genome editing in mammalian cells" Mol Ther. 2016;24:645-54. To determine whether Nme1Cas9 maintains its minimal off-target profile in mouse cells and in vivo, we screened for off-target sites in the mouse genome using genome-wide unbiased identification of DSBs enabled by sequencing (GUIDE-seq). Tsai et al., "Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases" Nat Rev Genet. 2016;17:300-12. Hepa1-6 cells were transfected with sgPcsk9, sgRosa26, sgRosa27, sgRosa28, sgRosa29 ... gHpd1, and sgHpd2 integrated AAV-sgRNA-hNme1Cas9 plasmids were transfected, and the resulting genomic DNA was subjected to GUIDE-seq analysis. Consistent with the observations in human cells (data not shown), GUIDE-seq revealed very few off-target (OT) sites in the mouse genome. Four potential OT sites were identified for sgPcsk9, and another six for sgRosa26. No off-target editing was detected with sgHpd1 and sgHpd2. See Figure 56A. These data further confirm that Nme1Cas9 is intrinsically ultra-accurate.

[0129] Some of the putative OT sites for sgPcsk9 and sgRosa26 are consistent with Nme1Cas9 PAM preference (i.e., N 4 GATT, N 4G.C.T.T., N. 4 G.T.T.T., N 4 GACT, N 4 GATA, N. 4 GTCT, and N 4 GACA). See Figure 56B. To confirm these OT sites, targeted deep sequencing was performed using genomic DNA from Hepa1-6 cells. This higher sensitivity read resulted in indels being undetectable above background at all of these OT sites except OT1 of Pcsk9, where the indel frequency was <2%. See Figure 56B. To confirm the high fidelity of Nme1Cas9 in vivo, indel formation was measured at these OT sites in liver genomic DNA from sgPcsk9-targeted and sgRosa26-targeted mice treated with AAV8-Nme1Cas9. Little or no detectable off-target editing was found at all sites except sgPcsk9 OT1, which showed a lesion efficiency of <2%, in mouse livers sacrificed on day 14. More importantly, the level of this OT editing remained below <2% even after 50 days, and remained undetectable or very low at all other candidate OT sites. These results suggest that long-term (50 day) expression of Nme1Cas9 in vivo does not compromise its targeting fidelity. See Figure 56C.

[0130] rAAV vectors are a promising delivery platform to achieve targeted delivery of Nme1Cas9 to various tissues in vivo because the small size of the Nme1Cas9 transgene allows delivery of Nme1Cas9 and its guide in an integrated format. The data presented herein validate this approach for targeting Pcsk9 and Rosa26 genes in adult mice, with efficient editing observed even 14 days after injection. Nme1Cas9 is endogenously accurate without extensive engineering required to reduce off-targeting by SpyCas9. Lee et al., "The Neisseria meningitidis CRISPR-Cas9 system enables specific genome editing in mammalian cells" Mol Ther. 2016;24:645-54;Bolukbasi et al., "Creating and evaluating accurate CRISPRCas9 scalpels for genomic surgery" Nat Methods 2016;13:41-50;Tsai et al., "Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases" Nat Rev Genet. 2016;17:300-12; and Tycko et al., "Methods for optimizing CRISPR-Cas9 genome editing specificity" Mol Cell. 2016;63:355-70.

[0131] Side-by-side comparison of Nme1Cas9 OT editing was performed in cultured cells and in vivo by targeted deep sequencing and found to have minimal off-targeting in both settings, with editing at the sgPcsk9 OT1 site (within an unannotated locus) being the most detectable at approximately 2%.

[0132] IV. Small Cas9 orthologs with cytosine-rich PAM As mentioned above, CRISPR system can be classified into at least six different types.Generally, type II system is categorized by the presence of Cas9 nuclease protein.For example, Cas9 nuclease protein is considered to be an RNA-guided nuclease that can be repurposed as genome editing platform in almost all organisms, including humans.Reports show that Cas9 genome editing has been used in medicine, agriculture, human gene therapy and many other applications.

[0133] In general, targeting of a specific gene locus within the human genome can be accomplished by a Cas9 nuclease protein bound to a single guide RNA (sgRNA) that targets the locus through interaction with a specific nucleic acid sequence (e.g., a protospacer adjacent motif; PAM). The sgRNA typically includes a 20-24 nucleotide segment that is complementary to the target nucleic acid sequence, followed by a constant region that interacts with (e.g., binds to) the Cas9 protein. To effect genome editing with a Cas9 nuclease protein, the Cas9:sgRNA complex first recognizes a protospacer adjacent motif (PAM) sequence that is typically found downstream of the target site sequence. Although it is not necessary to understand the mechanism of an invention, it is believed that each Cas9 nuclease protein has an affinity for a particular PAM (i.e., mediated by a protospacer adjacent motif recognition domain). In the absence of a PAM recognition domain that binds to the downstream PAM target nucleic acid sequence, double-stranded DNA (dsDNA) cannot be cleaved by the Cas9 nuclease.

[0134] Reports suggest that only a few Cas9 orthologues have been validated for human genome editing. Three of the reported CRISPR-Cas9 types include II-A, II-B, and II-C. Type II-A Cas9 (e.g., Streptococcus pyogenes (SpyCas9)) is the most commonly used Cas9 to date. However, SpyCas9 (and most other type II-A orthologues) have several properties that may make them unsuitable for certain applications. First, SpyCas9 is relatively large, which makes it unsuitable for efficient packaging into viral vectors. Second, SpyCas9 has a high rate of off-target activity (i.e., cleaving DNA at unintended loci in the human genome), although more specific variants have been created. Finally, the PAM (e.g., NGG) of SpyCas9 is of limited use for some sites in the human genome, or for applications where specific nucleotides should be recognized during editing. To overcome these shortcomings, other Cas9 orthologs have been repurposed by several groups to function in humans and other organisms. As discussed above, type II-C Cas9 orthologs (e.g., Nme1Cas9) are small enough for integral viral packaging (e.g., adeno-associated virus (AAV) vectors) that results in higher fidelity activity in mammalian cells. However, in contrast to the SpyCas9 PAM, which is usually two nucleotides long, wild-type Cas9 II-C PAMs are usually approximately four nucleotides long. This additional PAM length may limit the number of loci that can be targeted by wild-type Cas9 II-C PAM. This creates a need in the art to identify more Cas9 orthologs for genome editing.

[0135] Although there are thousands of Cas9 orthologs to choose from in the NCBI database, an empirical process is required to develop small type II-C Cas9 orthologs with less restrictive PAMs that result in improved functionality in mammalian cells. In one embodiment, the present invention contemplates improved type II-C Cas9 orthologs that allow for precise genome editing with a broader range of target sites. In one embodiment, the improved type II-C Cas9 orthologs have a compact size that allows for efficient viral delivery. In one embodiment, the improved type II-C Cas9 orthologs include, but are not limited to, Haemophilus parainfluenzae (HpaCas9), Simonsiella muelleri (SmuCas9), and Neisseria meningitidis strain De10444 (Nme2Cas9).

[0136] A. Short PAMs associated with type II-C Cas9 orthologs Data presented herein show characterization of short PAM targets for several type II-C Cas9 orthologs. Figure 17. For example, type II-C Cas9 orthologs can interact with short PAMs that contain between 1 and 4 required nucleotides. Although it is not necessary to understand the mechanism of an invention, it is believed that these short C-rich PAMs provide improved genome editing by Cas9 at target sites that were previously inaccessible even by smaller Cas9 orthologs (e.g., Nme1Cas9). In one embodiment, the Nme2Cas9 PAM has a sequence of NNNNCc, where "c" is the only partial preference. In one embodiment, the SmuCas9 PAM has a sequence of NNNNCT. Figure 18.

[0137] It is currently believed that there are no Cas9 orthologues with short C-rich PAMs that have been validated for genome editing, and that Nme2Cas9 is particularly convincing as a potential candidate for highly efficient gene editing activity in human cells.In one embodiment, the present invention contemplates Nme2Cas9 nuclease (e.g., Neisseria meningitidis 8013 Cas9; previously referred to as NmeCas9) combined with wild-type Nme1Cas9 sgRNA.Nme1Cas9 has been previously described.Sontheimer et al., "RNA-Directed DNA Cleavage and Gene Editing by Cas9 Enzyme From Neisseria Meningitidis", US Patent Publication No. 2014 / 0349,405 (hereby incorporated by reference).Although Nme1Cas9 can be useful for genome editing, its main limitation is that its PAM is relatively long, which limits the number of editable sites at any given genomic locus.

[0138] In some embodiments, the present invention contemplates shorter and less stringent PAM for type II-C Cas9 orthologs, including but not limited to Nme2Cas9.It is not necessary to understand the mechanism of the invention, but it is believed that a short and less stringent PAM can partially alleviate the limitation of target restriction, while still retaining many, if not most, of the advantages of Nme1Cas9, including but not limited to small size (e.g., compactness) and improved target accuracy (e.g., reduced off-target cleavage) for efficient integrated AAV delivery.In addition, the minimized sgRNA for Nme1Cas9 discussed above is also compatible with Nme2Cas9 constructs.Therefore, such shortened guide RNAs may be used for genome editing using Nme2Cas9 as well.

[0139] In one embodiment, the invention contemplates an HpaCas9 PAM having the sequence NNNNGNTTT. Despite the fact that longer PAMs limit the number of targetable sites in the human genome, it is believed that the HpaCas9 PAM can target sites with a high degree of accuracy, similar to the extremely accurate Nme1Cas9 (described above).

[0140] The data presented herein demonstrate the ability of type II-C Cas9 nucleases targeting short C-rich PAMs to perform genome editing in human (HEK293T) cells. Certo et al., "Tracking genome engineering outcome at individual DNA breakpoints" Nature Methods 8:671-676 (2011). For example, HpaC As9 and Nme2Cas9 were shown to result in efficient genome editing at specific loci, demonstrating that HpaCas9 and Nme2Cas9 are active in mammalian cells. Figure 19 and Table 2. [Table 2]

[0141] These data indicate that both Nme2Cas9 and HpaCas9 perform genome editing at the same genomic loci as previously validated Nme1Cas9 at comparable levels. For SmuCas9, the efficiency of editing is relatively low, but importantly, the activity is not zero, and efficiency improvement is predicted. Nme2Cas9 was then used to test 14 additional sites in the traffic light reporter (TLR) integrated into the genome of HEK293T cells. In these assays, each site matches the PAM template (i.e., NNNNCNNN), where "C" is the fifth nucleotide of the PAM region. Surprisingly, all 14 sites were edited by Nme2Cas9, indicating that this enzyme is consistently active with various guides in mammalian cells. The most successful guide RNA matches the NNNNCCN PAM consensus. Figure 20.

[0142] The II-C type Cas9 ortholog cleavage was tested for sensitivity to anti-CRISPR proteins. Anti-CRISPR proteins are naturally occurring proteins that can turn off Cas9 when Cas9 activity is no longer desired. The data show that all three II-C type Cas9 orthologs are inhibited by certain anti-CRISPRs. Figure 21. The controllability of these Cas9 orthologs by anti-CRISPRs may increase their potential utility in genome editing.

[0143] B. Nme2Cas9 gene editing Data presented herein demonstrate gene editing using Nme2Cas9-sgRNA complexes. The data uses the traffic light reporter (TLR) system to demonstrate that any CC dinucleotide in a gene target sequence in the context of the NNNNCC sequence (above) can function as a PAM. FIG. 22. Blue bars are % of cells showing fluorescence, red bars show % editing based on more precise sequencing ("TIDE analysis"). These data confirm that a dinucleotide is sufficient for Nme2Cas9 PAM binding, as opposed to the need for a trinucleotide sequence (e.g., "X" in the sequence NNNNCCX). While it is not necessary to understand the mechanism of an invention, it is believed that this means that the frequency of genomic target sites editable by Nme2Cas9 is at least similar to sites editable by SpyCas9, and greater than SauCas9, Nme1Cas9, or CjeCas9 and other current alternatives.

[0144] Furthermore, the T7E1 assay was used to analyze the editing of native genomic sites (e.g., not integrated artificial fluorescent reporters). These data suggest that in some situations, the second "C" may not even be necessary. See Figure 23. Note that DeTS1 and DeTS4, both target sites in the AAVS1 locus, allow editing at target sites with NNNNCA and NNNNCG candidate PAMs, respectively. Some of these Nme2Cas9 target sites are disclosed herein. See Table 3. [Table 3]

[0145] Although it is not necessary to understand the mechanism of an invention, it is believed that these data suggest that, on average, there may be a candidate editing site every 4-8 base pairs in a genome. These data also suggest that the majority of Cas9 sgRNAs have some functionality, and thus the need for sgRNA screening may have been overemphasized in the art.

[0146] C. Rapidly evolving PAM interaction domains The in vivo application of CRISPR-Cas9 has the potential to transform many areas of biotechnology and therapeutics. Thousands of Cas9 orthologs exist in nature, only a few of which have been validated for in vivo genome editing. Cas9 from Streptococcus pyogenes (SpyCas9) has been widely used due to its high efficiency and non-restrictive NGG protospacer adjacent motif (PAM). However, the relatively large size of SpyCas9 limits its use in in vivo therapeutic applications that use delivery shuttles with limited packaging capacity, such as adeno-associated viruses (AAVs). Although several smaller Cas9 orthologs are known to be active in mammalian cells, they have a restrictive, large PAM that limits the density of target sites. Natural variations in the PAM-interacting domain (PID) of closely related Cas9 orthologs can exploit the identification of genome editing enzymes that overcome these limitations. In some embodiments, the present invention provides a method for the identification of a genome editing enzyme that overcomes these limitations by using a Cas9 ortholog that is capable of integrating Cas9 with ... 4We intend to use Nme2Cas9 complex, which is a small, naturally ultra-high accuracy Cas9 using CC PAM.The data presented herein shows that Nme2Cas9 is a high fidelity mammalian genome editing platform that produces the same target site density as SpyCas9.Delivery of Nme2Cas9 with its guide RNA by integrated AAV vector leads to efficient genome editing in adult mice, and targeting of Pcsk9 gene in liver induces serum cholesterol reduction without significant off-targeting (see below).Nme2Cas9 also provides a unique combination of integrated AAV compatibility, natural ultra-high accuracy, and high target site density for in vivo genome editing in mammals.

[0147] In addition to target density, minimizing off-target activity of Cas9 (e.g., cleavage at undesired loci) is highly desirable for using Cas9 as a safe therapeutic agent. Wild-type (wt) SpyCas9 has a high degree of off-target activity due to unique hybridization kinetics (Klein et al., 2018). In particular, questions remain regarding their on-target editing efficiency, and these variants have not overcome the limitations regarding overall size discussed above. In contrast, it is shown herein that embodiments of Nme1Cas9 and CjeCas9 contain naturally accurate gene editing activity. Although it is not necessary to understand the mechanism of an invention, it is believed that there are no Cas9 orthologs previously reported that are i) active in human cells; ii) exhibit exceptionally high target site density of SpyCas9; iii) are small enough for deliverability by integrated AAV; and iv) are naturally ultra-accurate. In one embodiment, the invention contemplates Nme2Cas9 as a genome editing platform that includes all of the above properties. For example, Nme2Cas9 4It contains a binding site that contains high affinity for CC PAM, is ultra-precise, and functions efficiently in mammalian cells. In one embodiment, Nme2Cas9 is packaged into an integrated AAV delivery platform for therapeutic genome editing.

[0148] 1. Closely related Nme1Cas9 orthologs with rapidly evolving PIDs Nme1Cas9 (derived from Neisseria meningitidis strain 8013) has previously been reported as a small, highly accurate Cas9 for in vivo genome editing (Amrani et al., 2018). However, Nme1Cas9 lacks the ability to encode long PAMs (N 4 Nme1Cas9 binds to the PAM-binding domain (GMTT), limiting the use of Nme1Cas9 in certain situations where it may target a small window. PAM recognition by Cas9 occurs primarily through protein-DNA interactions between the PAM-interacting domain (PID) of Cas9 and the nucleotides adjacent to the PAM. The PID is subject to high selection pressure by phages and other mobile genetic elements (MGEs). For example, anti-CRISPR proteins have been shown to interact with the PID and inhibit Cas9 (below). This can result in closely related Cas9 orthologs with PIDs that recognize significantly different PAMs.

[0149] Recently, this principle was highlighted using two species of Geobacillus. G. sterothermpophilus, which inhibits N 4 It contains a PID specific to the CRAA PAM, but when exchanged for the LC300 strain PID, its affinity is increased to N 4 It was determined that N. meningitidis strains have a GMAA PAM mutation (Harrington et al., 2017). Considering that it is highly sequenced, it was hypothesized that closely related Cas9 orthologs could be found with rapidly evolved PIDs that recognize different PAMs.Since NmeCas9 8013 strain is well characterized for genome editing, small, and highly accurate, Cas9 orthologs with high sequence identity (>80%) to this Cas9 were investigated.Several Cas9 orthologs with different PID amino acid sequences compared to 8013 strain were identified.Figure 34A.

[0150] Three distinct groups of Cas9 orthologs with significantly different PIDs were found. Figure 35A. One strain was selected from each PID group, for example, De11444 from group 2 and 98002 from group 3. These two CRISPR loci have intact Cas9 open reading frames and CRISPR arrays with some spacers, suggesting that these two CRISPR loci are active loci. Interestingly, the crRNA and tracrRNA of these CRISPR loci are identical to the crRNA and tracrRNA of 8013 and can utilize the same sgRNA. Figure 35B.

[0151] To test whether these Cas9 orthologs indeed have PIDs with affinity for different PAMs, the 8013 PID was replaced with the 98002 PID and De11444 PID, since the rest of the proteins derived from these orthologs have high sequence identity. To identify the PAMs, these protein "chimeras" were recombinantly expressed, purified, and used for in vitro PAM identification, as previously described. Briefly, a DNA fragment containing the protospacer and a downstream 10-nucleotide randomized sequence was cleaved in vitro using recombinant Cas9 and an sgRNA targeting the protospacer. Figure 34B. Consistent with Nme1Cas9 8013 and other type II-C systems that have been tested, a G23 nucleotide spacer length was used for the sgRNA. PAM identification assays revealed that these different Cas9 chimeras have PIDs that recognize different PAMs. For example, the N 4 By recognizing a C residue at position 5 instead of the G recognized by Nme1Cas9 8013 using the GATT PAM. Figure 34C.

[0152] However, due to the low cleavage efficiency of the chimeric protein, the remaining nucleotides could not be characterized with confidence, suggesting that a few residues outside the PID may be responsible for efficient activity. Figure 35C. To further elucidate the PAM, in vitro assays were performed on libraries with a 7-nucleotide randomized PAM (e.g., NNNNCNNN) with a C at position 5. The results showed that NmeCas9-De11444 and NmeCas9-98002 cleaved the nucleotides NNNNCC(A), respectively. NmeCas9-De11444 had a strong preference for C at position 5, but less so for nucleotides 6 and 7. As used herein, the Cas9 De11444 ortholog is referred to as "Nme2Cas9" and the Cas9 98002 ortholog is referred to as "Nme3Cas9".

[0153] This assay was also performed using full-length (e.g., PID not replaced) Nme2Cas9 and similar results were observed (Figure 34E). These results suggest that Nme2Cas9 and Nme3Cas9 have PIDs that recognize PAMs that are significantly different from that of Nme1Cas9.

[0154] 2. Nme2Cas9 in Human Cells Since the Nme2Cas9 PID binds to small PAM sequences, this ortholog is useful for human genome editing, especially when high targeting density is involved. To characterize Nme2Cas9, full-length (no PID replacement) humanized Nme2Cas9 was cloned with the NLS into a CMV-driven plasmid for mammalian expression. For characterization in human cells, a traffic light reporter system similar to that previously described was used (Certo et al., 2011).

[0155] Induction of a +1 frameshift indel was created by imperfect repair by non-homologous end joining (NHEJ) at the TLR2.0 locus. In the absence of donor DNA, in-frame mCherry protein was generated, which could be quantified by flow cytometry. Figure 36A. As an initial test, the Nme2Cas9 plasmid was transfected into N 4 The 15 sgRNA plasmids with spacers targeting the protospacer with a CCX PAM were transfected together. As controls, SpyCas9 and Nme1Cas9 were transfected with NGG and N, respectively. 4 GATT protospacer was used with their cognate sgRNAs targeting the GATT protospacer. After 72 hours, cells were harvested and the number of mCherry-positive cells was quantified for each target site. SpyCas9 and Nme1Cas9 showed efficient editing at their respective targets (approximately 28% and 10% mCherry, respectively) (Figure 36B). For Nme2Cas9, Nme2Cas9 showed no significant difference in the number of mCherry-positive cells. 4All 15 targets with a CCX PAM were functional to various degrees (ranging from 4% mCherry to 20% mCherry), but were not functional after treatment with NmeCas9 without an accompanying sgRNA and / or NmeCas9. 4 The GATT control did not result in mCherry cells (Figure 36B). These data demonstrate that Nme2Cas9 mediates N expression in human cells. 4 It was suggested that it recognizes CC PAM.

[0156] To further elucidate the Nme2Cas9 PAM, we 5 CX and N 4 Target sites were also tested using CD (D=A, T, G). 5 CX PAM and N 4 No detectable editing at the target site was observed with CD PAM, suggesting that both the C nucleotide at position 5 and the C nucleotide at position 6 are required for Nme2Cas9 activity based on the TLR2.0 reporter (Figures 37A and 37B). These results demonstrate that Nme2Cas9 is able to selectively amplify N 4 It contains a PID that binds to the CC PAM and is consistently demonstrated to be functional at the TLR2.0 locus in mammalian cells.

[0157] The length of the spacer portion of the crRNA varies among different Cas9 orthologs. The optimal spacer length for SpyCas9 is 20 nucleotides, but it can be shortened to 17 nucleotides. Fu et al., Nature Biotechnology 32, 279 (2014). In contrast In contrast, Nme1Cas9 contains an sgRNA with a 24-nucleotide spacer, and can tolerate shortening to 18 nucleotides (Amrani et al., 2018). To test the spacer length for Nme2Cas9, sgRNA plasmids were created targeting the same locus but with varying spacer lengths (Figure 36C and Figure 37B). Comparable activity was observed when using G23, G22 and G21 spacers, and activity was significantly reduced when the guide was shortened to G20 and G19 (Figure 36C). These results suggest that the optimal spacer length for Nme2Cas9 is between 22 and 24 nucleotides, similar to Nme1Cas9, GeoCas9 and CjeCas9. Therefore, all experiments described below were performed with a spacer of 23 to 24 nucleotides.

[0158] Cas9 orthologues are believed to use the HNH and RuvC domains to induce double-stranded breaks in the complementary and non-complementary strands of the target DNA, respectively. Alternatively, Cas9 nickases have been used to improve genome editing specificity and homology-directed repair (HDR) by creating overhangs (Ran et al., 2013). However, this approach has only been successful with SpyCas9 due to its high target density. To use Nme2Cas9 as a nickase, Nme2Cas9, which results in mutations in the catalytic residues of the RuvC and HNH domains, respectively, has been used. D16A and Nme2Cas9 H588AWe created Nme2Cas9 nickases. TLR2.0 can also be used to test the efficiency of HDR, in which case the repaired locus expresses GFP when the donor is introduced, so we included donor DNA sequences to test HDR with these Nme2Cas9 nickases. Target sites within the TLR2.0 gene were selected to test the functionality of each nickase using guide RNAs targeting 32bp and 64bp spaced cut sites. As a control, wild-type Nme2Cas9 targeting a single site showed efficient editing, accompanied by induction of both NHEJ and HDR repair pathways. For nickases, Nme2Cas9 was shown to be highly efficient at 32bp and 64bp spaced cut sites. D16A (HNH nickase) was shown to be effective for editing, but Nme2Cas9 H588A No targets were nicked using Figure 36D.

[0159] Cas9 orthologs contain a seed sequence that typically hybridizes to the target sequence between 8-12 nucleotides proximal to the PAM. Mismatches (e.g., non-complementarity) between the seed sequence and the PAM can reduce Cas9 nuclease activity. A series of transient transfections targeting the same locus in the TLR2.0 gene were performed by moving single nucleotide mismatches along a 23-nucleotide spacer (Figure 37C). Similar to other Cas9 orthologs, the data suggest that Nme2Cas9 has a "seed sequence" within the first 8-9 nucleotides that hybridize to the target sequence proximal to the PAM, as inferred from the reduced number of mCherry-positive cells. Although tolerance to mismatches is highly dependent on the sequence of the sgRNA and the target locus, these results suggest that Nme2Cas9 has a very low tolerance for mismatches, especially in its seed sequence.

[0160] 3. Genome editing efficiency by Nme2Cas9 We used Nme2Cas9 to target 40 different target sites throughout the human genome in HEK293T cells using transient transfection. Table 4. [Table 4]

[0161] 72 hours after transfection, cells were harvested, followed by gDNA extraction and selective amplification of the targeted locus. Tracking of Indels by The indel rate at each locus was measured using TIDE analysis. Despite significant variation in indel rate depending on the target sequence and locus, efficient editing by Nme2Cas9 was observed (Figure 38A). Furthermore, the affinity of Nme2Cas9 for target sites at or near therapeutically relevant loci such as CYBB (mutations causing x-linked chronic granulomatous disease) and AGA (mutations causing aspartylglycosaminuria) suggests that Nme2Cas9 has therapeutic potential. Furthermore, the editing efficiency could be increased by increasing the quantity of Nme2Cas9 plasmid (Figure 39A). Taken together, these results demonstrate that Nme2Cas9 can be constructed to selectively edit specific target genomic sites in HEK293T cells.

[0162] In addition to HEK293T cells, we determined the gene editing efficiency of Nme2Cas9 in several other mammalian cells, including human leukemia K562 cells, human osteosarcoma U2OS cells, and mouse liver hepatoma Hepa1-6 cells. We created a lentiviral construct expressing Nme2Cas9 and transduced it into K562 cells to stably express Nme2Cas9 under the control of the SFFV promoter. This stable cell line did not show any significant difference in growth and morphology compared to untreated cells, suggesting that Nme2Cas9 is not toxic when stably expressed. These cells were transiently electroporated with a plasmid expressing sgRNAs targeting several target sites and analyzed for indel rates by TIDE after 72 hours. Efficient editing was observed at the three sites tested, demonstrating the ability of Nme2Cas9 to function in K562 cells. For Hepa1-6 cells, the plasmid encoding Nme2Cas9 and the plasmid encoding sgRNA were co-transfected using a technique similar to that described above for HEK293T. These data also show that Nme2Cas9 efficiently edited the Pcsk9 and Rosa26 sites in this mouse cell line. Figure 38B.

[0163] Previous studies suggest that delivery of Cas9 by ribonucleoprotein (RNP) instead of plasmid transfection may be an alternative choice for some genome editing applications. For example, RNP electroporation can significantly reduce the off-target effects of SpyCas9 compared to plasmid delivery. Kim et al., Genome Research 24:1012-1019 (2014). Nme2Cas9 is functionalized by RNP delivery. To test whether Nme2Cas9 can be used to target genomic DNA, His-tagged Nme2Cas9 was cloned into a bacterial expression construct along with three nuclear localization signals (NLSs) and purified recombinant protein. sgRNAs targeting several validated target sites were generated by T7 in vitro transcription. Electroporation of the Nme2Cas9:sgRNA complex induced successful editing at the target site as detected by TIDE. Figure 38C. These results suggest that Nme2Cas9 can be delivered as a plasmid or as an RNP complex. Overall, these results demonstrate that Nme2Cas9 is functional in a variety of cell types with different delivery modes.

[0164] 4. Anti-CRISPR Protein Inhibition It has been reported that five anti-CRISPR (Acr) protein families against Nme1Cas9 from various bacterial species inhibit Nme1Cas9 in vitro and in human cells (Pawluk et al. 2016, Lee et al., mBio, in press). Given the high sequence identity between Nme1Cas9 and Nme2Cas9, it seems possible that at least some species within these Acr families may also inhibit Nme2Cas9. All five Acr families were recombinantly expressed and purified, and the ability of Nme2Cas9 to cleave the target sequence in vitro was tested (Acr:Cas9 molar ratio 10:1). As a negative control, an inhibitor of the IE-type CRISPR system in E. coli (AcrE2) was used. As expected, all Arc families inhibited Nme1Cas9, but AcrE2 failed to inhibit Nme1Cas9. Notably, Acr IIC1 Nme , Acr IIC2 Nme , Acr IIC3 Nme and Acr IIC4 Hpa inhibited Nme2Cas9 gene editing activity. Figure 40A, top.

[0165] Remarkably, AcrIIC5Smu In vitro, even a 10-fold excess of AcrIIC5 did not inhibit Nme2Cas9. Smu These results suggest that AcrIIC5 may inhibit Nme1Cas9 by interacting with the PID. To further confirm this, we performed the same in vitro cleavage assay using a hybrid version of NmeCas9 (e.g., Nme1Cas9 with the PID of Nme2Cas9). As this hybrid has reduced activity, we used a higher concentration of Cas9 (~30x) to achieve a similar cleavage profile while maintaining a Cas9:Acr molar ratio of 10:1. Consistent with the initial results, AcrIIC5 for this protein chimera was significantly higher than that of the control. Smu No inhibition was observed by AcrIIC5. Smu The inability of AcrIIC5 to inhibit the hybrid protein Smu It is further suggested that may interact with the PID of Nme1Cas9.

[0166] From the in vitro data above, Acr-IIC1 Nme , Acr-IIC2 Nme , Acr-IIC3 Nme and Acr-IIC4 Hpa These results suggest that AcrIIC1 can be used as an off switch for genome editing by Nme2Cas9. To test this, transfections were performed as described above in the presence or absence of a plasmid encoding Acr driven by a mammalian promoter. As it has been reported that the majority of ACRs inhibit Nme1Cas9 at a 1:1:1 ratio (Pawluk et al., 2016), approximately 150 ng of each plasmid (e.g., sgRNA:Cas9:Acr ratio 1:1:1) was transfected. As predicted from in vitro experiments, AcrIIC1 inhibited Nme1Cas9 at a 1:1:1 ratio (Pawluk et al., 2016). Nme , AcrIIC2 Nme , AcrIIC3 Nme and AcrIIC4 Hpa Genome editing by Nme2Cas9 was inhibited by AcrIIC5 Smufailed to inhibit genome editing by Nme2Cas9 (Figure 40B. Furthermore, complete inhibition below detection levels was observed by Acr3Nme and Acr4Hpa, demonstrating that their potency is limited by AcrIIC1. Nme and AcrIIC2 Nme This suggests that the AcrIIC1 Nme and AcrIIC4 Hpa To further compare the efficacy of AcrIIC4, experiments were performed with different Acr to Cas9 ratios. Hpa was a highly potent inhibitor of Nme2Cas9, inhibiting Nme2Cas9 four-fold at concentrations as low as 25 ng:100 ng Acr:Cas9. Collectively, these data suggest that the Acr protein can be used as an off switch for Nme2Cas9-based applications.

[0167] 5. Ultra-high accuracy of Nme2Cas9 Off-target effects can potentially confound therapeutic applications during ex vivo and in vivo human gene therapy by creating unintended mutations. Because wild-type SpyCas9 has a relatively large number of off-target sites in human cells, several attempts have been made to create high-fidelity SpyCas9 variants with variable success. In contrast, Nme1Cas9 is naturally ultra-precise and has demonstrated remarkable fidelity in cell and mouse models. Previous studies have shown that Cas9 fidelity can be determined from hybridization kinetics that are not determined by PID, thus suggesting that Nme2Cas9 may also be ultra-precise.

[0168] To empirically evaluate the NmeCas9 off-target profile, we used the Genome-Wide, Unbiased Identification of double-stranded breaks as Enabled by Sequencing (GUIDE-Seq) technique to unbiasedly determine potential off-target sites. GUIDE-Seq relies on the incorporation of double-stranded oligodeoxynucleotides (dsODNs) into DNA double-stranded break sites throughout the genome. These break sites are detected by amplification and high-throughput sequencing.

[0169] Wild-type SpyCas9 was used as a reference for GUIDE-Seq. Notably, SpyCas9 and Nme2Cas9 could be cloned into the same backbone driven by the same promoter, and their PAMs were used to target the same sites since they do not overlap. This technique allows a side-by-side comparison of the two nucleases. Six duplex sites (DS) with the NGGNCCN sequence in VEGFA were targeted. Figure 42A. 72 hours after transfection, TIDE analysis was performed on the target sites. Nme2Cas9 induced indels at all six sites, although with low efficiency at two of them, while SpyCas9 induced indels at 4 / 6 sites. Figure 42B. At two of these four sites (DS1 and DS4), SpyCas9 induced about 7-fold more indels than Nme2Cas9, whereas at DS6, Nme2Cas9 induced about a 3-fold increase in indels. For GUIDE-seq, targets DS2, DS4 and DS6 were selected to determine off-target cleavage at sites where Nme2Cas9 is similarly efficient, less efficient, or more efficient, respectively, than SpyCas9.

[0170] In addition to the three dual target sites, the TS6 target site with 30-50% indel rate (depending on cell type) was subjected to GUIDE-Seq analysis along with mouse Pcsk9 and Rosa26 genes. As TS6 targets are known to undergo highly efficient gene editing, it was expected that the off-target profile would be more prominent. Furthermore, the mouse Pcsk9 and Rosa26 sites would then be tested to reveal the fidelity of Nme2Cas9 in different cell lines, as well as candidate loci for in vivo genome editing. Consequently, for each Cas9, transfections were performed along with their cognate sgRNAs, and dsODN and GUIDE-Seq libraries were prepared. GUIDE-Seq analysis demonstrated efficient on-target editing with both Cas9 orthologs, with similar patterns observed by TIDE. With regard to off-target identification, the analysis revealed that the three SpyCas9 sites had a large predicted number of off-target sites (e.g., ranging between approximately 10 and 1000), whereas Nme2Cas9 had a remarkably clean off-target profile. Specifically, Nme2Cas9 targeting the same dual site showed a maximum of one off-target site. See Figure 42C.

[0171] To corroborate the off-target sites detected by GUIDE-seq, targeted deep sequencing was performed to measure indel formation at the top off-target loci after GUIDE-seq-independent editing (i.e., without co-transfection of dsODN). SpyCas9 showed substantial editing (in some cases more efficient than editing at the corresponding on-target sites) at the majority of off-target sites tested, whereas Nme2Cas9 showed no detectable indels at isolated DS2 and DS6 candidate off-target sites. With Rosa26 sgRNA, Nme2Cas9 induced approximately 1% editing at the Rosa26-OT1 site in Hepa1-6 cells, compared with approximately 30% on-target editing. Figure 42D.

[0172] Next, to allow SpyCas9 to be used as a reference for GUIDE-seq, due to the fact that SpyCas9 and Nme2Cas9 have non-overlapping PAMs, they can potentially edit any duplex site (DS) flanked by 5'-NGGNCC-3' sequences that simultaneously fulfill the PAM requirements of the binding properties of both Cas9s. This allows a side-by-side comparison of off-targeting with sgRNAs that bind to the exact same on-target site. Matched plasmids expressing each Cas9 and their respective sgRNAs were used to target 28 DSs at multiple loci throughout the human genome. 72 hours after plasmid delivery, TIDE analysis was performed on the sites targeted by each nuclease. Nme2Cas9 induced indels at 19 target sites, albeit with low efficiency (<5%) in 4 of them, while SpyCas9 induced indels at 23 target sites, in some cases with efficiency <5%. Three dual target sites were refractory to editing by either nuclease. Although SpyCas9 appears to be more efficient overall, many of the sites had similar efficiency with both enzymes, and two of the 17 sites were edited by both nucleases, with Nme2Cas9 being more efficient under these conditions. See Figure 42E.

[0173] It is noteworthy that this off-target site has a consensus Nme2Cas9 PAM (ACTCCCT) with only three mismatches at the PAM-distal end of the region complementary to the guide (i.e., outside the seed). See Figure 42F. These data confirm and reinforce our GUIDE-seq results showing high accuracy of genome editing by Nme2Cas9 in mammalian cells.

[0174] On-target vs. off-target in these aspects were compared by targeted amplification of each locus followed by TIDE analysis. Figure 43A. Interestingly, efficient on-target editing was observed, although no indels could be detected by TIDE at the off-target sites for either sgRNA. Furthermore, the read counts for these off-targets were negligible compared to those observed for SpyCas9, suggesting that Nme2Cas9 is highly specific (Figure 43C, left vs. right, respectively). To further corroborate these GUIDE-Seq results, CRISPRseek was used to computationally predict potential off-target sites for two of the most active sgRNAs with highly similar sites in the genome (Zhu et al., 2014). These were compared with Nme2Cas9, which is a sgRNA with highly similar sites in the genome. 4 CX PAM and mostly PAM far-field Nme2Cas9 was assayed using 2 to 5 mismatches within the target region (Figure 43D). Taken together, these data suggest that Nme2Cas9 is a high-fidelity nuclease in mammalian cells.

[0175] 6.Clinical application In one embodiment, the present invention contemplates the Nme2Cas9 complex as the first compact, ultra-precise Cas9 with small, non-restrictive PAM for therapeutic genome editing by AAV delivery.Previously reported ultra-precise Cas9 orthologs, although small, have longer PAMs than those disclosed herein, which limits their therapeutic use due to limited target sites in a given gene (and off-target profile in the case of SauCas9).This disadvantage is exacerbated in loci that can only be targeted in specific windows or require precise block deletion.

[0176] The integrated AAV delivery platform established herein can be used to target any gene in any tissue.In addition, the ultra-high accuracy of Nme2Cas9 allows precise editing of target gene, thus improving the safety concerns caused by off-target activity previously observed.For this purpose, Nme2Cas9 has the potential to not only complement existing tools, but also become the preferred choice for therapeutic genome editing by viral delivery.

[0177] Furthermore, inhibition of Nme2Cas9 by various Acr suggests possible evolutionary pressure on Cas9 to rapidly evolve specific domains. Smu Since Nme2Cas9 is not inhibited in AcrIIC5, the mechanism of inhibition may be through PID. Smu Considering that AcrIIC5 is the most potent inhibitor of Nme1Cas9 to date, Smu It is contemplated herein that can be used to robustly turn off Nme1Cas9, but not Nme2Cas9, which is of particular interest in the cellular context where multiplexing is enhanced by the ability to control specific orthologs.

[0178] Finally, there are thousands of Cas9 orthologs in public databases, but only a few of them have been characterized. In some embodiments contemplated herein, we exploit natural variations in closely related Cas9 orthologs to create two new Cas9 nucleases, namely Nme2Cas9 and Nme3Cas9, which use N4CC and N4CAAA PAMs, respectively. The data presented herein demonstrates that even closely related orthologs can have far more different properties. For example, these orthologs use exactly the same sgRNA as Nme1Cas9, which avoids the difficulty of predicting tracrRNA and determining the correct spacer length for each ortholog. In addition, it may be possible to create shorter and more stable sgRNAs (such as chemical modifications) to extend to all three nucleases. These properties can facilitate genome editing efforts and reduce the costs associated with protein and RNA engineering.

[0179] It should be clear to those skilled in the art that the embodiments described herein are not limited to Cas9, but can be applied to other Cas proteins, such as Cas12 and Cas13.It should also be understood that the hypervariability of Cas9 is not limited to PID.It is considered herein that there are strains that share a high degree of homology with a given Cas9, but differ in other domains due to other types of selection pressure.In summary, Nme2Cas9 is a novel nuclease that improves the current CRISPR platform for therapeutic genome editing.

[0180] V. Nucleotide Delivery Platforms In addition to the AAV nucleotide delivery systems described above, the present invention contemplates several delivery systems compatible with nucleic acids that provide roughly uniform distribution and have controllable release rates. Some embodiments of the present invention contemplate a nucleic acid delivery system encoding the Type II-C Cas9-sgRNA complex described herein.

[0181] Below are described a variety of different media that are useful in creating nucleic acid delivery system.No single media or carrier limits the present invention.Please note that any media or carrier can be combined with another media or carrier.For example, in one embodiment, the polymer microparticle carrier attached to the compound can be combined with gel medium.

[0182] Carriers or vehicles contemplated by the present invention include materials selected from the group including gelatin, collagen, cellulose esters, dextran sulfate, pentosan polysulfate, chitin, sugars, albumin, fibrin sealant, synthetic polyvinylpyrrolidone, polyethylene oxide, polypropylene oxide, block polymers of polyethylene oxide and polypropylene oxide, polyethylene glycol, acrylates, acrylamides, methacrylates including but not limited to 2-hydroxyethyl methacrylate, poly(orthoesters), cyanoacrylates, gelatin-resorcin-aldehyde type bioadhesives, polyacrylic acid and copolymers and block copolymers thereof.

[0183] microparticles In one embodiment of the present invention, a nucleic acid delivery system comprising microparticles is contemplated. Microparticles preferably include liposomes, nanoparticles, microspheres, nanospheres, microcapsules, and nanocapsules. Some microparticles contemplated by the present invention include poly(lactide-co-glycolide), aliphatic polyesters including, but not limited to, polyglycolic acid and polylactic acid, hyaluronic acid, modified polysaccharides, chitosan, cellulose, dextran, polyurethane, polyacrylic acid, psuedo-poly(amino acid)s, and the like. Preferred are polyhydroxybutyric acid related copolymers, polyanhydrides, polymethyl methacrylate, poly(ethylene oxide), lecithin and phospholipids.

[0184] Liposomes In one embodiment of the present invention, liposomes capable of attaching and releasing nucleic acids as described herein are contemplated. Liposomes are microscopic spherical lipid bilayers that surround an aqueous core, composed of amphipathic molecules such as phospholipids. For example, liposomes can entrap nucleic acids between the hydrophobic tails of phospholipid micelles. Water-soluble agents can be trapped within the core, and lipid-soluble agents can be dissolved in the shell-like bilayer. Liposomes have special properties in that they allow water-soluble and water-insoluble chemicals to be used together in a medium without the use of surfactants or other emulsifiers. Liposomes can be formed spontaneously by intensively mixing phospholipids in an aqueous medium. Water-soluble compounds are dissolved in an aqueous solution that can hydrate the phospholipids. Thus, when liposomes are formed, these compounds are trapped in the aqueous liposomal center. The liposome wall is a phospholipid membrane that holds lipid-soluble materials such as oils. Liposomes provide a controlled release of incorporated compounds. Additionally, liposomes can be coated with water-soluble polymers such as polyethylene glycol to increase pharmacokinetic half-life. In one embodiment of the present invention, ultra-high shear technology is contemplated to improve liposome fabrication, resulting in stable unilamellar (single layer) liposomes with specially designed structural characteristics. These unique properties of liposomes allow for the simultaneous storage of normally immiscible compounds and their controlled release capabilities.

[0185] In some embodiments, the present invention contemplates cationic and anionic liposomes, as well as liposomes with neutral lipids. Cationic liposomes preferably contain negatively charged materials by mixing the materials with fatty acid liposome components and associating them with a charge. Obviously, the choice of cationic or anionic liposomes depends on the desired pH of the final liposome mixture. Examples of cationic liposomes include lipofectin, lipofectamine, and lipofectace. .

[0186] In one embodiment of the present invention, a nucleic acid delivery system is contemplated that includes liposomes that provide controlled release of at least one nucleic acid.Preferably, the liposomes capable of controlled release are i) biodegradable and non-toxic, ii) carry both water-soluble and oil-soluble compounds, iii) solubilize refractory compounds, iv) prevent oxidation of compounds, v) promote protein stabilization, vi) control hydration, vii) control compound release by variations in bilayer composition, such as, but not limited to, fatty acid chain length, fatty acid lipid composition, relative amount of saturated and unsaturated fatty acids, and physical configuration, viiii) solvent-dependent, iv) pH-dependent, and v) temperature-dependent.

[0187] The composition of liposomes is broadly categorized into two classes. Conventional liposomes are generally a mixture of stabilized natural lecithin (PC) that may contain synthetic single-chain phospholipids that may or may not contain glycolipids. Special liposomes may include: i) bipolar fatty acids; ii) the ability to attach antibodies for tissue-targeted therapy; iii) coated with materials such as, but not limited to, lipoproteins and carbohydrates; iv) multiple encapsulation and v) emulsion compatibility.

[0188] Liposomes can be easily created in the laboratory by methods such as, but not limited to, sonication and vibration. Alternatively, compound delivery liposomes are commercially available. For example, it is known that Collaborative Laboratories, Inc. manufactures custom-designed liposomes for specific delivery requirements.

[0189] Microspheres, Microparticles and Microcapsules Microspheres and microcapsules are useful because they generally maintain uniform distribution, can provide a stable controlled release of the compound, and are economical to produce and distribute. The associated delivery gel or compound-permeating gel is preferably clear, or the gel is colored for ease of visualization by medical personnel.

[0190] Microspheres are commercially available (Prolease®, Alkerme's: Cambridge, Mass.). For example, freeze-dried media containing at least one therapeutic agent is homogenized in a suitable solvent and sprayed to produce microspheres in the 20-90 μm range. Techniques are then followed to maintain the integrity of the sustained release during purification, encapsulation and storage steps. Scott et al., Improving Protein Therapeutics With Sustained Release Formulations, Nature Biotechnology, Volume 10, Issue 1, 2011, pages 1111-1115. 16:153-157 (1998).

[0191] Modification of the microsphere composition through the use of biodegradable polymers can provide the ability to control the rate of nucleic acid release. Miller et al., Degradation Rates of Oral Resorbable Implants {Polylactates and Polyglycolates: Rate Modification and Changes in PLA / PGA Copolymer Ratios, J. Biomed. Mater. Res., Vol. II:711-719 (1977).

[0192] Alternatively, sustained or controlled release microsphere preparations are prepared using the drying-in-water method, in which a solution of biodegradable polymer metal salt in an organic solvent is first prepared. Then, the medium in which the nucleic acid is dissolved or dispersed is added to the biodegradable polymer metal salt solution. The weight ratio of nucleic acid to biodegradable polymer metal salt may be, for example, about 1:100000 to about 1:1, preferably about 1:20000 to about 1:500, more preferably about 1:10000 to about 1:500. Next, the organic solvent solution containing the biodegradable polymer metal salt and nucleic acid is poured into the aqueous phase to prepare an oil / water emulsion. The solvent in the oil phase is then evaporated to result in microspheres. Finally, these microspheres are then collected, washed, and lyophilized. The microspheres can then be heated under reduced pressure to remove the remaining water and organic solvent.

[0193] Other methods useful for producing microspheres that are compatible with mixtures of biodegradable polymer metal salts and nucleic acids are by i) phase separation during the stepwise addition of a coacervation agent; ii) the underwater drying method or phase separation method, with the addition of an anti-agglomeration agent to prevent particle agglomeration, and iii) the spray drying method.

[0194] In one embodiment, the present invention contemplates a vehicle comprising microspheres or microcapsules capable of delivering a controlled release of nucleic acid for a duration of between approximately 1 day and 6 months. In one embodiment, the microspheres or microparticles may be colored to allow clear identification by medical practitioners when the vehicle is being dispensed. In another embodiment, the microspheres or microcapsules may be transparent. In another embodiment, the microspheres or microparticles are impregnated with a radiopaque fluoroscopic dye.

[0195] Controlled release microcapsules can be made by using known encapsulation techniques such as centrifugal extrusion, pan coating and air suspension. Such microspheres and / or microcapsules can be engineered to achieve the desired release rate. For example, Oliosphere® (Macromed) is a controlled release microsphere system. These particular microspheres are available in uniform sizes ranging between 5-500 μm and are composed of biocompatible and biodegradable polymers. The specific polymer composition of the microspheres can control the nucleic acid release rate, thereby allowing custom-designed microspheres, including effective management of the burst effect. ProMaxx® (Epic Therapeutics, Inc.) is a protein matrix delivery system. This system is aqueous in nature and adaptable to standard pharmaceutical delivery models. In particular, ProMaxx® is a bioerodible protein microsphere that delivers both small and large molecule drugs and can be customized with respect to both microsphere size and desired release characteristics.

[0196] In one embodiment, the microspheres or microparticles comprise a pH-sensitive encapsulating material that is stable at a pH lower than the pH in the mesentery. The typical range in the mesentery is pH 7.6 to pH 7.2. Thus, the microcapsules should be maintained at a pH below 7. However, if pH fluctuations are expected, a pH-sensitive material can be selected based on a different pH criterion that is required for dissolution of the microcapsules. Thus, the nucleic acid to be encapsulated is selected with respect to the pH environment in which dissolution is desired and stored at a preselected pH so that stability is maintained. Examples of pH-sensitive materials useful as encapsulants are: , Eudragit® L-100 or S-100 (Rohm GMBH), hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate phthalate, and cellulose acetate trimellitate. In one embodiment, the lipids constitute the inner coating of the microcapsules. In these compositions, the lipids may be edible fats such as, but not limited to, partial esters of fatty acids and hexitiol anhydrides, and triglycerides. Lew CW, Controlled-Release pH Sensitive Capsule And Adhesive System And Method., U.S. Pat. No. 5,364,634 (see references). (herein incorporated by reference).

[0197] In one embodiment, the present invention contemplates microparticles comprising gelatin or other polymeric cations having a similar charge density to gelatin (i.e., poly-L-lysine) that are used as a complex to form primary microparticles. Primary microparticles are made as a mixture of the following compositions: i) gelatin (60 bloom, type A from porcine skin), ii) chondroitin 4-sulfate (0.005%-0.1%), iii) glutaraldehyde (25%, grade 1), and iv) 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC hydrochloride), and ultrapure sucrose (Sigma Chemical Co., St. Louis, Mo.). The source of the gelatin is not believed to be critical and may be from bovine, porcine, human, or other animal sources. Generally, the polymeric cation is between 19,000 and 30,000 daltons. Chondroitin sulfate is then added to the complex along with sodium sulfate as a coacervation agent, or ethanol.

[0198] After the formation of the microparticles, the nucleic acid is directly bound to the surface of the microparticles or indirectly attached using a "bridge" or "spacer". The amino groups of gelatin lysine groups are easily derivatized to provide sites for direct coupling of compounds. Alternatively, spacers such as avidin-biotin (i.e., linking molecules and derivatized moieties on the targeting ligand) are also useful for indirectly coupling the targeting ligand to the microparticles. The stability of the microparticles is controlled by the amount of glutaraldehyde-spacer crosslinks induced by EDC hydrochloride. The controlled release medium is also empirically determined by the final density of glutaraldehyde-spacer crosslinks.

[0199] In one embodiment, the present invention contemplates microparticles formed by spray drying a composition comprising fibrinogen or thrombin with nucleic acid. These microparticles are preferably soluble, and the walls of the microparticle are created by the selected protein (i.e., fibrinogen or thrombin). Thus, the nucleic acid is incorporated within and between the protein walls of the microparticle. Heath et al., Microparticles and Their Use In Wound Therapy., U.S. Patent No. 6,113,948 (incorporated herein by reference). (Incorporated herein.) After application of the microparticles to living tissue, the subsequent reaction of fibrinogen with thrombin creates a tissue sealant, thereby releasing the incorporated compounds into the immediately surrounding area.

[0200] Those skilled in the art will appreciate that microspheres do not have to be precisely spherical in shape, but are simply very small particles that can be sprayed or spread into or over a surgical site (i.e., open or closed). In one embodiment, the microparticles are composed of a biocompatible and / or biodegradable material selected from the group consisting of polylactide, polyglycolide and copolymers of lactide / glycolide (PLGA), hyaluronic acid, modified polysaccharides, and any other known materials. EXAMPLES

[0201] Example I Construction of integrated sgRNA-Nme1Cas9-AAV vector plasmid The bacterial Nme1Cas9 gene was codon-optimized for expression in humans and cloned into an AAV2 plasmid under the U1a ubiquitous promoter. The guide RNA is under the U6 promoter. The cas9 gene contains four nuclear localization signals and three HA tag sequences in tandem. A spacer sequence was inserted into the crRNA cassette using annealed synthetic oligonucleotides and digesting the plasmid with SapI restriction enzyme to generate a duplex with overhangs compatible with those generated by the SapI-digested backbone.

[0202] The human codon-optimized Nme1Cas9 gene under the control of the U1a promoter and a sgRNA cassette driven by a U6 promoter were cloned into the AAV2 plasmid backbone. The NmeCas9 ORF was flanked by four nuclear localization signals - two at each end - in addition to a triple HA epitope tag. This plasmid is available from Addgene (plasmid ID 112139). See Figure 64. Oligonucleotides with spacer sequences targeting Hpd, Pcsk9, and Rosa26 were inserted into the sgRNA cassette by ligation into the SapI cloning site.

[0203] AAV vector production was performed at the Horae Gene Therapy Center at the University of Massachusetts Medical School. Briefly, plasmids were packaged into AAV8 capsids by triple plasmid transfection in HEK293 cells and purified by precipitation as previously described. Gao et al., "Introducing genes into mammalian cells: viral vectors" In: Green MR, Sambrook J, editors. Molecular cloning: a laboratory manual. Volume 2. 4th ed. New York: Cold Spring Harbor Laboratory Press;2012. p. 1209-13. The off-target profiles of these spacers were computationally predicted using the Bioconductor package CRISPRseek. Search parameters were adapted to the Nme1Cas9 settings: gRNA.size=24, PAM="NNNNGATT", PAM.-size=8, RNA.PAM.pattern="NNNNGNNN$", weights=c(0, 0, 0, 0, 0, 0.014, 0, 0, 0.395, 0.317, 0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804, 0.685, 0.583), max.mismatch=6, allowed.mismatch.PAM=7, topN=10,000, min.score=0.

[0204] Example II Cell culture and transfection Murine Hepa1-6 hepatoma cells were cultured in DMEM with 10% FBS and 1% penicillin / streptomycin (Gibco) at 5% CO 2The cells were cultured in a 37°C incubator. Human HEK293T and PLB985 cells were cultured in DMEM and RPMI medium, respectively, both supplemented with 10% FBS and 1% penicillin / streptomycin (Gibco). Transient transfection of Hepa 1-6 cells was performed using Lipofectamine LTX, whereas for HEK293T cells Polyfect transfection reagent (Qiagen) was used. For transient transfection, approximately 1 × 10 cells per well were transfected in 24-well plates 24 h prior to transfection. 5 Each well was transfected with 500ng of integrated sgRNA-Nme1Cas9-AAV plasmid using Lipofectamine LTX with Plus Reagent (ThermoFisher) according to the manufacturer's protocol. In a 24-well plate, HEK293T cells were transfected with 400ng of integrated plasmid expressing Nme1Cas9 and sgRNA according to the manufacturer's guidelines (e.g., Psck9&Rosa26).

[0205] All cell lines were incubated at 5% CO 2 The cells were cultured in DMEM with 10% FBS and 1% penicillin / streptomycin (Gibco) and kept in a 37°C incubator. Mouse Hepa1-6 hepatoma and HEK293T cells were cultured in DMEM with 10% FBS and 1% penicillin / streptomycin (Gibco). K562 cells were grown in the same conditions, but using IMDM. IMR-90 cells were cultured in EMEM and 10% FBS. Finally, HDFa cells were grown in DMEM and 20% FBS.

[0206] Example III Expression and purification of Nme1Cas9 Nme1Cas9 was cloned into the pMCSG7 vector, which contains a T7 promoter followed by a 6xHis tag and then a tobacco etch virus (TEV) protease cleavage site. This construct was transformed into the Rosetta2 DE3 strain of E. coli to express Nme1Cas9. Briefly, bacterial cultures were grown at 37°C until the OD600 reached 0.6. At this point, the temperature was reduced to 18°C, after which 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added. Cells were grown overnight and then harvested for purification.

[0207] Purification of Nme1Cas9 was performed in three steps: nickel affinity chromatography, cation exchange chromatography, and then molecular sieve chromatography, the detailed protocol of which can be found in a previous publication (Jinek et al., Science 337, 816-821, 2012).

[0208] Example IV Ribonucleoprotein (RNP)-mediated delivery of Nme1Cas9 RNP-mediated delivery of Nme1Cas9 was performed using the Neon transfection system (ThermoFisher). Approximately 20 pmoles of Nme1Cas9 and 25 pmoles of sgRNA were mixed in buffer R and incubated at room temperature for 20-30 minutes. This preassembled complex was then mixed with 50,000-100,000 cells and electroporated using 10 μL of a Neon tip. After electroporation, cells were plated into 24-well plates containing the appropriate culture medium without antibiotics.

[0209] Example V DNA isolation from cells and tissues Genomic DNA was isolated from the cells 72 hours after transfection using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's protocol. Mice were sacrificed 10 days after hydrodynamic injection or 50 days after tail vein vector administration, and liver tissue was collected and genomic DNA was isolated using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's protocol.

[0210] Example VI Indel analysis 50 ng of genomic DNA was used for PCR amplification using genomic site-specific primers and High Fidelity® 2x PCR Master Mix (New England Biolabs). For TIDE analysis, 30 μl of PCR product was purified using the QIAquick® PCR Purification Kit (Qiagen) and subjected to Sanger sequencing. Indel scores were obtained using the TIDE web tool (tide-calculator.nki.nl / ) as previously described. Brinkman et al., Nucl. Acids Res. (2014).

[0211] For T7 endonuclease I (T7EI) assay, 10 μl of PCR product was hybridized and treated with 0.5 μl of T7 endonuclease I (New England Biolabs) in 1×NEB Buffer 2 for 1 hour. Samples were run on a 2.5% agarose gel and quantified using the ImageMaster-TotalLab® program. Indel percentages were calculated as previously described. Guschin et al., Engineered Zinc Finger Proteins: Methods and Protocols (2010).

[0212] Example VII GUIDE-Seq for off-target analysis GUIDE-seq analysis was performed as previously described (Tsai et al., Nature Biotechnology (2014); Bolukbasi et al., Nature Methods (2015a); Amrani et al., biorxiv.org / content / early / 2017 / 08 / 04 / 172650 (2017).

[0213] Briefly, for the two spacers targeting the Pcsk9 and Rosa26 genes, Hepa1-6 cells were transfected with 500 ng of integrated sgRNA-Nme1Cas9-AAV plasmid and 7.5 pmol of annealed GUIDE-seq oligonucleotides using Lipofectamine LTX® with Plus® Reagent (ThermoFisher). 72 hours after transfection, DNeasy® Blood and Genomic DNA was extracted using the Qiagen Tissue kit according to the manufacturer's protocol. Library preparation, deep sequencing, and read analysis were performed as previously described (Tsai et al., Nature Biotechnology (2014); Bolukbasi et al., Nature Methods (2015a); Amrani et al., biorxiv.org / content / early / 2017 / 08 / 04 / 172650 (2017)).

[0214] Example IX AAV vector production Plasmids were packaged into AAV8 by triple plasmid transfection in HEK293 cells at the Horae Gene Therapy Center at the University of Massachusetts Medical School and purified by precipitation as previously described. Gao GP, Sena-Esteves M. Introducing Genes into Mammalian Cells: Viral Vectors. In: Green MR, Sambrook J, eds. Molecular Cloning, Volume 2: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 2012:1209-1313.

[0215] Example X Animals, AAV vector injections, and liver tissue processing All animal experiments were approved under the guidelines of the University of Massachusetts Medical School Institutional Animal Care and Use Committee. For hydrodynamic injections, 2.5 mL of 30 μg of endotoxin-free sgRNA-Nme1Cas9-AAV plasmid targeting Pcsk9 or 2.5 mL of PBS as a control were injected into 9-18 week-old female C57BL / 6 mice via the tail vein. For AAV8 vector injections, 9-18 week-old female C57BL / 6 mice were injected with 4 × 10 11 Genome copies were injected via the tail vein. Eight-week-old female C57BL / 6NJ mice were in For ex vivo experiments, embryos that had progressed to the two-cell stage were transferred into the oviducts of E0.5 pseudopregnant female mice.

[0216] Mice are CO 2 Mice were euthanized by IV and the livers were harvested. Tissues were fixed overnight in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E).

[0217] Example XI serum analysis Blood (approximately 200 μL) was drawn from the facial vein on days 0, 25, and 50 after vector administration. Serum was isolated using a serum separator (BD, Cat# 365967) and stored at -80°C until assayed. Serum cholesterol levels were measured by Infinity™ colorimetric endpoint assay (Thermo-Scientific) according to the manufacturer's protocol. Briefly, serial dilutions of Data-Cal™ Chemistry calibrators were prepared in PBS. 2 μL of mouse serum or calibrator dilutions were mixed with 200 μL of Infinity™ cholesterol liquid reagent in a 96-well plate and then incubated at 37°C for 5 minutes. Absorbance at 500 nm was measured using a BioTek Synergy® HT microplate reader.

[0218] Example XII Discovery of Cas9 orthologs using highly evolved PID The Nme1Cas9 sequence was blasted to find all Cas9 orthologs within Neisseria species. Orthologs with >80% identity to Nme1Cas9 were selected for the remainder of the analysis. Each PID was then aligned with the PID of Nme1Cas9 (amino acids 820 to 1082) using ClustalW2, and those with clusters of mutations in the PID were selected.

[0219] The Nme1Cas9 peptide sequence was used as a query in a BLAST search to find all Cas9 orthologs in Neisseria meningitidis strains. Orthologs with >80% identity to Nme1Cas9 were selected for testing. The PIDs were then aligned with the PID of Nme1Cas9 (residues 820-1082) using ClustalW2®, and those with clusters of mutations in the PIDs were selected for further analysis. An unrooted phylogenetic tree of NmeCas9 orthologs was constructed using FigTree (tree.bio.ed.ac.uk / software / figtree).

[0220] Example XIII Cloning and purification of Nme2 Cas9 and Nme3 Cas9 and Acr orthologues The PIDs for Nme2Cas9 and Nme3Cas9 were ordered as gBlocks (IDT) to replace the PID for Nme1Cas9 using Gibson Assembly (NEB) in the bacterial expression plasmid pMSCG7 with a 6xHis tag. Constructs were transformed into E. coli, expressed, and purified as previously described.

[0221] Briefly, Rosetta(DE3) cells containing the respective Cas9 plasmids were grown at 37° C. to an optical density of 0.6 and protein expression was induced with 1 mM IPTG for 16 h at 18° C. Cells were harvested and lysed by sonication in lysis buffer (50 mM Tris, pH 7.5, 500 mM NaCl, 5 mM imidazole, 1 mM DTT) supplemented with lysozyme and protease inhibitor cocktail (Sigma).

[0222] The lysate was then loaded onto a Ni-NTA agarose column (Qiagen) and bound proteins were eluted with 300 mM imidazole and dialyzed into storage buffer (20 mM HEPES, pH 7.5, 250 mM NaCl, 1 mM DTT). For the Acr protein, a 6xHis tagged protein was expressed in E. coli strain BL21 Rosetta (DE3). Cells were incubated at 37°C in a shaking incubator for 1 h at 4°C for 1 h at 5°C for 1 h at 5°C. 600nm) to 0.6. The bacterial culture was 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 (50 mM Tris, pH 7.5, 500 mM NaCl, 5 mM imidazole, 1 mM DTT) supplemented with 1 mg / mL lysozyme and protease inhibitor cocktail (Sigma), and the protein was purified using the same protocol as Cas9. The 6xHis tag was removed by incubation with tobacco etch virus (TEV) protease overnight at 4 °C to isolate successfully cleaved, tag-free Acr.

[0223] Example IVX In vitro PAM discovery assay A library of protospacers with randomized PAM sequences was generated using overlapping PCR with a forward primer containing a 10 nucleotide randomized PAM.

[0224] The library was gel purified and subjected to an in vitro cleavage reaction with purified Cas9 together with in vitro transcribed sgRNA. 300 nM of the target fragment was cleaved in 1×NE Buffer 3.1 (NEB) at 37° C. for 1 hour using 300 nM of Cas9:sgRNA complex. The reaction was then treated with proteinase K at 50° C. for 10 minutes and run on a 4% agarose gel with 1×TAE. The cleavage products were purified and subjected to library preparation. The library was sequenced and analyzed using the Illumina NextSeq500® sequencing platform. Sequence logos were generated using R.

[0225] Example XV Transfection and mammalian genome editing Humanized Nme2Cas9 was cloned using Gibson Assembly into the pCDest2 plasmid previously used for Nme1Cas9 and SpyCas9 expression. Transfection of HEK293T and HEK293T-TLR cells was performed as previously described (Amrani et al. 2018). Hepa1 For -6 transfection, 500 ng of the integrated AAV.sgRNA.Nme2Cas9 plasmid was transfected into 24-well plates using Lipofectamine LTX at approximately 1 × 10 cells per well, which were cultured 24 h prior to transfection. 5 For K562 cells stably expressing Nme2Cas9, 50,000–150,000 cells were electroporated with the 500 sgRNA plasmid using 10 µL of Neon chip.

[0226] To measure indels in all cells, cells were harvested 72 hours after transfection and genomic DNA was extracted using the DNaesy® Blood and Tissue kit (Qiagen). Targeted loci were amplified by PCR, Sanger sequenced (Genewiz®), and analyzed by TIDE (Brinkman et al. 2014).

[0227] Example XVI Lentiviral transduction of K562 cells to stably express Nme2Cas9 K562 cells stably expressing Nme2Cas9 were generated as previously described. For lentivirus production, lentiviral vectors were co-transfected with packaging plasmids (Addgene 12260&12259) into HEK293T cells in 6-well plates using TransIT-LT1 transfection reagent (Mirus Bio) as recommended by the manufacturer. After 24 hours, the medium was aspirated from the transfected cells and replaced with a fresh 1 mL of fresh DMEM medium.

[0228] The next day, virus-containing supernatant from the transfected cells was harvested and filtered through a 0.45 μm filter. 10 μL of undiluted supernatant was used with 2.5 μg of polybrene to transduce approximately one million K562 cells in 6-well plates. Transduced cells were selected using medium containing 2.5 μg / mL of puromycin.

[0229] Example XVII RNP-based delivery for mammalian genome editing For RNP experiments, the Neon electroporation system was used. 3xNLS Nme2Cas9 40 pmoles were assembled in Buffer R with 50 pmoles of in vitro transcribed sgRNA and electroporated using 10 μL of a Neon chip. After electroporation, cells were plated in 24-well plates containing pre-warmed appropriate culture medium without antibiotics. Electroporation parameters (voltage, width, number of pulses) were 1150v, 20ms, 2 pulses for HEK293T cells and 1000v, 50ms, 1 pulse for K562 cells.

[0230] Example XVIII GUIDE-Seq GUIDE-Seq experiments were performed as previously described ( Amrani et al., 2018 ) with minor modifications.

[0231] Briefly, HEK293T cells were transfected with 200 ng of Cas9, 200 ng of sgRNA, and 7.5 pmol of annealed GUIDE-seq oligonucleotides with SpyCas9 or Nme2Cas9 for guides targeting dual sites using Polyfect (Qiagen). Hepa1-6 cells were transfected as described above.

[0232] 72 hours after transfection, DNeasy® Blood and Genomic DNA was extracted using a Tissue kit (Qiagen) according to the manufacturer's protocol. Library preparation and sequencing were performed exactly as previously described.

[0233] For analysis, sites matching sequences possessing 10 mismatches with the target site were considered as potential off-target sites. Data were analyzed using the Bioconductor package GUIDEseq version 1.1.17 (Zhu et al., 2017).

[0234] Example XIX Targeted deep sequencing and analysis To confirm the GUIDE-Seq results and to measure the indel rate more quantitatively, we used targeted deep sequencing. Two-step PCR amplification was used to generate DNA fragments for each on-target and off-target site. For SpyCas9, the top off-target positions were selected.

[0235] In the first step, a primer specific for the locus with a universal overhang with a complementary end to the adapter was mixed with 2x Phusion® PCR Master Mix (NEB) to generate a fragment with an overhang. In the second step, a universal forward primer and an indexed reverse primer were mixed with the primers to generate a fragment with an overhang. The purified PCR products were amplified using

[0236] Full-size products (approximately 250 bp in length) were gel extracted and sequenced using a paired-end MiSeq run. MiSeq data analysis was performed exactly as previously described (Amrani 2018).

[0237] (Example XX) Off-target analysis using CRISPRseek Comprehensive off-target analysis for TS25 and TS47 was performed using the Bioconductor package CRISPRseek.

[0238] Minor changes were made to accommodate properties of Nme2Cas9 that are not shared with SpyCas9. Specifically, the following changes were used: gRNA.size=24, PAM="NNNNCC", PAM.size=6, RNA.PAM.pattern="NNNNCN", and off-target sites with fewer than six mismatches were harvested. The top potential off-target sites were selected based on the number and location of mismatches. gDNA from cells targeted by each sgRNA was used to amplify each off-target locus and analyze it by TIDE.

[0239] Example XXI AAV8.Nme2Cas9 delivery and liver tissue processing in vivo All animal procedures were performed at the Institutional Animal Care Center at the University of Massachusetts Medical School. The study was reviewed and approved by the International Agency for Clinical Trials and Use Committee (IACUC).

[0240] For AAV8 vector injections, 4 × 10 per mouse were administered to 8-week-old female C57BL / 6 mice targeting Pcsk9 or Rosa26. 11Genome copies were injected via the tail vein. Mice were sacrificed 28 days after vector administration and liver tissue was harvested for analysis. Liver tissue was fixed overnight in 4% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). Blood was drawn from the facial vein at 0, 14 and 28 days after injection and serum was isolated using a serum separator (BD, Cat# 365967) and stored at -80°C until assayed. Serum cholesterol levels were measured using the Infinity™ colorimetric endpoint assay (Thermo-Scientific) according to the manufacturer's protocol and as previously described (Ibraheim et al. al, 2018) was measured.

[0241] Example XXII Animals and liver tissue processing For hydrodynamic injections, 2.5 mL of 30 μg of endotoxin-free AAV-sgRNA-hNme1Cas9 plasmid targeting Pcsk9 or 2.5 mL of PBS were injected via the tail vein into 9-18 week-old female C57BL / 6 mice. Mice were euthanized 10 days later and liver tissue was collected. For AAV8 vector injections, 12-16 week-old female C57BL / 6 mice were injected with 4 × 10 vectors per mouse targeting Pcsk9 or Rosa26. 11 Genome copies were injected via the tail vein. Mice were sacrificed 14 and 50 days after vector administration, and liver tissue was harvested for analysis.

[0242] For Hpd targeting, 2 mL of PBS or 2 mL of 30 μg of endotoxin-free AAV-sgRNA-hNme1Cas9 plasmid were administered via the tail vein to 15-21 week-old tyrosinemia type 1 Fah knockout mice (Fahneo). The encoded sgRNA targeted a site within exon 8 (sgHpd1) or exon 11 (sgHpd2). HT1 mice were fed with 10 mg / L NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione) (Sigma-Aldrich, Cat# PHR1731-1G) in drinking water at the indicated times. Both sexes were used in these experiments. Mice were maintained on NTBC water for 7 days after injection and then switched to regular water. Body weight was monitored every 1-3 days. PBS-injected control mice were sacrificed when they became moribund after losing 20% ​​of their body weight following removal of NTBC treatment.

[0243] Mice were euthanized according to our protocol, liver tissue was cut into thin slices, and the slices were stored at -80°C. Some liver tissues were fixed overnight in 4% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E).

[0244] (XXIII) Western blot Liver tissue fractions were pulverized and resuspended in 150 μL of RIPA lysis buffer. Total protein content was estimated by Pierce™ BCA Protein Assay Kit (Thermo-Scientific) according to the manufacturer's protocol. 20 μg total protein from tissue or 2 ng of recombinant mouse proprotein convertase 9 / PCSK9 protein (R&D Systems, 9258-SE-020) were loaded onto 4–20% Mini-Rotean® TGX™ Precast Gel (Bio-Rad). Separated bands were transferred to PVDF membranes and blocked with 5% Blocking-Grade Blocker solution (Bio-Rad) for 2 h at room temperature. Membranes were incubated overnight at 4°C with rabbit anti-GAPDH antibody (Abcam ab9485, 1:2000) or goat anti-PCSK9 antibody (R&D Systems AF3985, 1:400). The membrane was washed five times with TBST and incubated with goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) (Bio-Rad 1,706,515, 1:4000) and donkey anti-goat secondary antibody (R&D Systems HAF109, 1:2000) at room temperature for 2 hours. The membrane was washed five times with TBST and visualized with Clarity™ western ECL substrate (Bio-Rad) using an M35A X-OMAT Processor (Kodak).

[0245] Example XXIV Humoral immune response Humoral IgG1 immune responses against Nme1Cas9 were measured by ELISA (Bethyl; Mouse IgG1 ELISA Kit, E99-105) according to the manufacturer's protocol with minor modifications. Briefly, expression and three-step purification of Nme1Cas9 and SpyCas9 were performed. A total of 0.5 μg of recombinant Nme1Cas9 or SpyCas9 protein suspended in 1× Coating Buffer (Bethyl) was used to coat 96-well plates (Corning) and incubated for 12 hours with shaking at 4°C. Wells were washed three times with shaking for 5 minutes with 1× Wash Buffer. Plates were blocked with 1× BSA Blocking Solution (Bethyl) for 2 hours at room temperature and then washed three times. Serum samples were diluted 1:40 using PBS and added in duplicate to each well. After incubating the samples for 5 hours at 4°C, the plate was washed three times for 5 minutes and 100 μL of biotinylated anti-mouse IgG1 antibody (Bethyl; 1:100,000 in 1×BSA Blocking Solution) was added to each well. After incubating for 1 hour at room temperature, the plate was washed four times and 100 μL of TMB substrate was added to each well. The plate was developed in the dark at room temperature for 20 minutes and then 100 μL of ELISA Stop Solution was added per well. After development of the yellow solution, the absorbance at 450 nm was recorded using a BioTek Synergy® HT microplate reader.

[0246] Example XXV Zygote incubation and transfection

[0247] Mouse strains and embryo collection All animal experiments were performed under the guidance of the Institutional Animal Care and Use Committee (IACUC) of the University of Massachusetts Medical School. C57BL / 6NJ (stock number 005304) mice were obtained from Jackson Laboratory. All animals were maintained on a 12-hour light cycle. The midpoint of the light cycle on the day when a copulatory plug was observed was considered as embryonic day 0.5 (E0.5) of gestation. Zygotes were harvested on E0.5 by tearing the ampulla with forceps and incubated in M2 medium containing hyaluronidase to remove cumulus cells.

[0248] AAV8.Nme2Cas9+sgRNA delivery and liver tissue processing in vivo For AAV8 vector injections, 8-week-old female C57BL / 6NJ mice were injected with 4 × 10 11 Genomic copies were injected via the tail vein along with sgRNAs targeting validated sites within either Pcsk9 or Rosa26. Mice were sacrificed 28 days after vector administration and liver tissue was harvested for analysis. Liver tissue was fixed overnight in 4% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Blood was drawn from the facial vein at 0, 14, and 28 days after injection and serum was isolated using a serum separator (BD, Cat# 365967) and stored at -80°C until assayed. Serum cholesterol levels were measured using the Infinity™ colorimetric endpoint assay (Thermo-Scientific) according to the manufacturer's protocol as previously described. Ibraheim et al., "All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo" Genome Biology 19:137 (2018).

[0249] For anti-PCSK9 Western blot, 40 μg of protein from tissue or 2 ng of Recombinant Mouse PCSK9 Protein (R&D Systems, 9258-SE-020) was loaded onto a MiniProtean® TGX™ Precast Gel (Bio-Rad). The separated bands were transferred to a PVDF membrane and blocked with 5% Blocking-Grade Blocker® solution (Bio-Rad) for 2 hours at room temperature. The membrane was then incubated overnight with rabbit anti-GAPDH antibody (Abcam ab9485, 1:2,000) or goat anti-PCSK9 antibody (R&D Systems AF3985, 1:400). The membrane was washed with TBST and incubated with goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) (Bio-Rad 1706515, 1:4,000) and donkey anti-goat secondary antibody (R&D Systems HAF109, 1:2,000) at room temperature for 2 hours. The membrane was washed again with TBST and incubated with Clarity™ western ECL substrate (Bio-Rad) for 2 hours at room temperature ... Visualization was performed using a XOMAT Processor (Kodak).

[0250] Ex vivo AAV6.Nme2Cas9 delivery in mouse zygotes The zygote was incubated at 3 × 10 9 or 3×10 8 GCs were incubated in 15 μl droplets of KSOM (Potassium-Supplemented Simplex Optimized Medium, Millipore, Cat# MR-106-D) containing the AAV6.Nme2Cas9.sgTyr vector for 5–6 h (four zygotes in each droplet). After incubation, zygotes were rinsed with M2 and transferred to fresh KSOM for overnight culture. The next day, embryos that had progressed to the 2-cell stage were transferred into the oviducts of pseudopregnant recipients and developed to term.

[0251] (Example XXVI) Quantification and statistical analysis Analysis of in vitro PAM discovery data was performed using R.GraphPad Prism 6® for all statistical analyses. For mammalian cell experiments using Nme2Cas9, three independent replicates were performed and indel percentages were calculated using TIDE software, with error bars showing sem. TIDE parameters were set such that indels of <20 nucleotides were quantified for all figures. For control comparisons of Nme2Cas9 and SpyCas9, the average indel percentages were calculated using Microsoft Excel®. For in vivo experiments in mice, n=5 for control and test subjects. P values ​​were calculated by unpaired two-tailed t-test.

Claims

1. A Neisseria meningitidis single guide ribonucleic acid (Nme sgRNA) comprising a shortened repeat:anti-repeat region and a shortened Stem 2 region relative to the full length Neisseria meningitidis single guide ribonucleic acid (Nme sgRNA) set forth in SEQ ID NO:

219.

2. The Nme sgRNA of claim 1, wherein the full length Nme sgRNA comprises a Stem 2 region corresponding to residues 102-143 of SEQ ID NO:

219.

3. The Nme sgRNA of claim 1, wherein the full length Nme sgRNA comprises a repeat:anti-repeat region corresponding to residues 25-76 of SEQ ID NO:

219.

4. The Nme sgRNA of claim 1 , wherein the shortened Stem 2 region is 24 nucleotides in length.

5. The Nme sgRNA of claim 4, wherein the truncated Stem 2 region comprises residues 78-101 of SEQ ID NO:

220.

6. 2. The Nmes sgRNA of claim 1, wherein the truncated repeat:anti-repeat region comprises a first nucleotide sequence corresponding to residues 25-36 of SEQ ID NO:219, a second nucleotide sequence corresponding to residues 49-52 of SEQ ID NO:219, and a third nucleotide sequence corresponding to residues 65-76 of SEQ ID NO:

219.

7. The sgRNA sequence of claim 1, further comprising a shortened spacer region compared to the full length Nme sgRNA set forth in SEQ ID NO:

219.

8. The Nme sgRNA of claim 7, wherein the full length Nme sgRNA comprises a spacer region corresponding to residues 1 to 24.

9. The Nme sgRNA of claim 1, further comprising a spacer region that is 24 nucleotides in length.

10. The Nme sgRNA of claim 1 , wherein the Nme sgRNA has a length of 101 nucleotides.

11. 2. The NmesgRNA of claim 1, wherein the length of the NmesgRNA is selected from 121 nucleotides, 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 101 nucleotides, 100 nucleotides, and 99 nucleotides.

12. The Nme sgRNA of claim 1, wherein the Nme sgRNA is an Nme1 sgRNA.

13. The Nme sgRNA of claim 1, wherein the Nme sgRNA is an Nme2 sgRNA.

14. The Nme sgRNA sequence of claim 1 , wherein the Nme sgRNA comprises at least one chemical modification.

15. The Nmes sgRNA sequence of claim 14, wherein the chemical modification comprises ribose 2'-O-methylation.

16. The Nmes sgRNA sequence of claim 14, wherein the chemical modification comprises a phosphorothioate linkage.

17. A Neisseria meningitidis single guide ribonucleic acid (Nme sgRNA) comprising a shortened Stem 2 region compared to the full length Neisseria meningitidis single guide ribonucleic acid (Nme sgRNA) set forth in SEQ ID NO: 219, wherein the shortened Stem 2 region is 24 nucleotides in length.

18. The Nme sgRNA of claim 17, wherein the full length Nme sgRNA comprises a Stem 2 region corresponding to residues 102-143 of SEQ ID NO:

219.

19. The Nme sgRNA of claim 17, wherein the truncated Stem 2 region comprises residues 78-101 of SEQ ID NO:

220.

20. The Nme sgRNA of claim 17, further comprising a shortened repeat:anti-repeat region compared to the full length Nme sgRNA set forth in SEQ ID NO:

219.

21. The Nme sgRNA of claim 20, wherein the full length Nme sgRNA comprises a repeat:anti-repeat region corresponding to residues 25-76 of SEQ ID NO:

219.

22. 21. The Nmes sgRNA of claim 20, wherein the truncated repeat:anti-repeat region comprises a first nucleotide sequence corresponding to residues 25-36 of SEQ ID NO:219, a second nucleotide sequence corresponding to residues 49-52 of SEQ ID NO:219, and a third nucleotide sequence corresponding to residues 65-76 of SEQ ID NO:

219.

23. 18. The Nme sgRNA sequence of claim 17, further comprising a shortened spacer region compared to the full length Nme sgRNA set forth in SEQ ID NO:

219.

24. The Nme sgRNA of claim 23, wherein the full length Nme sgRNA comprises a spacer region corresponding to residues 1-24.

25. The Nme sgRNA of claim 17, further comprising a spacer that is 24 nucleotides in length.

26. The Nme sgRNA of claim 17, wherein the Nme sgRNA is an Nme1 sgRNA.

27. The Nme sgRNA of claim 17, wherein the Nme sgRNA is an Nme2 sgRNA.

28. The Nme sgRNA sequence of claim 17, wherein the Nme sgRNA comprises at least one chemical modification.

29. 29. The Nmes sgRNA sequence of claim 28, wherein the chemical modification comprises ribose 2'-O-methylation.

30. 29. The Nmes sgRNA sequence of Claim 28, wherein the chemical modification comprises a phosphorothioate linkage.

31. 18. The NmesgRNA of claim 17, wherein the length of the NmesgRNA is selected from 121 nucleotides, 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 101 nucleotides, 100 nucleotides, and 99 nucleotides.

32. The Nme sgRNA of claim 17, wherein the Nme sgRNA has a length of 101 nucleotides.

33. 1. An adeno-associated virus (AAV) vector encoding a Neisseria meningitidis single guide ribonucleic acid (Nme sgRNA), wherein the Nme sgRNA comprises a shortened repeat:anti-repeat region and a shortened Stem 2 region relative to the full length Nme sgRNA set forth in SEQ ID NO:

219.

34. The AAV vector of claim 33, wherein the AAV vector comprises at least one promoter.

35. The AAV vector of claim 34, wherein the at least one promoter is selected from a U6 promoter and a U1a promoter.

36. The AAV vector of claim 33, wherein the AAV vector comprises a Kozak sequence.

37. The AAV vector of claim 33, wherein the Nme sgRNA comprises a spacer region complementary to a gene of interest selected from PCSK9 and ROSA26.

38. 34. The AAV vector of claim 33, wherein the Nme sgRNA has a length selected from 121 nucleotides, 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 101 nucleotides, 100 nucleotides, and 99 nucleotides.

39. The AAV vector of claim 33, wherein the Nme sgRNA has a length of 101 nucleotides.

40. The AAV vector of claim 33, wherein the full length Nme sgRNA comprises a Stem 2 region corresponding to residues 102-143 of SEQ ID NO:

219.

41. The AAV vector of claim 33, wherein the full length Nme sgRNA comprises a repeat:anti-repeat region corresponding to residues 25-76 of SEQ ID NO:

219.

42. The AAV vector of claim 33, wherein the shortened Stem 2 region is 24 nucleotides in length.

43. The AAV vector of claim 42, wherein the truncated Stem 2 region comprises residues 78-101 of SEQ ID NO:

220.

44. 43. The AAV vector of claim 42, wherein the truncated repeat:anti-repeat region comprises a first nucleotide sequence corresponding to residues 25-36 of SEQ ID NO:219, a second nucleotide sequence corresponding to residues 49-52 of SEQ ID NO:219, and a third nucleotide sequence corresponding to residues 65-76 of SEQ ID NO:

219.

45. The AAV vector of claim 33, wherein the Nme sgRNA further comprises a shortened spacer region compared to the full-length Nme sgRNA set forth in SEQ ID NO:

219.

46. The AAV vector of claim 45, wherein the full length Nme sgRNA comprises a spacer region corresponding to residues 1-24.

47. The AAV vector of Claim 33, wherein the Nme sgRNA further comprises a spacer region that is 24 nucleotides in length.

48. a) providing a patient exhibiting at least one symptom of a medical condition, the patient comprising a plurality of genes associated with said medical condition; b) administering an AAV vector to said patient under conditions such that said at least one symptom of said medical condition is alleviated; A composition comprising an AAV vector encoding a Neisseria meningitidis single guide ribonucleic acid (NmesgRNA) for use in a method comprising: The composition, wherein the Nme sgRNA comprises a nucleic acid sequence complementary to a portion of at least one of the plurality of genes, and the Nme sgRNA comprises a shortened repeat:anti-repeat region and a shortened Stem 2 region compared to the full-length Nme sgRNA set forth in SEQ ID NO:

219.

49. 49. The composition of claim 48, wherein the medical condition comprises hypercholesterolemia.

50. 49. The composition of claim 48, wherein said at least one of said plurality of genes is a PCSK9 gene.

51. 51. The composition of claim 50, wherein the Nme sgRNA is complementary to a portion of the PCSK9 gene.

52. The composition of Claim 48, wherein the Nme sgRNA further comprises a shortened spacer region.

53. 49. The composition of Claim 48, wherein the Nme sgRNA comprises a spacer region that is 24 nucleotides in length.

54. 49. The composition of claim 48, wherein the Nme sgRNA has a length of 101 nucleotides.

55. 49. The composition of Claim 48, wherein the NmesgRNA has a length selected from 121 nucleotides, 111 nucleotides, 107 nucleotides, 105 nucleotides, 103 nucleotides, 102 nucleotides, 100 nucleotides, and 99 nucleotides.

56. 49. The composition of claim 48, wherein the truncated Stem 2 region is 24 nucleotides in length.

57. 1. An adeno-associated virus (AAV) vector encoding a type II-C Cas9 nuclease protein, wherein the type II-C Cas9 nuclease protein is selected from a Neisseria meningitidis strain De10444 Nme2Cas9 nuclease protein, a Haemophilus parainfluenzae HpaCas9 nuclease protein, and a Simonsiella muelleri SmuCas9 nuclease protein, ４ C.N. ３ , N ４ C.T., N. ４ C.C.N., ４ CCA, and N ４ G.N.T. ３ An AAV vector comprising a protospacer adjacent motif recognition domain configured with a binding site for a protospacer adjacent motif sequence selected from:

58. 58. The AAV vector of claim 57, wherein the type II-C Cas9 nuclease protein is a Neisseria meningitidis strain De10444 Nme2Cas9 nuclease protein.

59. 59. The AAV vector of claim 57 or 58, wherein the Type II-C Cas9 nuclease protein comprises an amino acid sequence having at least 86% identity to SEQ ID NO:

307.

60. 58. The AAV vector of claim 57, wherein the AAV vector further encodes a shortened sgRNA comprising a shortened repeat:anti-repeat region relative to the full-length Nme sgRNA set forth in SEQ ID NO:

219.

61. 61. The AAV vector of claim 57 or 60, wherein the AAV vector further encodes a shortened sgRNA comprising a shortened Stem 2 region relative to the full-length Nme sgRNA set forth in SEQ ID NO:

219.

62. a) a patient exhibiting at least one symptom of a medical condition, the patient comprising a plurality of genes associated with the medical condition, the plurality of genes comprising: ４ C ３ , N ４ C.T., N. ４ C.C.N., ４ CCA, and N ４ G.N.T. ３ providing a patient comprising a protospacer adjacent motif selected from the group consisting of: b) administering the composition to said patient under conditions such that said at least one symptom of said medical condition is alleviated; A composition comprising a delivery platform comprising at least one nucleic acid encoding a type II-C Cas9 nuclease for use in a method comprising: said type II-C Cas9 nuclease protein is selected from Neisseria meningitidis strain De10444 Nme2Cas9 nuclease protein, Haemophilus parainfluenzae HpaCas9 nuclease protein, and Simonsiella muelleri SmuCas9 nuclease protein; The type II-C Cas9 nuclease protein is ４ C.N. ３ , N ４ C.T., N. ４ C.C.N., ４ CCA, and N ４ G.N.T. ３ A composition comprising a protospacer adjacent motif recognition domain configured with a binding site for a protospacer adjacent motif sequence selected from:

63. 63. The composition of claim 62, wherein the delivery platform comprises an adeno-associated virus (AAV) vector.

64. 63. The composition of claim 62, wherein the delivery platform comprises a microparticle.

65. 63. The composition of claim 62, further comprising a truncated sgRNA.

66. 63. The composition of claim 62, wherein the medical condition is selected from hyperlipidemia and tyrosinemia.

67. 1. A composition comprising a delivery platform comprising a nucleic acid encoding a type II-C Cas9 nuclease protein, wherein said type II-C Cas9 nuclease protein is selected from a Neisseria meningitidis Nme2Cas9 nuclease protein, a Neisseria meningitidis Nme3Cas9 nuclease protein, a Haemophilus parainfluenzae HpaCas9 nuclease protein, and a Simonsiella muelleri SmuCas9 nuclease protein, wherein said type II-C Cas9 nuclease protein is selected from a Neisseria meningitidis Nme2Cas9 nuclease protein, a Neisseria meningitidis Nme3Cas9 nuclease protein, a Haemophilus parainfluenzae HpaCas9 nuclease protein, and a Simonsiella muelleri SmuCas9 nuclease protein, ４ C.N. ３ , N ４ C.T., N. ４ C.C.N., ４ CCA, and N ４ G.N.T. ３ A composition comprising a protospacer adjacent motif recognition domain configured with a binding site for a protospacer adjacent motif sequence selected from:

68. 68. The composition of claim 67, wherein the type II-C Cas9 nuclease protein is a Neisseria meningitidis strain De10444 Nme2Cas9 nuclease protein.

69. 69. The composition of claim 67 or 68, wherein the Type II-C Cas9 nuclease protein comprises an amino acid sequence having at least 86% identity to SEQ ID NO:

307.

70. 68. The composition of claim 67, wherein the delivery platform is selected from a microparticle, a nanoparticle, a microsphere, a liposome, and an adeno-associated viral vector.

71. The composition of claim 67, further comprising a cognate sgRNA.

72. A method of in vitro or ex vivo gene editing, comprising delivering a delivery platform to a cell containing a gene, the gene being selected from the group consisting of N ４ C ３ , N ４ C.T., N. ４ C.C.N., ４ CCA, and N ４ G.N.T. ３ and the delivery platform comprises a protospacer adjacent motif selected from: i) at least one nucleic acid encoding a type II-C Cas9 nuclease protein, said type II-C Cas9 nuclease protein being selected from a Neisseria meningitidis Nme2Cas9 nuclease protein, a Neisseria meningitidis Nme3Cas9 nuclease protein, a Haemophilus parainfluenzae HpaCas9 nuclease protein and a Simonsiella muelleri SmuCas9 nuclease protein, wherein said type II-C Cas9 nuclease protein comprises a protospacer adjacent motif recognition domain configured with a binding site for a protospacer adjacent motif sequence; and ii) at least one cognate sgRNA A method comprising:

73. 73. The in vitro or ex vivo method of claim 72, wherein the type II-C Cas9 nuclease protein is a Neisseria meningitidis strain De10444 Nme2Cas9 nuclease protein.

74. 74. The in vitro or ex vivo method of claim 72 or 73, wherein the Type II-C Cas9 nuclease protein comprises an amino acid sequence having at least 86% identity to SEQ ID NO:307.

Images