Methods of editing single nucleotide polymorphism using programmable base editor systems

Abstract

To provide compositions and methods for altering mutations associated with Rett Syndrome (RTT).SOLUTION: Provided herein are compositions and methods of using base editors comprising a polynucleotide-programmable nucleotide binding domain and a nucleobase editing domain in conjunction with a guide polynucleotide. Also provided herein are base editor systems for editing nucleobases of target nucleotide sequences.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application was filed on May 11, 2018, each of which is incorporated herein by reference in its entirety. U.S. Provisional Patent Application No. 62 / 670,558, filed December 17, 2018; U.S. Provisional Patent Application No. 62 / 780,838, filed December 17, 2018; and claims priority to and benefit of U.S. Provisional Patent Application No. 62 / 817,986, filed March 13, 2019. [Background technology]

[0002] Rett syndrome (RTT or RETT) is a condition in which the coding transcription factor (TCR) is expressed in the central nervous system (CNS). Methyltransferases damage or abolish the ability of proteins to modify chromatin and the transcriptional state. It is caused by a heterogeneous group of mutations in CpG-binding protein 2 (Mecp2). Using gene therapy to deliver or RNA editing to repair endogenous Mecp2 mRNA transcripts If delivered broadly throughout the CNS, this represents a promising approach for therapeutic intervention. However, both approaches must overcome significant challenges to achieve therapeutic efficacy. Mecp2 gene therapy requires strict control of the gene dose delivered per cell. otherwise there is a risk of mimicking the phenotype of Mecp2 duplication syndrome. The platform precisely complements the most prevalent Mecp2 mutations, which account for over 45% of RTT diagnoses. It induces uncorrected and uninduced off-target editing with high efficiency.

[0003] The Mecp2 gene mutations that cause Rett syndrome are highly heterogeneous. The preferred strategy for this is delivery by recombinant adeno-associated virus (rAAV). The strategy was to deliver wild-type Mecp2. This strategy was to identify the causative mutation in each individual. Because gene therapy is agnostic to mutations, successful gene therapy approaches are limited to the RTT patient population. This could provide a treatment option for a large proportion of patients. However, to date, this strategy has been limited. RTT patients are almost always heterozygous females with random X-transfected genes. Chromophore inactivation results in the formation of distinct wild-type and mutant X chromosomes within the central nervous system (CNS). This results in mosaic expression of Mecp2 associated with the phenotype. rAAV delivery and wild-type Mecp2 expression in rhesus monkeys partially reverses the phenotype of Mecp2 duplication syndrome. Consistent with this, high transduction rates in the CNS of RTT model mice were observed. The efficiency resulted in expression of Mecp2 that was approximately twice as great as that seen in wild-type mice.

[0004] Thus, there is a need for novel compositions and methods for treating patients with Rett Syndrome. exists.

[0005] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety. The same patents and patent applications are specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, the inventions described herein are incorporated by reference to the extent permitted by law. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety. Summary of the Invention

[0006] As described below, the present invention utilizes programmable nucleobase editors to target pathogenic antigens. The present invention features compositions and methods for precisely correcting amino acids. The present invention provides adenosine agonists and methods useful for treating Rett syndrome (RTT). (A) Single nucleotide polymorphisms in the endogenous Mecp2 gene were identified using a base editor (ABE). Precision correction and correction of deleterious mutations (e.g., R133C, T158M, R255*, R270*, R306C) The present invention provides compositions and methods for treating Rett Syndrome.

[0007] In one aspect, the present invention provides a method for identifying single nucleotide polymorphisms (SNPs) associated with Rett Syndrome (RTT). The present invention provides a method for editing an MECP2 polynucleotide comprising: contacting the base editor complexed with one or more guide polynucleotides a base editor for generating a polynucleotide programmable DNA binding domain; and adenosine deaminase domains, and Several studies have targeted base editors to alter the A and T SNPs associated with RTT. This results in a change to G·C.

[0008] In another aspect, the invention provides a base editor or a polynucleotide encoding a base editor. A cell produced by introducing a protease into a cell or its precursor, Editors of polynucleotide-programmable DNA-binding domains and adenosine deoxyribonucleic acid A of SNPs associated with RTT by targeting the amine domain and base editors a cell containing one or more guide polynucleotides that result in a T to G C modification; to provide.

[0009] In another aspect, the invention provides a method of treating RTT in a subject, comprising administering to a subject a base editor or or a polynucleotide encoding a base editor to the subject. The base editor is a polynucleotide with a programmable DNA binding domain and adenosine S-related RTT targeting the deaminase domain, as well as base editors Contains one or more guide polynucleotides that result in an A·T to G·C modification of NP , a method is provided.

[0010] In another aspect, the present invention provides a method for the detection of inflammatory bowel diseases, comprising: (i) the amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R , R1335V, T1337R and L1111, D1135L, S1136R, G1218S, E1219V, D1332A, R1335Q, T one or more of 1337, T1337L, T1337Q, T1337I, T1337V, T1337F and T1337M; or a modified (altered) SpCas9 containing the corresponding amino acid substitutions, and (ii) Provided are base editors comprising adenosine deaminase.

[0011] In another aspect, the present invention provides a method for the detection of inflammatory bowel diseases, comprising: (i) the amino acid substitutions D1135L, S1136R, G1218S, E1219V, A1322R , R1335Q, and T1337, as well as L1111R, G1218R, E1219F, D1332A, D1332S, D1332T, D 1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T One or more of 1337K, T1337R, T1337H, T1337Q, and T1337M, or (ii) a modified SpCas9 containing the corresponding amino acid substitutions, and (iii) an adenosine deaminase. and a base editor comprising:

[0012] In various embodiments, the contacting step is in a cell, in a eukaryotic cell, in a mammalian cell, or in a human cell. In various embodiments, the cell is in vivo or ex vivo. In various embodiments, the modifications are R106W, R168*, R133C, T158M, R255*, R270*, and R306C. In various embodiments, the RTT-associated SNP is one or more of: A T to G The modification to C replaces the cysteine ​​in the methyl-CpG binding protein 2 (MeCP2) polypeptide. to arginine, methionine to threonine, or change the stop codon to arginine. In various embodiments, the SNPs associated with RTT are at amino acid positions 168, 133, 255, 270, or or expression of a Mecp2 polypeptide containing arginine at position 306 or threonine at position 158. In various embodiments, the polynucleotide programmable DNA binding domain is Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof. In this study, the polynucleotide-programmable DNA binding domain was engineered to bind to the modified protospacer In various embodiments, the present invention includes a modified SpCas9 with PAM specificity. In various embodiments, the modified PAM has specificity for the nucleic acid sequence 5'-NGT-3'. The modified SpCas9 contains the amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R, as well as L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D 1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1 One or more of 337F, T1337S, T1337N, T1337K, T1337H, T1337Q, and T1337M or its corresponding amino acid substitution. In various embodiments, the modified SpCas9 comprises: Amino acid substitutions D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, and T1337, and For L1111R, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R , T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q and T1337M, or the corresponding amino acid substitutions thereof. In one embodiment, the polynucleotide programmable DNA binding domain is a nuclease In various embodiments, the nickase variant is an inactive or variant nickase. contains the amino acid substitution D10A or its corresponding amino acid substitution. The adenosine deaminase domain deaminates adenosine in deoxyribonucleic acid (DNA). In various embodiments, the adenosine deaminase can be synthesized from a naturally occurring In various embodiments, the adenosine deaminase is a modified (altered) adenosine deaminase that does not In various embodiments, the deaminase is a TadA deaminase. , TadA*7.10. In various embodiments, the one or more guide RNAs are CRISPR RNA (c The crRNA contains the RTT-associated SNPs. In various embodiments, the base editor comprises a nucleic acid sequence complementary to a Mecp2 nucleic acid sequence comprising , a single guide RNA ( In various embodiments, the cell is a neuron. In embodiments, the neurons express a Mecp2 polypeptide. The cells are from subjects with RTT.

[0013] The features of the present disclosure are set forth with particularity in the appended claims. The following detailed description and accompanying drawings set forth illustrative embodiments, in which the principles of the present invention are utilized. A better understanding of the features and advantages of the present invention can be obtained by referring to: [Brief explanation of the drawings]

[0014] [Figure 1]

[0033] Figure 12 is a graph showing the percentage of accurate correction of the R255X RTT mutation using base editor variants with specificity for the NGT PAM.

[0015] [Figure 2]

[0033] Figure 12 is a graph showing the percentage of accurate correction of the R255X RTT mutation using base editor variants with specificity for the NGT PAM.

[0016] [Figure 3] 10 is a graph showing PAM variant optimization with the amino acid substitution T1337L. Figure 11 shows the percentage of accurate correction of the R255X RTT mutation using base editor variants with specificity for the NGT PAM.

[0017] [Figure 4]

[0023] Figure 12 is a graph showing the percentage of accurate correction of the R255X RTT mutation by PAM base editor variants with specificity for NGT PAMs generated by shuffling mutations from other characterized PAM variants. T1337Q was identified as important for editing efficiency.

[0018] [Figure 5] 12A-C are graphs showing the change in base editing efficiency when T1337 and D1332 are substituted with other amino acids. The percentage of accurate correction of the R255X RTT mutation using base editor variants with specificity for the NGT PAM is shown.

[0019] [Figure 6]

[0023] Figure 1 shows the importance of E1219V and R1335Q for base editing activity associated with T1337. Percentage of accurate correction of the R255X RTT mutation using base editor variants with specificity for the NGT PAM is shown.

[0020] [Figure 7]

[0023] Figure 12 is a graph showing the percentage of accurate correction of the R255X RTT mutation using PAM variant base editors with specificity for the NGT PAM variants listed in Table 8 and Table 9.

[0021] [Figure 8]

[0023] Figure 12 is a graph showing the percentage of accurate correction of the R255X RTT mutation using PAM variant base editors with specificity for the NGT PAM variants listed in Table 8 and Table 9.

[0022] [Figure 9]

[0023] Figure 11 is a graph showing the percentage of accurate correction of the R255X RTT mutation using PAM variant base editors with specificity for the NGT PAM variants listed in Table 10. DETAILED DESCRIPTION OF THE INVENTION

[0023] Rett syndrome (RTT), a progressive neurodevelopmental disorder, and its causally related symptoms One or more mutations in the methyl-CpG binding protein 2 (Mecp2) gene are identified. Described herein are compositions and methods for providing base edits and base editing systems that correct for RTT is an X-linked dominant disorder that primarily affects females, with 96% of affected individuals % of cases are associated with mutations in the Mecp2 gene, resulting in apparently normal early development followed by Regression, fixed movements, and apraxia, with loss of fine motor skills and effective communication or is characterized by a complete loss of ambulation. Additional clinical features of affected individuals include: Abnormal slowing of head growth after birth, periodic breathing, gastrointestinal dysfunction, epilepsy, and scoliosis.

[0024] The most common mutation causing RTT is a cytidine resulting in a C·G to T·A base pair substitution. This is a C to T transition from thymidine to thymidine. This transition catalyzes the A·T to G·C substitution. Adenosine base editors (ABEs) can be used to reverse the wild-type, non-pathogenic genome sequence. By extension, highly dominant mutations that cause RTT can be treated with gene therapy. ABE was used to express wild-type Mecp2 genes without the risk of inducing overexpression of the Mecp2 gene, which can occur in some cases. Therefore, A·T to G·C DNA base editing is a potential target for reversing the sequence. precisely corrects one or more of the most common mutations that cause RTT in the Mecp2 gene It has the potential to do so.

[0025] The following description and examples further illustrate embodiments of the present disclosure. It should be understood that the present invention is not limited to the specific embodiments described herein, as such may vary. Those skilled in the art will recognize that there are numerous variations and modifications to this disclosure that are encompassed within its scope. It is understood that.

[0026] The headings used herein are for organizational purposes only and do not limit the subject matter described. It should not be interpreted as such.

[0027] Although various features of the disclosure may be described in the context of a single embodiment, these features may also be used interchangeably. may be provided separately or in any suitable combination. Conversely, this disclosure is Although the present disclosure may be described herein in the context of separate embodiments, the present disclosure also provides a single embodiment. It can also be executed in this state.

[0028] definition Unless otherwise defined, all technical and scientific terms used herein refer to the The following references are provided to those skilled in the art: provides general definitions for many of the terms used in this invention. ., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridg e Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genet ics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham , The Harper Collins Dictionary of Biology (1991).

[0029] In this application, the use of the singular includes the plural unless specifically stated otherwise. In these cases, the singular forms "a," "an," and "the" are used unless the context clearly indicates otherwise. It should be noted that the use of "or" includes plural referents. The term "and / or" means "and / or" unless stated otherwise. " and other uses of "include," "includes," and "included" are expressly prohibited. It is not definitive.

[0030] As used in this specification and claims, the word "comprising" (and "comprise" and "comprises" and any other form of "comprising"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "including (containing)" (and any form of containing, such as "contains" and "contain" The research paper is comprehensive and open-ended, and does not include additional elements or methodologies not cited. Any embodiment discussed herein does not exclude any method or step of the present disclosure. It is contemplated that this may also be practiced with respect to compositions, and vice versa. The compositions of the present disclosure can be used to achieve the methods of the present disclosure.

[0031] The terms "about" or "approximately" are used to refer to a particular value as determined by one of ordinary skill in the art. This means that the value is within a margin of error, which depends in part on how it is measured or determined. For example, "about" may be used to mean any deviation from the actual value of the measurement system. It can mean that the results are within one or more standard deviations per experiment. "About" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term refers to a range within an order of magnitude of a value, preferably Preferably, it means within 5 times, more preferably within 2 times. When a specific value is listed, the tolerance for that particular value is assumed unless otherwise stated. The term "about" should be understood to mean within the expected margin of error.

[0032] Ranges provided herein are understood to be shorthand for all of the values ​​within that range. For example, the range 1 to 50 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 , 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 It is understood to include any number, combination of numbers, or sub-range.

[0033] "Some embodiments," "one embodiment," or "other" References in the specification to "an embodiment of" include specific features described in connection with the embodiment, Although a structure or feature may be included in at least some embodiments of the present disclosure, it is not necessarily the case that all This means that the embodiments are not included.

[0034] "Adenosine deaminase" is a enzyme that hydrolyzes the deamination of adenine or adenosine. In some embodiments, the term "catalyzed" refers to a polypeptide or fragment thereof that is capable of catalyzing The deaminase or deaminase domain converts adenosine to inosine, or deaminase Adenosine deoxygenase catalyzes the hydrolytic deamination of hydroxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase is a deoxyribonuclease. Catalyzes the hydrolytic deamination of adenine or adenosine in acid (DNA). adenosine deaminases provided herein (e.g., engineered adenosine deaminases, The evolved adenosine deaminase may be from any organism, such as a bacterium.

[0035] An "agent" is any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or means their fragments.

[0036] "Ameliorate" means to reduce, suppress, attenuate, diminish, or arrest the development or progression of a disease. , or stabilize.

[0037] "Alterations" can be detected by methods known in the standard art, such as those described herein. refers to a change (increase or decrease) in the expression level or activity of a gene or polypeptide As used herein, modification includes a 10% change in expression level, preferably a 25% change in expression level. more preferably a 40% change, and most preferably a 50% or greater change. do.

[0038] "Analog" means a molecule that is not identical but has similar functional or structural characteristics. For example, a polypeptide analog may possess the biological activity of a corresponding naturally occurring polypeptide. Certain analogs enhance the functionality of naturally occurring polypeptides while retaining their inherent properties. Such biochemical modifications can improve, for example, ligand binding. Increased protease resistance, membrane permeability, or half-life of the analog without altering the Analogs can include unnatural amino acids.

[0039] "Administering" as used herein refers to administering one or more compositions described herein to a patient or subject. By way of example and without limitation, administration of the composition, e.g., injection, can be by intravenous administration. intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) ) injection. One or more of these routes may be used. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration may be by the oral route.

[0040] "Cytidine deaminase" catalyzes the deamination reaction that converts amino groups into carbonyl groups. In one embodiment, the amino acid sequence of the cytidine amino acid sequence is a polypeptide or fragment thereof that can mediate the transcription of cytidine. Deaminases convert cytosine to uracil or 5-methylcytosine to thymine. PmCDA1 (Petromyzon marinus cytidine deaminase 1, derived from Petromyzon marinus) "PmCDA1"), derived from mammals (e.g., humans, pigs, cows, horses, monkeys, etc.) AID (activity-induced cytidine deaminase, AICDA), which is a cytidine deaminase, and APOBEC are exemplary cytidine deaminase enzymes. It is an aminase.

[0041] "Methyl-CpG binding protein 2 (Mecp2) protein" is NCBI Accession No. NP_00498 3 or a fragment thereof having at least about 95% amino acid sequence identity with In certain embodiments, the Mecp2 protein comprises one or more modifications relative to the following reference sequence: In certain embodiments, the Mecp2 protein associated with RTT comprises the mutations R106W, R168*, R133 Examples include one or more mutations selected from C, T158M, R255*, R270*, and R306C. A representative Mecp2 amino acid sequence is provided below. 1 mvagmlglre eksedqdlqg lkdkplkfkk vkkdkkeeke gkhepvqpsa hhsaepaeag 61 kaetsegsgs apavpeasas pkqrrsiird rgpmyddptl pegwtrklkq rksgrsagky 121 dvylinpqgk afrskvelia yfekvgdtsl dpndfdftvt grgspsrreq kppkkpkspk 181 apgtgrgrgr pkgsgttrpk aatsegvqvk rvlekspgkl lvkmpfqtsp ggkaegggat 241 tstqvmvikr pgrkrkaead pqaipkkrgr kpgsvvaaaa aeakkkavke ssirsvqetv 301 lpikkrktre tvsievkevv kpllvstlge ksgkglktck spgrkskess pkgrsssass 361 ppkkehhhhh hhsespkapv pllpplpppp pepessedpt sppepqdlss svckeekmpr 421 ggslesdgcp kepaktqpav ataataaeky khrgegerkd ivsssmprpn reepvdsrtp 481 vtervs

[0042] "Mecp2 polynucleotide" refers to a nucleic acid molecule that encodes a Mecp2 protein or a fragment thereof. An exemplary Mecp2 polynucleotide is available under NCBI Accession No. NM_004992. The sequences of the Mecp2 polynucleotides are provided below. In certain embodiments, the Mecp2 polynucleotides are In certain embodiments, the Mecp2 polypeptide associated with RTT comprises one or more modifications to the sequence. The nucleotide is selected from 316C>T, 397C>T, 473C>T, 763C>T, 808C>T and 916C>T. It contains one or more mutations that 1 ccggcgtcgg cggcgcgcgc gctccctcct ctcggagaga gggctgtggt aaaagccgtc 61 cggaaaatgg ccgccgccgc cgccgccgcg ccgagcggag gaggaggagg aggcgaggag 121 gagagactgc tccataaaaa tacagactca ccagttcctg ctttgatgtg acatgtgact 181 ccccagaata caccttgctt ctgtagacca gctccaacag gattccatgg tagctgggat 241 gttagggctc agggaagaaa agtcagaaga ccaggacctc cagggcctca aggacaaacc 301 cctcaagttt aaaaaggtga agaaagataa gaaagaagag aaagagggca agcatgagcc 361 cgtgcagcca tcagcccacc actctgctga gcccgcagag gcaggcaaag cagagacatc 421 agaagggtca ggctccgcccc cggctgtgcc ggaagcttct gcctccccca aacagcggcg 481 ctccatcatc cgtgaccggg gacccatgta tgatgacccc accctgcctg aaggctggac 541 601 caatccccag ggaaaagcct ttcgctctaa agtggagttg attgcgtact tcgaaaaggt 661 aggcgacaca tccctggacc ctaatgattt tgacttcacg gtaactggga gagggagccc 721 ctcccggcga gagcgagaaac cacctaagaa gcccaaatct cccaaagctc caggaactgg 781 841 gggtgtgcag gtgaaaaggg tcctggagaa aagtcctggg aagctccttg tcaagatgcc 901 ttttcaaact tcgccagggg gcaaggctga ggggggtggg gccaccacat ccacccaggt 961 catggtgatc aaacgccccg gcaggaagcg aaaagctgag gccgaccctc aggccattcc 1021 caagaaacgg ggccgaaagc cggggagatgt ggtggcagcc gctgccgccg aggccaaaaa 1081 gaaagccgtg aaggagtctt ctatccgatc tgtgcaggag accgtactcc ccatcaagaa 1141 gcgcaagacc cgggagacgg tcagcatcga ggtcaaggaa gtggtgaagc ccctgctggt 1201 gtccaccctc ggtgagaaga gcgggaaagg actgaagacc tgtaagagcc ctgggcggaa 1261 aagcaaggag agcagcccca aggggcgcag cagcagcgcc tcctcacccc ccaagaagga 1321 gcaccaccac catcaccacc actcagagtc cccaaaggcc cccgtgccac tgctcccacc 1381 cctgccccca cctccacctg agcccgagag ctccgaggac cccaccagcc cccctgagcc 1441 ccaggacttg agcagcagcg tctgcaaaga ggagaagatg cccagaggag gctcactgga 1501 gagcgacggc tgccccaagg agccagctaa gactcagccc gcggttgcca ccgccgccac 1561 ggccgcagaa aagtacaaac accgagggga gggagagcgc aaagacattg tttcatcctc 1621 catgccaagg ccaaacagag aggagcctgt ggacagccgg acgccccgtga ccgagagagt 1681 tagctgactt tacacggagc ggattgcaaa gcaaaccaac aagaataaag gcagctgttg 1741 tctcttctcc ttatgggtag ggctctgaca aagcttcccg attaactgaa ataaaaaaata 1801 tttttttttc tttcagtaaa cttagagttt cgtggcttca gggtgggagt agttggagca 1861 ttggggatgt ttttcttacc gacaagcaca gtcaggttga agacctaacc agggccagaa 1921 gtagctttgc acttttctaa actaggctcc ttcaacaagg cttgctgcag atactactga 1981 ccagacaagc tgttgaccag gcacctcccc tcccgcccaa acctttcccc catgtggtcg 2041 ttagagacag agcgacagag cagttgagag gacactcccg ttttcggtgc catcagtgcc 2101 ccgtctacag ctcccccagc tccccccacc tcccccactc ccaaccacgt tgggacaggg 2161 aggtgtgagg caggagagac agttggattc tttagagaag atggatatga ccagtggcta 2221 tggcctgtgc gatcccaccc gtggtggctc aagtctggcc ccacaccagc cccaatccaa 2281 aactggcaag gacgcttcac aggacaggaa agtggcacct gtctgctcca gctctggcat 2341 ggctaggagg ggggagtccc ttgaactact gggtgtagac tggcctgaac cacaggagag 2401 gatggcccag ggtgaggtgg catggtccat tctcaaggga cgtcctccaa cgggtggcgc 2461 tagaggccat ggaggcagta ggacaaggtg caggcaggct ggcctggggt caggccgggc 2521 agagcacagc ggggtgagag ggattcctaa tcactcagag cagtctgtga cttagtggac 2581 agggggggg gcaaagggggg agggggg aatgttcttc cagttacttt ccaattctcc 2641 tttagggaca gcttagaatt atttgcacta ttgagtcttc atgttcccac ttcaaaacaa 2701 acagatgctc tgagagcaaa ctggcttgaa ttggtgacat ttagtccctc aagccaccag 2761 atgtgacagt gttgagaact acctggattt gtatatatac ctgcgcttgt tttaaagtgg 2821 gctcagcaca tagggttccc acgaagctcc gaaactctaa gtgtttgctg caattttata 2881 aggacttcct gattggtttc tcttctcccc ttccattct gccttttgtt catttcatcc 2941 tttcacttct ttcccttcct ccgtcctcct ccttcctagt tcatcccttc tcttccaggc 3001 agccgcggtg cccaaccaca cttgtcggct ccagtcccca gaactctgcc tgccctttgt 3061 cctcctgctg ccgtaccag ccccaccctg ttttgagccc tgaggaggcc ttgggctctg 3121 ctgagtccga cctggcctgt ctgtgaagag caagagagca gcaaggtctt gctctcctag 3181 gtagccccct cttccctggt aagaaaaagc aaaaggcatt tcccaccctg aacaacgagc 3241 cttttcaccc ttctactcta gagaagtgga ctggaggagc tgggcccgat ttggtagttg 3301 aggaaagcac agaggcctcc tgtggcctgc cagtcatcga gtggcccaac aggggctcca 3361 tgccagccga ccttgacctc actcagaagt ccagagtcta gcgtagtgca gcagggcagt 3421 agcggtacca atgcagaact cccaagaccc gagctgggac cagtacctg gtccccagcc 3481 cttcctctgc tcccctttt ccctcggagt tcttcttgaa tggcaatgtt ttgctttgc 3541 tcgatgcaga cagggggcca gaacaccaca catttcactg tctgtctggt catagctgt 3601 ggtgtagggg cttagaggca tgggcttgct gtgggttttt aattgatcag ttttcatgtg 3661 ggatcccatc ttttaacct ctgttcagga agtccttatc tagctgcata tcttcatcat 3721 attggtatat ccttttctgt gttacagag atgtctctta tatctaaatc tgtccaactg 3781 agaagtacct tatcaaagta gcaaatgaga cagcagtctt atgcttccag aaacacccac 3841 aggcatgtcc catgtgagct gctgccatga actgtcaagt gtgtgttgtc ttgtgtattt 3901 cagttattgt ccctggcttc cttactatgg tgtaatcatg aaggagtgaa acatcataga 3961 aactgtctag cacttccttg ccagtcttta gtgatcagga accatagttg acagttccaa 4021 tcagtagctt aagaaaaaac cgtgtttgtc tcttctggaa tggttagaag tgagggagtt 4081 tgccccgttc tgtttgtaga gtctcatagt tggactttct agcatatatg tgtccatttc 4141 cttatgctgt aaaagcaagt cctgcaacca aactcccatc agcccaatcc ctgatccctg 4201 atcccttcca cctgctctgc tgatgacccc cccagcttca cttctgactc ttccccagga 4261 agggaagggg ggtcagaaga gagggtgagt cctccagaac tcttcctcca aggacagaag 4321 gctcctgccc ccatagtggc ctcgaactcc tggcactacc aaaggacact tatccacgag 4381 agcgcagcat ccgaccaggt tgtcactgag aagatgttta ttttggtcag ttgggttttt 4441 atgtattata cttagtcaaa tgtaatgtgg cttctggaat cattgtccag agctgcttcc 4501 ccgtcacctg ggcgtcatct ggtcctggta agaggagtgc gtggcccacc aggcccccct 4561 gtcacccatg acagttcatt cagggccgat ggggcagtcg tggttgggaa cacagcattt 4621 caagcgtcac tttatttcat tcgggcccca cctgcagctc cctcaaagag gcagttgccc 4681 agcctctttc ccttccagtt tattccagag ctgccagtgg ggcctgaggc tccttagggt 4741 tttctctcta tttccccctt tcttcctcat tccctcgtct ttcccaaagg catcacgagt 4801 cagtcgcctt tcagcaggca gccttggcgg tttatcgccc tggcaggcag gggccctgca 4861 gctctcatgc tgcccctgcc ttggggtcag gttgacagga ggttggaggg aaagccttaa 4921 gctgcaggat tctcaccagc tgtgtccggc ccagttttgg ggtgtgacct caatttcaat 4981 tttgtctgta cttgaacatt atgaagatgg gggcctcttt cagtgaattt gtgaacagca 5041 gaattgaccg acagctttcc agtacccatg gggctaggtc attaaggcca catccacagt 5101 ctcccccacc cttgttccag ttgttagtta ctacctcctc tcctgacaat actgtatgtc 5161 gtcgagctcc ccccaggtct acccctcccg gccctgcctg ctggtgggct tgtcatagcc 5221 agtgggattg ccggtcttga cagctcagtg agctggagat acttggtcac agccaggcgc 5281 tagcacagct cccttctgtt gatgctgtat tcccatatca aaagacacag gggacaccca 5341 gaaacgccac atcccccaat ccatcagtgc caaactagcc aacggcccca gcttctcagc 5401 tcgctggatg gcggaagctg ctactcgtga gcgccagtgc gggtgcagac aatcttctgt 5461 tgggtggcat cattccaggc ccgaagcatg aacagtgcac ctgggacagg gagcagcccc 5521 aaattgtcac ctgcttct gcccagcttt tcattgctt gagtgatg gcgaaagagg 5581 gtaatacca gacaacaact gccaagttgg gtggagaag gagttttctt agctgacaga 5641 atctctgaat ttaatcac ttagtaagcg gctcaagccc aggaggc agaggtac 5701 gagcggagtc ccctgcgcgg gaccatctgg aattggttta gcccagtgg agcctgacag 5761 ccagaactct gtgtccccg tctaccaca gctcctttc cagagcattc cagtcaggct 5821 ctctggctg actgggccag gggaggttac aggtaccagt tctttaagaa gatctttggg 5881 catatacatt tttagcctgt gtcattgccc caatggatt cctgtttcaa gttcacct 5941 gcagattcta ggactgtgt cctagacttc agggattcag ctgttctag agttcctacc 6001 atggagtggg tctggaggac ctgccggtg ggggggcaga gcctgctcc ctcgggtct 6061 tcctactctt ctctctgctc tgacggtt tgttgattct gtgtctttct 6121 cttttagata ttgtatcaat ctttagaaaa ggcatagtct acttgttata aatcgttagg 6181 atactgcctc ccccagggtc taaattaca tattagagggg gaaaagctga acactgaagt 6241 cagttctcaa caatttagaa ggaaaacta gaaaacattt ggcagaaaat tacatttcga 6301 tgtttttgaa tgaatacgag caagctttta caacagtgct gatctaaaaa tacttagcac 6361 ttggcctgag atgcctggtg agcattacag gcaaggggaa tctggaggta gccgacctga 6421 ggacatggct tctgaacctg tctttggga gtggtatgga aggtggagcg ttcaccagtg 6481 acctggaagg cccagcacca ccctccttcc cactcttctc atcttgacag agcctgcccc 6541 agcgctgacg tgtcaggaaa acacccaggg aactaggaag gcacttctgc ctgaggggca 6601 gcctgccttg cccactcctg ctctgctcgc ctcggatcag ctgagccttc tgagctggcc 6661 tctcactgcc tcccaaggc cccctgcctg ccctgtcagg aggcagaagg aagcaggtgt 6721 gagggcagtg caaggagggga gcacaacccc cagctcccgc tccgggctcc gacttgtgca 6781 caggcagagc ccagaccctg gaggaaatcc tacctttgaa ttcaagaaca tttggggaat 6841 ttggaaatct ctttgcccc aaacccccat tctgtcctac cttaatcag gtcctgctca 6901 gcagtgagag cagatgaggt gaaaaggcca agaggtttgg ctcctgccca ctgatagccc 6961 ctctccccgc agtgtttgtg tgtcaagtgg caaagctgtt cttcctggtg accctgatta 7021 tatccagtaa cacatagact gtgcgcatag gcctgctttg tctcctctat cctgggcttt 7081 tgttttgctt tttagttttg cttttagttt ttctgtccct tttatttaac gcaccgacta 7141 gacacacaaa gcagttgaat ttttatatat atatctgtat attgcacaat tataaactca 7201 ttttgcttgt ggctccacac acacaaaaa agacctgtta aaattatacc tgttgcttaa 7261 ttacaatatt tctgataacc atagcatagg acaaggaaa ataaaaaaag aaaaaaaaga 7321 aaaaaaaacg acaaatctgt ctgctggtca cttcttctgt ccaagcagat tcgtggtctt 7381 ttcctcgctt ctttcaaggg ctttcctgtg ccaggtgaag gaggctccag gcagcaccca 7441 ggttttgcac tcttgtttct cccgtgcttg tgaaagaggt cccaaggttc tgggtgcagg 7501 agcgctccct tgacctgctg aagtccggaa cgtagtcggc acagcctggt cgccttccac 7561 ctctgggagc tggagtccac tggggtggcc tgactccccc agtccccttc ccgtgacctg 7621 gtcagggtga gcccatgtgg agtcagcctc gcaggcctcc ctgccagtag ggtccgagtg 7681 tgtttcatcc ttcccactct gtcgagcctg ggggctggag cggagacggg aggcctggcc 7741 tgtctcggaa cctgtgagct gcaccaggta gaacgccagg gaccccagaa tcatgtgcgt 7801 cagtccaagg ggtcccctcc aggagtagtg aagactccag aaatgtccct ttcttctccc 7861 ccatcctacg agtaattgca tttgcttttg taattcttaa tgagcaatat ctgctagaga 7921 gtttagctgt aacagttctt tttgatcatc tttttttaat aattagaaac accaaaaaaa 7981 tccagaaact tgttcttcca aagcagagag cattataatc accagggcca aaagcttccc 8041 tccctgctgt cattgcttct tctgaggcct gaatccaaaa gaaaaacagc cataggccct 8101 ttcagtggcc gggctacccg tgagcccttc ggaggaccag ggctggggca gcctctgggc 8161 ccacatccgg ggccagctcc ggcgtgtgtt cagtgttagc agtgggtcat gatgctcttt 8221 cccacccagc ctgggatagg ggcagaggag gcgaggaggc cgttgccgct gatgtttggc 8281 cgtgaacagg tgggtgtctg cgtgcgtcca cgtgcgtgtt ttctgactga catgaaatcg 8341 acgcccgagt tagcctcacc cggtgacctc tagccctgcc cggatggagc ggggcccacc 8401 cggttcagtg tttctgggga gctggacagt ggagtgcaaa aggcttgcag aacttgaagc 8461 ctgctccttc ccttgctacc acggcctcct ttccgtttga tttgtcactg cttcaatcaa 8521 taacagccgc tccagagtca gtagtcaatg aatatatgac caaatatcac caggactgtt 8581 actcaatgtg tgccgagccc ttgcccatgc tgggctcccg tgtatctgga cactgtaacg 8641 tgtgctgtgt ttgctcccct tccccttcct tctttgccct ttacttgtct ttctggggtt 8701 tttctgtttg ggtttggttt ggtttttatt tctccttttg tgttccaaac atgaggttct 8761 ctctactggt cctcttaact gtggtgttga ggcttatatt tgtgtaattt ttggtgggtg 8821 aaaggaattt tgctaagtaa atctcttctg tgtttgaact gaagtctgta ttgtaactat 8881 gtttaaagta attgttccag agacaaatat ttctagacac tttttcttta caaacaaaag 8941 cattcggagg gagggggatg gtgactgaga tgagagggga gagctgaaca gatgacccct 9001 gcccagatca gccagaagcc acccaaagca gtggagccca ggagtcccac tccaagccag 9061 caagccgaat agctgatgtg ttgccacttt ccaagtcact gcaaaaccag gttttgttcc 9121 gcccagtgga ttcttgtttt gcttcccctc cccccgagat tattaccacc atccccgtgct 9181 tttaaggaaa ggcaagattg atgtttcctt gaggggagcc aggaggggat gtgtgtgtgc 9241 agagctgaag agctggggag aatggggctg ggcccaccca agcaggaggc tgggacgctc 9301 tgctgtgggc acaggtcagg ctaatgttgg cagatgcagc tcttcctgga caggccaggt 9361 ggtgggcatt ctctctccaa ggtgtgcccc gtgggcatta ctgtttaaga cacttccgtc 9421 acatcccacc ccatcctcca gggctcaaca ctgtgacatc tctattcccc accctcccct 9481 tcccagggca ataaaatgac catggagggg gcttgcactc tcttggctgt cacccgatcg 9541 ccagcaaaac ttagatgtga gaaacccct tcccattcca tggcgaaaac atctccttag 9601 aaaagccatt accctcatta ggcatggttt tgggctccca aaacacctga cagcccctcc 9661 ctcctctgag aggcggagag tgctgactgt agtgaccatt gcatgccggg tgcagcatct 9721 ggagagcta ggcagggtgt ctgccccctc ctgagttgaa gtcatgctcc cctgtgccag 9781 cccagaggcc gagagctatg gacagcattg ccagtaacac aggccaccct gtgcagaagg 9841 gagctggctc cagcctggaa acctgtctga ggttgggaga ggtgcacttg gggcacaggg 9901 agaggccggg acacacttag ctggagatgt ctctaaaagc cctgtatcgt attcaccttc 9961 agtttttgtg ttttgggaca attactttag aaaataagta ggtcgtttta aaaacaaaaa 10021 ttattgattg ctttttgta gtgttcagaa aaaaggttct ttgtgtatag ccaaatgact 10081 gaaagcactg atatatttaa aaacaaaagg caatttatta aggaaatttg taccatttca 10141 gtaaacctgt ctgaatgtac ctgtatacgt ttcaaaaaca cccccccccc actgaatccc 10201 tgtaacctat ttattatata aagagtttgc cttataaatt t

[0043] A "base editor (BE)" or "nucleobase editor (NBE)" is a In various embodiments, the term "agent" refers to an agent that binds to a base and has nucleobase modifying activity. The primer comprises a nucleobase-modified polymerase linked to a guide polynucleotide (e.g., a guide RNA). Polypeptides (e.g., deaminases) and polynucleotide programmable nucleases In various embodiments, the agent comprises a protein having base editing activity. Domains, i.e., bases (e.g., A, T, C, G, or U) in nucleic acid molecules (e.g., DNA), are modified. In some embodiments, the biomolecular complex comprises a domain that can be decorated with a polynucleotide. The oligonucleotide programmable DNA binding domain is fused to the deaminase domain and In one embodiment, the agent is a fusion tag comprising a domain with base editing activity. In another embodiment, the protein domain having base editing activity is a guide protein. RNA (e.g., RNA-binding motifs on guide RNAs and RNA-binding domains fused to deaminases) In some embodiments, the domains having base editing activity are linked together (through a domain). In some embodiments, the base is capable of deaminating a base in a nucleic acid molecule. The editor is capable of deaminating bases in a DNA molecule. Base editors deaminate cytosine (C) or adenosine (A) in DNA. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the adenosine deaminase is evolved from TadA. In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-associated In some embodiments, the base editor is a deoxyribonucleic acid (e.g., a Cas or Cpf1) enzyme. Catalytically dead Cas9 (dCas9) fused to an aminase domain. In its form, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. In some embodiments, the base editor is an inhibitor of base excision repair (BER). In some embodiments, the inhibitor of base excision repair is fused to a uracil-DNase inhibitor. A glycosylase inhibitor (UGI). In some embodiments, an inhibitor of base excision repair. are inosine base excision repair inhibitors. For details on base editors, see the respective references. International PCT Application No. PCT / 2017 / 045381 (WO2018 / 027078), which is incorporated herein in its entirety; and No. PCT / US2016 / 058344 (WO2017 / 070632), the entire contents of which are incorporated by reference. Komor, AC, et al., “Programmable editing of a target,” incorporated herein by reference. t base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in "Genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); Komor, AC, et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Scie nce Advances 3:eaao4774 (2017), and Rees, HA, et al., “Base editing: preci sion chemistry on the genome and transcriptome of living cells.” Nat Rev Genet . 2018 Dec;19(12):770-788. doi: 10.1038 / s41576-018-0059-1. By way of example, base editing compositions, systems, and methods described herein may be used to The cytidine base-editing CBE is synthesized using the following nucleic acid sequence (8877 base pairs) provided below: Addgene, Wa tertown, MA.; Komor AC, et al., 2017, Sci Adv., 30;3(8):eaao4774. doi: 10.1126 / s ciadv.aao4774. At least 95% or greater identity with the BE4 nucleic acid sequence Also encompassed are polynucleotide sequences having the following structure: 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag 421 actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 481 gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 541 gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 601 aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 661 acctggttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg 721 tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 781 ccccgcaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg 841 actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 901 gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 961 atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1021 ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1081 gccaccgggt tgaaatctgg tggttcttct ggtggttcta gcggcagcga gactcccggg 1141 acctcagagt ccgccacacc cgaaagttct ggtggttctt ctggtggttc tgataaaaag 1201 tattctattg gtttagccat cggcactaat tccgttggat gggctgtcat aaccgatgaa 1261 tacaaagtac cttcaaagaa atttaaggtg ttgggggaaca cagaccgtca ttcgattaaa 1321 aagaatctta tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg 1381 aaacgaaccg ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa 1441 atttttagca atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc 1501 ttccttgtcg aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat 1561 gaggtggcat atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac 1621 tcaactgata aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc 1681 cgtgggcact ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg 1741 ttcatccagt tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt 1801 ggcgtggatg cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac 1861 ctgatcgcac aattacccgg agagagaaa aatggttgt tcggtaacct tatagcgctc 1921 tcactaggcc tgacaccaaa tttagtcg aacttcgact tagctgaga tgccaattg 1981 cagcttagta aggacacgta cgatgacgat ctcgacaatc tactggcaca attggagat 2041 cagtagcgg acttatttt ggctgccaa aaccttagcg atgcaatcct cctactcgac 2101 atactgagag ttatactga gattaccaag gcgccgtta ccgctcaat gatcaaagg 2161 tacgatgaac atcaccaga cttgacactt ctcaagccc tagtccgtca gcaactgcct 2221 gagaaata aggaaatatt cttgatcag tcgaaaaacg ggtacgcagg ttatattgac 2281 ggcggagcga gtcagagga attctacaag tttatcaac ccatattaga gagatggat 2341 gggacggaag agttgcttgt aaaaccaat cgcgagatc tactgcgaaa gcagcggact 2401 ttcgacacg gtagcattcc acatcaatc cacttaggcg aattgcatgc tatacttaga 2461 aggcaggagg atttttacc gttcccaaa gatacgtg aaagattga gaaaatccta 2521 acctttcgca taccttacta tgtgggaccc ctggcccgag ggaactcg gttcgcatgg 2581 atgacagaa agtccgaaga aacgattact ccatggaatt tgaggaagt tgtcgataaa 2641 gtgcgtcag ctcaatcgtt catcgagagg atgaccact ttgacagaa tttaccgaac 2701 gaaaagtat tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc 2761 acgaaagtta agtatgtcac tgaggcatg cgtaaacccg cctttctaag cggagacag 2821 aagaaagcaa tagtagatct gttattcaag accaccgca aagtgacagt tagcattg 2881 aagaggact actttaagaa attgaatgc tcgattctg tcgagatc cggggtagaa 2941 gatcgattta atgcgtcact tgtacgtat catgacctcc taagataat taagataag 3001 gacttcctgg ataacgaaga gatgagat atcttagaag atagtgtt gactcttacc 3061 ctctttgaag atcgggaat gattgaggaa agactaaaa catacgctca cctgttcgac 3121 gataagtta tgaacagtt aaagaggcgt cgctatacgg gctgggacg attgtcgcgg 3181 aaacttatca acggataag agacaagcaa agtggtaaa ctattctcga ttttctaaag 3241 agcgacggct tcgccatag gaactttatg cagctgatcc atgatgactc tttaccttc 3301 aaagaggata tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt 3361 gcgaatcttg ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg 3421 gatgagctag ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca 3481 cgcgaaaatc aaacgactca gaagggcaa aaaaacagtc gagagcggat gaagagaata 3541 gaagggta ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc 3601 3661 3721 tccttttga aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga 3781 gggaaaagtg acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg 3841 3901 aggggtggct tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc 3961 cgccaaatca caaagcatgt tgcacagata ctagattccc gaatgaatac gaatacgac 4021 gagaacgata agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg 4081 gacttcagaa aggattttca attctataaa gttagggaga taaataacta ccaccatgcg 4141 cacgacgctt atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta 4201 gaaagtgagt ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa 4261 agcgaacagg agataggcaa ggctacagcc aatacttct tttattctaa cattatgaat 4321 ttctttaaga cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa 4381 accaatgggg agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga 4441 aaagttttgt ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg 4501 tttcaaagg aatcgattct tccaaaagg aatagtgata agctcatcgc tcgtaaaaag 4561 gactgggacc cgaaaaagta cggtggcttc gatagcccta cagttgccta ttctgccta 4621 gtagtggcaa aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg 4681 gggataacga tttggagcg ctcgtctttt gaaaagaacc ccatcgactt cttgaggcg 4741 aaaggttaca aggagtaa aaaggatctc aatattaac taccaaagta tagtctgttt 4801 gagttagaaa atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac 4861 gaactcgcac taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag 4921 ttgaaaggtt cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat 4981 tatctcgacg aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat 5041 gccaatctgg acaaagtatt aagcgcatac aacaagcaca gggataacc catacgtgag 5101 caggcggaa atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc 5161 aagtattttg acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac 5221 gcgacactga ttcaccaatc catcacggga ttattgaaa ctcggataga tttgtcacag 5281 cttggggggtg actctggtgg ttctggagga tctggtggtt ctactaatct gtcagatatt 5341 attgaaaagg agaccggtaa gcaactggtt atccaggaat ccatcctcat gctcccagag 5401 gaggtggaag aagtcattgg gaacaagccg gaaagcgata tactcgtgca caccgcctac 5461 gacgagagca ccgacgagaa tgtcatgctt ctgactagcg acgcccctga atacaagcct 5521 tgggctctgg tcatacagga tagcaacggt gagaacaaga ttaagatgct ctctggtggt 5581 tctggaggat ctggtggttc tactaatctg tcagatatta ttgaaaagga gaccggtaag 5641 caactggtta tccaggaatc catcctcatg ctcccagagg aggtggaaga agtcattggg 5701 aacaagccgg aaagcgatat actcgtgcac accgcctacg acgagagcac cgacgagaat 5761 gtcatgcttc tgactagcga cgcccctgaa tacaagcctt gggctctggt catacaggat 5821 agcaacggtg agaacaagat taagatgctc tctggtggtt ctcccaagaa gaagaggaaa 5881 gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg 5941 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 6001 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 6061 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 6121 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 6181 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc 6241 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 6301 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 6361 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 6421 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 6481 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 6541 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 6601 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 6661 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 6721 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 6781 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 6841 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 6901 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 6961 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 7021 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 7081 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 7141 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 7201 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 7261 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 7321 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 7381 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 7441 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 7501 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 7561 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 7621 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 7681 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 7741 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 7801 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 7861 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 7921 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 7981 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 8041 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 8101 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 8161 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 8221 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 8281 gcattatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaata 8341 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg 8401 gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat 8461 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca 8521 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg 8581 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat 8641 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt 8701 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 8761 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 8821 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatc BE4 amino acid sequence: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNT RCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYS PSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPPHILWATGLKSGGSSGGSSGS ETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE ATRLKRTARRRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK KLVDSTDKADLRIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDLNLLAQIGDQYADLFLAAKNLSDAI LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPIL EKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMN TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTK EVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILV HTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVE EVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRK

[0044] By way of example, base editing compositions, systems, and methods described herein may be used to The adenine base-editing ABE is synthesized from the following nucleic acid sequence (8877 base pairs) provided below (Addgene, Watertown, MA.; Gaudelli NM, et al., Nature. 2017 Nov 23;551(7681):464-471. doi: 10.1038 / nature24644; Koblan LW, et al., Nat Biotechnol. 2018 Oct;36(9):843-846. doi: 10.1038 / nbt.4172.) ABE nucleic acid sequences and at least 95% or more Polynucleotide sequences with high degrees of identity are also encompassed. ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT GACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGATTTCCAAGTCTCCACCCCATTG ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCC ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGT CAGATCCGCTAGAGATCCGCGGCCGCTAATACGACTCACTATAGGGAGAGCCGCCACCATGAAACGGACA GCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAGTCTCTGAAGTCGAGTTTAGCCACGAGT ATTGGATGAGGCACGCACTGACCCTGGCAAAGCGAGCATGGGATGAAAGAGAAGTCCCCGTGGGCGCCGT GCTGGTGCACAACAATAGAGTGATCGGAGAGGGATGGAACAGGCCAATCGGCCGCCACGACCCTACCGCA CACGCAGAGATCATGGCACTGAGGCAGGGAGGCCTGGTCATGCAGAATTACCGCCTGATCGATGCCACCC TGTATGTGACACTGGAGCCATGCGTGATGTGCGCAGGAGCAATGATCCACAGCAGGATCGGAAGAGTGGT GTTCGGAGCACGGGACGCCAAGACCGGCGCAGCAGGCTCCCTGATGGATGTGCTGCACCACCCCGGCATG AACCACCGGGTGGAGATCACAGAGGGAATCCTGGCAGACGAGTGCGCCGCCCTGCTGAGCGATTTCTTTA GAATGCGGAGACAGGAGATCAAGGCCCAGAAGAAGGCACAGAGCTCCACCGACTCTGGAGGATCTAGCGG AGGATCCTCTGGAAGCGAGACACCAGGCACAAGCGAGTCCGCCACACCAGAGAGCTCCGGCGGCTCCTCC GGAGGATCCTCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGG CACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGGTGCTGAACAATAGAGTGATCGGCGAGGGCTG GAACAGAGCCATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTG GTCATGCAGAACTACAGACTGATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTGATGTGCGCCG GCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGAGGAACGCAAAAACCGGCGCCGCAGG CTCCCTGATGGACGTGCTGCACTACCCCGGCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCA GATGAATGTGCCGCCCTGCTGTGCTATTTCTTTCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAAGG CCCAGAGCTCCACCGACTCCGGAGGATCTAGCGGAGGCTCCTCTGGCTCTGAGACACCTGGCACAAGCGA GAGCGCAACACCTGAAAGCAGCGGGGGCAGCAGCGGGGGGTCAGACAAGAAGTACAGCATCGGCCTGGCC ATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGA AACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGC TATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGT CCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGC CTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGAC CTGCGGCTGATCTATCTGGCGCTGGCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACC TGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGA GGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGA CGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGGAGAAAGAATGGCCTGTTCGGAAACCTGATTGCCC TGAGCCTGGGCCTGACCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAG CAAGGACACCTACGACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGGCCGACCTGTTTT CTGGCCGCCAAGAACCTGCTCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCA AGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGC TCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCC GGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGG ACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAA CGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTAC CCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCC CTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAA CTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAG AACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGC TGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGC CATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAG AAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACAT ACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGA AGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCC CACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCC GGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGG CTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAA GCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTA AGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGA ATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACA CCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGAC TCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAG AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTT CGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAG CTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACG ACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCG GAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAAC GCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACA AGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTT CTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGG CCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGC GGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAA AGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAG TACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGT CCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAA TCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAG TACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAA ACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGG CTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATC GAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCT ACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAA TCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAA GAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTC AGCTGGGAGGTGACTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAG GAAAGTCTAACCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTT CTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCT CTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTA ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA AGCATAAAGTGTAAAGCCTAGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGC CCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGG TTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGA GCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCT CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT ACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTA ACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTA CACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACACTCAGTGGAACGAAAACTC ACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTAC GATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGT TGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGA TCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTAC TGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAA AAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATAC TCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATG TATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGA TCGGGAGATCGATCTCCCGATCCCCTAGGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAA GCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAAC AAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGAT GTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCAT TAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCAT TGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC

[0045] "Base editing activity" acts to chemically modify bases within polynucleotides In one embodiment, the first base is converted to the second base. Base editing activity is a cytidine deaminase activity, e.g., the activity of converting the target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, For example, the activity of converting A·T to G·C.

[0046] The term "base editor system" refers to a system that edits nucleic acid bases of a target nucleotide sequence. In various embodiments, the base editor (BE) system comprises: (1) a polynucleotide sequence; Nucleotide-programmable nucleotide-binding domain and deamination of said nucleobases (2) a deaminase domain for polynucleotide programmability The nucleic acid sequence includes a guide polynucleotide (e.g., a guide RNA) linked to a nucleic acid binding domain. In some embodiments, the base editor system comprises: (1) a polynucleotide program; a programmable DNA binding domain and a deaminase domain for deaminating the nucleic acid bases main, and (2) a polynucleotide linked to a programmable DNA binding domain In some embodiments, the polynucleotide programmable nucleotide sequence comprises a guide RNA. The nucleotide binding domain is a polynucleotide-programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). is.

[0047] In some embodiments, a nucleobase editor system comprises two or more base editing components. For example, a nucleobase editor system may include two or more deaminases. In some embodiments, the nuclease base editor system comprises one or more cytidine deaminase and / or one or more adenosine deaminases In some embodiments, a single guide polynucleotide may be used to The deaminase may be targeted to a target nucleic acid sequence. A pair of guide polynucleotides is used to target different deaminases to a target nucleic acid sequence. Targeting may also be performed.

[0048] Nucleobase components of base editor systems and polynucleotide programmable nucleosides The peptide-binding moieties may be covalently or non-covalently bound to one another. In some embodiments, the polynucleotide programmable nucleotide binding domain Thus, the deaminase domain can be targeted to the target nucleotide sequence. In some embodiments, the polynucleotide programmable nucleotide binding driver The domain can be fused or linked to the deaminase domain. In this embodiment, the polynucleotide programmable nucleotide binding domain binds to a deaminase Deamidation occurs by non-covalently interacting with or binding to the domain. The enzyme domain can be targeted to a target nucleotide sequence, for example In some embodiments, the nucleobase editing component, e.g., the deaminase component, is Further heterologous moieties or portions thereof that are part of the nucleotide-programmable nucleotide-binding domain or domains that can interact with, associate with, or form complexes with In some embodiments, the additional heterologous moieties or domains may be included. The moiety is capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous moiety can be linked to the polynucleotide and interact with it. In some embodiments, the ATP-binding domain is capable of interacting with, associating with, or forming a complex with the ATP-binding domain. In some embodiments, the heterologous moiety can be attached to a guide polynucleotide. In some cases, further heterologous moieties can be attached to the polypeptide linker. In embodiments, the additional heterologous moiety can be attached to the polynucleotide linker The additional heterologous moiety may be a protein domain. In some embodiments, The heterologous portion consists of a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, and a PP7 coat protein domain. coat protein domain, SfMu Com coat protein domain, steryl alpha motif fu, telomerase Ku binding motif and Ku protein, telomerase Sm7 binding motif and and Sm7 protein, or an RNA recognition motif.

[0049] The base editor system may further comprise a guide polynucleotide component. The components of the inter-system may be bound by covalent bonds, non-covalent interactions, or their associations and interactions. It should be appreciated that the hydroxyl groups may be associated with each other via any combination of The guide polynucleotide aligns the deaminase domain with the target nucleotide sequence. For example, in some embodiments, base editors can be targeted. The nucleobase editing component of the system, e.g., a deaminase component, of the guide polynucleotide interacts with, associates with, or binds to a portion or segment (e.g., a polynucleotide motif) or a further heterologous moiety or domain (e.g., RNA or or a polynucleotide-binding domain such as a DNA-binding protein. In embodiments, additional heterologous moieties or domains (e.g., RNA or DNA binding proteins) may be included. a polynucleotide binding domain such as a nucleotide-binding domain (e.g., a nucleotide-binding domain) fused or linked to the deaminase domain In some embodiments, the additional heterologous moiety can be attached to the polypeptide. , interact, associate, or form a complex. The additional heterologous moiety binds to, interacts with, associates with, or complexes with the polynucleotide. In some embodiments, the additional heterologous moiety can form a guidepole. In some embodiments, the additional heterologous moiety can be linked to a nucleotide. can be attached to a polypeptide linker. In some embodiments, further The heterologous moiety can be attached to a polynucleotide linker. may be a protein domain. In some embodiments, the further heterologous moiety K homology (KH) domain, MS2 coat protein domain, PP7 coat protein domain In, SfMu Com coat protein domain, steryl alpha motif, telomerase Ku binding motif and Ku protein, telomerase Sm7 binding motif and Sm7 protein, Or it may be an RNA recognition motif.

[0050] In some embodiments, the base editor system inhibits base excision repair (BER) components. The components of the base editor system may be linked by covalent bonds, non-covalent interactions, or It is recognized that the proteins may associate with one another through any combination of these associations and interactions. Inhibitors of BER components may include base excision repair inhibitors. In some embodiments, Inhibitors of base excision repair can be uracil DNA glycosylase inhibitors (UGIs). In some embodiments, the inhibitor of base excision repair may be an inosine base excision repair inhibitor. In some embodiments, the polynucleotide-programmable nucleotide binding domain This allows for targeting of inhibitors of base excision repair to target nucleotide sequences. In some embodiments, the polynucleotide-programmable nucleotide binding domain The amine can be fused or linked to an inhibitor of base excision repair. In this paper, the polynucleotide programmable nucleotide binding domain is The inhibitors of base excision repair can be fused or linked to the nucleotide sequences of the target gene and the target gene. In embodiments, the polynucleotide programmable nucleotide binding domain is Inhibition of base excision repair by non-covalently interacting with or associating with inhibitors of repair. The inhibitor can be targeted to a target nucleotide sequence. In embodiments, the inhibitor of base excision repair components is a polynucleotide programmable nucleotide It interacts with a further heterologous moiety or domain that is part of the peptide-binding domain. Further heterologous moieties or domains that can be bound to, associated with, or complexed with In some embodiments, the guide polynucleotide may include base excision. Inhibitors of repair can be targeted to the target nucleotide sequence. For example, In some embodiments, the inhibitor of base excision repair is a portion of the guide polynucleotide or can interact with and associate with a segment (e.g., a polynucleotide motif). or a further heterologous moiety or domain (e.g., RNA) capable of forming a complex. A or a polynucleotide binding domain such as a DNA binding protein. In embodiments, a guide polynucleotide (e.g., a polynucleotide such as an RNA or DNA binding protein) is Further heterologous portions or domains of the nucleotide-binding domain (e.g., the nucleotide binding domain) may inhibit base excision repair. In some embodiments, the polypeptide may be fused or linked to an additional heterologous moiety. The molecule is capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous moiety can be linked to the guide polynucleotide. In some embodiments, the additional heterologous moiety can be a polypeptide linker. In some embodiments, the additional heterologous moiety can be linked to a polynucleotide. Further heterologous moieties may be protein domains. In some embodiments, the additional heterologous moiety may be a K homology (KH) domain. , MS2 coat protein domain, PP7 coat protein domain, SfMu Com coat protein Protein domain, steryl alpha motif, telomerase Ku binding motif and Ku protein protein, telomerase Sm7 binding motif and Sm7 protein, or RNA recognition motif. That's fine.

[0051] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nucleic acid sequence containing the Cas9 protein. a cleavage enzyme, or a fragment thereof (e.g., an active, inactive, or partially active DNA cleavage enzyme of Cas9) The Cas9 nuclease is a nucleotide sequence that binds to the cas9 gene. n1 nuclease or CRISPR (clustered regularly interspaced short palindromic reactivity) An exemplary Cas9 is the Cas9 enzyme of Streptococcus pyogenes. Cas9, the amino acid sequence of which is provided below. JPEG2024138268000002.jpg162167 (single underline: HNH domain, double underline: RuvC domain)

[0052] The term "conservative amino acid substitution" or "conservative mutation" refers to a mutation that replaces one amino acid with another that shares a common characteristic. This means replacing an amino acid with another amino acid that has a common characteristic between individual amino acids. A functional way to define sex is by amino acid changes between corresponding proteins of the same species. (Schulz, GE and Schirmer, RH, Princ Examples of Protein Structure, Springer-Verlag, New York (1979). According to this theory, amino acids within a group are preferentially exchanged with one another, thus resulting in a Define groups of amino acids that are most similar to each other in their effect on structure. (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include For example, the amino acid lysine is replaced by arginine, which maintains a positive charge, and the negative charge is replaced by arginine. Maintained glutamate to aspartate and vice versa, free -OH to serine to threonine, and glutamine to asparagine, which maintains the free -NH2 group. amino acid substitutions are included.

[0053] The terms "coding sequence" or "protein coding sequence" are used interchangeably herein. "coding sequence" means a segment of a polynucleotide that encodes a protein. This region or sequence is bounded by the initiation codon closer to the 5' end and by the 3' end. The coding sequence is separated by a stop codon near the open reading frame. It can also be called a room.

[0054] As used herein, the term "deaminase" or "deaminase domain" refers to a deaminase domain that In some embodiments, deaminase refers to a protein or enzyme that catalyzes a deaminase reaction. The enzyme or deaminase domain is a cytidine deaminase, and Catalyzes the hydrolytic deamination of cycytidine to uridine or deoxyuridine, respectively In some embodiments, the deaminase or deaminase domain aminase, which catalyzes the hydrolytic deamination of cytosine to uracil. In an embodiment, the deaminase is adenosine deaminase, and the deaminase is adenosine hypoxase. In some embodiments, the deaminase catalyzes the hydrolytic deamination of acetylcholine to acetylcholine. adenosine deaminase, which hydrolyzes adenosine or adenine (A) to inosine (I) In some embodiments, a deaminase or deaminase catalyzes degradative deamination. The ze domain is an adenosine deaminase that converts adenosine or deoxyadenosine into They catalyze the hydrolytic deamination of ATP to inosine or deoxyinosine, respectively. In this embodiment, the adenosine deaminase is The adenosine deaminases provided herein (e.g., Adenosine deaminase (e.g., engineered adenosine deaminase, evolved adenosine deaminase) is a type of enzyme that is expressed in bacteria, etc. In some embodiments, the adenosine deaminase is from any organism. E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentu In some embodiments, the adenosine deaminase is from a bacterium such as Tad In some embodiments, the deaminase or deaminase domain The inn is a living organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is a variant of a naturally occurring deaminase from the body. The enzyme or deaminase domain is not naturally occurring. For example, in some embodiments In this case, the deaminase or deaminase domain is at least as similar to a naturally occurring deaminase. 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 9 8%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or are at least 99.9% identical. For example, the deaminase domains are No. PCT Application No. PCT / 2017 / 045381 (WO2018 / 027078), which is incorporated herein in its entirety by reference thereto. and PCT / US2016 / 058344 (WO2017 / 070632), the entire contents of which are incorporated herein by reference. Komor, AC, et al., “Programmable editing of at arget base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420 -424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, AC, et al., “Improved base excision repair inhibition and bacteriophage Mu Gam pro tein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017)), and Rees, HA, et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec;19(12):770-788. doi: 10.1038 / s41576-018-0059-1.

[0055] A "detectable label" is a label that, when attached to a molecule of interest, can be used to detect that molecule by spectroscopy, photochemistry, biochemistry, or other methods. It means a composition that is detectable by chemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent Photochromic dyes, electron-dense reagents, enzymes (e.g., widely used in ELISA), biotin, dinucleotides, These include hydroxybenzoates, ...

[0056] A "disease" is any condition that damages or interferes with the normal function of a cell, tissue, or organ. "A disorder" refers to any condition or disorder of any kind. Examples of disorders include Rett Syndrome.

[0057] "Effective amount" means the amount required to ameliorate symptoms of the disease in an untreated patient. An effective amount of an active compound used to practice this invention for the therapeutic treatment of a disease is It will vary depending on the mode of administration, the age, weight, and general health of the subject. Your physician or veterinarian will determine the appropriate amount and dosage regimen. is referred to as an "effective" amount. In one embodiment, an effective amount is measured in a cell (e.g., in vitro or in vivo). The present invention provides a method for the preparation of a gene of interest (e.g., Mecp2) sufficient to introduce a modification into the gene of interest (e.g., Mecp2) in a cell of interest. In one embodiment, an effective amount is an amount of a base editor that provides a therapeutic effect (e.g., an increase in the level of Rett syndrome). or reduction or control of the group or its symptoms or pathology) Such a therapeutic effect is achieved by increasing the amount of Mecp in all of the cells of a subject, tissue, or organ. 2, but not necessarily sufficient to modify the activity of cells present in a subject, tissue, or organ. Mecp2 may be altered by about 1%, 5%, 10%, 25%, 50%, 75%, or more. In certain embodiments, the effective amount is sufficient to ameliorate one or more symptoms of Rett Syndrome. do.

[0058] "Fragment" means a portion of a polypeptide or nucleic acid molecule. This portion preferably At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 100% of the total length of the reference nucleic acid molecule or polypeptide %, 80%, or 90%. Fragments may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids It may include.

[0059] "Hybridization" refers to the Watson-Crick, Hoogewal-type, or Hoogewal-type reaction between complementary nucleobases. This means that the hydrogen bond may be a Hoogsteen, or reversed Hoogsteen hydrogen bond. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds. .

[0060] The term "inhibitor of base repair" or "IBR" refers to a nucleic acid repair enzyme, such as a base excision repair enzyme. In some embodiments, IBR refers to a protein that can inhibit the activity of an enzyme. is an inhibitor of inosine base excision repair. Exemplary base repair inhibitors include APE1, Endo I II, Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4PDG, UDG, hSMUGl In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is a catalytically inactive Endo V or catalytically inactive h In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the base repair inhibitor is catalytically inactive Endo V or catalytically inactive Endo V. In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor. UGI is a uracil-DNA glycosylase (UGI) inhibitor that inhibits base excision repair enzymes. In some embodiments, the UGI domain refers to a protein that can In some embodiments, the UGI proteins provided herein include fragments of native UGI. In some embodiments, the present invention includes a fragment of UGI and a protein homologous to UGI or a UGI fragment. The base repair inhibitor is an inhibitor of inosine base excision repair. Repair inhibitors are either "catalytically inactive inosine-specific nucleases" or "dead inosine-specific Without wishing to be bound by any particular theory, catalytic inactivity Inosine glycosylases (e.g., alkyladenine glycosylase (AAG)) Able to bind inosine but create abasic sites or remove inosine This prevents the newly formed inosine moiety from being sterically blocked from the DNA damage / repair machinery. In some embodiments, the catalytically inactive inosine-specific nuclease locks the nucleic acid. It can bind to inosine in the nucleic acid but does not cleave the nucleic acid. Non-limiting examples of allogeneic nucleases include catalytically inactive alkyladenosidases from, for example, humans. Synglycosylase (AAG nuclease) and catalytically inactive enzymes from, for example, E. coli. In some embodiments, catalytically inactive nucleases include Endo V nuclease. Active AAG nuclease is mutated with E125Q or a corresponding mutation in another AAG nuclease. Includes:

[0061] The terms "isolated," "purified," or "biologically pure" refer to a substance that is in its natural state. Substances that have been removed to varying degrees from components that normally accompany them when found in the natural state. "Isolate" refers to the degree of separation from the original source or surroundings. "Purified" or "biologically pure" indicates a higher degree of separation than isolation. The protein is free of any impurities that do not substantially affect the biological properties of the protein, or Other substances are sufficiently removed so as not to cause other harmful effects. The nucleic acid or peptide of interest may be derived from cellular material, if produced by recombinant DNA techniques, The viral material, or culture medium, or if chemically synthesized, the chemical precursor or A substance is purified if it is substantially free of other chemicals. Purity and homogeneity are typically analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" means that a nucleic acid or protein is purified by electrophoresis. This can mean that the protein gives rise to essentially one band in a running gel. For proteins that can be subjected to glycosylation, various modifications can be made to produce various Isolated proteins can be produced, which can be individually purified.

[0062] An "isolated polynucleotide" is a naturally occurring polynucleotide of the organism from which the nucleic acid molecule of the present invention is derived. It refers to the gene-free nucleic acid (e.g., DNA) that flanks genes in the genome of a living organism. The term therefore includes autonomously replicating plants, e.g., those incorporated into vectors. DNA integrated into a mide or virus, or genomic DNA of a prokaryote or eukaryote recombinant DNA incorporated into a molecule, or a separate molecule (e.g., PCR or cDNA or fragments of genomic or cDNA generated by restriction endonuclease digestion) and In addition, the term includes recombinant DNA that exists as a RNA molecule transcribed from a DNA molecule. A molecule, as well as portions of a hybrid gene encoding additional polypeptide sequences. It contains recombinant DNA.

[0063] An "isolated polypeptide" is a polypeptide that has been separated from components that naturally accompany it. Typically, the polypeptide refers to a polypeptide of the invention that is in a state where it is naturally associated. At least 60% by weight free from proteins and naturally occurring organic molecules Preferably, the preparation is at least 75% by weight, more preferably at least Both are 90% by weight, and most preferably at least 99% by weight, of the polypeptide of the present invention. An isolated polypeptide of the present invention is one obtained by isolating such polypeptide, for example, by extraction from a natural source. by expression of a recombinant nucleic acid encoding the polypeptide or by chemically synthesizing the protein Purity can be determined by any suitable method, for example, column chromatography. Measured by chromatography, polyacrylamide gel electrophoresis, or HPLC analysis can be done.

[0064] The term "linker," as used herein, refers to a covalent linker (e.g., a covalent bond). , a non-covalent linker, chemical group, or complex between two molecules or moieties, e.g., proteins Two components of a nucleic acid or ribonucleic acid complex, or a polynucleotide programmable a DNA-binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine Two domains of a fusion protein, such as ATP deaminase or cytidine deaminase, are A linker can refer to a molecule that connects different components of a base editor system, or Different parts of the moiety can be joined together. For example, in some embodiments, a linker The polynucleotide-programmable nucleotide-binding domain guides polynucleotides. The oxidase-binding domain can be joined to the catalytic domain of the deaminase. In this embodiment, the linker can join the CRISPR polypeptide and the deaminase. In some embodiments, the linker can join the Cas9 and the deaminase. In some embodiments, the linker can join dCas9 and the deaminase. In some embodiments, the linker can join the nCas9 and the deaminase. In some embodiments, a linker joins the guide polynucleotide and the deaminase. In some embodiments, the linker can be a deaminating moiety and a base editor. - Conjugating the polynucleotide of the system with the programmable nucleotide binding component In some embodiments, the linker can bind the RNA binding moiety of the deaminating component to the base end Conjugating the polynucleotide of the editor system with a programmable nucleotide binding component In some embodiments, the linker can be linked to the RNA binding portion of the deaminating component. RN of the polynucleotide programmable nucleotide binding component of the base editor system A linker is a bond between two groups, molecules, or other moieties. Located between or adjacent to each other and linked together via covalent or non-covalent interactions , thereby linking the two. In some embodiments, the linker is an organic It can be a molecule, group, polymer, or chemical moiety. In some embodiments, the linker is In some embodiments, the linker is a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, the linker may comprise an aptamer capable of binding to the ligand. In embodiments, the ligand may be a carbohydrate, peptide, protein, or nucleic acid. In some embodiments, the linker can include an aptamer derived from a riboswitch. The riboswitches from which aptamers are derived are theophylline riboswitch, thiazolinone riboswitch, and thiazolinone riboswitch. TPP riboswitch, adenosine cobalamin (AdoCbl) riboswitch switch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch, flavin mononucleotide Nucleotide (FMN) riboswitch, tetrahydrofolate riboswitch, lysine riboswitch switch, glycine riboswitch, purine riboswitch, GlmS riboswitch, or prekeo In some embodiments, the linker may be selected from a poly(Asp-Asp-Asp-Asp) riboswitch. Aptamers that bind to polypeptide or protein domains, such as peptide ligands In some embodiments, the polypeptide ligand may comprise a K homology (KH) domain. Main, MS2 coat protein domain, PP7 coat protein domain, SfMu Com coat Protein domains, steryl alpha motif, telomerase Ku binding motif and Ku Protein, telomerase Sm7 binding motif and Sm7 protein, or RNA recognition motif In some embodiments, the polypeptide ligand may be a base editor system. For example, the nucleobase editing component may be part of a deaminase domain and and RNA recognition motifs.

[0065] In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide). In some embodiments, the linker can be about 5 to 100 amino acids long. Length of acid, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 Length of 0-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids In some embodiments, the linker can be about 100-150, 150-200, 200-250, It can be 250-300, 300-350, 350-400, 400-450, or 450-500 amino acids in length. Longer or shorter linkers are also contemplated.

[0066] In some embodiments, the linker comprises an RNA program containing a Cas9 nuclease domain. The gRNA-binding domain of a recognizable nuclease and a nucleic acid editing protein (e.g., cytidine or In some embodiments, the ligase is ligated to the catalytic domain of adenosine deaminase. The linker connects dCas9 to the nucleic acid editing protein. For example, the linker consists of two groups: a molecule or other moieties and are located between or adjacent to each other via a covalent bond. In some embodiments, the linker is an amino acid or In some embodiments, the amino acid sequence is a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. , the linker is 5 to 200 amino acids long, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90 , 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190 or 200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES, which is Also referred to as a linker, in some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker is (SGGS) n , (GGGS) n , (GGGGS) n , (G) n , (EA AAK) n , (GGS) n , SGSETPGTSESATPES, or (XP) n motif, or any of these where n is independently an integer from 1 to 30, and X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises multiple proline residues, such as 5 to 21, 5 to 14, 5-9, 5-7 amino acids in length, e.g., PAPAP, PAPAPA, PAPAPAP, PAPAPAPA, P(AP)4 , P(AP)7, P(AP) 10 Such proline-rich linkers are called "rigid" linkers. It is also called.

[0067] In some embodiments, the base editor domain is SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG Amino acid sequence of SAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS In some embodiments, the domain of the base editor is fused via a linker comprising The amino acid sequence SGSETPGTSESATPES is fused to the ribozyme via a linker. is also referred to as an XTEN linker. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids in length. In the example, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids in length. The anchor has the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS In some embodiments, the linker is 92 amino acids in length. In one embodiment, the linker has the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAG Includes SPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0068] The term "mutation," as used herein, refers to a mutation in a sequence, e.g., a nucleic acid or amino acid sequence. substitution of a residue in the sequence by another residue, or deletion or deletion of one or more residues in the sequence means an insertion. Mutations are typically described herein by identifying the original residue followed by the sequence The residues are described by identifying the positions of the residues in the Various methods for making the amino acid substitutions (mutations) provided herein are known in the art, e.g. For example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Sp Provided by Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012) In some embodiments, the base editors disclosed herein eliminate a significant number of unintended The nucleic acid (e.g., in the genome of a subject) is modified without introducing unwanted mutations (e.g., unintended point mutations). It is possible to efficiently generate "intended mutations" such as point mutations in nucleic acids. In embodiments, the intended mutation is mediated by a guidepoietin specifically designed to produce the intended mutation. A specific base editor (e.g., cytidine salt) bound to a oligonucleotide (e.g., gRNA) Mutations caused by nucleotide editors (nucleotide base editors or adenosine base editors) are generally Mutations made or identified in a sequence (e.g., an amino acid sequence described herein) can be The numbers are relative to the reference (or wild-type) sequence, i.e., the sequence that does not contain the mutation. If so, how do you locate the mutations in the amino acid and nucleic acid sequences relative to the reference sequence? It is easy to understand why decisions are made based on the above.

[0069] The terms "nucleic acid" and "nucleic acid molecule" as used herein refer to a nucleic acid molecule having a nucleic acid base and an acid moiety. It refers to a compound containing, for example, a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules containing three or more nucleotides, are A linear molecule in which adjacent nucleotides are linked via phosphodiester linkages In some embodiments, a "nucleic acid" refers to individual nucleic acid residues (e.g., In some embodiments, "nucleoside" refers to a nucleotide and / or a nucleoside. "Nucleotide" means an oligonucleotide chain containing three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably. Possibly used, a polymer of nucleotides (e.g., a chain of at least three nucleotides) In some embodiments, "nucleic acid" refers to RNA and single-stranded and / or double-stranded nucleic acids. Nucleic acids include, for example, genomes, transcripts, mRNA, tRNA, rRNA, siR NA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nuclei Nucleic acid molecules can occur naturally in association with non-naturally occurring molecules, e.g. For example, recombinant DNA or RNA, artificial chromosomes, engineered genomes or fragments thereof, or Synthetic DNA, RNA, DNA / RNA hybrids, or non-naturally occurring nucleotides or nucleosides Additionally, the terms "nucleic acid," "DNA," "RNA," and / or similar terms may be used. The term includes nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, and optionally They can be purified, chemically synthesized, etc. Where appropriate, chemically synthesized molecules, e.g. In this case, nucleic acids may be analogs with chemically modified bases or sugar and backbone modifications. Nucleic acid sequences may contain nucleoside analogs of the following in the 5' to 3' direction unless otherwise indicated: In some embodiments, the nucleic acid is a nucleic acid that is composed of natural nucleosides (e.g., adenosines). thimidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine deoxyguanosine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine uridine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine , C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine cytidine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically Modified bases; biologically modified bases (e.g., methylated bases); intercalated bases modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose); ose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages) , including these.

[0070] The term "nuclear localization sequence," "nuclear localization signal," or "NLS" refers to a sequence that targets a target gene into the cell nucleus. Nuclear localization sequences are known in the art. and e.g., the contents of which are incorporated herein by reference for disclosure of exemplary nuclear localization sequences. Incorporated: Pla, filed November 23, 2000, published May 31, 2001 as WO / 2001 / 038547 In other embodiments, the NLS is For example, Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172 In some embodiments, the NLS is an optimized NLS with the amino acid sequence KRTADGSEFESPKK KRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK , PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0071] The terms "nucleobase," "nitrogenous base," or "base" are used interchangeably herein. " refers to nitrogen-containing biological compounds that form nucleosides, and nucleosides are It is a component of nucleotides. The ability of nucleic acid bases to form base pairs and stack one by one. Directly binds long helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleic acid bases are adenine (A), cytosine (C), guanine (G), and thiamine (C). Adenine (T), and uracil (U) are called primary or canonical. Aminomethyl- and guanine are derived from purines, while cytosine, uracil, and thymine are pyrimidines. DNA and RNA may contain other (non-primary) bases that are modified. Non-limiting exemplary modified nucleobases include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydroxy Hypoxanthine and xanthine can be converted to methylcytosine in the presence of mutagens. Both can be created by deamination (replacement of an amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be derived from the deamination of cytosine. A nucleoside consists of a nucleic acid base and a five-carbon sugar (ribose or deoxyribose) Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine, uridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine Examples of nucleosides containing modified nucleobases include: , inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Φ). A "nucleotide" consists of a nucleobase, a five-carbon sugar (ribose or deoxyribose), and at least one It consists of phosphate groups.

[0072] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" refers to a The term "nucleotide-programmable nucleotide-binding protein" is used interchangeably with "nucleotide-programmable nucleotide-binding protein" and is used interchangeably with "nucleotide-programmable nucleotide-binding protein." a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid that guides the pDNAbp to a specific nucleic acid sequence; For example, the Cas9 protein is a protein that guides the Cas9 protein. They can associate with guide RNAs that guide them to specific DNA sequences complementary to the RNA. In this embodiment, the napDNAbp contains a Cas9 domain, e.g., a nuclease-active Cas9, a Cas9 nickle domain, or a Nuclease-inactive Cas9 (nCas9), or nuclease-inactive Cas9 (dCas9). Examples of DNA binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl , Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Ca Other nucleic acid programmable DNA binding proteins are also specifically disclosed herein. Although not specifically listed, they are within the scope of this disclosure. For example, the entire contents of each are incorporated by reference. "Classification and Nomenclature of CRISP" by Makarova et al., incorporated herein by reference. R-Cas Systems: Where from Here?” CRISPR J. 2018 Oct;1:325-336. doi: 10.1089 / cri spr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Sc ience. 2019 Jan 4;363(6422):88-91. doi: 10.1126 / science.aav7271

[0073] The term "nucleobase-editing domain" or "nucleobase-editing protein" is used herein. In some cases, modifications of nucleic acid bases in RNA or DNA, such as cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine), and deamination of thiamin (or adenosine) to hypoxanthine (or inosine), and proteins that can catalyze the addition and insertion of non-templated nucleotides. or an enzyme. In some embodiments, the nucleobase-editing domain is a deaminase Main (e.g., cytidine deaminase, cytosine deaminase, adenine deaminase) In some embodiments, the nucleobase-editing domain is The nucleobase-editing domain can be a naturally occurring nucleobase-editing domain. The editing domain may be engineered or evolved from a naturally occurring nucleobase editing domain. The nucleobase editing domain may be a bacterial, human, chimpanzee, gorilla, or The cells may be from any organism, such as a monkey, cow, dog, rat, or mouse. For example, nucleobase editing proteins are described in the following sections: The international PCT applications PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), the entire contents of which are incorporated herein by reference. Komor, AC, et al., “Programmable editing of a target base in genomic DNA wi Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, AC, et al., “Improved b ase excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T: A base editors with higher efficiency and product purity” Science Advances 3:ea Please refer to ao4774 (2017).

[0074] As used herein, "obtaining" as in "obtaining a drug" means synthesizing a drug. This includes obtaining, purchasing, or otherwise acquiring

[0075] As used herein, a "patient" or "subject" refers to a person having or developing a disease or disorder. means a mammalian subject diagnosed or suspected of having In some embodiments, the term "patient" refers to a patient who has a higher than average likelihood of progressing to a disease or disorder. Exemplary patients include humans, non-human primates, cats, dogs, pigs, cattle, cats, horses, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), as well as other mammals that may benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female.

[0076] A "patient in need thereof" or a "subject in need thereof" is used herein to refer to, for example, means a patient diagnosed with or suspected of having a disease or disorder, Not limited to Rett Syndrome (RTT).

[0077] The terms "pathogenic mutation," "pathogenic variant," "disease-causing mutation," and "disease-causing variant" A "deleterious mutation," or "predisposing mutation" is a mutation that increases an individual's susceptibility to a disease or disorder. In some embodiments, a gene expression level is a level that increases the risk of developing a genetic disorder. , a pathogenic mutation is a mutation that results in at least one wild-type mutation in the protein encoded by the gene. The variant comprises a substitution of at least one pathogenic amino acid for a type amino acid.

[0078] The term "non-conservative mutation" refers to a change between different groups, e.g., a change in tryptophan for a lysine. or phenylalanine with serine. The complementary amino acid substitutions do not interfere with or inhibit the biological activity of the functional variant. Preferably, non-conservative amino acid substitutions enhance the biological activity of functional variants. thereby improving the biological activity of the functional variant compared to the wild-type protein. Increase.

[0079] The terms "protein," "peptide," "polypeptide," and their grammatical equivalents are used interchangeably herein and are linked to each other by peptide (amide) bonds. The term refers to a polymer of amino acid residues of any size, structure, or function. Typically, a protein is a protein, peptide, or polypeptide having A peptide or polypeptide will be at least three amino acids in length. A protein, peptide, or polypeptide is an individual protein or collection of proteins. It can mean one or more of the amino acids in a protein, peptide, or polypeptide. The plural groups may be, for example, carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, iodo groups, etc. For sofarnesyl groups, fatty acid groups, conjugation, functionalization, or other modifications These can be modified by the addition of linkers or other chemical entities. A peptide, or polypeptide, may be a single molecule or a multi-molecular complex. A protein, peptide, or polypeptide is a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be a naturally occurring protein or a simple fragment of a peptide. It may be produced, recombinantly, or synthetically, or any combination thereof. The term "fusion protein" as used herein refers to a protein formed from at least two different proteins. A hybrid polypeptide containing a domain is referred to as a fusion protein. Located at the amino-terminal (N-terminal) or carboxy-terminal (C-terminal) portion of the protein, to form an amino-terminal or carboxy-terminal fusion protein, respectively. The protein can have different domains, e.g., nucleic acid binding domains (e.g., target domains). The gRNA-binding domain of Cas9, which directs protein binding to the target site, and the nucleic acid cleavage domain In some embodiments, the target protein may comprise a target domain, or the catalytic domain of a nucleic acid editing protein. A protein consists of a protein portion, e.g., an amino acid sequence constituting a nucleic acid-binding domain, and and organic compounds, such as compounds that can act as nucleic acid cleaving agents. In this case, proteins are either in complex with nucleic acids, such as RNA or DNA, or are Any of the proteins provided herein can be synthesized by any method known in the art. For example, the proteins provided herein can be produced by recombinant transcription. can be produced via expression and purification of a protein containing a peptide linker. Fusion proteins are particularly suitable. Methods for expressing and purifying recombinant proteins are well known. Green and Sambrook, Molecular Biology, vol. 1, pp. 111-115, 1997, the entire contents of which are incorporated herein by reference. lar Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012).

[0080] The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) (including ants) contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexane. Carboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylamino Methylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine Lanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α- Naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2- Carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, amino Monomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxybenzoate Droxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexyl Xanthanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane ) carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine Polypeptides and proteins contain polypeptides, This can be combined with post-transcriptional modification of one or more amino acids of the peptide construct. Non-limiting examples of post-modifications include acylation, including phosphorylation, acetylation, and formylation. , glycosylation (including N-linked and O-linked), amidation, hydroxylation, methylation and alkylation, including ethylation, ubiquitylation, addition of pyrrolidone carboxylic acid, disulfide Formation of amide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation These include ionization, geranylation, glypiation, lipoylation, and iodination.

[0081] The term "polynucleotide programmable nucleotide binding domain" refers to a polynucleotide A guide polynucleotide that guides a programmable DNA binding domain to a specific nucleic acid sequence Proteins that associate with nucleic acids (e.g., DNA or RNA), such as nucleic acids (e.g., guide RNAs) In some embodiments, the polynucleotide programmable nucleotide The DNA-binding domain is a polynucleotide-programmable DNA-binding domain. In some embodiments, the polynucleotide-programmable nucleotide binding domain is In some embodiments, the polynucleotide is a programmable RNA binding domain. The oligo-programmable nucleotide binding domain is the Cas9 protein. The Cas9 protein is a guide RNA that guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the polynucleotide program can associate with the target RNA. The nucleotide-binding domain can be a Cas9 domain, e.g., a nuclease-active Cas9, s9 nickase (nCas9), or nuclease-inactive Cas9 (dCas9). Non-limiting examples of tunable DNA binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas 12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, etc. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, and Cas4. , Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9( Also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, C as12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, C sm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Cs x14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, type II Cas effectors Protein, Type V Cas effector protein, Type VI Cas effector protein, CARF, D inG, their homologs, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins may also be used, although not specifically listed in this disclosure. However, it is within the scope of this disclosure.

[0082] The term "recombinant" as used herein with respect to a protein or nucleic acid means a protein or nucleic acid that is not naturally produced. It refers to proteins or nucleic acids that are not the product of human manipulation, but are the product of human manipulation. For example, In some embodiments, the recombinant protein or nucleic acid is compared to any naturally occurring sequence. and at least one, at least two, at least three, at least four, at least five , an amino acid or nucleotide sequence containing at least six, or at least seven mutations Includes:

[0083] By "reduction" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

[0084] "Reference" refers to a standard or control condition. In one embodiment, the reference is a wild-type or Healthy cells,

[0085] A "reference sequence" is a defined sequence used as a basis for sequence comparison. a subset or the entirety of a given sequence, e.g., a fragment of a full-length cDNA or gene sequence; or the complete cDNA or gene sequence. The length of the peptide sequence is generally at least about 16 amino acids, preferably at least about 20 amino acids. more preferably at least about 25 amino acids, even more preferably about 35 amino acids, For nucleic acids, the length of a reference nucleic acid sequence is generally Preferably, the sequence is at least about 50 nucleotides, more preferably at least about 60 nucleotides, and even more preferably or at least about 75 nucleotides, more preferably about 100 nucleotides, or about 300 nucleotides. It can be a nucleotide, or any integer thereabout or therebetween.

[0086] The terms "RNA-programmable nuclease" and "RNA-guided nuclease" with (e.g., bind to or associate with) one or more RNAs that are not targets for cleavage. In some embodiments, the RNA programmable nuclease is used as an RNA In complex with the nuclease, it can be referred to as a nuclease:RNA complex. Typically, the bound RNA is gRNAs can be expressed as a complex of two or more RNAs or as a single RNA molecule. A gRNA that exists as a single RNA molecule is called a single-guide RNA. (sgRNA), although "gRNA" is used interchangeably and refers to both a single molecule and refers to a guide RNA that exists as a complex of two or more molecules. Typically, a single RNA A gRNA species consists of two domains: (1) a domain that shares homology with the target nucleic acid (i.e., (2) a domain that directs the binding of the Cas9 complex to its target; In some embodiments, domain (2) comprises a domain that binds to the tracrRNA known as tracrRNA. For example, in some embodiments, Domain (2) is described in Jinek et al., S. Science 337:816-821 (2012), and is identical to or homologous to tracrRNA. Other examples of gRNAs (e.g., those containing domain 2) are incorporated herein by reference in their entirety. "Switchable Cas9 Nucleases and No. 61 / 874,682, filed September 6, 2013, entitled "Description of Uses Thereof" and U.S. Provisional Patent Application No. No. 61 / 874,746, entitled "Library System For Functional Nucleases." In some embodiments, the gRNA can contain two or more of domains (1) and (2). For example, an extended gRNA can be any of the gRNAs described herein. As shown in Figure 1, two or more Cas9 proteins bind to the target nucleic acid at two or more distinct regions. The gRNA contains a nucleotide sequence complementary to the target site, which binds to the nuclease. It mediates the binding of the nuclease / RNA complex to the target site and regulates the sequence-specific binding of the nuclease:RNA complex. In some embodiments, the RNA-programmable nuclease (CRISPR) Related systems) Cas9 endonucleases, such as Streptococcus pyogenes Cas9 (Csnl ) (see, for example, "Complete genome sequence of an M1 strain of Streptococcus pyo" Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Ji a HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Ro e BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663(2001); "SPR RNA maturation by trans-encoded small RNA and host factor RNase III.", Delt cheva E., Chylinski K., Sharma CM., Gonzales K., Chao Y., Pirzada ZA, Eckert M .R., Vogel J., Charpentier E., Nature 471:602-607(2011)).

[0087] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide that occurs at a specific location in the genome. Nucleotide variations, each of which occurs to some discernible degree in the population (e.g. For example, at certain base positions in the human genome, Although the leiothide can occur in most individuals, in a few individuals its position is occupied by A. This means that there is a SNP at this particular position, with two possible nucleotide variations: This means that C or A is the allele for this position. SNPs are used to treat diseases, Underlies differences in human susceptibility to disease, severity of illness, and our body's treatment The response to a gene is also a manifestation of genetic variation. It may be contained within the non-coding regions of a gene or within intergenic regions (regions between genes). In some embodiments, SNPs in the coding sequence are located at positions that, due to the degeneracy of the genetic code, It does not necessarily change the amino acid sequence of the resulting protein. There are two types of SNPs: synonymous SNPs and non-synonymous SNPs. Synonymous SNPs are Although they do not affect the sequence, nonsynonymous SNPs do change the amino acid sequence of a protein. There are two types of NPs: missense and nonsense. Even small SNPs can affect gene splicing, transcription factor binding, messenger RNA degradation, and The gene expression affected by this type of SNP can affect the sequence of non-coding RNA. These are called eSNPs (expressed SNPs) and can be located upstream or downstream of the gene. Single nucleotide variants (SNVs) are single nucleotide variations with no frequency restriction and are associated with body single nucleotide mutations in somatic cells (e.g., caused by cancer) ) can also be referred to as a single nucleotide modification.

[0088] "Specifically bind" means to bind to an antigen that recognizes and binds to a polypeptide and / or nucleic acid molecule of the present invention. and bind to other molecules in the sample, e.g., biological sample, without substantially recognizing or binding to them. Nucleic acid molecules, polypeptides, or complexes thereof (e.g., nucleic acid programmable "DNA binding domain and guide nucleic acid"), compound, or molecule.

[0089] Nucleic acid molecules useful in the methods of the invention include those encoding a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules may be 100% identical to the endogenous nucleic acid sequence. Although there need not be any difference, the endogenous sequence will typically exhibit substantial identity. Polynucleotides that have a "specific identity" typically have at least one double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include those capable of hybridizing to strands of the present invention. Any nucleic acid molecule encoding an inventive polypeptide or fragment thereof is included. The nucleic acid molecule need not be 100% identical to the endogenous nucleic acid sequence, but typically exhibits substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically In other words, a nucleic acid molecule is capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" refers to the hybridization of complementary polynucleotide sequences under various stringency conditions. (e.g., a gene described herein) or a portion thereof, forming a double-stranded molecule. means (e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; See Kimmel, AR (1987) Methods Enzymol. 152:507).

[0090] For example, stringent salt concentrations are typically less than about 750 mM NaCl and less than 75 mM Cl. Trisodium enoate, preferably less than about 500 mM NaCl and less than 50 mM trisodium citrate more preferably less than about 250 mM NaCl and less than 25 mM trisodium citrate. Low stringency hybridization is achieved using organic solvents such as formamide. hybridization at high stringency is obtained in the absence of Both are obtained in the presence of about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions generally include at least about 30°C, and more preferably at least about 50°C. This includes temperatures of at least about 37°C, and most preferably at least about 42°C. The duration of the reaction, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the Further various parameters, such as the inclusion or exclusion of rear DNA, are known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In a preferred embodiment, hybridization is carried out in 750 mM N This occurs in aCl solution, 75 mM trisodium citrate, and 1% SDS at 30°C. Hybridization was performed in 500 mM NaCl, 50 mM trisodium citrate, 1% S DS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37°C. In a most preferred embodiment, hybridization is performed in 250 mM NaCl, 25 mM citrate Occurs at 42°C in trisodium chloride, 1% SDS, 50% formamide, and 200 μg / ml ssDNA Useful variations on these conditions will be readily apparent to those skilled in the art.

[0091] For most applications, the washing steps following hybridization are also stringent. The stringency of the wash varies depending on the salt concentration and temperature. As mentioned above, the stringency of the wash can be defined by the salt concentration. This can be increased by lowering or increasing the temperature, e.g., for a washing step. Stringent salt concentrations are preferably less than about 30 mM NaCl and less than 3 mM tricitric acid. Sodium, most preferably less than about 15 mM NaCl and less than 1.5 mM trisodium citrate Stringent temperature conditions for the wash step typically include at least about 25°C. More preferably, the temperature includes at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the wash step is performed with 30 mM NaCl, 3 mM trisodium citrate, In a more preferred embodiment, washing is carried out in 0.1% SDS at 25°C. The cleaning step was performed in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 42°C. In a more preferred embodiment, the wash step is performed with 15 mM NaCl, 1.5 mM The procedure will be carried out in trisodium citrate and 0.1% SDS at 68°C. Further variations of the subject matter will be readily apparent to those skilled in the art. Hybridization techniques are well known in the art. These methods are well known to those skilled in the art, for example, Benton and Davis (Science 196:180, 1977); Grunstein and d Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current P rotocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and K immel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harb. or Laboratory Press, New York.

[0092] A "subject" includes, but is not limited to, a human or a bovine, equine, canine, ovine, or nematode. It refers to mammals, including non-human mammals such as humans.

[0093] "Substantially identical" means that the amino acid sequence is substantially the same as the reference amino acid sequence (e.g., the amino acid sequence described herein). any one of the nucleic acid sequences described herein) or a nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein) By "sequence number" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to the sequence of the sequence. Such sequences may be identical in amino acid or nucleic acid sequence to the sequences used for comparison. At least 60%, more preferably 80% or 85%, more preferably 90%, 95%, or even 9 It has 9% identity.

[0094] Sequence identity is typically determined using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology C enter, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, COBALT, EMBO SS Needle, GAP, or PILEUP / PRETTYBOX programs). The software assigns degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically involve: The following: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid Amino acid, asparagine, glutamine; serine, threonine; lysine, arginine; and Substitutions within the group of phenylalanine, tyrosine, and phenylalanine are included. An exemplary approach uses the BLAST program, -3 and e -100 Probability between Scores indicate closely related sequences. COBALT can be used with the following parameters: There are. a) Alignment parameters: gap penalty -11, -1 and end gap penalty Nalty-5,-1 b) CDD parameters: Use RPS BLAST on; BLAST E-value 0.003; Find conserved columns and recalculate, and c) Query clustering parameters: Use query cluster on; Word size Size 4, max cluster distance 0.8; alphabet regular. An EMBOSS needle is used, for example, with the following parameters: a) Matrix: BLOSUM62 b) GAP OPEN: 10; c)GAP EXTEND: 0.5; d)OUTPUT FORMAT: Pair; e)END GAP PENALTY: FALSE; f)END GAP OPEN: 10, and g)END GAP EXTEND: 0.5.

[0095] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a nucleobase editor. In one embodiment, the target site is a deaminase or a fusion protein comprising a deaminase. determined by enzymes (e.g., cytidine deaminase or adenine deaminase) .

[0096] RNA-programmable nucleases (e.g., Cas9) target DNA cleavage sites Since RNA:DNA hybridization is used for the analysis, these proteins are in principle Any sequence specified by the RNA can be targeted for site-specific cleavage. using RNA-programmable nucleases such as Cas9 (e.g., to modify the genome) The methods for doing so are known in the art (e.g., the entire contents of each are incorporated herein by reference). Cong, L. et al., Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et ah, RNA-guided human genome en gineering via Cas9. Science 339, 823-826 (2013); Hwang, WY et ah, Efficient ge nome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 22 7-229 (2013); Jinek, M. et ah, RNA-programmed genome editing in human cells. eLi fe 2, e00471 (2013); Dicarlo, JE et ah, Genome engineering in Saccharomyces ce revisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et a h RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biote (See Technology 31, 233-239 (2013)).

[0097] As used herein, the terms "treat," "treating," and "treatment" are used interchangeably. ", etc., reduce or ameliorate a disorder and / or its associated symptoms, or provide a desired It means to obtain the pharmacological and / or physiological effects of a disorder or condition. Treating a disorder, condition, or its associated symptoms may not completely eliminate, but may not preclude, the disorder, condition, or its associated symptoms. It will be appreciated that this does not require the immediate removal of the effect. without limitation, the effect is therapeutic, i.e., the effect is to alleviate the disease and / or adverse symptoms resulting from the disease. Partially or completely reduce, alleviate, neutralize, attenuate, mitigate, or diminish the intensity of In some embodiments, the effect is preventative, i.e., the effect is prophylactic, i.e., the effect is curative, or curative. To this end, the presently disclosed The method includes the step of administering a therapeutically effective amount of a composition described herein.

[0098] "Uracil glycosylase inhibitor" refers to a drug that inhibits the uracil excision repair system. In one embodiment, the agent binds to the host's uracil DNA glycosylase and induces cleavage from DNA. A protein or fragment thereof that prevents the removal of uracil residues.

[0099] Reference to a list of groups of chemical substances in any definition of a variable herein Also includes the definition of any single group or combination of listed groups and its variables. References herein to embodiments of variables or aspects are not intended to be construed as limiting the scope of any single embodiment. or in combination with any other embodiment or part thereof Includes:

[0100] Any composition or method provided herein may be used in combination with any other composition or method provided herein. Any one or more of the methods may be combined.

[0101] DNA editing alters disease states by correcting pathogenic mutations at the gene level Until recently, all DNA editing platforms have been identified Induce DNA double-strand breaks (DSBs) at selected genomic sites and rely on endogenous DNA repair pathways to repair them semi-probably. The complexities of gene products are important because they function by determining the outcome of the product in a stochastic manner. The homology-directed repair (HDR) pathway provides accurate and user-defined While it is possible to achieve the desired repair outcome, it is possible to use HDR in therapeutically relevant cell types. Highly efficient repair has been hindered by many challenges. In fact, this pathway is dominated by competing HDR is less efficient than the error-prone non-homologous end-joining pathway. It is strictly restricted to the G1 and S phases of the nucleus and is able to accurately repair DSBs in postmitotic cells. As a result, user-defined programmable It has proven difficult or impossible to modify genome sequences in a systematic and highly efficient manner. It has been done.

[0102] Nucleobase Editor Salts for editing, modifying, or altering a target nucleotide sequence of a polynucleotide Nucleic acid base editors or nucleobase editors are disclosed herein. Nucleobase editing domains containing a grammable nucleotide-binding domain and a nucleobase-editing domain. Polynucleotide programmable filters or base editors are described herein. The nucleotide-binding domain is coupled to a bound guide polynucleotide (e.g., gRNA). This allows the nucleic acid to specifically bind to the target polynucleotide sequence (i.e., the salt of the bound guide nucleic acid). through complementary base pairing between the bases and the bases of the target polynucleotide sequence, thereby This allows the base editor to be localized to the target nucleic acid sequence where editing is desired. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.

[0103] Polynucleotide-programmable nucleotide-binding domains The terms "polynucleotide programmable nucleotide binding domain" or "nucleic acid programmable nucleotide binding domain" are used interchangeably. "Programmable DNA binding protein (napDNAbp)" is a polynucleotide-programmable A guide polynucleotide (e.g., a guide polynucleotide) that guides a suitable nucleotide-binding domain to a specific nucleic acid sequence. refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide RNA In some embodiments, the polynucleotide-programmable nucleotide binding domain In some embodiments, the polynucleotide is a programmable DNA binding domain. In the present invention, a polynucleotide-programmable nucleotide binding domain is In some embodiments, the polynucleotide is a programmable RNA binding domain. The programmable nucleotide-binding domain is the Cas9 protein. In embodiments, the polynucleotide programmable nucleotide binding domain is a Cpf1 protein. It's plagiarism.

[0104] CRISPRs are mobile genetic elements (viruses, transposable elements, and conjugative plasmids) The CRISPR cluster is a spacer, a CRISPR clusters contain sequences complementary to the preceding mobile element and the target-invasive nucleic acid. is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, The correct processing of pre-crRNA requires the trans-encoded small RNA (tracrRNA), endogenous ribosomal RNA (ERRNA), and nuclease 3 (rnc), and Cas9 protein are required. tracrRNA is a linker of pre-crRNA. It acts as a guide for ribonuclease 3-assisted processing followed by Cas9 / crRNA / trac rRNA endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer The target strand that is not complementary to the crRNA is first endonucleolytically cleaved, then In nature, DNA binding and cleavage typically occurs via the '-5' exonucleolytic trimming. However, both the crRNA and tracrRNA aspects are required for the protein and both RNAs. Single-guide RNA ("sgRNA," or simply "gRNA") is designed to integrate into a single RNA species. See, for example, the entire contents of which are incorporated herein by reference. Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Scien ce 337:816-821 (2012). Cas9 targets short motifs in CRISPR repeat sequences. (PAM or protospacer adjacent motif) and distinguish self from non-self. To assist.

[0105] Nucleobase editor Cas9 domain The nuclease sequence and structure of Cas9 are known to those skilled in the art (e.g., the full sequence of each). "Complete genome sequence of an M1 sequence," the contents of which are incorporated herein by reference. rain of Streptococcus pyogenes.” Ferretti et al., Natl. Acad. Sci. USA 98:4 658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host fact or RNase III.” Deltcheva E., Chylinski K., Sharma CM., Gonzales K., Chao Y., Pi rzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607(2011); “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial imm unity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier (See E. Science 337:816-821 (2012)). Cas9 orthologs are not limited to Although not yet described, it has been described in various species, including S. pyogenes and S. thermophilus. Suitable Cas9 nucleases and sequences comprising the above will be apparent to those skilled in the art based on this disclosure. Such Cas9 nucleases and sequences are incorporated herein by reference in their entirety. Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 fa milies of type II CRISPR-Cas immunity systems”(2013) RNA Biology 10:5, 726-737 These include Cas9 sequences from the organisms and loci disclosed in

[0106] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 driver. Non-limiting exemplary Cas9 domains are provided herein. Cas9 domains include Nuclease-active Cas9 domain, nuclease-inactive Cas9 domain, or Cas9 nicker In some embodiments, the Cas9 domain is a nuclease active domain. For example, the Cas9 domain can bind both strands of a double-stranded nucleic acid (e.g., both strands of a double-stranded DNA molecule). In some embodiments, the Cas9 domain may be a Cas9 domain that cleaves the nucleotide sequence of the target gene (the nucleotide sequence of the target gene). In some embodiments, the Cas9 domain is In some embodiments, the Cas9 polypeptide comprises any one of the amino acid sequences described herein. A domain may have at least 60% to any one of the amino acid sequences described herein, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 9 In some embodiments, the Cas sequence comprises an amino acid sequence that is at least 99.9%, at least 99.5%, identical to the Cas sequence. The 9 domains are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 3 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 47, 48, 49, 50, or more amino acid sequences. In embodiments, the Cas9 domain has a sequence similar to any one of the amino acid sequences described herein. At least 10, at least 15, at least 20, at least 30, at least 40, at least At least 50, at least 60, at least 70, at least 80, at least 90, at least 100, At least 150, at least 200, at least 250, at least 300, at least 350, at least At least 400, at least 500, at least 600, at least 700, at least 800, at least 9 00, at least 1000, at least 1100, or at least 1200 identical consecutive amino acids It comprises an amino acid sequence having residues.

[0107] In some embodiments, the Cas9 nuclease is capable of cleaving inactive (e.g., inactivated) DNA. Cas9 has a cleavage domain, i.e., the "nCas9" protein ("nickase" Cas9) The nuclease-inactivated Cas9 proteins are interchangeable. It may be referred to as a "dCas9" protein (for nuclease-dead Cas9). Methods for producing Cas9 proteins (or fragments thereof) having the same domain are known (e.g., See, for example, Jinek et al., Science. 33, the entire contents of each of which are incorporated herein by reference. 7:816-821(2012); Qi et al, “Repurposing CRISPR as an RNA-Guided Platform for Se quence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5): 1173-83 For example, the DNA cleavage domain of Cas9 consists of two subdomains: the HNH nucleotide and the cleavage domain of Cas9. It is known to contain the leucine subdomain and the RuvC1 subdomain. The RuvC1 subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within the subdomain can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Cas9 in S. pyogenes. (Jinek et al, Science. 337:816-821(2012); Qi et al, Cell. 28;152(5): 1173-83 (2 013)). In some embodiments, proteins comprising fragments of Cas9 are provided, for example In some embodiments, the protein comprises two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. Proteins containing Cas9 or fragments thereof are referred to as "Cas9 variants." The variants share homology with Cas9 or its fragments. For example, Cas9 variants At least about 70% identical, at least about 80% identical, at least about 90% identical to live-type Cas9; At least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical 1. At least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical In some embodiments, the Cas9 variant has 1, 2, 3, 4, 5, 6 ,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,21,24,25,26,2 7, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 4 In some embodiments, the amino acid sequence may have 7, 48, 49, 50 or more amino acid changes. Cas9 variants are fragments of Cas9 (e.g., gRNA binding domain or DNA cleavage domain) wherein the fragment is at least about 70% identical, at least about 80% identical to the corresponding fragment of wild-type Cas9. , at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least In some embodiments, the fragment is at least about 99.9% identical to the corresponding wild-type Cas9 fragment. At least 30%, at least 35%, at least 40%, at least 45%, or less of the length of the amino acid At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 7 5%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, At least 97%, at least 98%, at least 99%, or at least 99.5%.

[0108] In some embodiments, the fragment is at least 100 amino acids in length. In embodiments, the fragments are at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 60 0, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or In some embodiments, the wild-type Cas9 is at least 1300 amino acids in length. Corresponding to Cas9 of O. pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide sequence The amino acid sequence is as follows: ATGGATAAGAAATACTCAATGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGATTATAAGGT TCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTG GCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGT TATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGT GGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA CTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCAT ATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCA GTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTG CACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTTGGGAAT CTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTA AGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCA ATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTA TAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATA AATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC AAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGA AGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTC CATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAA GTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGT ACTACCAAAACATAGTTTGCTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAA TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACC GTTAAGCAATTAAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATT TAATGCTTCATTAGGCGCCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAG ATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCT CACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTA TGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGGCCATAGTTTA CATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACT GGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGA AAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTT GAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATT AGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCAATAGACAATA AGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAAC TATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTT GAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTT TGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAA TTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTA TCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAG TTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAAT ATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGA AACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTG TCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCT CGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAA GGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTG AAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATAT AGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCT GCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAA AACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATT TTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAA TATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAAC GATATACGTCTACAAAAGAAGTTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGAT TTGAGTCAGCTAGGAGGTGACTGA JPEG2024138268000003.jpg164170 (single underline: HNH domain, double underline: RuvC domain).

[0109] In some embodiments, the wild-type Cas9 is Corresponds to or contains an array. ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGT ACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAAGAATCTTATCGGTGCCCTCCTATTCG ATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGT TACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGT CGAAGAGGACAAAACATGAACGGCACCCCATCTTTGGAAACATAGGATGAGGTGGCATATCATGAAAAGTACCCAA CGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCAT ATGATAAAGTTCCGTGGGCACTTTCCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCA GTTAGTACAAACCTATAATCAGTTGTTTGAAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCG CCCGCCTCTCTAAATCCCGACGGCTAGAAAAACCTGATCGCACAATTACCCGGAGAGAAAAAAATGGTTGTTCGGTAAC CTTATAGCGCTCTCACTAGGCCTGACACCAAATTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAG TAAGGACACGTACGATGACGATCTCGCAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCA AAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCCGCTTCA ATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATA TAAGGAAATATTCTTTGATCAGTCGAAAACGGGTACGCAGGTTATATTGACGGGCGGAGCGAGTCAAGAGGAATTCTACA AGTTTATCAAACCCATATTAGAGAATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGA AAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGA GGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTACTATGTGGGAC CCCTGGCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAA GTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCACTTTGACAAGAATTTACCGAACGAAAAAGT ATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCA TGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACA GTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATT TAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGGAATGAAG ATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCT CACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTAT CAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTA TGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTG CACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCT AGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGC AAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCT GTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGA ACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACA ATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAG AACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGG CTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGA TACTAGATTCCCGAATGAATACGAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCA AAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGC TTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACA AAGTTTATGACGTCCGTAAGATGATCGCGAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCT AACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGG GGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACA TAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATC GCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGC AAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTT TTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAG TATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGC [[ID=⑥]]ACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAAC[[ID=⑦]] [[ID=⑧]]AGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTC[[ID=⑨]] [[ID=⑩]]ATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGA[[ID=⑪]] [[ID=⑫]]AAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCA[[ID=⑬]] [[ID=⑭]]AACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATA[[ID=⑮]] [[ID=⑯]]GATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGA[[ID=⑰]] [[ID=⑱]]TTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAGGA[[ID=⑲]] [[ID=⑳]] [[ID=㉑]]JPEG2024138268000004.jpg161168(Underline once: HNH domain, Underline twice: RuvC domain)[[ID=㉒]] [[ID=㉓]]

[0110] It should be noted that in the above translation, the tags -

[0110] are preserved as they are because they are specific tags that may have special meanings in the original context and are required to be kept unchanged according to the instructions. Also, the text "一本下線:HNHドメイン、二本下線:RuvCドメイン" is translated as "Underline once: HNH domain, Underline twice: RuvC domain" following the best understanding of the meaning in the context of patent text translation. And the "⑥", "⑦", "⑧", "⑨", "⑩", "⑪", "⑫", "⑬", "⑭", "⑮", "⑯", "⑰", "⑱", "⑲", "⑳", "㉑", "㉒", "㉓" are just to show the correspondence with the original tags for better clarity in the translation process and are not part of the final translated text in terms of the actual English output. The final translated text should be presented as shown above with the tags and text in the appropriate format. In some embodiments, wild-type Cas9 is Cas9 from Streptococcus pyogenes (NCBI Reference ce Sequence: NC_002737.2 (nucleotide sequence is as follows) and Uniprot Reference Sequence: Q99ZW2 (amino acid sequence is as follows) ATGGATAAGAAATACTCAATGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGT TCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTG ACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGT TATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGT GGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA CTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCAT ATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCA GTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTG CACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAAT CTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTA AGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCA ATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTA TAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATA AATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC AAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGA AGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTC CATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAA GTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGT ACTACCAAAACATAGTTTGCTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAA TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACC GTTAAGCAATTAAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATT TAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAG ATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCT CACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTA TGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAGGCACAAGTGTCTGGACAAGGCGATAGTTTA CATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATT GGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCC AGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCT GTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGA ATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACA ATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAA AACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGG TTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAA TTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCT AAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGC GTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATA AAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCT AATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGG GGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATA TTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTTACCAAAAGAAATTCGGACAAGCTTATT GCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGC TAAGGTGGAAAGGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCT TTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAGACTTAATCATTAAACTACCTAAA TATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAGGGAAATGAGCTGGC TCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAAC AAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTT ATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAACCAATACGTGAACAAGCAGA AAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATTTTTGATACAACAATTGATCGTA AACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATT GATTTGAGTCAGCTAGGAGGTGACTGA JPEG2024138268000005.jpg164169 (Original Files:HNH Lion, Printed Files:RuvC Lion)

[0111] One of the strains of Cas9 is Corynebacterium ulcerans(NCBI Refs: NC_015683.1, 10). NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI R). ef: NC_017861.1); Taiwanese Spiroplasma(NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychr oflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NC BI Ref: YP_002344900.1) or Neisseria meningitidis (NCBI Ref: YP_002342100. 1), or Cas9 from other organisms.

[0112] In some embodiments, dCas9 partially or completely inhibits the activity of the Cas9 nuclease. The Cas9 amino acid sequence may correspond to or contain one or more mutations that inactivate the Cas9 amino acid sequence. For example, in some embodiments, the dCas9 domain contains the D10A and H840A mutations or In some embodiments, the dCas9 comprises a corresponding mutation in another Cas9. Amino acid sequence of H840A and H840A Contains JPEG2024138268000006.jpg163170 (single underline: HNH domain, double underline: RuvC domain).

[0113] In some embodiments, the Cas9 domain comprises a D10A mutation, while residue 840 is The corresponding amino acid sequences in either the provided amino acid sequences or the amino acid sequences provided herein The histidine at the position where the amino acid is to be added remains.

[0114] In other embodiments, dCas9 variants are provided that have mutations other than D10A and H840A, This results in, for example, nuclease-inactivated Cas9 (dCas9). Such mutations are e.g. Other amino acid substitutions at D10 and H840, or within the nuclease domain of Cas9, other substitutions (e.g., in the HNH nuclease subdomain and / or the RuvC1 subdomain) In some embodiments, the amino acid sequence includes at least about 70% identity, at least about 80% identity, Identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least Variants of dCas9 that are about 99% identical, at least about 99.5% identical, or at least about 99.9% identical or homologs are provided. In some embodiments, about 5 amino acids, about 10 amino acids, About 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids amino acids, about 75 amino acids, about 100 amino acids, or more, with shorter or longer amino acid sequences Variants of dCas9 are provided.

[0115] In some embodiments, the Cas9 fusion proteins provided herein comprise a Cas9 protein. It may comprise a full-length amino acid sequence, such as one of the Cas9 sequences provided herein. In embodiments, the fusion proteins provided herein do not contain the full-length Cas9 sequence, but rather contain one or more of the Cas9 sequences. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments Further suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art. This becomes:

[0116] The Cas9 protein guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the polynucleotide protease can be associated with a guide RNA. The activatable nucleotide binding domain can be a Cas9 domain, e.g., a nuclease-active Cas9, Cas9 nickase (nCas9), or nuclease-inactive Cas9 (dCas9). Examples of programmable DNA binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), Cas X, CasY, Cpfl, Cas12b / C2cl, and Cas12c / C2c3.

[0117] Nuclease-inactivated Cas9 proteins are interchangeably referred to as "dCas9" proteins (nuclease-inactivated Cas9 proteins). The enzyme may be referred to as "dead" Cas9 or catalytically inactive Cas9. Methods for producing a Cas9 protein (or a fragment thereof) having an A cleavage domain are known. (See, e.g., Jinek et al., Sc, the entire contents of each of which are incorporated herein by reference. ience. 337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platf orm for Sequence-Specific Control of Gene Expression” (2013) Cell. 28;152(5):11 For example, the DNA cleavage domain of Cas9 consists of two subdomains: H It is known to contain the NH nuclease subdomain and the RuvC1 subdomain. The RuvC1 subdomain cleaves the strand complementary to the gRNA, and the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Cas9 in S. pyogenes. (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28;152(5):1173 -83 (2013).

[0118] In some embodiments, the Cas9 domain is a Cas9 nickase. Cas9 proteins that can cleave only one strand of a single-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 nickase can bind to a double-stranded nucleic acid. The Cas9 nickase cleaves the target strand of the molecule, which allows the Cas9 to bind to the gRNA (e.g., sgRNA). This means cleaving the complementary strand that has formed base pairs with the base pair. In some forms, the Cas9 nickase contains a D10A mutation and has a histidine at position 840. In embodiments, the Cas9 nickase is not targeted to the duplexed nucleic acid molecule and is not base-edited. The Cas9 nickase cleaves the long strand, which is the base pairing site of the Cas9-bound gRNA (e.g., sgRNA). In some embodiments, the Cas9 nick is used to cleave the strand that does not form a The enzyme contains the H840A mutation and has an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments, the Cas9 nickase is any of the Cas9 nickases provided herein. At least 60%, at least 65%, at least 70%, at least 75%, at least At least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% , an amino acid sequence that is at least 98%, at least 99%, or at least 99.5% identical to Further suitable Cas9 nickases include, based on this disclosure and knowledge in the art: This will be apparent to those skilled in the art and is within the scope of this disclosure.

[0119] In some embodiments, the Cas9 domain is a nuclease-inactive Cas9 domain (dCas9). For example, the dCas9 domain cleaves both strands of a double-stranded nucleic acid molecule. It can bind to the double-stranded nucleic acid molecule (e.g., via a gRNA molecule) without In some embodiments, the nuclease-inactive dCas9 domain comprises an amino acid sequence described herein. the D10X and H840X mutations in the amino acid sequence, or any of the amino acid sequences provided herein. where X is any amino acid change. In one embodiment, the nuclease-inactive dCas9 domain has the amino acid sequence D10A described herein. mutation and the H840A mutation, or its counterpart in any of the amino acid sequences provided herein. For example, a nuclease-inactive Cas9 domain can be inserted into a cloning vector The amino acid sequence provided in pPlatTET-gRNA2 (Accession No. BAV54124) includes: MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD (See, e.g., Qi et al., “Repurposing,” the entire contents of which are incorporated herein by reference.) ng CRISPR as an RNA-guided platform for sequence-specific control of gene expres sion." Cell. 2013; 152(5):1173-83).

[0120] Additional Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nicker nCas9, or nuclease-active Cas9, and their variants and homologs It should be appreciated that within the scope of this disclosure, exemplary Cas9 proteins include, but are not limited to: In some embodiments, the Cas9 protein is a nuclease In some embodiments, the Cas9 protein is a Cas9 nickeratinocyte. In some embodiments, the Cas9 protein is a nuclease-active Cas9. It is 9.

[0121] Exemplary catalytically inactive Cas9 (dCas9): MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD

[0122] Exemplary catalyst of Cas9ニッカーゼ(nCas9): MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD

[0123] Exemplary catalytically active Cas9: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD.

[0124] In some embodiments, Cas9 refers to a Cas9 from an Archaea (e.g., a Nanoarchaea), Archaea constitute the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, Nucleic acid programmable DNA binding proteins are described, for example, in the entire contents of the present invention. Burstein et al., "New CRISPR-Cas systems from uncultivated microRNAs," incorporated herein by reference. robes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21 CasY stands for "archaeal" and refers to the archaeal domain of the biological kingdom, determined by metagenomics. Several CRISPR-Cas systems have been identified, including Cas9, which was first reported in Diverse Cas9 proteins are active CRISPR-Cas in less studied nanoarchaea In bacteria, two previously unknown systems, CRISP R-CasX and CRISPR-CasY were discovered, which are the most compact proteins discovered to date. In some embodiments, the base editing systems described herein are included in In some embodiments, Cas9 is replaced with CasX, or a variant of CasX. In the base editing system described herein, Cas9 interacts with CasY or the barrier of CasY. Other RNA-guided DNA-binding proteins are also nucleic acid programmable. and can be used as a DNA binding protein (napDNAbp) within the scope of the present disclosure. I want to be recognized.

[0125] In some embodiments, the nucleic acid promoter of any of the fusion proteins provided herein The grammable DNA binding protein (napDNAbp) may be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. The napDNAbp is a CasY protein. In some embodiments, the napDNAbp is a naturally occurring Ca At least 85%, at least 90%, at least 91%, or at least at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least or at least 97%, at least 98%, at least 99%, or at least 99.5% identical amino acid sequence. In some embodiments, the napDNAbp comprises a sequence encoding a naturally occurring CasX or CasY protein. In some embodiments, the napDNAbp is any CasX or CasY described herein. Protein and at least 85%, at least 90%, at least 91%, at least 92%, at least At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , or at least 99%, or at least 99.5% identical amino acid sequence to other bacteria. It should be appreciated that CasX and CasY from any species may also be used in accordance with the present disclosure.

[0126] CasX (uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53) tr|F0NN87|F0NN87_S ULIH CRISPR-associated Casx protein OS = Sulfolobus islandicus (strain HVE10 / 4) GN = SiH_0402 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIIL PLSNVKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAA AGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAV GQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSSSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNL NSDDGKVRDLKLISAYVNGELIRGEG

[0127] >tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = Sulfolobus islandic us(strain REY15A) GN=SiRe_0771 PE=4 SV=1 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIIL PLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAA AGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAV GQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLN SDDGKVRDLKLISAYVNGELIRGEG Deltaproteobacteria CasX MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMDTDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAIL QVYWQEFKDDHVGLMCKFAQPASKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFWYKLEQVSEKGKAITNYFGRCNVA EHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESHTPVKPLAQIAGNRYASGPVGKALSDACMGTIASFL SKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLNLWQKLKLSRDDA KPLRLLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENP KKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEM DEKEFYACEIQLQKWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFT DGTDIKKSGKWQGLLYGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKI GRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQ AAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAK LAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKEL SAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSY KSGKQPFVGAWQAFYKRRLKEVWKPNA

[0128] CasY (ncbi.nlm.nih.gov / protein / APG80656.1) >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacte rium] MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPREIVSAINDDYVGLYGLSNFDDLYNAEKRNEE KVYSVLDFWYDCVQYGAVFSYTAPGLLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIKFLNKKEISRANGSLDKLKKD IIDCFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQKKLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNR NRGEVLFNKLKEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELKKAMMDITDAWRGQEQEEELE KRLRILAALTIKLREPKFDNHWGGYRSDINGKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVVS SLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQEALIKERLEAEKKKKPKKRKKKSDAEDEKET IDFKELFPHLAKPLKLVPNFYGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKDFFIKRLQKIF SVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQSRSRKSAAIDKNRVRLPSTENIAKAGIALARELSVAGFDWK DLLKKEEHEEYIDLIELHKTALALLLAVTETQLDISALDFVENGTVKDFMKTRDGNLVLEGRFLEMFSQSIVFSELRGLA GLMSRKEFITRSAIQTMNGKQAELLYIPHEFQSAKITTPKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHY FGYELTRTGQGIDGGVAENALRLEKSPVKKREIKCKQYKTLGRGQNKIVLYVRSSYYQTQFLEWFLHRPKNVQTDVAVSG SFLIDEKKVKTRWNYDALTVALEPVSGSERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEITGDSAKILDQN FISDPQLKTLREEVKGLKLDQRRGTFAMPSTKIARIRESLVHSLRNRIHHLALKHKAKIVYELEVSRFEEGKQKIKKVYA TLKKADVYSEIDADKNLQTTVWGKLAVASEISASYTSQFCGACKKLWRAEMQVDETITTQELIGTVRVIKGGTLIDAIKD FMRPPIFDENDTPFPKYRDFCDKHHISKKMRGNSCLFICPFCRANADADIQASQTIALLRYVKEEKKVEDYFERFRKLKN IKVLGQMKKI

[0129] Polynucleotide-programmable nucleotide-binding domains include nuclear domains that bind to RNA. It should be appreciated that acid-programmable proteins are also included. For example, polynucleosides The polynucleotide-programmable nucleotide binding domain It can associate with a nucleic acid that guides the nucleotide-binding domain to the RNA. Programmable DNA binding proteins, although not specifically listed in this disclosure, are also within the scope of this disclosure. It is inside. Cas proteins that can be used herein include class 1 and class 2. Non-limiting examples of as proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5 t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), and Cas10 , Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1 , Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO , Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and and Cas12i, CARF, DinG, their homologs, or modified versions thereof. Unmodified CRISPR enzymes contain, for example, two functional endonuclease domains, R CRISPR enzymes can have DNA cleavage activity, such as Cas9, which has UVC and HNH. and / or one or both strands in the target sequence, such as in the complement of the target sequence. For example, CRISPR enzymes can direct cleavage of the first or last nucleotide in a target sequence. Nucleotide to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or The cleavage of one or both strands can be directed within 1 base pair of the target site or within 2 base pairs of the target site.

[0130] mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme: CRISPR that lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence A vector encoding the enzyme can be used. Cas9 is an exemplary wild-type Cas9 polypeptide. at least (approximately) 50%, 60%, 70%, 80%, 9% for peptides (e.g., Cas9 in S. pyogenes) 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or or a polypeptide having sequence homology thereto. Cas9 is an exemplary wild-type Cas9 polypeptide (e.g., Cas9 of S. pyogenes) and at most (approximately) 50%, 60%, 70%, 80%, or 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or can refer to a polypeptide with sequence homology. Cas9 can be wild-type or contain deletions, insertions, Amino acid modifications such as substitutions, variants, mutations, fusions, chimeras, or any combination thereof. The term "Cas9 protein" may refer to a modified form of the Cas9 protein, which may include cleavage.

[0131] In some embodiments, the methods described herein utilize engineered Cas proteins. Guide RNA (gRNA) contains a scaffold sequence required for Cas binding and a sequence that encodes the modified RNA. A user-defined spacer of approximately 20 nucleotides that defines the genomic target to be decorated Therefore, those skilled in the art can easily identify the genomic target of the Cas protein. Specificity can be varied, in part, by the specificity of the gRNA target sequence relative to other parts of the genome. The specificity of a gene is determined by how specific it is for its genomic target compared to other genes.

[0132] Cas9 nuclease contains two functional endonuclease domains, RuvC and HNH. Upon binding to its target, Cas9 induces nuclease activity to cleave the opposite strand of the target DNA. The final step in Cas9-mediated DNA cleavage is a second conformational change that positions the cleavage domain. The result is a double-strand break (DSB) in the target DNA (approximately 3–4 nucleotides upstream of the PAM sequence). The resulting DSBs then undergo two general repair pathways: (1) efficient but error-prone the non-homologous end joining (NHEJ) pathway, or (2) the low-efficiency but high-fidelity homology-directed repair pathway. It is repaired by one of the HDR paths.

[0133] The "efficiency" of non-homologous end joining (NHEJ) and / or homology-directed repair (HDR) is It can be calculated in a convenient way. For example, in some cases, the efficiency For example, the HDR obtained by the Surveyor nuclease assay can be expressed as a percentage of the HDR obtained by the The cleavage products can be generated using the enzyme, and the ratio of product to substrate is used to calculate the percentage. For example, the newly incorporated restriction sequence as a result of successful HDR can be Surveyor nuclease enzymes can be used to directly cleave DNA containing The more cleaved the substrate, the greater the percentage of HDR (greater HDR efficiency). The fraction (percentage) of HDR is calculated using the following formula: [(cleavage product) / (substrate + cleavage product)] (for example, For example, (b+c) / (a+b+c), where "a" is the band intensity of the DNA substrate, "b" and "c" are the cleavage products. The band intensity of the product can be calculated using the

[0134] In some cases, efficiency can be expressed as the percentage of successful NHEJ. The cleavage products can be generated using a T7 endonuclease I assay, and the products and The ratio of substrates can be used to calculate the percentage of NHEJ. Mismatched heteroduplex DNA resulting from hybridization of the normal and mutant DNA strands NHEJ creates small random insertions or deletions (indels) at the original break site. More cleavages indicate a greater percentage of NHEJ (greater efficiency of NHEJ). As an illustrative example, the fraction (percentage) of NHEJ is calculated using the following formula: (1-(1-(b+c) / (a+b+c)) 1 / 2 ) × 100, where "a" is the band intensity of the DNA substrate, "b" and "c" is the band intensity of the cleavage product (Ran et al., Cell. 2013 Sep. 12; 154(6):1380 -9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11): 2281-2308).

[0135] The NHEJ repair pathway is the most active repair mechanism, which often repairs small nucleic acids at DSB sites. Randomness of NHEJ-mediated DSB repair, which results in nucleotide insertions or deletions (indels) This has important practical implications. This is because the population of cells expressing NHEJ can result in a variety of mutations. are small inserts that result in amino acid deletions, insertions, or frameshift mutations in the target DNA. This results in a premature deletion within the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation in the target gene.

[0136] While NHEJ-mediated DSB repair often disrupts gene open reading frames, Homology-directed repair (HDR) allows for the generation of DNA fragments ranging from single nucleotide changes to fluorophores. or resulting in specific nucleotide changes ranging from large insertions such as tag additions. can be done.

[0137] To utilize HDR for gene editing, a DNA repair template containing the desired sequence is introduced into the gRNA and It can be delivered to the cell type of interest along with Cas9 or Cas9 nickase. The rate is determined by the desired edit and the presence of additional homologous sequences directly upstream or downstream of the target (left and right The length of each homology arm is The size of the inserted alteration may depend on the size of the alteration; a larger insertion requires a larger homology arm. The template may be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded The efficiency of HDR can be determined by the combination of Cas9, gRNA, and an exogenous repair template. Even in cells that express the gene, the expression is generally low (less than 10% of the altered alleles). HDR occurs during the S phase of the cell cycle. Because HDR occurs during the G1 and G2 phases, the efficiency of HDR can be improved by synchronizing cells. Chemical or genetic inhibition of genes involved in NHEJ also increases the frequency of HDR. It is possible.

[0138] In some embodiments, the Cas9 is a modified Cas9. A given gRNA target sequence may be a partial There may be additional sites throughout the genome where there is substantial homology. These are called target genes and must be taken into consideration when designing gRNAs. In addition, the specificity of CRISPR can be increased by modifying Cas9. By combining the activities of the cleavage domains, RuvC and HNH, double-stranded cleavage is achieved. Cas9 nickase is a D10A mutant of SpCas9 that binds one nuclease dopant to generate a double strand break (DSB). For specific gene editing, nicks are used. The case system can also be combined with HDR-mediated gene editing.

[0139] In some cases, the Cas9 is a variant Cas9 protein. The peptides differ in amino acid sequence from the wild-type Cas9 protein by one amino acid. amino acid sequence (e.g., deletions, insertions, substitutions, fusions). In some instances, the variant Cas9 polypeptide has reduced nuclease activity of the Cas9 polypeptide. have amino acid changes (e.g., deletions, insertions, or substitutions) that result in In some cases, variant Cas9 polypeptides exhibited less than 50% of the corresponding wild-type Cas9 protein. less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% nuclease activity In some cases, the variant Cas9 protein has substantial nuclease activity. A variant in which the subject Cas9 protein does not have substantial nuclease activity. In the case of the Cas9 protein, it may be referred to as "dCas9."

[0140] In some cases, the variant Cas9 protein has reduced nuclease activity. For example, a variant Cas9 protein may be a wild-type Cas9 protein, e.g., a wild-type Cas9 protein. Less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the protein It exhibits nuclease activity of 1000 kJ / ml.

[0141] In some cases, the variant Cas9 protein cleaves the complementary strand of the guide target sequence. However, they have a lower ability to cleave the non-complementary strand of the double-stranded guide target sequence. The variant Cas9 protein has a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant Cas9 protein may be D1 OA (aspartic acid to alanine at amino acid position 10), and therefore double-stranded It is capable of cleaving the complementary strand of the guide target sequence but not the non-complementary strand of the double-stranded guide target sequence. (hence, the ability of the variant Cas9 protein to cleave double-stranded target nucleic acid. When cleaved, single-strand breaks (SSBs) are generated instead of double-strand breaks (DSBs) (for example, See, for example, Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21).

[0142] In some cases, the variant Cas9 protein binds to the non-complementary strand of the double-stranded guide target sequence. However, they have a low ability to cleave the complementary strand of the guide target sequence. The variant Cas9 protein contains mutations (amino acid substitutions) that reduce the function of the HNH domain. (RuvC / HNH / RuvC domain motif). As a non-limiting example, some embodiments In this state, the variant Cas9 protein is H840A (histidine to acetylcholine at amino acid position 840). lanin) mutation and therefore capable of cleaving the non-complementary strand of the guide target sequence However, the ability to cleave the complementary strand of the guide target sequence is low (hence the variant Cas9 protein When the protein cleaves the double-stranded guide target sequence, an SSB is generated instead of a DSB. The Cas9 protein cleaves the guide target sequence (e.g., a single-stranded guide target sequence). Lower capacity, but retains the ability to bind to a guide target sequence (e.g., a single-stranded guide target sequence) is doing.

[0143] In some cases, the variant Cas9 protein is capable of cleaving complementary and non-complementary strands of double-stranded target DNA. As a non-limiting example, in some cases, The ant Cas9 protein has both the D10A and H840A mutations, and therefore the polypeptide These have a low ability to cleave both the complementary and non-complementary strands of double-stranded target DNA. Although the Cas9 protein has a low ability to cleave target DNA (e.g., single-stranded target DNA), it has a low ability to cleave target DNA. A retains the ability to bind to A (e.g., single-stranded target DNA).

[0144] As another non-limiting example, in some cases, the variant Cas9 protein comprises W476A and W The polypeptide has a mutation at 1126A, and therefore has a reduced ability to cleave target DNA. Although Cas9 proteins such as It retains the ability to bind to target DNA (eg, single-stranded target DNA).

[0145] As another non-limiting example, in some cases, the variant Cas9 protein may be P475A, W, It has mutations 476A, N477A, ​​D1125A, W1126A, and D1127A, and therefore Such Cas9 proteins have a low ability to cleave target DNA. binds to target DNA (e.g., single-stranded target DNA) but has low ability to cleave double-stranded target DNA retains the ability.

[0146] As another non-limiting example, in some cases, the variant Cas9 protein is H840A, W 476A and W1126A mutations, so that the polypeptide cleaves the target DNA. Such Cas9 proteins cleave target DNA (e.g., single-stranded target DNA) Although the ability to bind to target DNA (e.g., single-stranded target DNA) is low, the ability to bind to target DNA is maintained. As a non-limiting example, in some cases, the variant Cas9 protein is H840A, D10A, W 476A and W1126A mutations, so that the polypeptide cleaves the target DNA. Such Cas9 proteins cleave target DNA (e.g., single-stranded target DNA) Although they have low binding capacity, they retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, the variant Cas9 contains a catalytic His residue at position 840 of the HNH domain of Cas9. Restoring (A840H).

[0147] As another non-limiting example, in some cases, the variant Cas9 protein is H840A, P 475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, and therefore Such Cas9 proteins have low ability to cleave target DNA (e.g., It has low ability to cleave target DNA (e.g., single-stranded target DNA), but As another non-limiting example, in some cases, variants The Cas9 proteins were D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A. The polypeptide has a mutation in the target DNA, and therefore has a reduced ability to cleave the target DNA. Although such Cas9 proteins have a low ability to cleave target DNA (e.g., single-stranded target DNA), retains the ability to bind to target DNA (e.g., single-stranded target DNA). , if the variant Cas9 protein has the W476A and W1126A mutations, or AntCas9 protein harbors the P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations. If the variant Cas9 protein has a PAM sequence, it will not bind efficiently to the PAM sequence. Therefore, in some cases, such variants may differ in the way they bind. When using the s9 protein, this method does not require a PAM sequence. In such cases, when using such variant Cas9 proteins in methods of conjugation, Although this method can include a guide RNA, the method is performed in the absence of a PAM sequence. (Thus, the specificity of binding is provided by the target segment of the guide RNA. Other residues (i.e., inactive residues or other nucleic acids) may be added to achieve the above effects. Non-limiting examples include residues D10, G12, G17, E762, H Modify (i.e., replace) 840, N854, N863, H982, H983, A984, D986, and / or A987 Mutations other than alanine substitutions are also suitable.

[0148] In some embodiments, variant Cas9 proteins with reduced catalytic activity (e.g., For example, the Cas9 protein is D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D98 6, and / or A987 mutations, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A , H982A, H983A, A984A, and / or D986A), this interacts with the guide RNA As long as it retains its ability to act (which is guided to the target DNA sequence by the guide RNA), However, it can still bind to the target DNA in a site-specific manner.

[0149] Alternatives to Cas9 in S. pyogenes include the Cpf1 family of proteins that exhibit cleavage activity in mammalian cells. RNA-guided endonucleases from Prevotella and Francisella 1 are included. PR (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is a class I I. The RNA-guided endonuclease of the CRISPR / Cas system. This adaptive immune mechanism is Prevo The Cpf1 gene is found in the bacteria Tella and Francisella. and encodes an endonuclease that uses a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9 and is involved in the CRISPR / Cas9 system. Unlike Cas9 nuclease, Cpf1-mediated DNA cleavage is The result is a double-stranded break with a short 3' overhang. The cleavage pattern of Cpf1 is zigzag. The fragment structure allows for the directional cloning of genes in the same way as conventional restriction enzyme cloning. It can expand the possibilities for gene transfer, thereby increasing the efficiency of gene editing. Similar to the Cas9 variants and orthologs mentioned above, Cpf1 acts in tandem with SpCas9. CRISPR-mediated targeting of AT-rich regions or genomes lacking preferred NGG PAM sites This expands the number of sites that can be targeted. The Cpf1 locus contains a mixed α / β domain. In, it contains RuvC-I, RuvC-II, and zinc finger-like domains followed by a helical region The Cpf1 protein contains a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, Cpf1 does not have an HNH endonuclease domain, and the N-terminus of Cpf1 is the α-terminal domain of Cas9. The construction of the CRISPR-Cas domain of Cpf1 demonstrates that Cpf1 is functionally unrelated to the CRISPR-Cas domain. This indicates that the Cpf1 gene is unique and classified as a Class 2, Type V CRISPR system. The Cas1, Cas2, and Cas4 proteins are more similar to type I and III than to type II systems. Functional Cpf1 does not require trans-activating CRISPR RNA (tracrRNA) and Therefore, only CRISPR (crRNA) is required. Cpf1 is not only smaller than Cas9, but also It contains a small sgRNA molecule (almost half the nucleotides of Cas9), which allows for genome editing. The Cpf1-crRNA complex favors protozoal transcription, in contrast to the G-rich PAM targeted by Cas9. Cleavage of target DNA or RNA by identifying the pacer adjacent motif 5'-YTN-3' After the identification of the PAM, Cpf1 was found to have a sticky-end-like structure with a 4–5 nucleotide overhang. introduces a DNA double-strand break.

[0150] Some embodiments of the present disclosure act as nucleic acid programmable DNA binding proteins. fusion proteins containing domains that bind proteins such as base editors to specific It can be used to guide to nucleic acid (e.g., DNA or RNA) sequences. In embodiments, the fusion protein comprises a nucleic acid programmable DNA binding protein domain and and a deaminase domain. DNA binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Ca These include s12g, Cas12h, and Cas12i, which are programmable with PAM specificities distinct from those of Cas9. One example of a suitable polynucleotide-binding protein is the α- and β-glucan-binding protein from Provotella and Francisella 1. Clustered Regularly Interspaced Short Palindromic Repeats (Cpf1). Cas9 and Similarly, Cpf1 is also a class 2 CRISPR effector. Cpf1 has characteristics that distinguish it from Cas9. Cpf1 mediates robust DNA interference by inhibiting the transcription of a single RNA genome lacking tracrRNA. It is a nucleotide endonuclease that contains a T-rich protospacer adjacent motif (TTN, Furthermore, Cpf1 breaks DNA via zigzag DNA double-strand breaks. Among the 16 Cpf1 family proteins, Acidaminococcus and Lachnospira Two enzymes from the ceae family have been shown to have efficient genome editing activity in human cells. Cpf1 proteins are known in the art, for example, see, e.g., Yamano et al., "Crystal structure of Cpf1 in complex with g guide RNA and target DNA.” Cell (165) 2016, pp. 949-962.

[0151] Nuclease-inactive Cpf1 (dCpf1) variants are also used in the compositions and methods of the present invention. This is useful for guide nucleotide sequence-programmable polynucleotide-binding proteins. The Cpf1 protein is identical to the RuvC domain of Cas9. It has a RuvC-like endonuclease domain, but does not contain an HNH endonuclease domain. The N-terminus of Cpf1 does not contain the α-helical recognition lobe of Cas9. Zetsche et al., Cell, 163, 759-771, 2015 (which is incorporated herein by reference) The C-like domain is involved in cleavage of both DNA strands, and inactivation of the RuvC-like domain results in the cleavage of the Cpf1 nucleotide. For example, in the case of Cpf1 of Francisella novicida, Mutations corresponding to D917A, E1006A, or D1255A in the Cpf1 gene inactivate the nuclease activity of Cpf1. In some embodiments, the dCpf1 of the present disclosure comprises D917A, E1006A, D1255A, D917A / E1006A, Contains mutations corresponding to D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. Any Mutations in the RuvC domain of Cpf1, such as substitution mutations, deletions, or insertions that inactivate the RuvC domain of Cpf1, are disclosed herein. It should be understood that the present invention may be used in accordance with the present invention.

[0152] In some embodiments, the nucleic acid promoter of any of the fusion proteins provided herein The grammable nucleotide-binding protein may be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is nuclease-inactive Cpf1 (dCpf1). , Cpf1, nCpf1, or dCpf1 is at least 85% identical to the Cpf1 sequences disclosed herein; At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least In some embodiments, dCpf1 comprises an amino acid sequence that is at least 99.5% identical to at least 85%, at least 90%, at least 91% relative to the Cpf1 sequences disclosed herein; At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, at least 99%, or at least 99.5% identity. The amino acid sequence includes D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A or containing mutations corresponding to D917A / E1006A / D1255A. Cpf1 from other bacterial species may also be used in accordance with the present disclosure. It should be recognized that the

[0153] Cpf1 of wild-type Francisella novicida (D917, E1006, and D1255 are in bold and underlined) There are MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIAANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI D RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFEFSFDYKNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN

[0154] Cpf1 D917A of Francisella novicida (A917, E1006, and D1255 are in bold and underlined) There are MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIAANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI A RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFEFSFDYKNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN

[0155] Cpf1 E1006A of Francisella novicida (D917, A1006, and D1255 are bold and underlined) (I'm MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEKELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPCKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI D RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF ADLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFEFSFDYKNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN

[0156] Francisella novicida Cpf1 D1255A (D917, E1006, and A1255 are bold and underlined) (I'm MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEKELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPCKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI D RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFESFDYKNFGDKAAKKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN

[0157] Francisella novicida Cpf1 D917A / E1006A (A917, A1006, and D1255 are bold and underlined) (marked with MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIAANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI A RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF A DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFEFSFDYKNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN

[0158] Francisella novicida Cpf1 D917A / D1255A (A917, E1006, and A1255 are bold and underlined) (marked with MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEKELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIAANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI A RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFEFSFDYKNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN

[0159] Cpf1 E1006A / D1255A of Francisella novicida (D917, A1006, and A1255 are bold and underlined) (marked with MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEKELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPCKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI D RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF ADLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFEFSFDYKNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN

[0160] Francisella novicida Cpf1 D917A / E1006A / D1255A (A917, A1006, and A1255 are in bold) (underlined with ). MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEKELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPCKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI A RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF A DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFESFDYKNFGDKAAKKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN

[0161] In some embodiments, one of the Cas9 domains present in the fusion protein contains a PAM sequence. Guide nucleotide sequence-programmable DNA-binding protein domains without requirements for may be replaced with

[0162] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) comprises: It is a single effector of the microbial CRISPR / Cas system. Effectors include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Typically, microbial CRISPR / Cas systems are divided into class 1 and class 2 systems. Class 1 systems involve multisubunit effector complexes, whereas class 2 systems involve single For example, Cas9 and Cpf1 are class 2 effectors. In addition to Cas9 and Cpf1, there are three distinct class 2 CRISPR / Cas systems (Cas12b, / C2c1, and Cas12c / C2c3) are described in Sh, the entire contents of which are incorporated herein by reference. makov et al., “Discovery and Functional Characterization of Diverse Class 2 CRI SPR Cas Systems”, Mol. Cell, 2015 Nov. 5; 60(3): 385-397 Two effectors in the system, Cas12b / C2c1 and Cas12c / C2c3, act on Ruv related Cpf1. The third system contains two described HEPN ribonucleotides and a C-like endonuclease domain. It contains an effector with a nuclease domain. The generation of mature CRISPR RNA is controlled by Cas1. Unlike CRISPR RNA production by Cas12b / C2c1, it does not depend on tracrRNA. It relies on both CRISPR RNA and tracrRNA for cleavage.

[0163] The crystal structure of the chimeric Cas12b / C2c1 (AacC2c1) from Alicyclobaccillus acidoterrastris It has been reported in complex with a single guide RNA (sgRNA). See Liu et al., “C2c1-sgRNA Complex Structure R,” the contents of which are incorporated herein by reference. eveals RNA-Guided DNA Cleavage Mechanism”, Mol. Cell, 2017 Jan. 19; 65(2):310-3 22. Alicyclobacillus acidoterrest bound to target DNA as a ternary complex. The crystal structure of C2c1 from ris has also been reported, e.g., the entire contents of which are incorporated herein by reference. Yang et al., "PAM-dependent Target DNA Recognition and Cleavage" See "C2C1 CRISPR-Cas endonuclease", Cell, 2016 Dec. 15; 167(7):1814-1828. The catalytically competent conformation of AacC2c1 is different between the target DNA strand and the non-target DNA strand. Both strands are independently captured within a single RuvC catalytic pocket, where they are then transported to Cas12b / C2c. Cas12b / C2c1 ternary complex: Cas12b / C2c1-mediated cleavage leads to a zigzag seven-nucleotide cut in the target DNA. Structural comparison of the CRISPR-Cas9 system with the previously identified Cas9 and Cpf1 counterparts confirmed that the CRISPR-Cas9 system The diversity of mechanisms employed is demonstrated.

[0164] In some embodiments, the nucleic acid promoter of any of the fusion proteins provided herein The grammable DNA-binding protein (napDNAbp) binds to the Cas12b / C2c1 or Cas12c / C2c3 proteins. In some embodiments, the napDNAbp is a Cas12b / C2c1 protein. In some embodiments, the napDNAbp is a Cas12c / C2c3 protein. In morphology, napDNAbp is less abundant than naturally occurring Cas12b / C2c1 or Cas12c / C2c3 proteins. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% In some embodiments, the amino acid sequence is at least 99.5% identical to napDNAbp is a naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In embodiments, the napDNAbp has at least one sequence similar to any one of the napDNAbp sequences provided herein. At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 9 4%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or contains an amino acid sequence that is at least 99.5% identical to Cas12b / C2 from other bacterial species. It should be appreciated that c1 or Cas12c / C2c3 may also be used in accordance with the present disclosure. Cas12b / C2c1 (uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2| / C2C1_ALIAG CRISPR-associated endo-nuclease C2c1 OS = =Alicyclobacillu s acido-terrestris ((strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B ) GN=c2c1 PE=1 SV=1 MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECDKTAEECKAELLERLRARQ VENGHRGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAAGNKPRWVRMREAGEPGWE EEKEKAETRKSADRTADVLRALADFGLKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQ EYAKLVEQKNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDA EIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLNHAKMFATTFTLPDATAHPIWTRFDKLGGN LHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAK IQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGL LSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQR TLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVR RVWRHMGKQVRDWRKDVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREH IDHAKEDRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELI NQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADDLIPTGEGEIF VSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLRCDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYE RERGKKRRKVFAQEKLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMV NQRIEGYLVKQIRSRVPLQ DSACENTGDI BhCas12b (Bacillus hisashii) NCBI Reference Sequence: WP_095142515 MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAE IQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRW YNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQAL ERFLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWL KMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPIN HPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEKGKVDIVLLPSRQFYNQIFLDIEEEKGKH AFTYKDESIKFPLKTGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKE LTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGET LVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWV AFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHL NALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGE IYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDR KCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIK KGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSS KQSMKRPAATKKAGQAKKKK

[0165] In some embodiments, the Cas12b is BvCas12B, which is a variant of BhCas12b, Contains the following changes relative to BhCas12B: S893R, K846R, and E837G. BvCas12b (Bacillus s p. V3-13) NCBI Reference Sequence: WP_101661451.1 MAIRSIKLKMKTNSGTDSIYLRKALWRTHQLINEGIAYYMNLLTLYRQEAIGDKTKEAYQAELINIIRNQQRNNGSSEEH GSDQEILALLRQLYELIIPSSIGESGDANQLGNKFLYPLVDPNSQSGKGTSNAGRKPRWKRLKEEGNPDWELEKKKDEER KAKDPTVKIFDNLNKYGLLPLFPLFTNIQKDIEWLPLGKRQSVRKWDKDMFIQAIERLLSWESWNRRVADEYKQLKEKTE SYYKEHLTGGEEWIEKIRKFEKERNMELEKNAFAPNDGYFITSRQIRGWDRVYEKWSKLPESAPEELWKVVAEQQNKMS EGFGDPKVFSFLANRENRDIWRGHSERIYHIAAYNGLQKKLSRTKEQATFTLPDAIEHPLWIRYESPGGTNLNLFKLEEK QKKNYYVTLSKIIWPSEEKWIEKENIEIPLAPSIQFNRQIKLKQHVKGKQEISFSDYSSRISLDGVLGGSRIQFNRKYIK NHKELLGEGDIGPVFFNLVVDVAPLQETRNGRLQSPIGKALKVISSDFSKVIDYKPKELMDWMNTGSASNSFGVASLLEG MRVMSIDMGQRTSASVSIFEVVKELPKDQEQKLFYSINDTELFAIHKRSFLLNLPGEVVTKNNKQQRQERRKKRQFVRSQ IRMLANVLRLETKKTPDERKKAIHKLMEIVQSYDSWTASQKEVWEKELNLLTNMAAFNDEIWKESLVELHHRIEPYVGQI VSKWRKGLSEGRKNLAGISMWNIDELEDTRRLLISWSKRSRTPGEANRIETDEPFGSSLLQHIQNVKDDRLKQMANLIIM TALGFKYDKEEKDRYKRWKETYPACQIILFENLNRYLFNLDRSRRRENSRLMKWAHRSIPRTVSMQGEMFGLQVGDVRSEY SSRFHAKTGAPGIRCHALTEEDLKAGSNTLKRLIEDGFINESELAYLKKGDIIPSQGGELFVTLSKRYKKDSDNNELTVI HADINAAQNLQKRFWQQNSEVYRVPCQLARMGEDKLYIPKSQTETIKKYFGKGSFVKNNTEQEVYKWEKSEKMKIKTDTT FDLQDLDGFEDISKTIELAQEQQKKYLTMFRDPSGYFFNNETWRPQKEYWSIVNNIIKSCLKKKILSNKVEL.

[0166] In some embodiments, the Cas9 domain is a Cas9 domain from Staphylococcus aureus (SaC In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, nuclease-inactive SaCas9 (SaCas9d) or SaCas9 nickase (SaCas9n). In some embodiments, SaCas9 has an N579A mutation or It includes corresponding mutations in either

[0167] In some embodiments, a SaCas9 domain, a SaCas9d domain, or a SaCas9n domain can bind to nucleic acid sequences with non-canonical PAMs. SaCas9 domain, SaCas9d domain, or SaCas9n domain, NNGRRT or NNNRRT In some embodiments, the SaCas9 domain can bind to a nucleic acid sequence having a PAM sequence. The main mutations include one or more of E781X, N967X, and R1014X, or any of the mutations provided herein. and corresponding mutations in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SaCas9 domain contains the following mutations: E781K, N967K, and R1014H. One or more of the variants, or any of the amino acid sequences provided herein or multiple corresponding mutations. In some embodiments, the SaCas9 domain contains E781K , N967K, or R1014H mutations, or in any of the amino acid sequences provided herein. Contains the corresponding mutations in

[0168] In some embodiments, the variant Cas protein is SpCas9, SpCas9-VRQR, SpCas9-V RER, xCas9 (sp), SaCas9, SaCas9-KKH, SpCas9-MQKSER, SpCas9-LRKIQK, or SpCas9- It can be LRVSQL.

[0169] Exemplary SaCas9 Sequences KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTHS ELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKD GEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPGFGWKDIKEWYEMLMGHCTYFP EELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKP EFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELARE KNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPR SVSFDNSFNNKVLVKQEE N SKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDF INRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKL DKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIK YYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAE FIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEV KSKKHPQIIKKG Residue N579, shown above in bold and underlined, was mutated (e.g., to A579) to form the SaCas9 nick A gene may be obtained.

[0170] Exemplary SaCas9n Sequences KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTHS ELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKD GEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPGFGWKDIKEWYEMLMGHCTYFP EELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKP EFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELARE KNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPR SVSFDNSFNNKVLVKQEE A SKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDF INRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKL DKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIK YYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAE FIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEV KSKKHPQIIKKG

[0171] Residue A579 above is underlined and can be mutated from N579 to yield SaCas9 nickase. It is shown in bold.

[0172] Exemplary SaKKH Cas9 KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTHS ELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKD GEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKWEYEMLMGHCTYFP EELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKP EFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIAIFNRKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIQUINAIIKKYGLPNDIIIELARE KNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPR SVSFDNSFNNKVLVKQEE A SKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTTKKEYLLEERDINRFSVQKDF INRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKKERNKGYKHHAEDALIIANADFIFKEWKKL DKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNR K LINDTLYSTRKDDKGNTLI VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIK YYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAE FIASFY K NDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPP H IIKTIASKTQSIKKYSTDILGNLYEV KSKKHPQIIKKG. Residue A579 above is underlined and in bold, which can be mutated from N579 to obtain SaCas9 nickase. E781, N967, and R1014 can be mutated to obtain SaKKH Cas9. Residues K781, K967, and H1014 shown above are underlined and in italics.

[0173] The polynucleotide-programmable nucleotide-binding domain of the base editor It itself contains one or more domains. For example, a polynucleotide programmable The nucleotide binding domain may comprise one or more nuclease domains. In some embodiments, the nucleic acid of the polynucleotide-programmable nucleotide binding domain The nuclease domain may comprise an endonuclease or an exonuclease. The term "exonuclease" refers to an enzyme that digests nucleic acids (e.g., RNA or DNA) from their free ends. The term "endonuclease" refers to a protein or polypeptide that is capable of can catalyze (e.g., cleave) an internal region of a nucleic acid (e.g., DNA or RNA). In some embodiments, the term "endonuclease" refers to a protein or polypeptide that can be used to synthesize a polypeptide. The enzyme is capable of cleaving one strand of a double-stranded nucleic acid. Nucleases are capable of cleaving both strands of a double-stranded nucleic acid molecule. In this form, the polynucleotide programmable nucleotide binding domain is a deoxyribonucleotide. In some embodiments, the polynucleotide programmable The nucleotide binding domain may be a ribonuclease.

[0174] In some embodiments, the polynucleotide-programmable nucleotide binding domain The nuclease domain of the target polynucleotide can cleave zero, one, or two strands. In some cases, polynucleotide programmable nucleotide binding can be achieved. The domain may comprise a nickase domain. As used herein, the term "nickase" refers to a double-stranded It is possible to cleave only one of the two strands of a formed nucleic acid molecule (e.g., DNA). Polynucleotide-programmable nucleotide-binding domains containing nuclease domains In some embodiments, the nickase refers to a polynucleotide programmable from a fully catalytically active (e.g., native) form of the nucleotide-binding domain capable of Polynucleotide-programmable nucleotide-binding domains containing one or more mutations For example, a polynucleotide program Possible nucleotide-binding domains include the nickase domain derived from Cas9. The Cas9-derived nickase domain contains a D10A mutation and a histidine at position 840. In such cases, residue H840 retains catalytic activity and thus is involved in the synthesis of nucleic acid duplexes. In another example, a Cas9-derived nickase domain can cleave a single strand of a target molecule. The amino acid may contain an H840A mutation, but the amino acid residue at position 10 remains D. In this embodiment, the nickase is a polynucleotide-programmable nucleotide-binding domain. From a fully catalytically active (e.g., native) form, nucleases not required for nickase activity can be removed. It can be derived by removing all or part of the ze domain. The polynucleotide-programmable nucleotide-binding domain is derived from Cas9. In the case of the RuvC domain, the nickase domain derived from Cas9 is similar to the RuvC domain. The HNH domain may include a deletion of all or part of the HNH domain.

[0175] Thus, polynucleotides containing nickase domains can be used as programmable nucleotides. The base editor, which includes a base-binding domain, binds to the complementary sequence of the bound guide nucleic acid (e.g., a single-stranded DNA break (nick) at a specific polynucleotide target sequence (determined by In some embodiments, a nickase domain (e.g., from Cas9) can be used to generate Nucleic acid duplexes cleaved by base editors containing a nickase domain derived from The strand of the target polynucleotide sequence is the strand that is not edited by the base editor (i.e. The strand cleaved by the base editor is the opposite strand to the strand containing the base to be edited. In other embodiments, a nickase domain (e.g., a nickase derived from Cas9) is used. Base editors containing a nucleotide sequence (nucleotide domain) open the strand of a DNA molecule that is targeted for editing. In such cases, the non-targeted strand is not cleaved.

[0176] Catalytically dead (i.e., unable to cleave target polynucleotide sequences) polynucleotides Base editors containing nucleotide-programmable nucleotide-binding domains are also described herein. As used herein, the terms "catalytically dead" and "nuclease dead" are used interchangeably. Used interchangeably, one or more mutations that result in the loss of the ability to cleave a strand of nucleic acid. Polynucleotides with programmable nucleotide binding domains and / or deletions In some embodiments, the catalytically dead polynucleotide programmable The base editors of the various nucleotide-binding domains are designed to interact with one or more nuclease domains. Nuclease activity can be lacking as a result of specific point mutations in the In the case of base editors containing the Cas9 domain, Cas9 contains both the D10A and H840A mutations. Such a mutation would inactivate both nuclease domains, thereby reducing the nuclease activity. In another embodiment, catalytically dead polynucleotide proteases result in a loss of proteases activity. The grammable nucleotide binding domain may be a catalytic domain (e.g., RuvC1 and / or The present invention may include one or more deletions of all or part of the HNH domain. In catalytically dead polynucleotide programmable nucleotide binding domains, Point mutations (e.g., D10A or H840A) and all or part of the nuclease domain Contains deletions.

[0177] Previously functional versions of polynucleotide programmable nucleotide binding domains Catalytically dead polynucleotide programmable nucleotide binding domains from Mutations that can be generated are also contemplated herein. For example, catalytically dead Ca In the case of s9 ("dCas9"), variants with mutations other than D10A and H840A are provided. , resulting in a nuclease-inactivated Cas9. Other amino acid substitutions at D10 and H840, or in the nuclease domain of Cas9, Other substitutions in (e.g., the HNH nuclease subdomain and / or the RuvC1 subdomain) (substitution in

[0178] Additional suitable nuclease-inactive dCas9 domains are described in this disclosure and in the art. Such modifications would be obvious to those skilled in the art based on their own knowledge and are within the scope of the present disclosure. Other exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A D10A / D839A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains ( See, for example, Prashant et al., CAS9 tran, the entire contents of which are incorporated herein by reference. scriptional activators for target specificity screening and paired nickases for Cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838 In some embodiments, the dCas9 domain is a dCas9 domain provided herein. At least 60%, at least 65%, at least 70%, at least 75% %, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical In some embodiments, the Cas9 domain comprises an amino acid sequence described herein. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more In some embodiments, the Cas9 domain comprises an amino acid sequence having the amino acid sequence of any one of the amino acids described herein. at least 10, at least 15, at least At least 20, at least 30, at least 40, at least 50, at least 60, at least 70, At least 80, at least 90, at least 100, at least 150, at least 200, at least 2 50, at least 300, at least 350, at least 400, at least 500, at least 600, At least 700, at least 800, at least 900, at least 1000, at least 1100, or or an amino acid sequence having at least 1200 identical contiguous amino acid residues.

[0179] Polynucleotide programmable nucleotide sequences that can be incorporated into base editors Non-limiting examples of domain-binding domains include domains derived from CRISPR proteins, restriction nucleases, and the like. , meganucleases, TAL nucleases (TALENs), and zinc finger nucleases In some cases, base editors include CRISPR (i.e., Clustal Genetic Factors) for nucleic acids. During modified Regularly Interspaced Short Palindromic Repeats (ERRs), the bound Natural or modified proteins that can bind to nucleic acid sequences via nucleotide sequences. a polynucleotide containing a programmable nucleotide binding domain or a portion thereof Such proteins are referred to herein as "CRISPR proteins." Polynucleotides containing all or part of a CRISPR protein. Programmable nucleotides. base editors containing a base-binding domain (i.e., base-binding all or part of a CRISPR protein) The editor contains a base sequence called the "CRISPR protein-derived domain." Disclosed herein are CRISPR proteins that are incorporated into the base editors. The CRISPR protein domain may be modified relative to the wild-type or naturally occurring version of the CRISPR protein. For example, as described below, CRISPR protein-derived domains can be used to one or more mutations, insertions, deletions, rearrangements relative to the wild-type or naturally occurring version of a gene The sequences may include substitutions, and / or recombinations.

[0180] In some embodiments, the CRISPR protein-derived domain incorporated into the base editor The nucleic acid is capable of binding to a target polynucleotide when linked to an attached guide nucleic acid. Endonucleases (e.g., deoxyribonucleases or ribonucleases) In some embodiments, the domains from the CRISPR proteins incorporated into the base editors The nucleic acid is a nucleic acid capable of binding to a target polynucleotide together with a bound guide nucleic acid. In some embodiments, the CRISPR protein is integrated into a base editor. The derived domain is capable of binding to a target polynucleotide together with the bound guide nucleic acid. In some embodiments, the base editor is a CRISPR domain. The target polynucleotide bound by the protein-derived domain is DNA. In some embodiments, the base editor is bound by a domain derived from a CRISPR protein. The target polynucleotide is RNA.

[0181] In some embodiments, the CRISPR protein-derived domain of the base editor is bacterium ulcerans(NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphthe ria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: N C_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanen se(NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliell a baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1) ; Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref : NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria menin gitidis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus The Cas9 gene may comprise all or part of the Cas9 gene of S. aureus.

[0182] In some embodiments, the Cas9 induction domain of the base editor is Staphylococcus aureus In some embodiments, the SaCas9 domain is a Cas9 domain of nucleic acid sequence encoding the nucleic acid sequence of ... nuclease-active SaCas9, nuclease-inactive SaCas9 (SaCas9d), or SaCas9 nickase (S In some embodiments, the SaCas9 domain comprises an N579X mutation. In some embodiments, the SaCas9 domain comprises an N579A mutation. The SaCas9 domain, SaCas9d domain, or SaCas9n domain is a nucleic acid having a non-canonical PAM. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9d domain can bind to the sequence. The SaCas9n domain can bind to nucleic acid sequences containing the NNGRRT PAM sequence. In some embodiments, the SaCas9 domain contains the E781X, N967X, and R1014X mutations. Contains one or more.

[0183] Base editors use domains derived from all or part of the high-fidelity Cas9 gene. In some embodiments, the high-fidelity Cas9 domain of the base editor may comprise a corresponding The electrostatic interactions between the Cas9 domain and the sugar-phosphate backbone of DNA are significantly different from those of the wild-type Cas9 domain. The Cas9 domain is an engineered domain containing one or more mutations that reduce the activity of the sugars in DNA. High-fidelity Cas9 domains with reduced electrostatic interactions with the phosphate backbone have fewer oligands. In some embodiments, the Cas9 domain (e.g., wild-type Cas9 domain) is one or more of the following that reduce the association between the Cas9 domain and the sugar-phosphate backbone of DNA: In some embodiments, the Cas9 domain comprises a Cas9 domain and a DNA at least 1%, at least 2%, at least 3%, at least At least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least at least 55%, at least 60%, at least 65%, at least 70%, or more It contains one or more mutations that

[0184] In some embodiments, the modified Cas9 is a high-fidelity Cas9 enzyme. In embodiments, the high-fidelity Cas9 enzyme is SpCas9(K855A), eSpCas9(1.1), SpCas9-HF1, or or a hyper-accurate Cas9 variant (HypaCas9). The modified Cas9, eSpCas9(1.1), is a nucleotide sequence encoding the HNH / R Contains alanine substitutions that weaken the interaction between the UVC groove and the non-target DNA strand, preventing strand separation and off-targeting. Similarly, SpCas9-HF1 prevents cleavage at the target site. Alanine substitutions that disrupt the interaction reduce off-target editing. Mutations in the REC3 domain of Cas9 that increase proofreading and target discrimination (SpCas9 All three high-fidelity enzymes contain less cleavage sites than wild-type Cas9. An exemplary high-fidelity Cas9 is provided below. High-fidelity Cas9 domain mutations to Cas9 are shown in bold and underlined. MDKKYSIGL A IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMT A FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWG A LSRKLINGIRDKQSGKTILDFLKSDGFANRNFM A LIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR A ITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD

[0185] Guide polynucleotide

[0186] As used herein, the term "guide polynucleotide" refers to a polynucleotide that is specific for a target sequence. and a polynucleotide-programmable nucleotide-binding domain protein. The term "polynucleotide" refers to a polynucleotide that can form a complex with a protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA. In this case, the term "guide RNA (gRNA)" and its grammatical equivalents refer to a gene that is specific for a target DNA. RN can refer to RNA that is capable of synthesizing and forming a complex with a Cas protein. The A / Cas complex may help "guide" the Cas protein to the target DNA. The tracrRNA targets linear or circular dsDNA targets complementary to the spacer with endonuclease activity. The target strand that is not complementary to the crRNA is first cleaved by endonuclease digestion. is cleaved by 3'-5' exonucleolytic degradation and then trimmed. However, DNA binding and cleavage typically require both a protein and RNA. , a single-guide RNA ("s") that incorporates both crRNA and tracrRNA aspects into a single RNA species. The entire contents of which are incorporated herein by reference. See Jinek M. et al., Science 337:816-821 (2012), which is incorporated herein by reference. Cas9 targets short motifs (PAM or protospacer) in the CRISPR repeat sequence. The Cas9 nuclease recognizes the nucleotide sequence (the nucleotide sequence of the self-binding domain) and helps distinguish self from non-self. The sequences and structures are known to those skilled in the art (see, for example, the entire contents of each of these references). "Complete genome sequence of an M1 strain of Streptococcus" incorporated herein by reference. pyogenes.” Ferretti, JJ et al., Natl. Acad. Sci. USA 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E. et al., Nature 471:602-607(2011); and “Programmable dual-RNA-gu ided DNA endonuclease in adaptive bacterial immunity.” Jinek M. et al, Science 3 37:816-821 (2012). Cas9 orthologs include, but are not limited to, S. pyogenes. nes and S. thermophilus. Additional suitable Cas9 Nucleases and sequences will be apparent to those skilled in the art based on this disclosure, and such Such Cas9 nucleases and sequences are incorporated herein by reference in their entirety. Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type I I CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737 In some embodiments, the Cas9 nuclease sequence includes a Cas9 sequence from a target gene and a locus. Cas9 has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase. is.

[0187] In some embodiments, the guide polynucleotide comprises at least one single guide R In some embodiments, the guide polynucleotide is a sgRNA or a gRNA. In some embodiments, the guide polynucleotide is at least one tracrRNA. The tid is a polynucleotide-programmable DNA binding domain (e.g., Cas9 or Cpf1). does not require a PAM sequence to guide the target nucleotide sequence.

[0188] Polynucleotide-programmable nucleotide base editors disclosed herein The binding domain (e.g., CRISPR guide domain) associates with the guide polynucleotide. The target polynucleotide sequence can be recognized by the guide polynucleotide. The guide RNA is typically single-stranded and is a target sequence of a polynucleotide. site-specific binding (e.g., via complementary base pairing) to the guide nucleic acid The associated base editor can be programmed to guide it to the target sequence. The polynucleotide may be DNA. The guide polynucleotide may be RNA. In some cases, the guide polynucleotide contains a natural nucleotide (e.g., adenosine). In some cases, the guide polynucleotide may be a non-naturally occurring (or unnatural) nucleotide. In some cases, the nucleic acid may be a nucleotide analogue (e.g., a peptide nucleic acid or a nucleotide analogue). the target region of the guide nucleic acid sequence is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, The target region of the guide nucleic acid can be 25, 26, 27, 28, 29, or 30 nucleotides in length. 10-30 nucleotides in length, or 15-25 nucleotides in length, or 15-20 nucleotides in length The length of the

[0189] In some embodiments, the guide polynucleotide comprises two or more distinct polynucleotides. which can interact with each other, for example, through complementary base pairing ( For example, a guide polynucleotide can be a CRISPR R For example, a transactivating CRISPR RNA (crRNA) may be included. The polynucleotide may comprise one or more trans-activating CRISPR RNAs (tracrRNAs). do.

[0190] Type II CRISPR systems involve targeting of nucleic acids by CRISPR proteins (e.g., Cas9). The synthesis typically involves the synthesis of a first RNA molecule (crRNA) that contains a sequence that recognizes the target sequence, and a guide RNA (guide RNA). -Contains repeat sequences that form a scaffold region that stabilizes the CRISPR protein complex Such a duplex requires complementary base pairing between the first and second RNA molecules (trRNA) that contain the first and second RNA molecules. The RNA system can be used to link the base editors disclosed herein to a target polynucleotide sequence. It can be used as a guide polynucleotide.

[0191] In some embodiments, the base editors provided herein are capable of encoding single-guide polypeptides. In some embodiments, the methods provided herein utilize nucleotides (e.g., gRNAs). A base editor that utilizes dual guide polynucleotides (e.g., dual gRNAs). In some embodiments, the base editors provided herein comprise one or more guide In some embodiments, a polynucleotide (e.g., multiple gRNAs) is utilized. Guide polynucleotides are utilized for the various base editors described herein. For example, a single guide polynucleotide can be engineered with a cytidine base editor and an adenine base editor. It can be used for syngeneic base editors.

[0192] In other embodiments, the guide polynucleotide comprises a polynucleotide sequence of a nucleic acid in a single molecule. It may contain both a guide targeting portion and a nucleic acid scaffold portion (i.e., a single-molecule guide nucleic acid). For example, a single-molecule guide polynucleotide may be a single-guide RNA (sgRNA or gRNA). As used herein, the term guide polynucleotide sequence can be a base editor. any single, double, or multi-stranded nucleic acid capable of interacting with and directing the nucleic acid to a target polynucleotide sequence. Alternatively, multiple molecules of nucleic acid are contemplated.

[0193] Typically, the guide polynucleotide (e.g., the crRNA / trRNA complex or the gRNA) A "polynucleotide" is a molecule that contains a sequence that can recognize and bind to a target polynucleotide sequence. nucleotide target segment" and the base editor polynucleotide programmable nucleotide Protein binding domain components stabilize the guide polynucleotide. In some embodiments, the polynucleotide of the guide polynucleotide comprises a "segment." The targeting segment recognizes and binds to a DNA polynucleotide, In other cases, the guide polynucleotide The polynucleotide targeting segment recognizes and binds to an RNA polynucleotide. As used herein, a "segment" refers to a fragment of a nucleic acid molecule. A segment or region of a guide polynucleotide, e.g., a continuous stretch of nucleotides in a guide polynucleotide A segment may also be a molecule such that the segment may comprise a region of two or more molecules. It can refer to a region / section of a complex, e.g., a nucleic acid molecule containing multiple guide polynucleotides. , the protein-binding segment may hybridize along the region of complementarity, e.g. In some embodiments, the two The protein-binding segment of the DNA-targeting RNA comprises two individual molecules: (i) 100% ribonucleotides; (ii) the 40th to 75th base pairs of the first RNA molecule, which is 50 base pairs in length; and The term "segment" may refer to the 10th to 25th base pairs of the RNA molecule of the present invention. Unless otherwise specifically defined, the base pairs of a given RNA molecule are not limited to a particular number of total base pairs. is not limited to any particular number of pairs, nor is it limited to any particular number of individual molecules in the complex; It can include regions of RNA molecules of any length, including regions that have complementarity with other molecules. obtain.

[0194] A guide RNA or guide polynucleotide may be composed of two or more RNAs, such as CRISPR RNAs (crR The guide RNA or guide polynucleotide may comprise a guide RNA (guide RNA) and a transactivating crRNA (tracrRNA). Nucleotides are single-stranded RNAs or portions (e.g., functional portions) of crRNA and tracrRNA. It may also contain a single guide RNA (sgRNA) formed by fusion. Alternatively, the guide polynucleotide can be a duplex RNA comprising a crRNA and a tracrRNA. Furthermore, the crRNA can hybridize with the target DNA.

[0195] As discussed above, a guide RNA or guide polynucleotide can be an expression product. For example, the DNA encoding the guide RNA may be inserted into a vector containing a sequence encoding the guide RNA. The guide RNA or guide polynucleotide can be an isolated guide RNA or is a plasmid DNA containing a sequence encoding a guide RNA and a promoter that transfects cells. The guide RNA or guide The polynucleotide may be delivered into the cell by other methods, such as using viral-mediated gene delivery. You can also do this.

[0196] The guide RNA or guide polynucleotide can be isolated. The RNA can be transfected into a cell or organism in the form of isolated RNA. The guide RNA can be transcribed in vitro using any in vitro transcription system known in the art. The guide RNA can be prepared by a method comprising the steps of: The vector can be delivered into cells in the form of isolated RNA rather than in the form of a mide.

[0197] The guide RNA or guide polynucleotide targets three regions: a first region at the 5' end that may be complementary to a site; a second region that may form a stem-loop structure; The first region of each guide RNA may comprise an internal region, and a third 3' region, which may be single-stranded. The regions of each guide RNA can be different, so that each guide RNA targets the fusion protein to a specific target. Furthermore, the second and third regions of each guide RNA guide all the guide RNAs to the target site. The RNA may be identical in nature.

[0198] The first region of the guide RNA or guide polynucleotide is targeted to a target site in the chromosomal sequence. The first region of the guide RNA may be complementary to a sequence in the target site. In some cases, the first region of the guide RNA is approximately 10 nucleotides long. nucleotide to 25 nucleotides (i.e., 10 nucleotides to nucleotides, or about 10 nucleotides) 10 to about 25 nucleotides, or 10 to about 25 nucleotides, or about 10 nucleotides For example, the first region of the guide RNA may comprise a sequence of 1 to 25 nucleotides (or more). The region of base pairing between the target site in the chromosomal sequence is approximately 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length. Sometimes, the first region of the guide RNA is approximately 19, 20, or 21 nucleotides in length. obtain.

[0199] The guide RNA or guide polynucleotide may also include a second region that forms a secondary structure. For example, the secondary structure formed by the guide RNA is a stem (or hairpin) and The length of the loop and stem may vary. The length of the nucleotide sequence can range from approximately 3 to 10 nucleotides, and the stem can range from approximately 6 to 20 base pairs in length. The stem may contain one or more bulges of 1 to 10 or about 10 nucleotides. The total length of the region can range from approximately 16 to 60 nucleotides in length. The loop may be approximately 4 nucleotides in length and the stem may be approximately 12 base pairs.

[0200] The guide RNA or guide polynucleotide may include a third region that may be essentially single-stranded. For example, the third region can sometimes be located at any chromosomal sequence in the cell of interest. and sometimes not complementary to the rest of the guide RNA. The length of the region can vary. The third region can be greater than about 4 nucleotides in length. For example, the length of the third region can range from approximately 5 to 60 nucleotides in length.

[0201] The guide RNA or guide polynucleotide can be inserted into any exon or intron of the gene target. In some cases, the guide can target exon 1 or In other cases, the guide may target exon 3 or 2 of the gene. The composition can target multiple genes all targeting the same exon. may contain nucleotide sequences and, in some cases, may target different exons. Multiple guide RNAs may be included. Exons and introns of a gene may be targeted.

[0202] The guide RNA or guide polynucleotide targets a nucleic acid sequence of approximately 20 nucleotides. The target nucleic acid may be less than approximately 20 nucleotides. At most approximately 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or 1 to 100 The target nucleic acid may be at most about 5, 10, 15, 100, 15 ... 6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or between 1 and 100 nucleotides The target nucleic acid sequence is adjacent to and 5' of the first nucleotide of the PAM. The guide RNA can target a nucleic acid sequence. The target nucleic acid can be at least 20 bases long. At most approximately 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or can be 1 to 100 nucleotides.

[0203] A guide polynucleotide, e.g., a guide RNA, may be coupled to another nucleic acid, e.g., a target nucleic acid or It may refer to a nucleic acid that can hybridize to a protospacer in the genome of a cell. The guide polynucleotide can be RNA. The guide polynucleotide can be DNA. The guide polynucleotide is programmed or designed to bind to a nucleic acid sequence in a site-specific manner. A guide polynucleotide may comprise one polynucleotide strand. A guide polynucleotide can be referred to as a single guide polynucleotide. The polynucleotide may contain two polynucleotide strands and may be referred to as a double-guide polynucleotide. Guide RNAs can be introduced into cells or embryos as RNA molecules. The gene can be transcribed in vitro and / or chemically synthesized. The resulting DNA molecule can be transcribed from a gBlocks® gene fragment. Guide RNA can be introduced into cells or embryos as an RNA molecule. Guide RNAs are non-RNA nucleic acid molecules. It can also be introduced into a cell or embryo in the form of a molecule, for example a DNA molecule. The DNA encoding the guide RNA is used as a promoter for expression of the guide RNA in the cell or embryo of interest. The RNA coding sequence can be operably linked to a control sequence. It is operably linked to a promoter sequence recognized by Pol III. Plasmid vectors that can be used to express guide RNA include: This includes, but is not limited to, the px330 vector and the px333 vector. For this purpose, the plasmid vector (e.g., the px333 vector) must contain at least two guide RNA sequences. The nucleic acid sequence may include a coding DNA sequence.

[0204] A guide polynucleotide, e.g., a guide RNA, and a target sequence are selected, designed, and detected. Methods for determining the identity of nucleic acid salts are described herein and are known to those skilled in the art. Potential of deaminase domains (e.g., AID domains) in base editor systems To minimize the effects of unwanted substrate hybridization, residues that may be unintentionally targeted for deamination (e.g., (e.g., off-target C residues that may be present on ssDNA within the target nucleic acid locus) Furthermore, in order to optimize the gRNA corresponding to the target nucleic acid sequence, for example, For example, software tools are used to minimize all off-target activity across the genome. For example, each possible target domain using S. pyogenes Cas9 can be up to a certain number of mismatches (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) All off-target sequences containing the selected PAM, e.g., NAG or NGG, containing the missing base pair The first region of the gRNA that is complementary to the target site may be identified throughout the genome. and all first regions (e.g. crRNA) can be determined based on their overall predicted off-target sequences. The top ranked target domains can be ranked according to their target score. represents those that may have the greatest on-target and least off-target activity. Candidate targeting gRNAs can be identified using methods known in the art and / or described herein. This allows functional evaluation.

[0205] As a non-limiting example, a target DNA fragment in the crRNA of a guide RNA for use with Cas9 can be used. Hybridizing sequences may be identified using DNA sequence search algorithms. ae S., Park J., & Kim J.-S. Cas-OFFinder: A fast and versatile algorithm that se arches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinfor A custom-built gRN based on the public tool cas-offinder described in matics 30, 1473-1475 (2014) This may be done using A design software, which allows for genome-wide off-target analysis. The guide is scored by calculating the target's propensity. Typically, it is a perfect match to seven Consider matches up to mismatches, with guide lengths ranging from 17 to 24. Off Once the target sites are determined, a combined score is calculated for each guide. The web interface aggregates the possible sequences adjacent to the PAM sequence in a tabular output. In addition to identifying target sites, the software can identify 1, 2, 3, or 3 fragments from the selected target sites. It also identifies all PAM flanking sequences that differ by more than one nucleotide. The genomic DNA sequence for the target gene is obtained and analyzed using publicly available tools, such as RepeatM The repeat element is screened using the RepeatMasker program. Search the input DNA sequence for identified elements and regions of low complexity. A detailed annotation of the repeats present in the Lee sequence is printed.

[0206] Following identification, the first region of the guide RNA, e.g., crRNA, is sequenced based on its distance to the target site, its Orthogonality and the presence of a 5' nucleotide for a close match with the related PAM sequence (e.g. For example, related PAMs, e.g., NGG PAM for S. pyogenes, NNGRRT for S. aureus or NNGRRV PAM, based on the identification of a close match in the human genome containing a 5' G As used herein, orthogonality refers to the ability to achieve the highest degree of orthogonality to a target sequence. "High level of orthogonality" refers to the number of sequences in the human genome that contain a low number of mismatches. Or "good orthogonality" means, for example, that a gene has identical sequences in the human genome in addition to the intended target. and does not have any sequence containing one or two mismatches in the target sequence. To minimize off-target DNA cleavage, Orthogonal targeting domains can be selected.

[0207] In some embodiments, the base editing activity of the candidate guide polynucleotides is tested. A reporter system may be used to test for The reporter gene system is a system in which base editing activity leads to reporter gene expression. For example, a reporter system may include an assay based on an inactivated start codon. reporter genes containing mutations in the template strand, e.g., from 3'-TAC-5' to 3'-CAC-5' If the target C is successfully deaminated, the corresponding mRNA may contain 5' -AUG-3', allowing translation of the reporter gene. Non-limiting examples of reporter genes include green fluorescent protein (GFP) and (GFP), red fluorescent protein (RFP), luciferase, secreted alkaline phosphatase (S EAP), as well as any other gene whose expression is detectable and apparent to one skilled in the art. For example, each deaminase determines which residues in the target DNA sequence Many different gRNAs are used with reporter systems to determine which are targeting the target gene. Specific base-editing proteins, such as Cas9 deaminase fusion proteins, can be tested. sgRNA targeting the non-template strand to assess off-target effects of the protein In some embodiments, such gRNAs can be used to test for mutated initiation codons. The guide polynucleotide can be designed so that it does not base-pair with the gRNA. are standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine ), ribonucleotide isomers, and / or ribonucleotide analogs In some embodiments, the guide polynucleotide comprises at least one detectable label. The detectable label may include a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Instruments Incorporated). Red, Oregon Green, Alexa Fluors, Halo Tags, or suitable fluorescent dyes), detection tags (e.g. For example, it can be biotin, digoxigenin, etc.), quantum dots, or gold particles.

[0208] The guide polynucleotide may be chemically synthesized, enzymatically synthesized, or a combination thereof. For example, guide RNAs can be synthesized using standard phosphoramidite-based solid-phase synthesis. Alternatively, the guide RNA can be inserted into the phage RNA polymerase, which encodes the guide RNA. by operably linking to a promoter regulatory sequence recognized by methylase Examples of suitable phage promoter sequences include T7, T3, SP 6 promoter sequence, or a variant thereof. The guide RNA is separated into two separate molecules ( In embodiments involving, for example, crRNA and tracrRNA, crRNA is chemically synthesized and tracrRNA is synthesized enzymatically. It can be synthesized.

[0209] In some embodiments, the base editor system comprises multiple guide polynucleotides, For example, the gRNA may comprise one of the base editor systems. or multiple target loci (e.g., at least one gRNA, at least two gRNAs, at least At least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 gRNAs A, at least 50 gRNAs). The multiple gRNA sequences may be arranged in tandem. The sequences may be separated by a direct repeat, preferably by a direct repeat.

[0210] The DNA sequence encoding the guide RNA or guide polynucleotide is part of a vector. Additionally, the vector may contain additional expression control sequences (e.g., enhancer sequences). , Kozak sequence, polyadenylation sequence, transcription termination sequence, etc.), selectable marker sequence (e.g., GFP or antibiotic resistance genes such as puromycin), origins of replication, etc. The DNA molecule encoding the guide RNA may be linear. The DNA molecule encoding the nucleotide may be circular.

[0211] In some embodiments, one or more components of the base editor system are Such DNA sequences may be incorporated into an expression system, e.g., a cell, together with For example, polynucleotides can be introduced into the programmable nucleotide chains. DNA sequences encoding the binding domain and guide RNA are introduced into cells, and the respective DNA sequences The individual molecular parts (e.g., polynucleotide programmable nucleotide binding domains) One vector contains a sequence encoding a guide RNA, and the second vector contains a sequence encoding a guide RNA. vector), or both may be the same molecule (e.g., a polynucleotide program Coding (and control) for both the possible nucleotide-binding domain and the guide RNA ) sequences).

[0212] The guide polynucleotide may be one or more sequences that provide new or improved characteristics to the nucleic acid. The guide polynucleotide may comprise a nucleic acid affinity tag. Synthetic nucleotides, synthetic nucleotide analogs, and nucleotide derivatives are used for polynucleotide synthesis. The nucleic acid may contain nucleotides, derivatives, and / or modified nucleotides.

[0213] In some cases, the gRNA or guide polynucleotide may contain a modification. It can be created at any position in the NA or guide polynucleotide. can make two or more modifications to the guide polynucleotide. Polynucleotides can be subjected to quality control after modification. Controls may include PAGE, HPLC, MS, or any combination thereof.

[0214] Modifications of the gRNA or guide polynucleotide can include substitutions, insertions, deletions, chemical modifications, physical modifications, and It may be modified, stabilized, purified, or any combination thereof.

[0215] The gRNA or guide polynucleotide is a 5' adenylate, 5' guanosine triphosphate 5'N7 methylguanosine triphosphate cap, 5'N7 methylguanosine triphosphate cap, 5'triphosphate cap, 3' phosphate, 3' thiophosphate, 5' phosphate, 5' thiophosphate Cis-Syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer , d spacer, PC spacer, r spacer, spacer 18, spacer 9, 3'-3' modification, 5'-5' modified, non-basic, acridine, azobenzene, biotin, biotin BB, biotin TEG, Cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, Dua Rubiotin, PC-biotin, Psoralen C2, Psoralen C6, TINA, 3'DABCYL, Black H Black Hole Quencher 1, Black Hole Quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1 , QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linker, 2'-deoxyribonucleotide Purine 2'-deoxyribonucleoside analogs, pyrimidinyl 2'-deoxyribonucleoside analogs Ribonucleoside analogues, 2'-O-methylribonucleoside analogues, sugar-modified analogues RNA, wobble / universal bases, fluorescent dye labeling, 2'-fluoro RNA, 2'-O-methyl RNA, thiylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA , phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, 5'-methylsilyl They may also be modified by thiamine-5'-triphosphate, ... do.

[0216] In some cases, the modification is permanent. In other cases, the modification is transient. In some cases, multiple modifications are made to the gRNA or guide polynucleotide. Modifications of the guide polynucleotide may affect the physicochemical properties of the nucleotide, e.g., conformational ionization, polarity, hydrophobicity, chemical reactivity, base pairing interactions, or any combination thereof The combination can be modified.

[0217] The modification may be a phosphorothioate substitution. In some cases, the natural phosphodiesterase Ester bonds can be susceptible to rapid degradation by cellular nucleases, while phosphoro Modification of internucleotide linkages using thioate (PS) bond substitutions is subject to cellular degradation The modification may make the gRNA or guide polynucleotide more stable to hydrolysis. The stability of the protein may be increased. Modifications may also enhance biological activity. Holothioate-enhanced RNA gRNA is cleaved by ribonuclease A, ribonuclease T1, and ribonuclease B. These features can inhibit cytotoxicity, ... In applications where exposure to nucleases is likely in vivo or in vitro due to The PS-RNA used in gRNAs can be used, for example, by adding a PS-RNA at the 5' or 5' end of the gRNA. Phosphorothioate (PS) bonds can be introduced between the last 3 to 5 nucleotides. This can inhibit exonucleolytic degradation. Holothioate linkages are added throughout the gRNA to reduce endonuclease attack It is possible.

[0218] Protospacer adjacent motif The term "protospacer adjacent motif (PAM)" or PAM-like motif refers to a CRISPR-bacterial A 2-6 base pair DNA sequence directly following the DNA sequence targeted by Cas9 nuclease in the immune response system In some embodiments, the PAM can be a 5' PAM (i.e., a protospacer In other embodiments, the PAM can be a 3' PAM (i.e., located upstream of the 5' end of the proto located downstream of the 5' end of the spacer).

[0219] Protospacer adjacent motifs (PAMs) or PAM-like motifs are essential for CRISPR-mediated bacterial adaptive immune systems In this context, it refers to the 2-6 base pair DNA sequence that directly follows the DNA sequence targeted by Cas9 nuclease. In some embodiments, the PAM can be a 5' PAM (i.e., the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (i.e., located upstream of the protospacer). The PAM sequence is important for target binding, but its exact location is unknown. The sequence depends on the type of Cas protein.

[0220] The base editors provided herein can be used to generate canonical or non-canonical protospacers. CRISPR proteins that can bind to nucleotide sequences containing a protein-adjacent motif (PAM) sequence. The PAM site may comprise a nucleotide sequence adjacent to the target polynucleotide sequence. Some embodiments of the present disclosure provide CRISPR proteins with various PAM specificities. For example, a base editor that contains all or part of a protein, typically S. pyogenes, is provided. Cas9 proteins, such as Cas9 (SpCas9) from nes, bind to specific nucleic acid regions and therefore canonical N GG PAM sequence is required. Here, "N" in "NGG" is adenine (A), thymine (T), It can be either guanine (G) or cytosine (C), where G is guanine. PAM is a CRISPR protein. Different base editors can be gene-specific and contain domains from different CRISPR proteins. The PAM can be 5' or 3' of the target sequence. The PAM can be upstream or The PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. PAMs are often 2-6 nucleotides in length.

[0221] In some embodiments, the Cas9 domain is a Cas9 domain from Streptococcus pyogenes (Sp In some embodiments, the SpCas9 domain is a nuclease-active SpCas9, ... The first is a cleavage-inactive SpCas9 (SpCas9d), or a Cas9 nickase (SpCas9n). In some embodiments, the SpCas9 has a D9X mutation or any of the amino acid sequences provided herein. or a corresponding mutation in any of the following, where X is any amino acid except D. In this case, SpCas9 does not have a D9A mutation or a counterpart in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain, the SpCas9d domain, or The SpCas9n domain can bind to nucleic acid sequences with non-canonical PAMs. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain is a NG It can bind to nucleic acid sequences having a G, NGA, or NGCG PAM sequence. In this form, the SpCas9 domain contains one or more of the following mutations: D1135X, R1335X, and T1137X; or a corresponding mutation in any of the amino acid sequences provided herein, where X are any amino acids. In some embodiments, the SpCas9 domain comprises D1135E, R1335Q, and T1137R mutations, or any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises D1135E, R1 335Q, and T1137R mutations, or counterparts in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises the mutations D1135X, R1335X, and one or more of the T1137X mutations or in any of the amino acid sequences provided herein In some embodiments, S The pCas9 domain may contain one or more of the following mutations: D1135V, R1335Q, and T1337R, or any of the mutations described herein. In some embodiments, the amino acid sequence of any of the amino acid sequences provided in In one embodiment, the SpCas9 domain contains the D1135V, R1335Q, and T1337R mutations, or the nucleotides of the sequences provided herein. In some embodiments, the SpCas9 domain contains a corresponding mutation in either of the amino acid sequences. The main mutations are one or more of D1135X, G1218X, R1335X, and T1337X, or and corresponding mutations in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SpCas9 domain is D1135V, G1218R, R1335Q, and and T1337R mutations, or any of the amino acid sequences provided herein. In some embodiments, the SpCas9 comprises a corresponding mutation in In this example, the SpCas9 domain contains the D1135V, G1218R, R1335Q, and T1337R mutations, or the nucleotide sequences provided herein. It includes corresponding mutations in any of the amino acid sequences provided.

[0222] In some embodiments, the Cas9 domain of any of the fusion proteins provided herein The polypeptides may be at least 60%, at least 65%, or at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least In some embodiments, the amino acid sequence of the present invention is at least 99.5% identical to the amino acid sequence of the present invention. The Cas9 domain of any of the provided fusion proteins can be any of the Cas9 domains described herein. In some embodiments, the fusion proteins provided herein comprise the amino acid sequence of a polypeptide. The Cas9 domain of any of the Cas9 proteins may be used in conjunction with any of the Cas9 polypeptides described herein. It consists of an amino acid sequence.

[0223] Exemplary SpCas9 protein sequences that can bind to PAM sequences are as follows: .

[0224] Exemplary SpCas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD

[0225] Exemplary SpCas9n MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD

[0226] Exemplary SpEQR Cas9 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGF ESPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRI DLSQLGGD Residue E11, as described above, can be mutated from D1135, R1335, and T1337 to obtain SpEQR Cas9 35, Q1335, and R1337 are underlined and in bold.

[0227] Exemplary SpVQR Cas9 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGF V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRI DLSQLGGD Residue V11, as mentioned above, can be mutated from D1135, R1335, and T1337 to obtain SpVQR Cas9 35, Q1335, and R1337 are underlined and in bold.

[0228] Exemplary SpVRER Cas9 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGF V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELINGRKRMLASA R ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK E Y R STKEVLDATLIHQSITGLYETRI DLSQLGGD. Exemplary SpVRQR Cas9 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETEATRLKRTARRRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDLNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGF V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASA R ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRI DLSQLGGD. D1135, G1218, R1335, and T1337 can be mutated to obtain SpVRQR Cas9 as described above. Residues V1135, R1218, Q1335, and R1337 are underlined and in bold.

[0229] In some embodiments, the Cas9 domain is a recombinant Cas9 domain. In some embodiments, the recombinant Cas9 domain is a SpyMacCas9 domain. The SpyMacCas9 domain is responsible for the nuclease-active SpyMacCas9 and the nuclease-inactive SpyMacCas9. (SpyMacCas9d), or SpyMacCas9 nickase (SpyMacCas9n). In the form, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain is a non-canonical In some embodiments, the SpyMacCas9 driver can bind to nucleic acid sequences with a PAM. The main, SpCas9d, or SpCas9n domain binds to a nucleic acid sequence containing a NAA PAM sequence. can be combined. Exemplary SpyMacCas9 MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFGSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGHSL HEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEGIGELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEIQTVGQNGGLFDDNPKSPLEVT PSKLVPLKKELNPKKYGGYQKPTTAYPVLLITDTKQLIPISVMNKKQFEQNPVKFLRDRGYQQVGKNDFIKLPKYTLVDI GDGIKRLWASSKEIHKGNQLVVSKKSQILLYHAHHLDSDLSNDYLQNHNQQFDVLFNEIISFSKKCKLGKEHIQKIENVY SNKKNSASIEELAESFIKLLGFTQLGATSPFNFLGVKLNQKQYKGKKDYILPCTEGTLIRQSITGLYETRVDLSKIGED.

[0230] High-fidelity Cas9 domain Some aspects of the present disclosure provide high-fidelity Cas9 domains. High-fidelity Cas9 domains have a higher fidelity than the corresponding wild-type Cas9 domains. Contains one or more mutations that reduce the electrostatic interaction between An engineered Cas9 domain. Without wishing to be bound by any particular theory, High-fidelity Cas9 domains with reduced electrostatic interactions with the sugar-phosphate backbone of DNA have fewer In some embodiments, the Cas9 domain (e.g., The native Cas9 domain reduces the association between the Cas9 domain and the sugar-phosphate backbone of DNA. In some embodiments, the Cas9 domain comprises a Cas9 domain and a The sugar-phosphate backbone of DNA is reduced by at least 1%, at least 2%, at least 3%, or 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25% , at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, one or more drugs that reduce the risk of death by at least 55%, at least 60%, at least 65%, or at least 70% contains multiple mutations.

[0231] In some embodiments, any of the Cas9 fusion proteins provided herein comprises N497 X, R661X, Q695X, and / or Q926X mutations, or one or more of the mutations provided herein. and corresponding mutations in any of the amino acid sequences, where X is any amino acid. In some embodiments, any of the Cas9 fusion proteins provided herein comprises N497 A, R661A, Q695A, and / or Q926A mutations, or one or more of the mutations provided herein. In some embodiments, the Cas9 The domain may contain the D10A mutation or the corresponding amino acid sequence in any of the amino acid sequences provided herein. High fidelity Cas9 domains are known in the art and will be apparent to those skilled in the art. For example, high-fidelity Cas9 domains are described in detail in US Pat. No. 6,49 ... the entire contents of each of which are incorporated herein by reference. Kleinstiver, BP, et al. "High-fidelity CRISPR-Cas9 nucleases with n o detectable genome-wide off-target effects.” Nature529, 490-495 (2016); and Sl aymaker, IM, et al. “Rationally engineered Cas9 nucleases with improved speci ficity.” Science 351, 84-88 (2015). High-fidelity Cas9 domain mutations to Cas9 are shown in bold and underlined. MDKKYSIGL A IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEE VVDKGASAQSFIERMT A FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWG A LSRKLINGIRDKQSGKTILDFLKSDGFANRNFM A LIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR A ITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD.

[0232] In some cases, the variant Cas9 protein is H840A, P475A, W476A, N477A, ​​D112 5A, W1126A, and D1127A mutations, so that the polypeptide binds to the target DNA and Such Cas9 proteins have low ability to cleave target DNA (e.g., single-stranded targets) and RNA. ability to bind to target DNA (e.g., single-stranded target DNA) but low ability to cleave target DNA As another non-limiting example, in some cases, variant Cas9 proteins The protein had the following mutations: D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A. Therefore, the polypeptide has a reduced ability to cleave the target DNA. Proteins have a low ability to cleave target DNA (e.g., single-stranded target DNA), but In some cases, variants retain the ability to bind to target DNA (e.g., single-stranded DNA). If the Cas9 protein has the W476A and W1126A mutations, or if the variant Cas9 The protein has the following mutations: P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A In some cases, the variant Cas9 protein does not bind efficiently to the PAM sequence. In some such cases, such variant Cas9 proteins may be used in a manner that binds to the target protein. When using a PAM sequence, this method does not require a PAM sequence. When using such variant Cas9 proteins in methods of conjugation, Although the method can include a guide RNA, this method can be performed in the absence of a PAM sequence. (Thus, specificity of binding is provided by the target segment of the guide RNA). Other residues (i.e., inactive residues or other nuclease moieties) may be added to achieve the above effects. Non-limiting examples include residues D10, G12, G17, E762, H840, and N854. N863, H982, H983, A984, D986, and / or A987 can be modified (i.e., substituted). Furthermore, mutations other than alanine substitution are preferred.

[0233] In some embodiments, the CRISPR protein-derived domain of the base editor is canonical. The Cas9 protein may comprise all or part of a Cas9 protein having a CAL PAM sequence (NGG). In this case, the Cas9-guided domain of the base editor may employ a non-canonical PAM sequence. Such sequences are known in the art and will be apparent to those skilled in the art. For example, non-canonical PAMs The Cas9 domain binding sequences are incorporated herein by reference in their entirety. Kleinstiver, B.P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B.P., et al. ., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modi "Fying PAM recognition" Nature Biotechnology 33, 1293-1298 (2015) do.

[0234] In some examples, the CRISPR protein-derived domains of the base editors disclosed herein The PAM recognized by the insert (e.g., AAV insert) encoding the base editor The nucleotide sequence may be provided to the cell on a separate oligonucleotide from the nucleotide sequence (sert). By providing PAM on a separate oligonucleotide, the target sequence and A target sequence that is not cleaved due to the absence of an adjacent PAM on the same polynucleotide is called a cleavage target. It becomes possible to split it.

[0235] In one embodiment, S. pyogenes Ca is used as a CRISPR endonuclease for genome engineering. s9 (SpCas9) can be used, but others may also be used. Different endonucleases can be used to target different genomic targets. In some cases, synthetic SpCas9 derivatives with non-NGG PAM sequences are used. Furthermore, other Cas9 orthologues from various species have been identified, and these "non- SpCas9 can bind to a variety of PAM sequences that may be useful in the present disclosure. For example, However, due to its relatively large size (approximately 4 kb coding sequence), SpCas9 is not expressed efficiently in cells. Conversely, the expression of SpCas9 in Staphylococcus aureus may be inhibited by the plasmid carrying the SpCas9 cDNA. The coding sequence of Cas9 (SaCas9) from Reus is approximately 1 kilobase shorter than SpCas9, which allows This likely allows for efficient expression in cells. Nucleases can modify target genes in vitro in mammalian cells or in vivo in mice. In some cases, Cas proteins target different PAM sequences. In some cases, the target gene can be adjacent to a Cas9 PAM, e.g., 5'-NGG. In other cases, other Cas9 orthologs may have different PAM requirements. For example, S. thermophilus philus (5'-NNAGAA for CRISPR1 and 5'-NGGNG for CRISPR3) and Neisseria me Other PAMs, such as ningiditis (5'-NNNNGATT), may also be found adjacent to target genes. do.

[0236] In some embodiments, for S. pyogenes systems, the target gene sequence is preceded by a 5'-NGG PAM. The 20-nt guide RNA sequence can base-pair with the opposing strand (i.e., can be at its 5'-). In some cases, the adjacent cleavage occurs at the PAM site. In some cases, the adjacent cleavage may be approximately 10 base pairs upstream of the PAM. In some instances, the adjacent cleavage may be approximately 0-20 base pairs upstream of the PAM. For example, adjacent cuts are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 of the PAM. , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs upstream The adjacent cleavage may be 1 to 30 base pairs downstream of the PAM. Fusion proteins containing nuclear localization sequences (NLS) In some embodiments, the fusion proteins provided herein comprise one or more It further comprises a nuclear targeting sequence, e.g., 2, 3, 4, 5), such as a nuclear localization sequence (NLS). In embodiments, a two-component NLS is used. In some embodiments, the NLS is a tandem NLS-containing amino acids that promote the import of proteins into the cell nucleus (e.g., by nuclear transport) In some embodiments, any of the fusion proteins provided herein comprises a In some embodiments, the fusion protein further comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the Cas9 domain. In some embodiments, the NLS is fused to the C-terminus of the nCas9 domain or dCas9 domain. In some embodiments, the NLS is fused to the N-terminus of the deaminase. In some embodiments, the NLS is fused to the C-terminus of the deaminase. In some embodiments, the NLS is one or more In some embodiments, NL is fused to the fusion protein via multiple linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. The amino acid sequence of any one of the NLS sequences provided or referenced herein. Localization sequences are known in the art and will be apparent to those skilled in the art. For example, NLS sequences are exemplified by See Plank et al., P.M., the contents of which are incorporated herein by reference for their disclosure of suitable nuclear localization sequences. In some embodiments, the NLS is the amino acid sequence PKKKR KVEGADKRTADGSEFES PKKKRKV, KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTK KL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRKPKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGR In some embodiments, the NLS is present in the linker or the NLS is present in the linker. In some embodiments, the linker is located adjacent to a linker, such as a linker described herein. In the case of N- or C-terminal NLS, the NLS is a two-component NLS. Two-component NLSs consist of two basic amino acid clusters. These are separated by a relatively short spacer sequence (hence the two components (One-component NLSs are not.) The nucleoplasmic NLS, KR[PAATKKAGQA]KKKK, is a ubiquitous two-component signaling It is a prototype of a nucleotide, and has basic amino acids separated by a spacer of about 10 amino acids. The sequence of an exemplary two-component NLS is: PKKKRKVEGAD KRTADGSEFES PKKKRKV

[0237] In some embodiments, the fusion proteins of the invention do not include a linker sequence. In some embodiments, there is a linker sequence between one or more of the domains or proteins. There is.

[0238] It is recognized that the fusion proteins of the present disclosure may include one or more additional features. For example, in some embodiments, the fusion protein may comprise an inhibitor, a cytoplasmic-localized sequence, or a sequences, export sequences such as nuclear export sequences, or other localization sequences, as well as fusion sequences. The conjugated protein may contain sequence tags useful for solubilizing, purifying, or detecting the conjugated protein. Suitable protein tags provided herein include, but are not limited to, biotin carboxylates. Enzyme carrier protein (BCCP) tag, myc tag, calmodulin tag, FLAG tag, Polyhistidine, also known as HA tag, histidine tag, or His tag Tag, maltose-binding protein (MBP) tag, nus tag, glutathione-S-transferase GST tag, green fluorescent protein (GFP) tag, thioredoxin tag, S tag, Softag (e.g. Softag 1, Softag 3), Streptag, Biotin Ligase Further suitable sequences are known to those skilled in the art. In s...

Claims

1. 1. An in vitro or ex vivo method of editing an MECP2 polynucleotide comprising a single nucleotide polymorphism (SNP) associated with Rett Syndrome (RTT), comprising contacting the MECP2 polynucleotide with a base editor complexed with one or more guide polynucleotides, wherein the base editor comprises a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain, and one or more of the guide polynucleotides targets the base editor to result in an A.T to G.C modification of the SNP associated with RTT, wherein the SNP associated with RTT results in expression of a MECP2 polypeptide comprising the amino acid modification R168X, T158M, R255X, or R270X, where X is a stop codon.

2. The method described in claim 1, wherein the A / T to G / C modification in the SNP associated with RTT changes a methionine to a threonine or a stop codon to an arginine in the MECP2 polypeptide.

3. The method described in claim 1, wherein the modification in the SNP associated with RTT results in expression of a MECP2 polypeptide containing arginine at amino acid position 168, 255, or 270, or threonine at position 158.

4. The method described in claim 1, wherein the napDNAbp domain is Cas9 (SpCas9) of Streptococcus pyogenes.

5. The napDNAbp domain of SpCas9 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:42, and comprising the following amino acid substitutions as numbered in SEQ ID NO:42: D1135V or D1135L; G1218R or G1218S; E1219F or E1219V; A1322R; R1335V or R1335Q; and T1337F, T1337H, T1337I, T1337K, T1337L, T1337M, T1337N, T1337Q, T1337R, T1337S, or T1337V The method of claim 4 further comprising:

6. The napDNAbp domain of SpCas9, a) further comprising the amino acid substitution L1111R and an amino acid substitution selected from the group consisting of D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, and D1332R, as numbered in SEQ ID NO: 42; or b) the following amino acid substitutions, numbered as in SEQ ID NO:42: D1135L; S1136R; G1218S or G1218R; E1219V or E1219F; A1322R; R1335Q; and T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, or T1337M and further comprising an amino acid substitution selected from the group consisting of D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, and D1332R. The method according to claim 5.

7. The adenosine deaminase domain comprising the amino acid sequence: SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD The method of claim 1, comprising an amino acid sequence having at least 90% identity to 8. The method of claim 1, wherein the one or more guide RNAs comprise a CRISPR RNA (crRNA) and a transcoding small RNA, the crRNA comprises a nucleic acid sequence complementary to the MECP2 polynucleotide, and / or the base editor is complexed with a single guide RNA comprising a nucleic acid sequence complementary to the MECP2 polynucleotide.

9. A base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain, or a polynucleotide encoding said base editor; one or more guide polynucleotides that target the base editor to result in an A.T to G.C modification of a SNP associated with RTT, wherein the SNP associated with RTT results in expression of a MECP2 polypeptide comprising the amino acid modification R168X, T158M, R255X, or R270X, where X is a stop codon; A cell or a precursor thereof, in vitro or ex vivo.

10. The cell described in claim 9, wherein the A / T to G / C modification in the SNP associated with RTT changes methionine to threonine or a stop codon to arginine in the MECP2 polypeptide.

11. The cell described in claim 9, wherein the modification in the SNP associated with RTT results in expression of a MECP2 polypeptide containing arginine at amino acid position 168, 255, or 270, or threonine at position 158.

12. The cell described in claim 9, wherein the napDNAbp domain is Cas9 (SpCas9) of Streptococcus pyogenes.

13. The napDNAbp domain of SpCas9 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:42, and comprising the following amino acid substitutions as numbered in SEQ ID NO:42: D1135V or D1135L; G1218R or G1218S; E1219F or E1219V; A1322R; R1335V or R1335Q; and T1337F, T1337H, T1337I, T1337K, T1337L, T1337M, T1337N, T1337Q, T1337R, T1337S, or T1337V The cell of claim 12, further comprising:

14. The napDNAbp domain of SpCas9, a) further comprising the amino acid substitution L1111R and an amino acid substitution selected from the group consisting of D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, and D1332R, as numbered in SEQ ID NO: 42; or b) the following amino acid substitutions, numbered as in SEQ ID NO:42: D1135L; S1136R; G1218S or G1218R; E1219V or E1219F; A1322R; R1335Q; and T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, or T1337M and further comprising an amino acid substitution selected from the group consisting of D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, and D1332R. The cell of claim 13.

15. The adenosine deaminase domain comprising the amino acid sequence: SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD The cell of claim 9, comprising an amino acid sequence having at least 90% identity to 16. A composition comprising a cell according to any one of claims 9 to 15 for use in a method of treating RTT in a subject, the method comprising administering the cell to the subject.

17. A composition comprising a base editor or a polynucleotide encoding same for use in a method of treating RTT in a subject, the method comprising: the base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain, or a polynucleotide encoding the base editor; one or more guide polynucleotides that target the base editor to result in an A.T to G.C modification of a SNP associated with RTT, wherein the SNP associated with RTT results in expression of a MECP2 polypeptide comprising the amino acid modification R168X, T158M, R255X, or R270X, where X is a stop codon; to said subject. composition.

18. The composition of claim 17, wherein the A / T to G / C modification in the RTT-associated SNP changes a methionine to a threonine or a stop codon to an arginine in the MECP2 polypeptide.

19. The composition described in claim 17, wherein the A / T to G / C modification in the SNP associated with RTT results in expression of a MECP2 polypeptide containing arginine at amino acid position 168, 255, or 270, or threonine at position 158.

20. The adenosine deaminase domain comprising the amino acid sequence: SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD The composition of claim 17, comprising an amino acid sequence having at least 90% identity to

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