Methods for suppressing pathogenic mutations using programmable base editor systems

Abstract

To provide a composition and a method using a base editor comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain conjunction with a guide polynucleotide, as well as a base editor system for editing neucleobases of a target nucleotide sequence.SOLUTION: Disclosed is a method for editing SERPINA1 polynucleotide having single nucleobase polymorphism related to alpha-1 antitrypsin deficiency (A1AD), where the SERPINA1 polynucleotide is contacted with a base editor conjugated to one or more guide polynucleotides, the base editor comprises a polynucleotide programmable DNA binding domain and a cytidine deaminase domain, where the one or more guide polynucleotides target the base editor to effect an alteration of a single nucleotide polymorphism (SNP) related to A1AD.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] Related Applications This application is a continuation of U.S. Provisional Application No. 62 / 670,498, filed May 11, 2018, and U.S. Provisional Application No. 62 / 670,498, filed December 17, 2018. This application claims the benefit of U.S. Provisional Application No. 62 / 780,864, the entire contents of each of which are incorporated herein by reference. is incorporated herein by reference. Incorporation by Reference All publications, patents, and patent applications mentioned herein are the property of their respective owners. All publications, patents, or patent applications are specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety. be incorporated into the book.

[0002] For most known genetic disorders, there are several methods to study or address the underlying cause of the disease. , which requires correction of a point mutation at the target locus rather than stochastic disruption of the gene. [Background technology]

[0003] Clustered regularly interspaced short palindromic repeat (CRISPR) system Current genome editing techniques induce double-stranded DNA breaks at target loci as the first step in gene correction. In response to double-stranded DNA breaks, the cell's DNA repair process mostly involves repairing the DNA break. Non-homologous end joining at the break site results in random insertions or deletions (indels). Most genetic disorders result from point mutations, but current approaches to point mutation correction These methods are inefficient and typically result from a cellular response to dsDNA breaks at the target locus. Induce more random insertions and deletions (indels) in the genome, thus making it more efficient and , and unwanted products such as stochastic insertions or deletions (indels) or translocations are much more likely to occur. Fewer and improved forms of genome editing are needed.

[0004] Alpha-1 antitrypsin deficiency (A1AD) is a condition characterized by the loss of the alpha-1 antitrypsin (A1AT) protein. Pathogenic variants in the SERPINA1 gene, which encodes the protein A1AT is a genetic disorder that results in decreased production of neutrophil elastase. A1 is a factor that protects tissues and organs, such as the lungs, from elastin degradation. Elastin in the lungs of patients with AD is more readily degraded by neutrophil elastase. Over time, loss of lung elasticity develops into chronic obstructive pulmonary disease (COPD). In humans, A1AT is produced in hepatocytes within the liver and secreted into the systemic circulation, where it The protein functions as a protease inhibitor.

[0005] The most common pathogenic A1AT variant is a guanine to adenine variant in the SERPINA1 gene. A mutation to (G → A) results in a glutamic acid at amino acid 342 of the A1AT protein. This substitution results in a lysine to lysine substitution, which leads to protein misfolding and hepatocyte This leads to its polymerization and ultimately to toxic aggregates that can lead to liver damage and cirrhosis. Hepatotoxicity can be controlled by gene knockout (CRISPR / ZFN / TALEN) or gene knockdown (siRNA) Although these approaches may address pathology in the lungs, Pulmonary pathology can be managed with protein replacement therapy, but this therapy is hepatotoxic. Gene therapy is also inadequate for treating genetic deficiencies of A1AT. The liver of patients with A1AD already harbors a heavy disease burden caused by endogenous A1AT aggregation. Therefore, gene therapy to increase A1AT in the liver would be counterproductive. There is a need for methods of treating patients with A1AD that address both the associated pulmonary pathology and liver toxicity. Summary of the Invention

[0006] Provided herein are methods for treating a genetic disorder in a subject, the methods comprising administering a base

[0023] Injecting the base editor, or a polynucleotide encoding the base editor, into a subject in need thereof. administering (wherein the base editor is a polynucleotide programmable nucleotide a deaminase domain) and a guide polynucleotide; administering the guide polynucleotide to a target nucleic acid sequence of interest, Targeting to the nucleotide sequence; Upon targeting, base editors deaminate the target nucleotides. Editing the nucleic acid bases of a nucleic acid sequence, thereby changing the nucleic acid bases to different nucleic acid bases. and treating a genetic disorder by the nucleic acid is in a protein-coding region and the nucleic acid base is not the cause of the genetic disorder (i.e., the The nucleobases do not code for the mutations that cause the genetic disease).

[0007] Provided herein are methods for producing cells, tissues, or cells for treating genetic disorders in a subject in need thereof. Also provided is a method for producing an organ, the method comprising: contacting the base editor with a polynucleotide encoding the base editor (wherein The base editor comprises a polynucleotide programmable nucleotide binding domain and a deaminase domain); and contacting the cell, tissue, or organ with the guide polynucleotide; targeting a base editor to a target nucleotide sequence; By deaminating nucleobases upon targeting base editors to sequences. Editing the nucleic acid base of the target nucleotide sequence, thereby changing the nucleic acid base to another nucleic acid base and producing cells, tissues, or organs for treating genetic disorders by inducing wherein said nucleobase is in a protein coding region of a polynucleotide, In some embodiments, the method further comprises: In some embodiments, the method comprises administering the cell, tissue, or organ to a subject. Or the organ is autologous to the subject. The tissue, or organ is allogeneic to the subject. The cells, tissues, or organs are xenogenic to the subject.

[0008] In some embodiments, changing the nucleobase to another nucleobase comprises modifying the polynucleobase. In some embodiments, the activity of a protein encoded by the nucleotide sequence of the present invention is increased. , changing a nucleobase to another nucleobase is encoded by a polynucleotide This results in improved protein folding and / or increased stability. In this context, changing one nucleobase to another nucleobase is a step in the translation of the nucleic acid encoded by a polynucleotide. In some embodiments, the expression of the protein The increase in protein translation rate is due to improved translation rate of the protein. Increased expression of a protein is associated with increased expression of the protein in the organelle or cellular compartment that contains it. In some embodiments, increased expression of the protein is due to increased release rate from the This is due to improved processing of the protein's signal peptide. In some embodiments, increased expression of a protein is due to a correlation between the protein and another protein. This is due to changes in interactions.

[0009] In some embodiments, the nucleobase is located in a gene that causes a genetic disorder. In some embodiments, the editing comprises editing a plurality of nucleobases located in the gene. In some embodiments, the plurality of nucleobases is not responsible for a genetic disorder. The collection may include editing one or more additional nucleic acid bases located in at least one other gene. In some embodiments, the gene and at least one other gene In some embodiments, the nucleic acid encodes one or more subunits of a protein. The acid-base is in a gene listed in Tables 3A and 3B herein, and the edit is in a gene listed in Table 3A and result in amino acid changes in the proteins encoded by the genes shown in 3B. .

[0010] In some embodiments, the genetic disorder is retinitis pigmentosa, Usher syndrome, sickle cell disease, or the like. Hemocytosis, beta-thalassemia, alpha-1-antitrypsin deficiency (A1AD), hepatic porphyrins deficiency, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency , phenylketonuria, hemochromatosis, von Gierke disease, Pompe disease, Gaucher disease In some embodiments, the condition is a chronic condition such as rheumatoid arthritis, ... The genetic disorder is alpha 1-antitrypsin deficiency (A1AD). The base edits are selected from the group consisting of F51L, M374I, A348V, A347V, K387R, T59A, and T68A. This results in amino acid changes in the α1-antitrypsin (A1AT) protein. In embodiments, the base editing results in an M374I amino acid change in A1AT.

[0011] In one embodiment, the genetic disorder is sickle cell disease. In one embodiment, the editing is In one embodiment, the amino acid changes reduce the ability of the A / HbS tetramer to polymerize. The nucleobase is located in the HBB gene, which encodes the beta subunit of hemoglobin (HbB). In some embodiments, the HBB gene is the sickle cell hemoglobin allele (HbS). In this form, editing results in amino acid changes in the beta subunit of hemoglobin. In some embodiments, the amino acid changes in the beta subunit of hemoglobin are A70T, A70V, In one embodiment, the nucleotide sequence includes L88P, F85L, F85P, E22G, G16D, G16N, or a combination thereof. The nucleic acid base is a member of the HBA1 or HBA2 gene encoding the α subunit of hemoglobin (HbA). In one embodiment, the edit is located at an amino acid in the α subunit of hemoglobin. In one embodiment, the amino acid changes in the α subunit result in a sickle cell It is located at the interface between the α and β subunits of hemoglobin. In some embodiments, the amino acid changes in the alpha subunit of hemoglobin are K11E, D47G, Includes Q54R, N68D, E116K, H20Y, H50Y or combinations thereof.

[0012] In one embodiment, a programmable nucleobase editor is used to suppress pathogenic mutations. The present invention provides compositions and methods for using base editors (e.g., BE4) to The present invention provides a method for treating A1AD by inducing modifications to the endogenous SERPINA1 gene using a modified SERPINA1 gene. The SERPINA1 gene stabilises E342K in the α1-antitrypsin protein, M37 Introduction of M374I using BE4 alleviates liver toxicity and improves circulation of A1AT to the lung. This may result in a concomitant increase in ring size, thereby compensating for the presence of the deleterious E342K mutation. This strategy aims to simultaneously remove pathogenic protein burden from the liver and deliver functional protein to the lungs. Restores quality.

[0013] In another aspect, the present invention provides a method for treating alpha 1-antitrypsin deficiency (A1AD)-associated monosodium glutamate (MSG)-related alpha 1-antitrypsin deficiency (A1AD). The present invention provides a method for editing a SERPINA1 polynucleotide containing a single base polymorphism (SNP), and the method The present invention relates to a method for the preparation of a SERPINA1 polynucleotide comprising: wherein the base editor is a polynucleotide program. a cytidine deaminase domain and a DNA binding domain capable of binding to one or more guide The polynucleotide targets a base editor to identify a single nucleotide polymorphism ( In one embodiment, the contacting results in an alteration of the nucleotide sequence (SNP) in the cell, in a eukaryotic cell, in a mammalian cell, In another embodiment, the cells are in vivo or ex vivo. is.

[0014] In another aspect, the present invention provides base editors, polynucleotides encoding the base editors, and nucleotides (where the base editor is a polynucleotide programmable DNA binding domain) and a cytidine deaminase domain); and targeting base editors to produce SEs one or more nucleotides that deaminate the cytidine at nucleic acid position 1455 of the RPINA1 polynucleotide and a guide polynucleotide; In one embodiment, the cells produced are hepatocytes. In another embodiment, The cells or their precursors are embryonic cells, induced pluripotent stem cells, or hepatocytes. In one embodiment, the hepatocytes express an A1AT polypeptide. In another embodiment, the cells have an A1AD polypeptide. In another embodiment, the cell is a mammalian cell or a human cell.

[0015] In another aspect, the invention provides a method for treating a cancer, comprising administering to a subject a cell of any of the preceding aspects. In one embodiment, the cells are In another embodiment, the cells are allogeneic (i.e., allogeneic) to the subject. allogeneic).

[0016] In another aspect, the invention provides a method for producing a cell line comprising growing or expanding a cell of any of the preceding aspects. An isolated cell or cell population is provided.

[0017] In another aspect, the present invention provides a method of treating A1AD in a subject, the method comprising: A base editor, or a polynucleotide encoding a base editor (wherein the base editor Deter is a polynucleotide-programmable DNA-binding domain and cytidine deaminase domain and include ); targeting a base editor to modify a nucleotide sequence at nucleic acid position 1455 of the SERPINA1 polynucleotide; and one or more guide polynucleotides that modify the cytidines present in the to a subject.

[0018] In one embodiment of the above-described aspects, the subject is a mammal or a human. In embodiments of the present invention, the method further comprises: and delivering the nucleic acid sequence and one or more guide polynucleotides to cells of the subject. In another embodiment, the cells are hepatocytes. In another embodiment, the cells are hepatocytes. In another embodiment, the hepatocyte expresses the A1AT protein.

[0019] In another aspect, a method for producing hepatocytes or progenitor cells thereof is provided, the method comprising: (a) Transfection of liver progenitor cells containing an A1AD-associated SNP with a base editor or its coding sequence Polynucleotide (wherein the base editor is a polynucleotide programmable nucleotide a nucleotide-binding domain and a cytidine deaminase domain; and one or more guide a polynucleotide (wherein one or more guide polynucleotides target a base editor) SERPINA1 polynucleotide. (resulting in thiazolidine deamination), and (b) differentiating the hepatic progenitor cells into stem cells. In one embodiment, the method comprises differentiating hepatic progenitor cells into stem cells. In one embodiment, the hepatic progenitor cells express an A1AT polypeptide. In another embodiment, the hepatic progenitor cells are derived from a mammalian subject with A1AD. cells or human cells.

[0020] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: 5'-CAAUCAUUAAGAAGACAAAGGGUUU-3' 5'-UCAAUCAUUAAGAAGACAAAGGGUUU-3' 5'-UUCAAUCAUUAAGAAGACAAAGGGUUU-3' 5'-GUUCAAUCAUUAAGAAGACAAAGGGUUU-3' 5'-UGUUCAAUCAUUAAGAAGACAAAGGGUUU-3' 5'-UUGUUCAAUCAUUAAGAAGACAAAGGGUU-3' 5'-UUCAAUCAUUAAGAAGACAAAG-3' 5'-UUCAAUCAUUAAGAAGACAAAGG-3' 5'-UCAAUCAUUAAGAAGACAAAGGG-3' 5'-AAUCAUUAAGAAGACAAAGGGU-3' The present invention provides a guide RNA comprising a nucleic acid sequence selected from:

[0021] In another aspect, the present invention provides a method for producing a method of manufacturing a device according to any of the embodiments described herein or otherwise illustrated. Provide a guide RNA comprising 18, 19, 20, 21, or 22 nucleotides of the guide RNA.

[0022] In another aspect, the invention provides a base editor according to the embodiments described herein and a method for the preparation of a base editor according to the invention. and a guide RNA as described in

[0023] In any of the above aspects or any other aspect of the invention described herein, The base editor deaminates the cytidine at position 1455 of the SERPINA1 polynucleotide thereby converting methionine to isoleucine at amino acid position 374 of the A1AT protein. Any of the above aspects or any of the inventions described herein In other embodiments, the A1AT polypeptide comprises a lysine at amino acid position 342, and and / or contains a lysine at amino acid position 376. In another embodiment of any of the inventions described herein, a polynucleotide programmable The DNA-binding domain is Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof In any of the above aspects or any other aspect of the invention described herein, SpCas9 has specificity for PAM sequences selected from 5'-NGG-3' or 5'-GGG-3'. do.

[0024] In any of the above aspects or any other aspect of the invention described herein, Polynucleotide-programmable DNA-binding domains are nuclease-inactive variants or a nickase variant. In any other embodiment, the nickase variant has the amino acid substitution D10A or and the corresponding amino acid substitutions. In other embodiments of any of the above, the cytidine deaminase domain is a deoxyribonucleic acid ( Any of the above aspects can deaminate cytidine in DNA. or any other embodiment of the invention described herein, a cytidine deaminase is a non-naturally occurring, modified cytidine deaminase. or any other embodiment of the invention described herein, a cytidine deaminase is an APOBEC deaminase. In any other embodiment, the base editor is BE4. In other embodiments of any of the inventions described herein, the one or more guide RNAs are It contains small trans-coding RNAs (crRNAs) and trans-coding small RNAs (tracrRNAs), and crRNAs are associated with A1AD. Any of the above embodiments includes a nucleic acid sequence complementary to a SERPINA1 nucleic acid sequence containing a SNP that or any other embodiment of the invention described herein, the base editor is A single guide R containing a nucleic acid sequence complementary to the SERPINA1 nucleic acid sequence encoding thionine 374 It forms a complex with NA (sgRNA).

[0025] In some embodiments, any of the methods provided herein further comprises the step of: In some cases, the additional nucleobases are not the cause of the genetic disorder. In some cases, the additional nucleobase is the cause of the genetic disorder. .

[0026] In some embodiments, the deaminase domain is a cytidine deaminase domain. In some embodiments, the deaminase domain is a deaminase domain. The enzyme domain is a cytidine deaminase domain. The deaminase domain is an adenosine deaminase domain. In this study, the adenosine deaminase domain binds to adenosine in deoxyribonucleic acid (DNA). In some embodiments, the guide poly Nucleotides include ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In embodiments, the guide polynucleotide comprises a CRISPR RNA (crRNA) sequence, a transactivating mediated CRISPR RNA (tracrRNA) sequences, or combinations thereof.

[0027] In some embodiments, any of the methods provided herein further comprises a second guide In some embodiments, the second guide polynucleotide The nucleic acid may be ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In this embodiment, the second guide polynucleotide comprises a CRISPR RNA (crRNA) sequence, a transactivating In some embodiments, the gene includes a recombinant CRISPR RNA (tracrRNA) sequence, a modified CRISPR RNA (tracrRNA) sequence, or a combination thereof. and a second guide polynucleotide directs the base editor to a second target nucleotide sequence. In some embodiments, the polynucleotide programmable The DNA-binding domains that can be used include the Cas9 domain, Cpf1 domain, CasX domain, CasY domain, and C In some embodiments, the as12b / C2c1 domain or the Cas12c / C2c3 domain is included. The polynucleotide programmable DNA binding domain is nuclease-dead. In some embodiments, the polynucleotide-programmable DNA-binding domain In some embodiments, the polynucleotide programmable In some embodiments, the DNA binding domain comprises a Cas9 domain. nuclease-inactive Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active C In some embodiments, the Cas9 domain comprises a Cas9 nickase. In some embodiments, the polynucleotide programmable DNA binding domain is or a modified polynucleotide programmable DNA binding domain.

[0028] In some embodiments, any of the methods provided herein further comprises adding a second base enzyme. In some embodiments, the second base editor further comprises a first base editor. or comprises a deaminase domain distinct from the primary base editor.

[0029] In some embodiments, the base editing results in less than 20% indel formation. In some embodiments, the editing results in less than 15% indel formation. In some embodiments, the editing results in less than 10% indel formation. In some embodiments, the editing results in less than 5% indel formation. The editing results in less than 4% indel formation. In some embodiments, the editing results in less than 3% indel formation. In some embodiments, the editing results in less than 2% indel formation. In some embodiments, the editing results in less than 1% indel formation. In some embodiments, the editing results in less than 0.5% indel formation. In some embodiments, the editing results in less than 0.1% indel formation. In this embodiment, the editing does not result in a translocation.

[0030] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of this point can be obtained by reference to the following detailed description. The accompanying drawings illustrate exemplary embodiments, in which the principles of the present disclosure are utilized, and in which: That's right. [Brief explanation of the drawings]

[0031] [Figure 1] Figure 1 shows a schematic diagram comparing a healthy individual with a patient with antitrypsin deficiency (A1AD). In healthy individuals, alpha-1 antitrypsin (A1AT) protects the lungs from protease damage, and the liver releases alpha-1 antitrypsin into the bloodstream. In patients with A1AD, the lack of functioning A1AT protein leads to lung tissue damage. Furthermore, accumulation of abnormal A1AT in liver cells leads to liver cirrhosis.

[0032] [Figure 2] Figure 2 is a graph showing the typical range of serum alpha-1 antitrypsin (A1AT) levels for different genotypes (normal (MM); heterozygous carriers of alpha-1 antitrypsin deficiency (MZ, SZ); homozygous deficiency (SS, ZZ)). Serum alpha-1 antitrypsin (AAT) concentrations are expressed in μM on the left "y" axis, as is common in the literature. The right "y" axis shows the approximate conversion of serum AAT concentrations to mg / dL, as commonly reported in clinical laboratories and by various measurement techniques (turbidimetry and radial immunodiffusion).

[0033] [Figure 3] Figure 3 shows the sequence of the target site for introducing the suppressor mutation M374I into SERPINA1. The standard spCas9 NGG PAM and the target C, whose editing results in the desired codon change M374I, are highlighted. The off-target C, which, if edited, results in the undesired codon change E376K, is also labeled.

[0034] [Figure 4] Figure 4 shows the levels of secreted proteins in the culture supernatant of HEK293T cells transiently transfected with plasmids encoding different variants of the A1AT protein. A1AT concentrations were determined by ELISA as published in Borel, Florie & Mueller, Christian. (2017) Alpha-1 Antitrypsin Deficiency: Methods and Protocols. 10.1007 / 978-1-4939-7163-3 (the entire contents of which are incorporated by reference). The two most common clinical variants (i.e., pathogenic mutations) of A1AT are E264V (PiS allele) and E342K (PiZ allele). PiS and PiZ proteins are produced in lower amounts than the wild-type proteins. Addition of the M374I suppressor mutation, referred to as the "complementing mutation" in Figure 4, appears to increase the levels of secreted PiS and PiZ A1AT proteins. Therefore, we hypothesized that introducing the M374I mutation using the base editors and base editing methods described herein could increase A1AT secretion from hepatocytes, alleviating liver toxicity while also increasing A1AT circulation to the lungs. A1AT: alpha-1 antitrypsin; A1AD: alpha-1 antitrypsin deficiency; "Z mutation" is E342K (PiZ allele); "S mutation" is E264V (PiS allele).

[0035] [Figure 5]Figure 5 is a bar graph showing the efficiency of base editing of the M374I mutation in HEK293T. Use of bpNLS outperformed the SV40 nuclear localization signal. Compared to the original codon usage, codon optimization 2 resulted in higher mutation efficiency both when delivered as a plasmid and as mRNA + gRNA.

[0036] [Figure 6] Figure 6 shows a schematic diagram of the strategy for evolving DNA deoxyadenosine deaminase starting from TadA. The E. coli library contained a plasmid library of mutant ecTadA (TadA*) genes fused to dCas9 and a selection plasmid requiring a targeted A·T to G·C mutation to repair the antibiotic resistance gene. Mutations from surviving TadA* variants were imported into an ABE construct for base editing in humans.

[0037] [Figure 7] 7 shows a graph depicting the predicted functional elastase activity of base-edited A1AT variants. The percent elastase activity relative to the wild-type (WT) A1AT is shown for the A1AT variant with the E342K (PiZ) mutation, the A1AT variant with the E342K mutation and the complementary M374I mutation, the A1AT variant with the E264V (PiS) mutation, and the A1AT variant with the E264V mutation and the complementary M374I mutation.

[0038] [Figure 8] Figures 8A-8C provide three graphs showing the percentage of base edits observed in HEK293 cells (Figure 8A) and induced pluripotent stem cells (iPSCs) (Figure 8B), respectively, transfected with the base editor BE4. Figure 8C shows the percent of edits achieved when wild-type primary hepatocytes were transfected.

[0039] [Figure 9]Figure 9 shows the percent base editing achieved and A1AT secretion in hepatocytes derived from BE4-edited IPSCs. DETAILED DESCRIPTION OF THE INVENTION

[0040] 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 particular embodiments described herein, as such may vary. Those skilled in the art will recognize that this disclosure has numerous variations and modifications that are encompassed within its scope. You will recognize it.

[0041] All terms are intended to be understood as would be understood by a person skilled in the art. Unless otherwise defined, all technical and scientific terms used herein are defined by the principles of the present disclosure. The designations have the same meaning as commonly understood by one of ordinary skill in the art to which they pertain.

[0042] Practice of the several embodiments disclosed herein is within the skill of those of ordinary skill in the art, unless otherwise indicated. Within the scope of skill, immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics Use conventional techniques of chemistry and recombinant DNA. See, e.g., Sambrook and Green, Molecul ar Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocol s in Molecular Biology (FM Ausubel, et al. eds.); the series Methods In Enzym ology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and See Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0043] The section headings used herein are for organizational purposes only and are not intended to be limiting unless otherwise stated. This document should not be construed as limiting the subject matter covered.

[0044] Various features of the disclosure may be described in the context of a single embodiment, but these features may also be combined in a single embodiment. They may also be provided separately or in any suitable combination. Although for clarity, may be described in the context of separate embodiments, the present disclosure also provides a single embodiment. It can also be implemented in this state.

[0045] definition The following definitions are supplemental to those in the art and are directed to this application: and any related or unrelated cases, e.g., any co-owned patents or No patent application should be attributed to this application. Any method and material can be used in the practice of the present disclosure for testing. However, preferred methods and materials are described herein. The terminology is for the purpose of describing particular embodiments only and is not intended to be limiting. .

[0046] Unless otherwise defined, all technical and scientific terms used herein are defined by the It has the meaning commonly understood by one of ordinary skill in the art to which the invention pertains. , provides those skilled in the art with general definitions of many of the terms used in this invention: n et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The C ambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary o f Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0047] In this application, the use of the singular includes the plural unless specifically stated otherwise. Where used, the singular forms "a," "an," and "the" shall be used unless the context clearly indicates otherwise. It should be noted that unless specifically indicated, plural referents are included. In this context, the use of "or" means "and / or" unless stated otherwise. The terms "including," as well as "include," "includes," and " The use of other forms such as "included" is non-limiting.

[0048] As used in this specification and claims, the terms "comprising" (and " "comprise" and "comprises" and all its forms), "having" g) (any of its forms, such as "have" and "has"), "include "including" ("include" and "includes" and any of its forms) or "including containing (any of its forms, such as "contains" and "contain") ) is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Any embodiment discussed herein may be used with respect to any method or composition of the present disclosure. It is believed that the same can be done, and vice versa. The method of the present disclosure can be achieved by

[0049] The terms "about" or "approximately" refer to a range of values ​​as determined by one of ordinary skill in the art. This means that the value is within an acceptable margin of error for a particular value, which indicates how How it is measured or determined depends in part on the limitations of the measurement system. For example, "about" means, according to practice in the art, within 1 or more than 1 standard deviation. Alternatively, "about" can mean up to 20%, up to 10%, up to 5%, or Alternatively, it may refer to a range of up to 1% of the total mass of a biological system or process. This term means values ​​within the same order of magnitude, preferably within 5-fold, more preferably within 2-fold. Where specific values ​​are described in the application and claims, Unless otherwise stated, "about" means within an acceptable margin of error for that particular value. The term should be inferred.

[0050] In the specification, "some embodiments," "an embodiment," "one embodiment," or Reference to "another embodiment" may include any particular features, structures, or features are included in at least some embodiments of the present disclosure, but not necessarily all. This means that the embodiments are not necessarily included.

[0051] "Administering" refers to providing one or more compositions described herein to a patient or subject. By way of example and not limitation, administration of a composition For example, injections can be intravenous (iv), subcutaneous (sc), intradermal (id), or intraperitoneal (i. p.) injection or intramuscular (i.m.) injection. Using more than one such route. Parenteral administration can be, for example, by bolus injection or by gradual infusion over time. Alternatively, or concurrently, administration may be by oral route. It is possible.

[0052] "Adenosine deaminase" is an enzyme that hydrolyzes adenine (A) to inosine (I). It refers to a deaminase that catalyzes deamination. In some embodiments, a deaminase or The deaminase domain converts adenosine or deoxyadenosine to inosine or deoxyadenosine, respectively. or adenosine deaminase, which catalyzes the hydrolytic deamination to deoxyinosine. In some embodiments, adenosine deaminase binds to adenosine in deoxyribonucleic acid (DNA). The adenosine deamination enzymes provided herein catalyze the hydrolytic deamination of adenosine. adenosine deaminase (e.g., engineered adenosine deaminase, evolved adenosine deaminase) The enzyme (adenosine) can be from any organism, such as a bacterium. Deaminase is a nucleotide deaminase that can be used in, for example, E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, In some embodiments, the antibody is from a bacterium such as C. nzae, or C. crescentus. The adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase The deaminase is E. coli TadA (ecTadA) deaminase or a fragment thereof.

[0053] For example, truncated ecTadA lacks one or more N-terminal amino acids relative to full-length ecTadA. In some embodiments, the truncated ecTadA may be missing the full-length ecTadA. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19 or 20 for In some embodiments, the truncated amino acid sequence may be deleted. The ecTadA fragments were 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 relative to the full-length ecTadA fragment. 15, 6, 17, 18, 19 or 20 C-terminal amino acid residues may be deleted. In some embodiments, the ecTadA deaminase does not contain an N-terminal methionine. In an embodiment, the TadA deaminase is an N-terminally truncated TadA. In embodiments, TadA is described in PCT / US2017 / 045, which is incorporated herein by reference in its entirety. 381.

[0054] An "agent" is any small molecule compound, antibody, nucleic acid molecule, or polypeptide. , or fragments thereof.

[0055] "Ameliorate" means to reduce, inhibit, or attenuate the occurrence or progression of a disease. means to cause, decrease, stop, or stabilize.

[0056] "Alteration" refers to any alteration that can be detected by standard art known methods such as those described herein. Such a change (increase or decrease) in the expression level or activity of a gene or polypeptide is As used herein, modification means a 10% change in expression level, preferably a 25% change. A change of 10% or more, more preferably a change of 40%, and most preferably a change of 50% or more in expression levels.

[0057] "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 the corresponding naturally occurring polypeptide. specific biochemical properties that enhance the function of the analog compared to the native polypeptide while retaining the Such biochemical modifications may occur, for example, by altering ligand binding. It can increase the protease resistance, membrane permeability, or half-life of the analogue. The analogs may include unnatural amino acids.

[0058] "Alpha-1 antitrypsin (A1AT) protein" is under UniProt accession number P01009 The present invention also refers to a polypeptide or fragment thereof having at least about 95% amino acid sequence identity to the In certain embodiments, the A1AT protein has one or more of the following reference sequences: In one particular embodiment, the A1AT protein associated with A1AD contains multiple modifications, including E342 An exemplary A1AT amino acid sequence is provided below: >sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens OX=9606 GN=SERPINA1 PE =1 SV=3: MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFS LYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGF QELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQ INDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTV KVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFL ENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKA VLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK

[0059] The term "base editor (BE)" refers to the modification of nucleic acid bases within a nucleic acid sequence (e.g., DNA or RNA). Agents containing polypeptides that can provide modifications to groups (e.g., A, T, C, G, or U) In some embodiments, the base editor represents a guide polynucleotide (e.g., a polynucleotide with a programmable nucleotide-binding domain and a nucleic acid Base editing domains (e.g., cytidine deaminase domains or adenosine deaminase In some embodiments, the base editor is a fusion protein comprising a is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). A programmable DNA binding domain is fused or linked to a deaminase domain. In some embodiments, the base editor is a guide polynucleotide (e.g., a guide Polynucleotide programmable DNA binding domain and deaminase domain together with In some embodiments, the polynucleotide-programmable DNA-binding domain The base is a CRISPR-associated (e.g., Cas or Cpf1) enzyme. The editor may further include a Cas9 protein that contains a deaminase domain (e.g., adenosine deaminase or cytidine deaminase). In some embodiments, the base The editor consists of a catalytically inactive (dead) Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9 ) fused to the deaminase domain. In some embodiments, the salt is fused to an inhibitor of base excision repair (BER). The inhibitor of base excision repair is uracil DNA glycosylase inhibitor (UGI). In this embodiment, the inhibitor of base excision repair is an inosine base excision repair inhibitor. In some embodiments, the base editor is capable of deaminating a base in a nucleic acid. In some embodiments, the base editor deaminates bases in a DNA molecule. In some embodiments, the base editor can deaminate bases in an RNA molecule. In some embodiments, the base editor can de-adenine (A). In some embodiments, the adenosine deaminase can be adenosine deaminase from TadA. In some embodiments, the base editor is evolved from a guanine (G) In some embodiments, the base editor can deaminate adenine In some embodiments, the base editor can deaminate (A). The base editor can deaminate tosine (C). For details, please refer to the international application PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), each of which is incorporated herein by reference in its entirety. Komor, AC, et al., "P rogrammable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable ba se editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 46 4-471 (2017); and Komor, AC, et al., “Improved base excision repair inhibit ion and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher See also “Efficiency and product purity” Science Advances 3:eaao4774 (2017) (among which The entire contents of which are incorporated herein by reference in their entirety.

[0060] In some embodiments, the base editing compositions, systems, and The cytidine base editor BE4 used in the method is represented by the following nucleic acid sequence provided below: (8877 base pairs) (Addgene, Watertown, MA; Komor AC, et al., 2017, Sci Adv ., 30;3(8):eaao4774. doi: 10.1126 / sciadv.aao4774). At least one nucleotide sequence corresponding to the BE4 nucleic acid sequence is Polynucleotide sequences having 95% or more identity to the sequence are also encompassed. 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 tcttagccc cgcctctcta atcccgacg gctagaaac 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 tttaccgac 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 tcgccaatag gaactttatg cagctgatcc atgatgactc tttaaccttc 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 gaaatacgac 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 ttatggagcg ctcgtcttttt gaaaagaacc ccatcgactt ccttgaggcg 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 gcaaaaagg 8221 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 8281 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 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

[0061] In some embodiments, a cytidine base editor has the sequence: ATGagctcagagactggcccagtggctgtggaccccacattgagacggcggatcgagccccatgagtttgaggtattctt cgatccgagagagctccgcaaggagacctgcctgctttacgaaattaattgggggggccggcactccatttggcgacata catcacagaacactaacaagcacgtcgaagtcaacttcatcgagaagttcacgacagaaagatatttctgtccgaacaca aggtgcagcattacctggtttctcagctggagcccatgcggcgaatgtagtagggccatcactgaattcctgtcaaggta tccccacgtcactctgtttatttacatcgcaaggctgtaccaccacgctgacccccgcaatcgacaaggcctgcgggatt tgatctcttcaggtgtgactatccaaattatgactgagcaggagtcaggatactgctggagaaactttgtgaattatagc ccgagtaatgaagcccactggcctaggtatccccatctgtgggtacgactgtacgttcttgaactgtactgcatcatact gggcctgcctccttgtctcaacattctgagaaggaagcagccacagctgacattctttaccatcgctcttcagtcttgtc attackgcgactgcccccacacattctctgggccaccgggttgaaatctggtggttcttctggtggttctagcggcagc gagactcccgggacctcagagtccgccacacccgaaagttctggtggttcttctggtggttctgataaaaagtattctat tggtttagccatcggcactaattccgttggatgggctgtcataaccgatgaatacaaagtaccttcaaagaaatttaagg tgttggggaacacagaccgtcattcgattaaaaagaatcttatcggtgccctcctattcgatagtggcgaaacggcagag gcgactcgcctgaaacgaaccgctcggagaaggtatacacgtcgcaagaaccgaatatgttacttacaagaaatttttag caatgagatggccaaagttgacgattctttctttcaccgtttggaagagtccttccttgtcgaagaggacaagaaacatg aacggcaccccatctttggaaacatagtagatgaggtggcatatcatgaaaagtacccaacgatttatcacctcagaaaa aagctagttgactcaactgataaagcggacctgaggttaatctacttggctcttgcccatatgataaagttccgtgggca ctttctcattgagggtgatctaaatccggacaactcggatgtcgacaaactgttcatccagttagtacaaacctataatc agttgtttgaagagaaccctataaatgcaagtggcgtggatgcgaaggctattcttagcgcccgcctctctaaatcccga cggctagaaaacctgatcgcacaattacccggagagaagaaaaatgggttgttcggtaaccttatagcgctctcactagg cctgacaccaaattttaagtcgaacttcgacttagctgaagatgccaaattgcagcttagtaaggacacgtacgatgacg atctcgacaatctactggcacaaattggagatcagtatgcggacttatttttggctgccaaaaaccttagcgatgcaatc ctcctatctgacatactgagagttaatactgagattaccaaggcgccgttatccgcttcaatgatcaaaaggtacgatga acatcaccaagacttgacacttctcaaggccctagtccgtcagcaactgcctgagaaatataaggaaatattctttgatc agtcgaaaaacgggtacgcaggttatattgacggcggagcgagtcaagaggaattctacaagtttatcaaacccatatta gagaagatggatgggacggaagagttgcttgtaaaactcaatcgcgaagatctactgcgaaagcagcggactttcgacaa cggtagcattccacatcaaatccacttaggcgaattgcatgctatacttagaaggcaggaggatttttatccgttcctca aagacaatcgtgaaaagattgagaaaatcctaacctttcgcataccttactatgtgggacccctggcccgagggaactct cggttcgcatggatgacaagaaagtccgaagaaacgattactccatggaattttgaggaagttgtcgataaaggtgcgtc agctcaatcgttcatcgagaggatgaccaactttgacaagaatttaccgaacgaaaaagtattgcctaagcacagtttac tttacgagtatttcacagtgtacaatgaactcacgaaagttaagtatgtcactgagggcatgcgtaaacccgcctttcta agcggagaacagaagaaagcaatagtagatctgttattcaagaccaaccgcaaagtgacagttaagcaattgaaagagga ctactttaagaaaattgaatgcttcgattctgtcgagatctccggggtagaagatcgatttaatgcgtcacttggtacgt atcatgacctcctaaagataattaaagataaggacttcctggataacgaagagaatgaagatatcttagaagatatagtg ttgactcttaccctctttgaagatcgggaaatgattgaggaaagactaaaaacatacgctcacctgttcgacgataaggt tatgaaacagttaaagaggcgtcgctatacgggctggggacgattgtcgcggaaacttatcaacgggataagagacaagc aaagtggtaaaactattctcgattttctaaagagcgacggcttcgccaataggaactttatgcagctgatccatgatgac tctttaaccttcaaagaggatatacaaaaggcacaggtttccggacaaggggactcattgcacgaacatattgcgaatct tgctggttcgccagccatcaaaaagggcatactccagacagtcaaagtagtggatgagctagttaaggtcatgggacgtc acaaaccggaaaacattgtaatcgagatggcacgcgaaaatcaaacgactcagaaggggcaaaaaaacagtcgagagcgg atgaagagaatagaagagggtattaaagaactgggcagccagatcttaaaggagcatcctgtggaaaatacccaattgca gaacgagaaactttacctctattacctacaaaatggaagggacatgtatgttgatcaggaactggacataaaccgtttat ctgattacgacgtcgatcacattgtaccccaatcctttttgaaggacgattcaatcgacaataaagtgcttacacgctcg gataagaaccgagggaaaagtgacaatgttccaagcgaggaagtcgtaaagaaaatgaagaactattggcggcagctcct aaatgcgaaactgataacgcaaagaaagttcgataacttaactaaagctgagaggggtggcttgtctgaacttgacaagg ccggatttattaaacgtcagctcgtggaaacccgccaaatcacaaagcatgttgcacagatactagattcccgaatgaat acgaaatacgacgagaacgataagctgattcgggaagtcaaagtaatcactttaaagtcaaaattggtgtcggacttcag aaaggattttcaattctataaagttagggagataaataactaccaccatgcgcacgacgcttatcttaatgccgtcgtag ggaccgcactcattaagaaatacccgaagctagaaagtgagtttgtgtatggtgattacaaagtttatgacgtccgtaag atgatcgcgaaaagcgaacaggagataggcaaggctacagccaaatacttcttttattctaacattatgaatttctttaa gacggaaatcactctggcaaacggagagatacgcaaacgacctttaattgaaaccaatggggagacaggtgaaatcgtat gggataagggccgggacttcgcgacggtgagaaaagttttgtccatgccccaagtcaacatagtaaagaaaactgaggtg cagaccggagggttttcaaaggaatcgattcttccaaaaaggaatagtgataagctcatcgctcgtaaaaaggactggga cccgaaaaagtacggtggcttcgatagccctacagttgcctattctgtcctagtagtggcaaaagttgagaagggaaaat ccaagaaactgaagtcagtcaaagaattattggggataacgattatggagcgctcgtcttttgaaaagaaccccatcgac ttccttgaggcgaaaggttacaaggaagtaaaaaaggatctcataattaaactaccaaagtatagtctgtttgagttaga aaatggccgaaaacggatgttggctagcgccggagagcttcaaaaggggaacgaactcgcactaccgtctaaatacgtga atttcctgtatttagcgtcccattacgagaagttgaaaggttcacctgaagataacgaacagaagcaactttttgttgag cagcacaaacattatctcgacgaaatcatagagcaaatttcggaattcagtaagagagtcatcctagctgatgccaatct ggacaaagtattaagcgcatacaacaagcacagggataaacccatacgtgagcaggcggaaaatattatccatttgttta ctcttaccaacctcggcgctccagccgcattcaagtattttgacacaacgatagatcgcaaacgatacacttctaccaag gaggtgctagacgcgacactgattcaccaatccatcacgggattatatgaaactcggatagatttgtcacagcttggggg tgactctggtggttctggaggatctggtggtctactaatctgtcagatattattgaaaggagaccggtaagcaactgg ttatccaggaatccatcctcatgctcccagaggaggtggaagaagtcattgggaacaagccggaaagcgatatactcgtg cacaccgcctacgacgagagcaccgacgagaatgtcatgcttctgactagcgacgcccctgaatacaagccttgggctct ggtcatacaggatagcaacggtgagaacaagattaagatgctctctggtggttctggaggatctggtggttctactaatc tgtcagatattattgaaaaggagaccggtaagcaactggttatccaggaatccatcctcatgctcccagaggaggtggaa gaagtcattgggaacaagccggaaagcgatatactcgtgcacaccgcctacgacgagagcaccgacgagaatgtcatgct tctgactagcgacgcccctgaatacaagccttgggctctggtcatacaggatagcaacggtgagaacaagattaagatgc tctctggtggttctAAAAGGACGGCGGACGGATCAGAGTTCGAGAGTCCGAAAAAAAAACGAAAGGTCGAAtaa

[0062] In some embodiments, the basic editor has the following sequence. ATGTCATCCGAAACCGGGCCAGTGGCCGTAGACCAACACTCAGGAGGCGGATAGAACCCCATGAGTTTGAAGTGTTCTT CGACCCCAGAGAGCTGCGCAAAGAGACTTGCCTCCTGTATGAAATAAATTGGGGGGGTCGCCATTCAATTTGGAGGCACA CTAGCCAGAATACTAACAAACACGTGGAGGTAAATTTTATCGAGAAGTTTACCACCGAAAGATACTTTTGCCCCAATACA CGGTGTTCAATTACCTGGTTTCTGTCATGGAGTCCATGTGGAGAATGTAGTAGAGCGATAACTGAGTTCCTGTCTCGATA TCCTCACGTCACGTTGTTTATATACATCGCTCGGCTTTATCACCATGCGGACCCGCGGAACAGGCAAGGTCTTCGGGACC TCATATCCTCTGGGGTGACCATCCAGATAATGACGGAGCAAGAGAGCGGATACTGCTGGCGAAACTTTGTTAACTACAGC CCAAGCAATGAGGCACACTGGCCTAGATATCCGCATCTCTGGGTTCGACTGTATGTCCTTGAACTGTACTGCATAATTCT GGGACTTCCGCCATGCTTGAACATTCTGCGGCGGAAACAACCACAGCTGACCTTTTTCACGATTGCTCTCCAAAGTTGTC ACTACCAGCGATTGCCACCCCACATCTTGTGGGCTACTGGACTCAAGTCTGGAGGAAGTTCAGGCGGAAGCAGCGGGTCT GAAACGCCCGGAACCTCAGAGAGCGCAACGCCCGAAAGCTCTGGAGGGTCAAGTGGTGGTAGTGATAAGAAATACTCCAT CGGCCTCGCCATCGGTACGAATTCTGTCGGTTGGGCCGTTATCACCGATGAGTACAAGGTCCCTTCTAAGAAATTCAAGG TTTTGGGCAATACAGACCGCCATTCTATAAAAAAAAACCTGATCGGCGCCCTTTTGTTTGACAGTGGTGAGACTGCTGAA GCGACTCGCCTGAAGCGAACTGCCAGGAGGCGGTATACGAGGCGAAAAAACCGAATTTGTTACCTCCAGGAGATTTTCTC AAATGAAATGGCCAAGGTAGATGATAGTTTTTTTCACCGCTTGGAAGAAAGTTTTCTCGTTGAGGAGGACAAAAAGCACG AGAGGCACCCAATCTTTGGCAACATAGTCGATGAGGTCGCATACCATGAGAAATATCCTACGATCTATCATCTCCGCAAG AAGCTGGTCGATAGCACGGATAAAGCTGACCTCCGGCTGATCTACCTTGCTCTTGCTCACATGATTAAATTCAGGGGCCA TTTCCTGATAGAAGGAGACCTCAATCCCGACAATTCTGATGTCGACAAACTGTTTATTCAGCTCGTTCAGACCTATAATC AACTCTTTGAGGAGAACCCCATCAATGCTTCAGGGGTGGACGCAAAGGCCATTTTGTCCGCGCGCTTGAGTAAATCACGA CGCCTCGAGAATTTGATAGCTCAACTGCCGGGTGAGAAGAAAAACGGGTTGTTTGGGAATCTCATAGCGTTGAGTTTGGG ACTTACGCCAAACTTTAAGTCTAACTTTGATTTGGCCGAAGATGCCAAATTGCAGCTGTCCAAAGATACCTATGATGACG ACTTGGATAACCTTCTTGCGCAGATTGGTGACCAATACGCGGATCTGTTTCTTGCCGCAAAAAATCTGTCCGACGCCATA CTCTTGTCCGATATACTGCGCGTCAATACTGAGATAACTAAGGCTCCCCTCAGCGCGTCCATGATTAAAAGATACGATGA GCACCACCAAGATCTCACTCTGTTGAAAGCCCTGGTTCGCCAGCAGCTTCCAGAGAAGTATAAGGAGATATTTTTCGACC AATCTAAAAACGGCTATGCGGGTTACATTGACGGTGGCGCCTCTCAAGAAGAATTCTACAAGTTTATAAAGCCGATACTT GAGAAAATGGACGGTACAGAGGAATTGTTGGTTAAGCTCAATCGCGAGGACTTGTTGAGAAAGCAGCGCACATTTGCAA TGGTAGTATTCCACACCAGATTCATCTGGGCGAGTTGCATGCCATTCTTAGAAGACAAGAAGATTTTTATCCGTTTCTGA AAGATAACAGAGAAAAAGATTGAAAAGATACTTACCTTTCGCATACCGTATTATGTAGGTCCCCTGGCTAGAGGGAACAGT CGCTTCGCTTGGATGACTCGAAAATCAGAAGAAAACAATAACCCCCTGGAATTTTGAAAGAAGTGGTAGATAAAGGTGCGAG TGCCCAATCTTTTATTGAGCGGATGACAAATTTTGACAAGAATCTGCCTAACGAAAAGGTGCTTCCCAAGCATTCCCTTT TGTATGAATACTTTACAGTATATAATGAACTGACTAAAGTGAAGTACGTTACCGAGGGGATGCGAAAGCCAGCTTTTTCTC AGTGGCGAGCAGAAAAAGCAATAGTTGACCTGCTGTTCAAGACGAATAGGAAGGTTACCGTCAAACAGCTCAAAGAAGA TTACTTTAAAAGATCGAATGTTTTGATTCAGTTGAGATAAGCGGAGTAGAGGATAGATTTAACCCAAGTCTTGGGAACTT ATCATGACCTTTTGAAGATCATCAAGGATAAAGATTTTTTGGACAACGAGGAGAATGAAGATATCCTGGAAGATATAGTA CTTACCTTGACGCTTTTTGAAGATCGAGAGATGATCGAGGAGCGACTTAAGACGTACGCACATCTCTTTGACGATAAGGT TATGAAACAATTGAAACGCCGGCGGTATACTGGCTGGGGCAGGCTTTCTCGAAAGCTGATTAATGGTATCCGCGATAAGC AGTCTGGAAAGACAATCCTTGACTTTCTGAAAAGTGATGGATTTGCAAATAGAAACTTTATGCAGCTTATACATGATGAC TCTTTGACGTTCAAGGAAGACATCCAGAAGGCACAGGTATCCGGCCAAGGGGATAGCCTCCATGAACACATAGCCAACCT GGCCGGCTCACCAGCTATTAAAAAGGGAATATTGCAAACCGTTAAGGTTGTTGACGAACTCGTTAAGGTTATGGGCCGAC ACAAACCAGAGAATATCGTGATTGAGATGGCTAGGGAGAATCAGACCACTCAAAAAGGTCAGAAAAATTCTCGCGAAAGG ATGAAGCGAATTGAAGAGGGAATCAAAGAACTTGGCTCTCAAATTTTGAAAGAGCACCCGGTAGAAAACACTCAGCTGCA GAATGAAAAGCTGTATCTGTATTATCTGCAGAATGGTCGAGATATGTACGTTGATCAGGAGCTGGATATCAATAGGCTCA GTGACTACGATGTCGACCACATCGTTCCTCAATCTTTCCTGAAAGATGACTCTATCGACAACAAAGTGTTGACGCGATCA GATAAGAACCGGGGAAAATCCGACAATGTACCCTCAGAAGAAGTTGTCAAGAAGATGAAAAACTATTGGAGACAATTGCT GAACGCCAAGCTCATAACACAACGCAAGTTCGATAACTTGACGAAAGCCGAAAGAGGTGGGTTGTCAGAATTGGACAAAG CTGGCTTTATTAAGCGCCAATTGGTGGAGACCCGGCAGATTACGAAACACGTAGCACAAATTTTGGATTCACGAATGAAT ACCAAATACGACGAAAACGACAAATTGATACGCGAGGTGAAAGTGATTACGCTTAAGAGTAAGTTGGTTTCCGATTTCAG GAAGGATTTTCAGTTTTACAAAGTAAGAGAAATAAACAACTACCACCACGCCCATGATGCTTACCTCAACGCGGTAGTTG GCACAGCTCTTATCAAAAAATATCCAAAGCTGGAAAGCGAGTTCGTTTACGGTGACTATAAAGTATACGACGTTCGGAAG ATGATAGCCAAATCAGAGCAGGAAATTGGGAAGGCAACCGCAAAATACTTCTTCTATTCAAACATCATGAACTTCTTTAA GACGGAGATTACGCTCGCGAACGGCGAAATACGCAAGAGGCCCCTCATAGAGACTAACGGCGAAACCGGGGAGATCGTAT GGGACAAAGGACGGGACTTTGCGACCGTTAGAAAAGTACTTTCAATGCCACAAGTGAATATTGTTAAAAAGACAGAAGTA CAAACAGGGGGGTTCAGTAAGGAATCCATTTTGCCCAAGCGGAACAGTGATAAATTGATAGCAAGGAAAAAAGATTGGGA CCCTAAGAAGTACGGTGGTTTCGACTCTCCTACCGTTGCATATTCAGTCCTTGTAGTTGCGAAAGTGGAAAAGGGGAAAA GTAAGAAGCTTAAGAGTGTTAAAGAGCTTCTGGGCATAACCATAATGGAACGGTCTAGCTTCGAGAAAAATCCAATTGAC TTTCTCGAGGCTAAAGGTTACAAGGAGGTAAAAAAGGACCTGATAATTAAACTCCCAAAGTACAGTCTCTTCGAGTTGGA GAATGGGAGGAAGAGAATGTTGGCATCTGCAGGGGAGCTCCAAAAGGGGAACGAGCTGGCTCTGCCTTCAAAATACGTGA ACTTTCTGTACCTGGCCAGCCACTACGAGAAACTCAAGGGTTCTCCTGAGGATAACGAGCAGAAACAGCTGTTTGTAGAG CAGCACAAGCATTACCTGGACGAGATAATTGAGCAAATTAGTGAGTTCTCAAAAAGAGTAATCCTTGCAGACGCGAATCT GGATAAAGTTCTTTCCGCCTATAATAAGCACCGGGACAAGCCTATACGAGAACAAGCCGAGAACATCATTCACCTCTTTA CCCTTACTAATCTGGGCGCGCCGGCCGCCTTCAAATACTTCGACACCACGATAGACAGGAAAAGGTATACGAGTACCAAA GAAGTACTTGACGCCACTCTCATCCACCAGTCTATAACAGGGTTGTACGAAACGAGGATAGATTTGTCCCAGCTCGGCGG CGACTCAGGAGGGTCAGGCGGCTCCGGTGGATCAACGAATCTTTCCGACATAATCGAGAAAGAAACCGGCAAACAGTTGG TGATCCAAGAATCAATCCTGATGCTGCCTGAAGAAGTAGAAGAGGTGATTGGCAACAAACCTGAGTCTGACATTCTTGTC CACACCGCGTATGACGAGAGCACGGACGAGAACGTTATGCTTCTCACTAGCGACGCCCCTGAGTATAAACCATGGGCGCT GGTCATCCAAGATTCCAATGGGGAAAACAAGATTAAGATGCTTAGTGGTGGGTCTGGAGGAGCGGTGGGTCCACGAACC TCAGCGACATTATTGAAAAAGAGACTGGTAAACAACTTGTAATACAAGAGTCTATTCTGATGTTGCCTGAAGAGGTGGAG GAGGTGATTGGGAACAAACCGGAGTCTGATATACTTGTTCATACCGCCTATGACGAATCTACTGATGAGAATGTGATGCT TTTaACGTCAGACGCTCCCGAGTACAAACCCTGGGCTCTGGTGATTCAGGACAGCAATGGTGAGAATAAGATTAAAATGT TGAGTGGGGGCTCAAAGCGCACGGCTGACGGTAGCGAATTTGAGAGCCCCAAAAAAAAACGAAAGGTCGAAtaa

[0063] "Base editing activity" refers to the ability to chemically modify bases within a polynucleotide. In one embodiment, the first base is converted to the second base. The base editing activity is a cytidine deaminase activity that converts the target C·G to T·A, for example. In another embodiment, the base editing activity is adenosine deaminase, e.g., converting A·T to G·C. Enzyme activity.

[0064] The term "base editor system" refers to a system for editing nucleic acid bases of a target nucleotide sequence. In some embodiments, the base editor system comprises: (1) a polynucleotide sequence; A programmable nucleotide-binding domain and a nucleotide-binding domain for deaminating nucleobases (2) a base editor (BE) containing a deaminase domain for the translation of polynucleotides A guide polynucleotide (e.g., a guide polynucleotide) associated with a programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleic acid The nucleic acid 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 adenosine base editor (ABE). be.

[0065] In some embodiments, the nucleobase editor system comprises a plurality of base editing components. For example, the nucleobase editor system can include multiple deaminases. In some embodiments, the nuclease base editing system can include one or more cytogenes. In some embodiments, the enzyme may comprise one or more adenosine deaminases. In embodiments, a single guide polynucleotide is utilized to target different deaminases. In some embodiments, a pair of guide polynucleotides can be targeted to a nucleic acid sequence. The nucleic acids can be used to target different deaminases to target nucleic acid sequences. do.

[0066] Nucleic acid base components of base editor systems and polynucleotide programmable Nucleotide binding moieties can be covalently or non-covalently linked to one another. For example, in some embodiments, the deaminase domain is Targeting to target nucleotide sequences by a ramifiable nucleotide-binding domain In some embodiments, polynucleotides can be programmable to bind nucleotides. The binding domain may be fused or linked to the deaminase domain. In the present invention, the polynucleotide programmable nucleotide binding domain is a deaminase by non-covalently interacting or binding with the deaminase domain. The enzyme can be targeted to a target nucleotide sequence. For example, some In embodiments, the nucleobase editing component, e.g., the deaminase component, is a polynucleotide protease. Further heterologous moieties or domains that are part of the grammable nucleotide binding domain. Further heterologous moieties or moieties capable of interacting with, associating with, or forming a complex with the In some embodiments, the additional heterologous moiety can comprise a poly It can bind to, interact with, associate with, or form a complex with a peptide. In some embodiments, the additional heterologous moiety binds to, interacts with, or associates with the polynucleotide. In some embodiments, additional heterologous The moiety is capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous moiety can be attached to the polypeptide linker. In some embodiments, the additional heterologous moiety can be attached to the polynucleotide linker. The additional heterologous moiety may be a protein domain. The additional heterologous portion contains the K homology (KH) domain, the MS2 coat protein domain, and the PP7 coat protein domain. Protein domain, SfMu Com coat protein domain, steryl α motif, telomerase Ku-binding motif and Ku protein, telomerase Sm7-binding motif and Sm7 protein , or an RNA recognition motif.

[0067] The base editing system can further comprise a guide polynucleotide component. The components of the assembly system are linked by covalent bonds, non-covalent interactions, or their binding and It should be understood that the molecules may be coupled to one another via any combination of interactions. In embodiments, the deaminase domain is cleaved by the guide polynucleotide to the target nucleoside. For example, in some embodiments, the base edit The nucleobase editing component of the target system, e.g., the deaminase component, is interacts with, binds to, or binds to a portion or segment of a polypeptide (e.g., a polynucleotide motif) or further heterologous moieties or domains (e.g., RNA or DNA binding proteins) that can form complexes. In some embodiments, the polypeptide may comprise a polynucleotide binding domain (e.g., a protein-like polynucleotide binding domain). In addition, additional heterologous moieties or domains (e.g., polynucleotides such as RNA or DNA binding proteins) can be added. The protease-binding domain may be fused or linked to the deaminase domain. In an embodiment of the present invention, the additional heterologous moiety binds to, interacts with, or associates with the polypeptide, or can form a complex with a polypeptide. The additional heterologous moiety may bind to, interact with, associate with, or otherwise interact with the polynucleotide. In some embodiments, the additional heterologous moiety is capable of forming a complex with the tide. In some embodiments, additional The heterologous moiety can be attached to a polypeptide linker. In this case, the additional heterologous moiety can be attached to the polynucleotide linker. The species portion may be a protein domain. In some embodiments, additional heterologous sequences may be present. The seed portion contains the K homology (KH) domain, the MS2 coat protein domain, and the PP7 coat protein domain. Main, SfMu Com coat protein domain, sterile alpha motif, telomerase Ku bond binding motif and Ku protein, telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0068] In some embodiments, the base editing system inhibits base excision repair (BER) components. The components of the base editing system can further include covalently or non-covalently bound factors. or any combination of these bonds and interactions. It should be understood that inhibitors of BER components may also inhibit base excision repair. In some embodiments, the inhibitor of base excision repair is uracil DNA glycosylase. In some embodiments, the inhibitor of base excision repair can be an inosine In some embodiments, the inhibitor of base excision repair is a polynucleotide. Nucleotide-programmable nucleotide-binding domains allow target nucleotide sequences to be identified. In some embodiments, polynucleotides can be programmable. A suitable nucleotide binding domain can be fused or linked to an inhibitor of base excision repair. In some embodiments, the polynucleotide programmable nucleotide binding domain comprises: It can be fused or linked to a deaminase domain and an inhibitor of base excision repair. In some embodiments, the polynucleotide programmable nucleotide binding domain is , non-covalently interacting with an inhibitor of base excision repair or inhibiting base excision repair By associating with the inhibitor, the inhibitor targets the base excision repair inhibitor to the target nucleotide sequence. For example, in some embodiments, a base excision repair component can be targeted. The inhibitor of and a further heterologous moiety or domain that is capable of interacting with, associating with, or complexing with a further heterologous moiety or domain that is In some embodiments, the inhibitor of base excision repair comprises a heterologous portion or domain comprising The target nucleotide sequence can be targeted by a guide polynucleotide. For example, in some embodiments, the inhibitor of base excision repair is a guide polynucleotide interacts with, associates with, or binds to a portion or segment (e.g., a polynucleotide motif) of Further heterologous moieties or domains that can be complexed (e.g., RNA or DNA binding proteins) In some embodiments, the polypeptide may comprise a polynucleotide binding domain (e.g., a polynucleotide binding domain). Additional heterologous moieties or domains of the guide polynucleotide (e.g., RNA or DNA binding) Protein-like polynucleotide-binding domain) fused to an inhibitor of base excision repair In some embodiments, the additional heterologous moiety may be a polynucleotide. In some embodiments, the antibody can bind to, interact with, associate with, or complex with the antibody. In some cases, an additional heterologous moiety can be attached to the guide polynucleotide. In this embodiment, the additional heterologous moiety can be attached to the polypeptide linker. In some embodiments, the additional heterologous moiety is attached to the polynucleotide linker. The additional heterologous moiety may be a protein domain. In embodiments, the additional heterologous moiety is a K homology (KH) domain, an MS2 coat protein domain, or a nucleotide sequence encoding the nucleotide sequence of interest. PP7 coat protein domain, SfMu Com coat protein domain, sterile alpha motif, telomerase Ku binding motif and Ku protein, telomerase Sm7 binding motif The Sm7 and Sm8 proteins, or RNA recognition motifs.

[0069] The term "Cas9" or "Cas9 domain" refers to a Cas9 protein or a fragment thereof (e.g., Cas9 Cas9 active, inactive, or partially active DNA cleavage domains, and / or gRNA binding domains of Cas9. Cas9 nuclease refers to an RNA-guided nuclease containing a nucleotide sequence (a protein containing a binding domain). , casnl nuclease or CRISPR (clustered regularly interspaced short palindromic An exemplary Cas9 is the nucleotide sequence of ... The Streptococcus pyogenes Cas9 provided: JPEG2025013768000002.jpg162167 (single underline: HNH domain; double underline: RuvC domain).

[0070] The term "conservative amino acid substitution" or "conservative variation" refers to a mutation in which an amino acid is replaced by another amino acid that shares a common characteristic. It refers to the substitution of an amino acid with another amino acid that has the same properties. It defines the common properties between individual amino acids. A functional method for determining the normality of amino acid changes between corresponding proteins of homologous organisms is The goal is to analyze the frequency of the results (Schulz, GE and Schirmer, RH, Principles of f Protein Structure, Springer-Verlag, New York (1979). According to such analysis, Amino acids within a group are preferentially exchanged with each other, thus affecting the overall protein structure. Define the group of amino acids that are most similar to each other in their effect on the (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include: For example, the amino acids arginine to lysine and the like that can maintain a positive charge are used. Reverse; aspartic acid to glutamic acid and vice versa, which can maintain a negative charge; free threonine to serine, which maintains the -OH; and asparagine to serine, which maintains the free -NH Examples of amino acid substitutions include glutamine.

[0071] The term "Cas9" or "Cas9 domain" refers to a Cas9 protein or a fragment thereof (e.g., Cas9 Cas9 active, inactive, or partially active DNA cleavage domains, and / or gRNA binding domains of Cas9. Cas9 nuclease refers to an RNA-guided nuclease containing a nucleotide sequence (a protein containing a binding domain). , casnl nuclease or CRISPR (clustered regularly interspaced short palindromic An exemplary Cas9 is the nucleotide sequence of ... The Streptococcus pyogenes Cas9 provided: JPEG2025013768000003.jpg162167 (single underline: HNH domain; double underline: RuvC domain).

[0072] The terms "coding sequence" or "protein-coding sequence" are used interchangeably herein. This region or sequence represents a segment of a polynucleotide that encodes a protein. is bounded by a start codon near the 5' end and a stop codon near the 3' end. The coding sequence is also called the open reading frame.

[0073] The term "conservative amino acid substitution" or "conservative variation" refers to a mutation in which an amino acid is replaced by another amino acid that shares a common characteristic. It refers to the substitution of an amino acid with another amino acid that has the same properties. It defines the common properties between individual amino acids. A functional method for determining the normality of amino acid changes between corresponding proteins of homologous organisms is The goal is to analyze the frequency of the results (Schulz, GE and Schirmer, RH, Principles of f Protein Structure, Springer-Verlag, New York (1979). According to such analysis, Amino acids within a group are preferentially exchanged with each other, thus affecting the overall protein structure. Define the group of amino acids that are most similar to each other in their effect on the (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include: For example, the amino acids arginine to lysine and the like that can maintain a positive charge are used. Reverse; aspartic acid to glutamic acid and vice versa, which can maintain a negative charge; free threonine to serine, which maintains the -OH; and asparagine to serine, which maintains the free -NH Examples of amino acid substitutions include glutamine.

[0074] "Cytidine deaminase" is a enzyme that converts amino groups into carbonyl groups through a deamination reaction. In one embodiment, the term "catalyzed polypeptide" refers to a polypeptide or fragment thereof that is capable of catalyzing , cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine PmCDA1 (Petromyzon marinus cytosine deaminase) from Petromyzon marinus 1, "PmCDA1"), AID (active AID) derived from mammals (e.g., humans, pigs, cows, horses, monkeys, etc.) Activation-induced cytidine deaminase (AICDA), and APOBEC are exemplary cytidine deaminases. It is Ze.

[0075] As used herein, the terms "deaminase" or "deaminase domain" and "deaminase domain" refer to refers to a protein or enzyme that catalyzes a deamination reaction. The enzyme or deaminase domain binds to uridine or deoxyuridine, respectively. Cytidine deaminase catalyzing the hydrolytic deamination of cytidine or deoxycytidine In some embodiments, the deaminase or deaminase domain is a cytosine deaminase. enzyme, which catalyzes the hydrolytic deamination of cytosine to uracil. In this case, the deaminase catalyzes the hydrolytic deamination of adenine to hypoxanthine. It is adenine deaminase.

[0076] In some embodiments, the deaminase or deaminase domain is selected from the group consisting of nucleotides of interest, e.g., human, human thymocyte, Natural deamins derived from living organisms such as humans, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is a variant of the deaminase. For example, in some embodiments, a deaminase or The deaminase domain may be at least 50%, at least 55%, or At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least Also, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, 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 at least For example, the deaminase domain may be identified by International PCT Application No. PCT 2004 / 0100666. T / 2017 / 045381 (International Publication No. 2018 / 027078) and PCT / US 2016 / 058344 (International Publication No. 2017 / 07 No. 0632, each of which is incorporated herein by reference in its entirety. It will be done. Komor, AC, et al., “Programmable editing of a target base in genomic DN A without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N .M., 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 ba se editors with higher efficiency and product purity”Science Advances 3:eaao477 4 (2017), and Rees, HA, et al., “Base editing: precision chemistry on the gen ome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec;19(12):770-788 See also doi: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference. nothing).

[0077] A "detectable label" means a label that, when attached to a molecule of interest, is detectable by spectroscopic, photochemical, or biochemical means. By "detectable" is meant a composition that renders it detectable through biological, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent Dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, zygotes xygenin, or hapten.

[0078] "Disease" means any condition that damages or interferes with the normal function of a cell, tissue, or organ. or disorders. Examples of diseases include retinitis pigmentosa, Usher syndrome, sickle cell disease, β-thalassemia, α1-antitrypsin deficiency (A1AD), hepatic porphyria, medium-chain acyl-CoA Dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenylketonuria , hemochromatosis, Von Gierke's disease, Pompe's disease, Gaucher's disease, Hurler's syndrome, cystic fibrosis In one particular embodiment, the disease is A1AD.

[0079] "Effective amount" means the amount of a drug required to improve the symptoms of a disease compared to an untreated patient. or the amount of an active compound (e.g., a base editor as described herein). The effective amount of the active compound used to practice this invention for therapeutic treatment will depend on the mode of administration, This will vary depending on the age, weight, and general health of the subject. A veterinarian will determine the appropriate amount and dosage. Such an amount is called an "effective" amount. In this embodiment, an effective amount is a dose of a gene of interest in a cell (e.g., a cell in vitro or in vivo). In one embodiment, the amount of a base editor of the invention is sufficient to introduce an alteration into The amount is adjusted to achieve a therapeutic effect (e.g., retinitis pigmentosa, Usher syndrome, sickle cell disease, β-thalassemia, α-1 antitrypsin deficiency (A1AD), hepatic porphyria, medium-chain acyl C oA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenylketone Tonuria, hemochromatosis, von Gierke disease, Pompe disease, Gaucher disease, Hurler disease Syndrome, cystic fibrosis, or reducing or controlling chronic pain Such a therapeutic effect is achieved by suppressing the progression of the disease in all cells of a subject, tissue, or organ. It need not be sufficient to alter a gene that is essential for the development of a particular cell type, but rather to alter the cell's ability to differentiate from other cells present in the subject, tissue, or organ. altering virulence genes in approximately 1%, 5%, 10%, 25%, 50%, 75% or more of the cells In one embodiment, the effective amount is a dose that is sufficient to treat a disease (e.g., retinitis pigmentosa, Usher syndrome, sickle erythrocytosis, beta-thalassemia, alpha-1 antitrypsin deficiency (A1AD), hepatic porphyria, Mono-chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenylalanine Ketonuria, hemochromatosis, von Goerke disease, Pompe disease, Gaucher disease, Hurricane Illness sufficient to improve one or more symptoms of rheumatoid arthritis (rheumatoid arthritis, cystic fibrosis, or chronic pain) .

[0080] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. is at least 10%, 20%, 30%, 40%, 50%, 60% of the entire length of the reference nucleic acid molecule or polypeptide , 70%, 80%, or 90%. The fragments may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 1 00, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids It may contain an acid.

[0081] "Hybridization" means hydrogen bonding between complementary nucleobases, as defined by Watson- It can be a Crick, Hoogsteen or reversed Hoogsteen hydrogen bond. For example, Adenine and thymine are complementary nucleobases that form hydrogen bonds to form pairs.

[0082] The terms "inhibitor of base repair," "inhibitor of base repair," "IBR," or other grammatical equivalents The substance is a protein capable of inhibiting the activity of a nucleic acid repair enzyme, such as a base excision repair enzyme. Non-limiting examples of inhibitors of base repair include APE1, Endo III, Endo IV, Endo Inhibition of V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4 PDG, UDG, hSMUGL and hAAG In one embodiment, the base repair inhibitor is an inhibitor of Endo V or hAAG. In certain embodiments, the base repair inhibitor is a catalytically inactive EndoV or catalytic In one embodiment, the base repair inhibitor is uracil glycosyltransferase. UGI is a base excision repair enzyme of uracil-DNA glycosylase. In some embodiments, a UGI domain refers to a protein that can inhibit In some embodiments, the polypeptides provided herein comprise wild-type UGI or fragments thereof. The UGI proteins include fragments of UGI and proteins homologous to UGI or UGI fragments. In certain embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. In some embodiments, the base repair inhibitor is a "catalytically inactive inosine-specific nucleotide The enzymes are "dead inosine-specific nuclease" or "dead inosine-specific nuclease."

[0083] Without wishing to be bound by any particular theory, catalytically inactive inosinic acid glycosylases (e.g., alkyladenine glycosylase (AAG)) bind to inosine However, it cannot create abasic sites or remove inosine, which This sterically blocks the newly formed inosine moiety from the DNA damage / repair mechanism. In some embodiments, the catalytically inactive inosine-specific nuclease is capable of detecting inosine in a nucleic acid. A typical catalytically inactive inoculum can bind to inoculum but does not cleave nucleic acids. Non-limiting examples of synthase-specific nucleases include catalytically inactive (e.g., human) Alkyl adenosine glycosylase (AAG nuclease), and (e.g., from E. coli) Catalytically inactive endonuclease V (EndoV nuclease) is one example. In embodiments, the catalytically inactive AAG nuclease is an AAG nuclease with an E125Q mutation or another AAG containing the corresponding mutation in the nuclease.

[0084] The terms "isolated," "purified," or "biologically pure" refer to a substance that is in its native state. Substances that have been removed to varying degrees from components normally associated with them when found in the natural state. "Isolated" refers to the degree of separation from the original source or surrounding environment. "Purified" refers to the degree of separation from the original source or surrounding environment. A "purified" or "biologically pure" protein is one that is free from impurities. that the substance does not materially affect the biological properties of the protein or cause other adverse consequences. In other words, the nucleic acid or peptide of the present invention is or, if produced by recombinant DNA technology, cellular material, viral material, or culture medium. If the substance is not naturally contained in the substance, or if it is chemically synthesized, chemical precursors or other chemical substances Purity and homogeneity are typically determined by analytical methods. Chemical techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography The term "purified" refers to the degree to which a nucleic acid or protein is purified by electrophoresis. This can mean that the resulting protein essentially produces one band. For proteins that can undergo glycosylation, the different modifications are purified separately. This can result in different isolated proteins that can be isolated.

[0085] An "isolated polynucleotide" is a nucleic acid molecule that does not occur in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. It means nucleic acid (e.g., DNA) that does not contain the genes adjacent to the gene. The term refers to, for example, those incorporated into vectors; autonomously replicating plasmids or viruses. integrated into the genomic DNA of prokaryotes or eukaryotes; or independently of other sequences another molecule created (e.g., cDNA generated by PCR or restriction endonuclease digestion) or genomic or cDNA fragments). RNA molecules transcribed from DNA molecules, as well as hybrids encoding additional polypeptide sequences. It contains recombinant DNA that is part of the hybrid gene.

[0086] An "isolated polypeptide" is a polypeptide of the invention separated from components that naturally accompany it. Typically, a polypeptide is a polypeptide that is a protein with which it is naturally associated. A substance is isolated if it is at least 60% by weight free from substances and naturally occurring organic molecules. Preferably, the preparation comprises at least 75% by weight of soluble fiber, more preferably at least 90% by weight of soluble fiber, most preferably at least 10% by weight of soluble fiber. or at least 99% of the isolated polypeptide of the present invention. can be prepared by, for example, extraction from a natural source, expression of a recombinant nucleic acid encoding such a polypeptide, or the like. or by chemically synthesizing the protein. Purity can be achieved by any suitable method. Suitable methods, such as column chromatography, polyacrylamide gel electrophoresis, or can be measured by HPLC analysis.

[0087] As used herein, the term "linker" refers to a molecule that binds two molecules or moieties (e.g., a Two components of a protein or ribonucleocomplex, or two domains of a fusion protein In, for example, polynucleotides containing programmable DNA binding domains (e.g., dCas9) and deamidation enzyme domains (e.g., adenosine deaminase or cytidine deaminase) refers to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule that The linker can be used to link different components or components of the base editor system. For example, in some embodiments, The linker is a guide pole of the polynucleotide-programmable nucleotide binding domain. The nucleotide binding domain and the catalytic domain of the deaminase can be joined. In some embodiments, the linker connects the CRISPR polypeptide and the deaminase. In some embodiments, a linker can join the Cas9 and the deaminase. In some embodiments, a linker can join the dCas9 and the deaminase. In some embodiments, a linker can join the nCas9 and the deaminase. In embodiments, the linker can connect the guide polynucleotide and the deaminase. In some embodiments, the linker is a deaminase inhibitor of the base editor system. Linking the Polynucleotide Programmable Nucleotide Binding Component and the Polynucleotide In some embodiments, the linker can be used to deamidate the base editor system. The RNA-binding portion of the polynucleotide component and the programmable nucleotide-binding component of the polynucleotide In some embodiments, the linker can link a base editor The RNA-binding portion of the deamination component of the system and the polynucleotide programmable nucleic acid The linker can link the RNA-binding portion of the nucleotide-binding component to the RNA-binding portion of the nucleotide-binding component. A molecule or other moiety that is located between or sandwiched by the molecule and that may be covalently or non-covalently bonded They are linked to each other through bonded interactions, thus linking the two. In some embodiments, the linker can be an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be 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 some embodiments, the ligand is a carbohydrate, peptide, protein, or nucleic acid. In some embodiments, the linker can be an aptamer derived from a riboswitch. The riboswitch from which the aptamer is derived may contain theophylline riboswitch. riboswitch, thiamine pyrophosphate (TPP) riboswitch, adenosine cobalamin (AdoCbl) riboswitch switch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch, flavin moiety Mononucleotide (FMN) riboswitch, tetrahydrofolate riboswitch, lysine riboswitch switch, glycine riboswitch, purine riboswitch, GlmS riboswitch, or prequel In some embodiments, the linker may be selected from the group consisting of poly(Asp-1, Asp-2, Asp-3, Asp-4, Asp-5, Asp-6, Asp-7, Asp-8, Asp-9, Asp-10, Asp-11, Asp-12, Asp-13, Asp-14, Asp-15, Asp-16, Asp-17, Asp-18, Asp-19, Asp-20 Aptamers bound to protein domains such as peptide or polypeptide ligands In some embodiments, the polypeptide ligand may comprise a K homology (KH) domain, an MS2 domain, or a nucleotide sequence. coat protein domain, PP7 coat protein domain, SfMu Com coat protein domain In, sterile α motif, telomerase Ku binding motif and Ku protein, telomerase The Sm7-binding motif and the Sm7 protein may be an RNA recognition motif. In the present invention, the polypeptide ligand can be part of a base editor system component. For example, the nucleobase editing component can include a deaminase domain and an RNA recognition motif.

[0088] In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or In some embodiments, the linker may be about 5 to 100 amino acids in length. For example, lengths of approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-30 , 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids. In some embodiments, the linker has a length of about 100-150, 150-200, 200-250, 250-300, It can be 300-350, 350-400, 400-450, or 450-500 amino acids. Shorter linkers are also contemplated.

[0089] In some embodiments, the linker comprises an RNA programmable The gRNA binding domain of a nuclease and a nucleic acid editing protein (e.g., cytidine or adenine) In some embodiments, the linker connects the catalytic domain of dCas9 to the catalytic domain of dCas9. and the nucleic acid editing protein. For example, a linker can be a linker that connects two groups, molecules, or other Located between or flanked by two groups, molecules, or other moieties, are linked to each other via a covalent bond, thus linking the two. The anchor is an amino acid or multiple amino acids (eg, a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5 to 200 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 95, 100, 150, 100, 250, 350, 400, 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 7 0, 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. Longer or shorter linkers are also possible. In some embodiments, the linker is an amino acid sequence, which may also be referred to as an XTEN 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, (EAAAK) n , (GGS) n , SGSETPGTSESATPES, or (XP) n motif, or any combination thereof, wherein n is independently an integer from 1 to 30, and X is any amino acid. wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In the above, the linker contains multiple proline residues and has a length of 5 to 21, 5 to 14, 5 to 9, 5 to 7 amino acids. amino acids, such as PAPAP, PAPAPA, PAPAPAP, PAPAPAPA, P(AP)4, P(AP)7, P(AP) 10 Such proline-rich linkers are also referred to as "rigid" linkers. Also called.

[0090] In some embodiments, the base editor domain is SGGSSGSETPGTSESATPESSGGS, SGGSSG GSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSP Contains the amino acid sequence AGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS In some embodiments, the base editor domain is fused via a linker. The linker contains the amino acid sequence SGSETPGTSESATPES, which may also be referred to as a linker. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In one embodiment, the linker has the amino acid sequence SGGSSGGSSSGSETPGTES In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker has the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS In one embodiment, the linker is 92 amino acids in length and In some embodiments, the linker has the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPG Contains TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0091] As used herein, the term "mutation" refers to a change in a sequence, e.g., a nucleic acid or amino acid sequence. The substitution of a residue in a sequence of amino acids by another residue, or the deletion of one or more residues in the sequence. Mutations, as used herein, are typically made by identifying the original residue and then and identifying the newly substituted residue, Various methods for making amino acid substitutions (mutations) are provided herein. are well known in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012). In some embodiments, the present disclosure The proposed base editor generates a significant number of unintended mutations, e.g., unintended point mutations. without introducing a "purposeful mutation," e.g., a point mutation, into a nucleic acid (e.g., a nucleic acid in a subject's genome) In some embodiments, the intended mutation can be efficiently generated. The mutation is a guide polynucleotide specifically designed to produce the intended mutation. A specific base editor (e.g., a cytidine base editor or an alpha base editor) bound to the gRNA This mutation is caused by adenosine base editor (DNA).

[0092] Generally, the amino acid sequence of the present invention is made or identified in a sequence (e.g., an amino acid sequence described herein). The mutations detected are numbered relative to the reference (or wild-type) sequence, i.e., the sequence that does not contain the mutation. Those skilled in the art will recognize mutations in amino acid and nucleic acid sequences relative to a reference sequence. It will be easy to understand how to determine the position.

[0093] The term "nuclear localization sequence," "nuclear localization signal," or "NLS" refers to the localization of a protein. The term "nuclear localization sequence" refers to an amino acid sequence that promotes import into the cell nucleus. For example, WO / 2001 / 038547 filed on November 23, 2000 and published on May 31, 2001 is incorporated herein by reference. and Plank et al. in published international PCT application PCT / EP 2000 / 011690, in which No. 6,239,493, which is incorporated herein by reference for its disclosure of exemplary nuclear localization sequences. In embodiments, the NLS may be any of the NLSs described, for example, in Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4 172. In some embodiments, the NLS is Sequence KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR , RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0094] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a nucleic acid molecule comprising a nucleobase and Compounds containing an acidic moiety, such as nucleosides, nucleotides, or polynucleotides Typically, a polymeric nucleic acid, e.g., a nucleic acid molecule containing three or more nucleotides is a linear sequence in which adjacent nucleotides are linked to each other via phosphodiester bonds. In some embodiments, a "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or or nucleosides). In some embodiments, a "nucleic acid" refers to three or more individual nucleosides. As used herein, the term "oligonucleotide" refers to an oligonucleotide chain containing nucleotide residues. "Nucleotide," "polynucleotide," and "polynucleic acid" refer to a polymer of nucleotides. can be used interchangeably to refer to a chain (e.g., a chain of at least three nucleotides). In certain embodiments, "nucleic acid" encompasses RNA and single- and / or double-stranded DNA. Nucleic acids include, for example, genomes, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, cos naturally occurring in the context of a sequence, chromosome, chromatid, or other naturally occurring nucleic acid molecule On the other hand, a nucleic acid molecule can be, for example, a non-naturally occurring molecule, recombinant DNA or RNA, Artificial chromosomes, engineered genomes, or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids non-naturally occurring nucleotides or nucleosides, Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or the like may be used interchangeably. The term analogous includes nucleic acid analogs, eg, analogs having other than a phosphodiester backbone. Nucleic acids may be purified from natural sources, produced using recombinant expression systems, and optionally purified. , chemically synthesized, etc. In the case of chemically synthesized molecules, the nucleic acid may be In some cases, for example, analogs with chemically modified bases or sugars and backbone modifications may be used. Nucleic acid sequences may contain nucleoside analogs such as 5'-3' nucleotides unless otherwise indicated. In some embodiments, the nucleic acid is a nucleotide sequence containing natural nucleosides (e.g., adenosine, thiamine, thiamin ... adenosine, cytidine, uridine, deoxyadenosine, deoxythymidine, oxyguanosine, and deoxycytidine; nucleoside analogs (e.g., 2-aminoadenosine, adenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methyl Cytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodo Uridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2- Aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- Oxoguanine, O 6 -methylguanine, and 2-thiocytidine); chemically modified bases; biological modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibonucleotides); ribose, 2'-deoxyribose, arabinose, and hexose); and / or is a modified phosphate group (e.g., phosphorothioate and 5'-N-phosphoramidite linkages) In some embodiments, the RNA is or comprises an RNA associated with a Cas9 system. For example, RNA can be CRISPR RNA (crRNA), transcoding small RNA (tracrRNA), single It may be a guide RNA (sgRNA), or a guide RNA (gRNA).

[0095] The terms "nucleobase," "nitrogenous base," or "base" are used interchangeably herein. refers to nitrogen-containing biological compounds that form nucleosides, which are nucleotides The ability of nucleobases to form base pairs and stack with each other is directly related to the synthesis of ribonucleic acid ( Adenine (A) is a nucleotide that gives rise to long helical structures such as RNA and deoxyribonucleic acid (DNA). The five nucleic acid bases are cytosine (C), guanine (G), thymine (T), and uracil (U). They are called primary or standard bases. Adenine and guanine are derived from purines, while cytosine, Uracil and thymine are derived from pyrimidines. DNA and RNA contain modified other (non-primary) bases. Non-limiting exemplary modified nucleobases include hypoxanthine, xanthine, , 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydroxybenzoates. Hypoxanthine and xanthine are produced in the presence of mutagens. Both can be produced 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 generated by deamination of cytosine. A "nucleoside" is a nucleic acid base and a five-carbon atom. They consist of a sugar (ribose or deoxyribose). Examples of nucleosides include adenosine , guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, These include deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Nucleosides with modified nucleobases include inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), psoriasis Nucleotides consist of a nucleic acid base, a pentose sugar (ribose or is deoxyribose), and at least one phosphate group.

[0096] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" refers to nucleotide-programmable nucleotide-binding domain" and napDNA A nucleic acid (e.g., DNA or RNA) that guides a nucleotide sequence to a specific nucleic acid sequence, such as a guide nucleic acid. For example, the Cas9 protein binds to a specific DNA sequence complementary to a guide RNA. The sequence can be linked to a guide RNA that guides the Cas9 protein. , napDNAbp is a Cas9 domain, e.g., nuclease-active Cas9, Cas9 nickase (nCas9) , or nuclease-inactive Cas9 (dCas9). Nucleic acid programmable DNA-binding protein Examples of proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, and Cas12c / Cpfl. 2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure. Although they may not be specifically described in this disclosure, for example, Maka rova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct;1:325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4;363(6422 ):88-91. doi: 10.1126 / science.aav7271, and each The entire contents of which are incorporated herein by reference.

[0097] The terms "nucleobase editing domain" or "nucleobase editing protein" are used herein. When used in the formula, cytosine (or cytidine) to uracil (or uridine) or hypoxia to thymine (or thymidine) and from adenine (or adenosine) Deamination to sansine (or inosine), and non-templated nucleotide addition and insertion Proteins or enzymes that can catalyze nucleobase modifications in RNA or DNA, such as refers to an enzyme. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., For example, cytidine deaminase, cytosine deaminase, adenine deaminase, or adenine deaminase. In some embodiments, the nucleobase-editing domain is a naturally occurring In some embodiments, the nucleobase-editing domain may be present in The main focus is on nucleobase editing domains engineered or evolved from naturally occurring nucleobase editing domains. The nucleobase editing domain can be a domain found in bacteria, humans, chimpanzees, gorillas, monkeys, and the like. The nucleic acid may be derived from any organism, such as a human, cow, dog, rat, or mouse. The base-editing proteins are disclosed in International PCT Application No. PCT / 2017 / 045381 (International Publication No. WO 2018 / 027078) and and PCT / US 2016 / 058344 (International Publication No. 2017 / 070632), each of which is , the entire contents of which are incorporated herein by reference. Komor, AC, et al., “Program mable editing of a target base in genomic DNA without double-stranded DNA cleava ge” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base edi ting of A·T to G·C in genomic DNA without DNA cleavage”Nature 551, 464-471 ( 2017); and Komor, AC, et al., “Improved base excision repair inhibition and d bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency See also “Efficiency and product purity” Science Advances 3:eaao4774 (2017) (the entire contents (which is incorporated herein by reference).

[0098] As used herein, "obtaining," as in "obtaining a drug," means This includes synthesizing, purchasing, or otherwise obtaining the drug.

[0099] As used herein, a "patient" or "subject" refers to a person who has been diagnosed with a disease or disorder. Mammals at risk of developing them or suspected of having or developing them In certain embodiments, the term "patient" refers to a subject or individual suffering from a disease or disorder. refers to a mammalian subject with a higher than average likelihood of developing a disease. Exemplary patients include humans, non-human primates, cats, dogs, pigs, cattle, horses, camels, llamas, goats, sheep, rodents (e.g. mice, rabbits, rats, guinea pigs) and will benefit from the treatments disclosed herein. Exemplary human patients include males and / or females. could be.

[0100] A "patient in need thereof" or a "subject in need thereof" is used herein to refer to, for example, Diseases or disorders, including but not limited to, alpha-1 antitrypsin deficiency (A1AD) Refers to a patient diagnosed with or suspected of having a disease or disorder.

[0101] "Pathogenic mutation," "pathogenic variant," "disease-causing mutation," "disease-causing variant" The terms "deleterious mutation" or "predisposing mutation" refer to a mutation that is associated with a particular disease or disorder. Refers to a genetic change or mutation that increases an individual's susceptibility or predisposition. A pathogenic variant is a mutation that results in at least one wild-type mutation in the protein encoded by the gene. This includes those in which an amino acid has been replaced with at least one pathogenic amino acid.

[0102] The terms "peptide," "polypeptide," "protein," and their grammatical equivalents are used interchangeably herein and are linked to each other by a peptide (amide) bond. The term refers to a polymer of amino acid residues bound together in a single chain. It typically refers to a protein, peptide, or polypeptide. A protein, peptide, or polypeptide is at least three amino acids in length. A polypeptide can refer to an individual protein or a group of proteins. One or more amino acids in a protein, peptide, or polypeptide may have, for example, a carbohydrate group, a hydrophobic group, xyl group, phosphate group, farnesyl group, isofarnesyl group, fatty acid group, linkage They may be modified by the addition of chemical entities such as anchors, functionalization, or other modifications. Proteins, peptides, or polypeptides may also be single molecules or multi-molecular. The protein, peptide, or polypeptide may be a naturally occurring It may be merely a fragment of a protein or peptide. Peptides may be naturally occurring, recombinant, or synthetic, or any of the foregoing. As used herein, the term "fusion protein" refers to a combination of: Hybrid polypeptides containing protein domains from at least two different proteins One protein is the amino-terminal (N-terminal) portion of the fusion protein or the capsid. can be located at the carboxy-terminus (C-terminus) of a protein, thus The protein forms a terminal or carboxy-terminal fusion protein. a nucleic acid binding domain (e.g., a nucleic acid binding domain that induces binding of a protein to a target site) Cas9 gRNA binding domain and nucleic acid cleavage domain, or nucleic acid editing protein In some embodiments, the protein may comprise a proteinaceous moiety, e.g., a catalytic domain of For example, an amino acid sequence constituting a nucleic acid binding domain and an organic compound, such as a nucleic acid cleaving agent In some embodiments, proteins include compounds that can act as nucleic acids (e.g., RNA). or DNA) are complexed with or associated with nucleic acids. Any protein can be produced by any method known in the art. For example, the proteins provided herein can be prepared via recombinant protein expression and purification. This is particularly suitable for fusion proteins containing peptide linkers. Methods for recombinant protein expression and purification are well known and are described in Green and Samb rook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Labora and those described by the University of California Press, Cold Spring Harbor, NY (2012). the entire contents of which are incorporated herein by reference.

[0103] The polypeptides and proteins disclosed herein (including functional portions thereof and their functional variants) (including riboflavin) contains 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, aminocyclo Hexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylacetone aminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenyl phenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine phenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-Naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline 2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, Aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyl-lysine, 6- Hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, aminocyclohexyl cyclohexanecarboxylic acid, aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diamino These include monopropionic acid, homophenylalanine, and α-tert-butylglycine. Polypeptides and proteins are derived from post-translational modifications of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications include phosphorylation, acetylation, and the like. and acylation, including formylation, glycosylation (including N-linked and O-linked), amino Derivatization, hydroxylation, alkylation including methylation and ethylation, ubiquitination, pyrolysis, Addition of lidocaine carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation ylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation and iodination.

[0104] 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 A protein attached to a nucleic acid (such as DNA or RNA) such as a nucleic acid protease (e.g., guide RNA). In some embodiments, the polynucleotide-programmable nucleotide binding domain is In one embodiment, the polynucleotide is a programmable DNA binding domain. The polynucleotide programmable nucleotide binding domain In one embodiment, the polynucleotide programmable nucleic acid is a RNA-binding domain. The nucleotide binding domain is the Cas9 protein. The Cas9 protein binds to the guide RNA. It can bind to a guide RNA that guides the Cas9 protein to a specific complementary DNA sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is s9 domain, e.g., nuclease-active Cas9, Cas9 nickase (nCas9), or nuclease Non-limiting examples of nucleic acid programmable DNA binding proteins include enzyme-inactive Cas9 (dCas9) and Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, and Cas12d / Ca Non-limiting examples of Cas enzymes include sY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7 , Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, C as12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Csc1, Csc1, Csm2, Csm3, Csm3, Csm4, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, 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 effector proteins, type V Cas effector proteins, type VI Cas effector proteins, CARF, DinG, and Other nucleic acid fragments include homologs of these fragments, or modified or altered versions thereof. Programmable DNA binding proteins are also within the scope of this disclosure, but are not specifically included in this disclosure. It may not be listed.

[0105] The term "recombinant" as used herein with respect to a protein or nucleic acid means that the protein or nucleic acid is It refers to proteins or nucleic acids that do not occur in nature but are the product of human engineering. For example, In some embodiments, the recombinant protein or nucleic acid molecule is any naturally occurring At least one, at least two, at least three, at least four, or at least at least five, at least six, or at least seven amino acid or nucleotide mutations Contains an octide sequence.

[0106] "Decrease" means a negative change of at least 10%, 25%, 50%, 75%, or 100% .

[0107] "Reference" refers to a standard or control condition. In one embodiment, the reference is a wild-type or or healthy cells.

[0108] A "reference sequence" is a defined sequence used as a basis for sequence comparison. It can be a subset or the entirety of a particular sequence; for example, a full-length cDNA or gene sequence. A segment of a gene, or the complete cDNA or gene sequence. For polypeptides, see the reference polypeptide. The length of the peptide sequence is generally at least about 16 amino acids, preferably at least about 20 amino acids. 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 about 50 amino acids, or about 100 amino acids. Generally, at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably Preferably, at least about 75 nucleotides, even more preferably at least about 100 nucleotides or It is about 300 nucleotides or any integer thereabout or therebetween.

[0109] The terms "RNA programmable nuclease" and "RNA-guided nuclease" refer to Used in conjunction with (e.g., bound to or associated with) one or more non-target RNAs In some embodiments, the RNA-programmable nuclease, when complexed with RNA, Typically, the bound RNA is called a guide RNA (gRNA). gRNAs can exist 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 sometimes referred to as "gRNA," "gRNA" can refer to a single molecule or a complex of two or more molecules. are used interchangeably to refer to any of the guide RNAs present in a single R gRNAs, which exist as NA species, contain two domains: (1) a domain that shares homology with the target nucleic acid (e.g., (2) a domain that binds to the Cas9 protein; In one embodiment, domain (2) comprises a sequence known as tracrRNA. and includes a stem-loop structure. For example, in some embodiments, the domain (2) is identical to or similar to the tracrRNA provided in Jinek et al., Science 337:816-821 (2012). gRNAs (e.g., containing domain 2) that are homologous to gRNAs containing domain 2. Another example of such a method is the "Switchable Cas9 Nucleases and Uses" application filed on September 6, 2013. Thereof" and U.S. Provisional Patent Application No. 61 / 874,682, filed September 6, 2013. U.S. Provisional Patent Application No. 61 / 874,7 entitled "Delivery System For Functional Nucleases" 46. ​​In some embodiments, the gRNA comprises two or more domains (1) and (2), which may be referred to as an "extended gRNA." For example, The gRNA may be coupled to two or more Cas9 proteins as described herein, and may be used to generate two or more different The gRNA binds to the target nucleic acid at a region different from the target site. The nuclease complements the target site, mediating its binding and providing sequence specificity for the nuclease:RNA complex. In some embodiments, the RNA programmable nuclease comprises a CRISPR-associated sequence. stem) Cas9 endonuclease, e.g., Cas9 from Streptococcus pyogenes (Csnl) (For example, "Complete genome sequence of an M1 strain of Streptococcus pyogenes" Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Pri. meaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia H. G., Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe B. A., McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663(2001); "CRISPR R NA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E ., Chylinski K., Sharma CM., Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vo gel J., Charpentier E., Nature 471:602-607 (2011)).

[0110] "SERPINA1 polynucleotide" refers to a nucleic acid that encodes an A1AT protein or a fragment thereof. An exemplary SERPINA1 polypeptide available at NCBI Accession No. NM_000295 is also included. The nucleotide sequence is provided below: JPEG2025013768000004.jpg54166JPEG2025013768000005.jpg250160JPEG2025013768000006.jpg25163The position of the base complementary to the PAM sequence is shown in italics and double underlined. The G complementary to the target C at position 1455 is shown in bold and underlined.

[0111] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide variation that occurs at a specific position in the genome. where each mutation is present in the population to a noticeable extent (e.g., >1%). For example, At certain base positions in the human genome, C nucleotides can occur in most individuals, In a small number of individuals, the position is occupied by A. This means that there is a SNP at this particular position, and C or A, meaning that two nucleotide variations are alleles at this position. SNPs underlie differences in disease susceptibility; they underlie a wide range of human diseases. The severity of the disease and the body's response to treatment are also a reflection of genetic variation. in coding regions, non-coding regions of genes, or intergenic regions (regions between genes) In some embodiments, SNPs within a coding sequence may be present due to the degeneracy of the genetic code. SNPs in the coding region do not necessarily change the amino acid sequence of the protein produced. There are two types of SNPs: synonymous and non-synonymous. Synonymous SNPs do not affect the protein sequence, whereas non-synonymous SNPs P changes the amino acid sequence of a protein. There are two types of nonsynonymous SNPs: missense and nonsense. SNPs that are not in protein-coding regions affect gene splicing, transcription, and They can affect factor binding, messenger RNA degradation, or the sequence of non-coding RNA. Gene expression affected by this type of SNP is called eSNP (expressed SNP), and Single nucleotide variants (SNVs) are single nucleotide variants with no frequency restriction. Somatic single nucleotide variations (e.g., caused by cancer) can occur in somatic cells. A single base modification (caused by a single base mutation) may also be called a single base modification.

[0112] "Specifically bind" means to recognize and bind to the polypeptide and / or nucleic acid molecule of the present invention. and bind to other molecules in the sample (e.g., biological sample) but do not substantially recognize or bind to other molecules in the sample. Nucleic acid molecules, polypeptides, or complexes thereof (e.g., nucleic acid programmable DNA) A binding domain and guide nucleic acid), compound, or molecule.

[0113] Nucleic acid molecules useful in the methods of the present invention may encode a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules include any nucleic acid molecule that is 100% identical to the endogenous nucleic acid sequence. Typically, but not necessarily, substantial identity is shown. A polynucleotide having a nucleotide sequence typically hybridizes with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention can be modified to The present invention also includes any nucleic acid molecule encoding an endogenous nucleotide sequence or a fragment thereof. It need not be 100% identical to the nucleic acid sequence, but typically will show substantial identity. Polynucleotides having "substantial identity" to a double-stranded nucleic acid molecule typically have a small number of double-stranded nucleic acid molecules. "Hybridize" means to hybridize with at least one of the strands of a given molecule. Complementary polynucleotide sequences (e.g., those described herein) are synthesized under various stringency conditions. This means that a double-stranded molecule is formed between the two genes (or a part of them). For example, Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R (1987) Methods Enzymol. 152:507).

[0114] For example, a stringent salt concentration is typically less than about 750 mM NaCl and 75 mM citrate. trisodium, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, more preferably Preferably, the concentration is less than about 250 mM NaCl and 25 mM trisodium citrate. See hybridization can be obtained in the absence of organic solvents, e.g., formamide. whereas high stringency hybridization requires at least about 35% formaldehyde. It can be obtained in the presence of at least about 50% formamide. Stringent temperature conditions are typically at least about 30°C, more preferably at least about 37°C. 0 C, and most preferably at least about 42°C. The concentration of detergent (e.g., sodium dodecyl sulfate (SDS)) and carrier DNA content Various additional parameters, such as inclusion or exclusion, are well known to those skilled in the art. By combining these various conditions, various levels of stringency can be achieved. In a preferred embodiment, hybridization is carried out at 30° C. in 750 mM N In a more preferred embodiment, the reaction takes place in 75 mM trisodium citrate, 75 mM aCl, and 1% SDS. Hybridization was performed at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% S DS, 35% formamide and 100 μg / ml denatured salmon sperm DNA (ssDNA). In a preferred embodiment, hybridization is carried out at 42°C in 250 mM NaCl, 25 mM Cl. This occurs in trisodium enoate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0115] For most applications, the washing steps that follow hybridization are also stringent. Wash stringency conditions are defined by salt concentration and temperature. As mentioned above, wash stringency can be increased by decreasing the salt concentration or increasing the temperature. This can be increased by increasing the thickness of the strip for the cleaning process. The appropriate salt concentration is preferably less than about 30 mM NaCl and 3 mM trisodium citrate. and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the process are typically at least about 25°C, more preferably This includes temperatures of at least about 42°C, and even more preferably at least about 68°C. In this configuration, the wash steps consisted of 30 mM NaCl, 3 mM trisodium citrate, and 0. In a more preferred embodiment, the wash steps are performed in 1% SDS at 42° C. in 15 mM NaCl , 1.5 mM trisodium citrate, and 0.1% SDS. The wash steps were performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% S. Further variations of these conditions will be readily apparent to one skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Be nton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biolog y, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cl oning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molec ular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York It is described in.

[0116] A "subject" includes, but is not limited to, a cow, horse, dog, sheep, or cat. "A mammal" means a mammal, including a human or non-human mammal.

[0117] "Substantially identical" means that the amino acid sequence of a reference amino acid sequence (e.g., an amino acid sequence described herein) is substantially identical to the amino acid sequence of a reference amino acid sequence (e.g., an amino acid sequence described herein). any one of the sequences) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein) It means a polypeptide or nucleic acid molecule that shows at least 50% identity to Or, 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 99% But it has the same identity.

[0118] Sequence identity is typically determined using sequence analysis software (e.g., Genetics Computer Group , University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705 Sequence Analysis Software Package, BLAST, BESTFIT, GAP, or PILE UP / PRETTYBOX program). Such software is available with various permutations. By assigning degrees of homology to the sequences, deletions, and / or other modifications, it is possible to determine whether the sequences are identical or Similar sequences are matched. Conservative substitutions typically include substitutions within the following groups: leucine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, Paragine, glutamine; serine, threonine; lysine, arginine; phenylalanine In an exemplary approach to determining the degree of identity, the BLAST program You can use the RAM, -3 and e -100 Probability scores between indicate closely related sequences .

[0119] 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 sequence that is targeted by a deaminase or a fusion enzyme containing the same. Deaminated by a ligase protein (e.g., cytidine or adenine deaminase) .

[0120] RNA-programmable nucleases (e.g., Cas9) use RNA:D to target DNA cleavage sites. Since NA hybridization is used, these proteins can, in principle, be used as guide RNAs. Any sequence specified by A can be targeted. For site-specific cleavage, C Methods using RNA-programmable nucleases such as as9 (e.g., to modify genomes) For this purpose, methods for generating multiplexed genomic DNA (e.g., Cong, L. et al., Multiplex gene expression) are known in the art (see, e.g., Cong, L. et al., Multiplex gene expression). ome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013 ); Hwang, WY et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programme d genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al., G enome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. See Nature biotechnology 31, 233-239 (2013). the entire contents of each of which are incorporated herein by reference).

[0121] As used herein, the terms "treatment," "treating," and and its grammatical equivalents refer to obtaining a desired pharmacological and / or physiological effect. In some embodiments, the effect is therapeutic, i.e., the effect is directed to treating a disease and / or In some embodiments, the effect is prophylactic. The effect is to prevent the occurrence or recurrence of a disease or condition. To this end, the methods of the present disclosure comprise administering a therapeutically effective amount of a composition as described herein. This includes:

[0122] "Uracil glycosylase inhibitor" means a factor that inhibits the uracil excision repair system. In one embodiment, the factor binds to the host uracil-DNA glycosylase to form D It is a protein or a fragment thereof that prevents the removal of uracil residues from NA.

[0123] Ranges provided herein are understood to be shorthand for all values ​​within the 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 subrange.

[0124] The recitation of a list of chemical groups in any definition of a variable herein means that the listed group The variables herein include definitions of the variables as any single group or combination of The description of an embodiment or aspect may be used interchangeably with any single embodiment or with any other embodiment. This includes any embodiment in combination with any embodiment or portion thereof.

[0125] Any composition or method provided herein may be used in combination with any other composition or method provided herein. The present invention may be combined with one or more of the compositions and methods.

[0126] DNA editing is a promising approach to modifying disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms have focused on specific genomes. It functions by inducing DNA double-strand breaks (DSBs) at genomic sites and is dependent on endogenous DNA repair pathways. The product outcome was determined in a semi-probabilistic manner, resulting in a complex population of genetic products. Achieve precise, user-defined repair results via homogeneous directed repair (HDR) pathway However, many challenges hinder efficient repair using HDR in therapeutically relevant cell types. In practice, this pathway is more efficient than the competing, error-prone non-homologous end-joining pathway. Furthermore, HDR is strictly restricted to the G1 and S phases of the cell cycle and does not occur after mitosis. This prevents accurate repair of DSBs in these cells, resulting in highly efficient repair of DSBs in these populations. , making it difficult or impossible to modify genome sequences in a user-defined, programmable manner. It has been revealed that this is the case.

[0127] [Nucleobase Editor] A base editor for editing, modifying or altering a target nucleotide sequence of a polynucleotide. Disclosed herein are nucleic acid base editors or nucleobase editors. Polynucleotide-programmable nucleotide-binding domains and nucleobase-editing domains A polynucleotide program is a nucleic acid base editor or base editor that includes Possible nucleotide binding domains are those that bind to the guide polynucleotide (e.g., gRNA). When the bases of the bound guide nucleic acid and the target polynucleotide sequence are specifically binds to a target polynucleotide sequence (through complementary base pairing between thereby localizing the base editor to the target nucleic acid sequence desired to be edited. In some embodiments, the target polynucleotide sequence can be single-stranded DNA or In some embodiments, the target polynucleotide sequence comprises double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In an embodiment, the target polynucleotide sequence comprises a DNA-RNA hybrid.

[0128] [Polynucleotide-programmable nucleotide-binding domain] The term "polynucleotide programmable nucleotide binding domain" refers to a guide Proteins that associate with nucleic acids (e.g., DNA or RNA) such as oligonucleotides (e.g., guide RNAs) a protein, the nucleic acid of which is a polynucleotide-programmable nucleotide binding domain In some embodiments, a polynucleotide is The programmable nucleotide binding domain is a polynucleotide-programmable DNA A binding domain. In one embodiment, the polynucleotide programmable nucleoside The tide-binding domain is a polynucleotide-programmable RNA-binding domain. In embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 target. In some embodiments, the polynucleotide is a programmable nucleotide binding protein. The binding domain is the Cpf1 protein.

[0129] CRISPR provides defense against mobile genetic elements (viruses, transposable elements, conjugative plasmids) CRISPR clusters are composed of spacers, sequences that are complementary to the preceding mobile element. The CRISPR cluster contains a sequence of target nucleic acids, and the target invader nucleic acid. The CRISPR cluster is transcribed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of pre-crRNA is essential for transcription. transcoding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein tracrRNA guides the processing of pre-crRNA by ribonuclease 3. Cas9 / crRNA / tracrRNA then binds to a linear or circular dsDNA target complementary to the spacer. The target strand that is not complementary to the crRNA is first cleaved by the endonuclease. It is cleaved protease-wise and then exonucleolytically trimmed to 3'-5'. In the case of mitochondrial DNA, both the protein and the RNA are required for DNA binding and cleavage. A single guide RNA ("sgRNA", or simply "gRNA") can be produced. See, for example, Jinek M., Chylinski K., Fonfa See ra I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821(2012) Cas9 is a CRISPR repeat sequence. It recognizes a short motif (PAM or protospacer adjacent motif) in the sequence to distinguish self from non-self. Helps you distinguish yourself.

[0130] The sequence and structure of Cas9 nuclease are well known to those skilled in the art (see, e.g., "Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al., J .J., McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS., Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L.,. Natl. Acad. Sci. USA 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.”Deltcheva E., Chylinski K., Sharma CM., Gonzales K., Chao Y., Pirzada ZA, Eckert MR, V ogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual- “RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylins ki K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821(201 2), the entire contents of which are incorporated herein by reference. Cas9 orthologs include: It has been described in a variety of species, including but not limited to S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be readily apparent to those skilled in the art based on the present disclosure. Such Cas9 nucleases and sequences are described in Chylinski, Rhun, and C harpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity syst Ca from organisms and loci disclosed in "ems" (2013) RNA Biology 10:5, 726-737 s9 sequence, the entire contents of which are incorporated herein by reference.

[0131] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting exemplary Cas9 domains are provided herein. The Cas9 domains are selected from nuclease-active Cas9 domains, nuclease-inactive Cas9 domains, and Cas9 domains. In some embodiments, the Cas9 domain may have nuclease activity. For example, the Cas9 domain binds both strands of a double-stranded nucleic acid (i.e., a double-stranded DNA). In some embodiments, the Cas9 domain cleaves both strands of the C molecule. The as9 domain comprises any one of the amino acid sequences described herein. In some embodiments, the Cas9 domain may have at least one amino acid sequence similar to any of the amino acid sequences described herein. at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least At least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 9 8%, at least 99%, or at least 99.5% identity. In some embodiments, the Cas9 domain comprises any of the amino acid sequences described herein. Compared to 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 amino acids with mutations In some embodiments, the Cas9 domain comprises an amino acid sequence described herein. at least 10, at least 15, at least 20, at least 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 250, 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 at least 12 00 identical consecutive amino acid residues.

[0132] In some embodiments, the Cas9 nuclease comprises an inactive (e.g., inactivated) DNA cleavage domain. Cas9 has a nuclease-inactivated nucleotide sequence, i.e., Cas9 is a nickase. may be interchangeably referred to as "dCas9" protein (for nuclease-dead Cas9). Methods for generating Cas9 proteins (or fragments thereof) with suitable DNA cleavage domains are known ( For example, Jinek et al, Science. 337:816-821(2012); Qi et al, “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013 ) Cell. 28; 152(5): 1173-83 (the contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 consists of an HNH nuclease subdomain and a RuvC1 subdomain. The HNH subdomain is known to contain two subdomains, the HNH subdomain and the HNH subdomain. The RuvCl subdomain cleaves the non-complementary strand, while the RuvCl subdomain cleaves the non-complementary strand. Mutations can suppress the nuclease activity of Cas9. For example, mutations D10A and H840A inhibit the nuclease activity of S. pyogenes. completely inactivates the nuclease activity of Cas9 (Jinek et al., Science. 337:81 6-821(2012); Qi et al, Cell. 28;152(5): 1173-83 (2013)). In one embodiment, Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase called the "nCas9" protein (for "nickase" Cas9). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, In this form, the protein contains one of two Cas9 domains: (1) Cas9 (2) a DNA cleavage domain of Cas9. In some embodiments, Cas9 or a fragment thereof Proteins containing Cas9 or its derivatives are referred to as "Cas9 variants." For example, a Cas9 variant may share at least about 70% homology with a fragment of wild-type Cas9. Identical, at least about 80% identical, 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, In some embodiments, the sequences are about 99.5% identical, or at least about 99.9% identical to each other. Cas9 mutants exhibited several advantages over wild-type Cas9: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 4, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 3 4, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or higher In some embodiments, the Cas9 variant may have an amino acid change in the fragment of Cas9. The fragment contains a fragment of wild-type Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain), At least about 70% identical to the corresponding fragment, at least about 80% identical, 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 In some embodiments, the fragment is about 99.5% identical, or at least about 99.9% identical. , at least 30%, at least 35%, at least 40% of the amino acid length of the corresponding wild-type Cas9; At least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least or at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 50 0, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 12 50, or at least 1300 amino acids in length.

[0133] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCB Reference sequence: NC_17053.1, with the following nucleotide and amino acid sequence:

[0134] ATGGATAAGAAATACTCAATGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGATTATAAGGT TCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTG GCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGT TATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGT GGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA CTATCTATCATCTGCGAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCAT ATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCA GTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTG CACGATTGAGTAAATCAAGACGATTAGAAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTTGGGAAT CTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAATTAACAGCTTTC AAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTTGGCAGCTA AGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCA ATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTA TAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATA AATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC AAGCAACGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGA 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

[0135] JPEG2025013768000007.jpg164170 (single underline: HNH domain; double underline: RuvC domain).

[0136] In some embodiments, wild-type Cas9 contains the following nucleotides and / or amino acids: corresponding to or including the amino acid sequence: ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGT ACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAAGAATCTTATCGGTGCCCTCCTATTCG ATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGTCCGGAGAAGGTATACACGTCGCAAGAACCGAATATGT TACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGT CGAAGAGGACAAAACATGAACGGCACCCCATCTTTGGAAACATAGGATGAGGTGGCATATCATGAAAAGTACCCAA CGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCAT ATGATAAAGTTCCGTGGGCACTTTCCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCA GTTAGTACAAACCTATAATCAGTTGTTTGAAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCG CCCGCCTCTCTAAATCCCGACGGCTAGAAAAACCTGATCGCACAATTACCCGGAGAGAAAAAAATGGTTGTTCGGTAAC CTTATAGCGCTCTCACTAGGCCTGACACCAAATTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAG TAAGGACACGTACGATGACGATCTCGCAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCA AAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCCGCTTCA ATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATA TAAGGAAATATTCTTTGATCAGTCGAAAACGGGTACGCAGGTTATATTGACGGGCGGAGCGAGTCAAGAGGAATTCTACA AGTTTATCAAACCCATATTAGAGAATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGA AAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGA GGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTACTATGTGGGAC CCCTGGCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAA GTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCACTTTGACAAGAATTTACCGAACGAAAAAGT ATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCA TGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACA GTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATT TAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAG ATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCT CACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTAT CAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTA TGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTG CACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCT AGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGC AAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCT GTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGA ACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACA ATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAG AACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGG CTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGA TACTAGATTCCCGAATGAATACGAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCA AAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGC TTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACA AAGTTTATGACGTCCGTAAGATGATCGCGAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCT AACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGG GGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACA TAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATC GCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGC AAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTT TTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAG TATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGC ACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAAC AGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTC ATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGA AAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCA AACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATA GATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGA TTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAGGA JPEG2025013768000008.jpg161168 (single underline: HNH domain; double underline: RuvC domain).

[0137] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NC BI reference sequence: NC_002737.2 (nucleotide sequence below); and Uniprot reference sequence: Q99ZW2 ( The 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 AAAAACAATTGTTTGTGGAGCAGCATAAGCATTTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTT ATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAACCAATACGTGAACAAGCAGA AAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATTTTTGATACAACAATTGATCGTA AACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATT GATTTGAGTCAGCTAGGAGGTGACTGA JPEG2025013768000009.jpg164169 (Public File:HNH File; Public File:RuvC File).

[0138] Meanwhile, Cas9 is Corynebacterium ulcerans (NCBI Refs: NC_01). 5683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016). 786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Intermediate prevotella (NCBI Ref: NC_017861.1); Spiroplasm taiwan (NCBI Ref: NC_021846.1); Strept ococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1) ; Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NC BI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter je juni (NCBI Ref: YP_002344900.1) or Neisseria meningitidis (NCBI Ref: YP_00 2342100.1), or represents Cas9 from other organisms.

[0139] In some embodiments, dCas9 is a nucleotide sequence that inactivates the nuclease activity of Cas9. Corresponding to, or containing part or all of, the Cas9 amino acid sequence having the above mutations Unless otherwise stated, mutations in Cas9 are expressed relative to the wild-type reference sequence. For example, in some embodiments, the dCas9 domain comprises or includes the D10A and H840A mutations. or a corresponding mutation in another Cas9. In some embodiments, the dCas9 is dCas9(D1 0A and H840A) contains the amino acid sequence: JPEG2025013768000010.jpg163170 (single underline: HNH domain; double underline: RuvC domain).

[0140] In some embodiments, the Cas9 domain comprises a D10A mutation, while the the residue at position 840 in the amino acid sequence provided herein, or The residue at the corresponding position in either sequence remains a histidine.

[0141] In other embodiments, D10A, e.g., resulting in nuclease-inactivated Cas9 (dCas9), and dCas9 variants having mutations other than H840A. For example, other amino acid substitutions at D10 and H840, or the nuclease domain of Cas9 may be used. Other substitutions within the domain (e.g., HNH nuclease subdomain and / or RuvC1 subdomain) In some embodiments, a variant or homolog of dCas9 is , at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least In some embodiments, those having at least about 99.9% identity are provided. About 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids or more, or longer. Variants of dCas9 having amino acid sequences are provided.

[0142] In some embodiments, the Cas9 fusion proteins provided herein comprise a Cas9 protein. The full-length amino acid sequence of the protein, for example, one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein contain a full-length Cas9 sequence. Examples of Suitable Cas9 Domains and Cas9 Fragments Suitable amino acid sequences are provided herein, and further suitable sequences for Cas9 domains and fragments are , will be apparent to those skilled in the art.

[0143] The Cas9 protein guides the protein to a specific DNA sequence complementary to its guide RNA. In one embodiment, the polynucleotide protease is capable of binding to 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 tunable DNA-binding proteins include Cas9 (e.g., dCas9 and nCas9), CasX, C These include, but are not limited to, asY, Cpf1, Cas12b / C2c1, and Cas12c / C2c3.

[0144] The nuclease-inactivated Cas9 protein is interchangeably referred to as a "dCas9" protein (nuclease-inactivated Cas9 protein). This enzyme may be referred to as "dead" Cas9, or catalytically inactive Cas9. Methods for generating Cas9 proteins (or fragments thereof) having domains are known (e.g., , Jinek et al., Science. 337:816-821(2012); Qi et al., “Repurposing CRISPR as a n RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) See Cell. 28;152(5):1173-83, the contents of each of which are incorporated herein by reference. The DNA cleavage domain of Cas9 consists of two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. It is known that the HNH subdomain cleaves the strand complementary to the gRNA. The RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains enhance Cas9 activity. For example, mutations D10A and H840A inhibit the nuclease activity of S. pyogenes Cas9. Completely inactivates nuclease activity (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83 (2013)). One example is nuclease-inactive Cas9 The domains were provided in the cloning vector pPlatTET-gRNA2 (Accession No. BAV54124). It includes the amino acid sequence.

[0145] The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows: MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD

[0146] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows: MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD

[0147] An exemplary amino acid sequence of a catalytically active Cas9 is as follows: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD.

[0148] In some embodiments, Cas9 is expressed in the Archaea (Archaea), which comprise the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, the term refers to a programmable nucleoprotein (e.g., Cas9) derived from a nanoarchaea. Tide-binding proteins are described, for example, in Burstein et al., "New CRISPR-Cas systems from uncu ltivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21 The CasX or CasY protein may be a CasX or CasY protein, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, the first reported Many CRISPR-Cas systems have been identified, including the divergent Cas9 protein. It was discovered as part of an active CRISPR-Cas system in little-studied nanoarchaea. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, were discovered. These are among the most compact systems ever discovered. In embodiments, in the base editor systems described herein, Cas9 is or a variant of CasX. In the base editor systems described herein, Cas9 is substituted with CasY or a variant of CasY. Nucleic acid programmable DNA binding protein (napDNAbp) is another It should be understood that RNA-guided DNA binding proteins such as is.

[0149] In some embodiments, a programmable nucleotide binding protein (as described herein) Nucleic acid programmable DNA-binding protein (also called napDNAbp in the literature) is CasX In some embodiments, the programmable nucleotide binding protein In some embodiments, the programmable nucleoprotein is a CasY protein. The tide-binding protein binds at least 85% of naturally occurring CasX or CasY proteins. %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or In some embodiments, the amino acid sequence has at least 99.5% identity. The programmable nucleotide-binding protein is a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide binding protein The quality is at least 85% for any CasX or CasY protein described herein. %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or Contains amino acid sequences with at least 99.5% identity. CasX and Cas from other bacterial species It is to be understood that Y may also be used in accordance with the present disclosure.

[0150] Exemplary CasX ((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53) tr|F0NN87 |F0NN87_SULIHCRISPR-associatedCasx protein OS = Sulfolobus islandicus (strain HV The amino acid sequence of E10 / 4) GN = SiH_0402 PE=4 SV=1) is as follows: MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIIL PLSNVKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAA AGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAV GQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSSSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNL NSDDGKVRDLKLISAYVNGELIRGEG

[0151] Exemplary CasX (>tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = Sulfo lobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1) It's the way it is. MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIIL PLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAA AGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAV GQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLN SDDGKVRDLKLISAYVNGELIRGEG

[0152] Deltaproteobacteria CasX MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAIL QVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVA EHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFL SKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLNLWQKLKLSRDDA KPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENP KKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEM DEKEFYACEIQLQKWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFT DGTDIKKSGKWQGLLYGGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKI GRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQ AAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAK LAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKEL SAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSY KSGKQPFVGAWQAFYKRRLKEVWKPNA

[0153] Exemplary CasY ((ncbi.nlm.nih.gov / protein / APG80656.1) >APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacte The amino acid sequence of [trium] is as follows: MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPREIVSAINDDYVGLYGLSNFDDLYNAEKRNEE KVYSVLDFWYDCVQYGAVFSYTAPGLLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIKFLNKKEISRANGSLDKLKKD IIDCFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQKKLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNR NRGEVLFNKLKEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELKKAMMDITDAWRGQEQEEELE KRLRILAALTIKLREPKFDNHWGGYRSDINGKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVVS SLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQEALIKERLEAEKKKPKKRKKKSDAEDEKET IDFKELFPHLAKPLKLVPNFYGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKDFFIKRLQKIF SVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQSRSRKSAAIDKNRVRLPSTENIAKAGIALARELSVAGFDWK DLLKKEEHEEYIDLIELHKTALALLLAVTETQLDISALDFVENGTVKDFMKTRDGNLVLEGRFLEMFSQSIVFSELRGLA GLMSRKEFITRSAIQTMNGKQAELLYIPHEFQSAKITTPKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHY FGYELTRTGQGIDGGVAENALRLEKSPVKKREIKCKQYKTLGRGQNKIVLYVRSSYYQTQFLEWFLHRPKNVQTDVAVSG SFLIDEKKVKTRWNYDALTVALEPVSGSERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEITGDSAKILDQN FISDPQLKTLREEVKGLKLDQRRGTFAMPSTKIARIRESLVHSLRNRIHHLALKHKAKIVYELEVSRFEEGKQKIKKVYA TLKKADVYSEIDADKNLQTTVWGKLAVASEISASYTSQFCGACKKLWRAEMQVDETITTQELIGTVRVIKGGTLIDAIKD FMRPPIFDENDTPFPKYRDFCDKHHISKKMRGNSCLFICPFCRANADADIQASQTIALLRYVKEEKKVEDYFERFRKLKN IKVLGQMKKI

[0154] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a microbial CRI The single effector of the microbial CRISPR-Cas system is Cas. 9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Microbial CRISPR-Cas systems are divided into class 1 and class 2 systems. Class 1 systems are multisubsystems. Class 1 systems have unit effector complexes, while Class 2 systems have single protein effectors. For example, Cas9 and Cpf1 are class 2 effectors. In addition to Cas9 and Cpf1, three Different class 2 CRISPR-Cas systems (Cas12b / C2c1 and Cas12c / C2c3) were described by Shmakov et al. , “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Syst ems”, Mol. Cell, 2015 Nov. 5; 60(3): 385-397 (in its entirety) (The contents of which are incorporated herein by reference.) Two systems, Cas12b / C2c1 and Cas12c / C2c3, The vector contains a RuvC-like endonuclease domain related to Cpf1. The third system is a hybrid of two predicted It contains an effector with a HEPN RNase domain that is used to inhibit CRISPR RNA transcription by Cas12b / C2c1. Unlike the production of CRISPR, the production of mature CRISPR RNA is tracrRNA-independent. Transfection depends on both CRISPRRNA and tracrRNA.

[0155] The crystal structure of Alicyclobaccillus acidoterrastris Cas12b / C2c1 (AacC2c1) was revealed to be a chimeric monolayer. It has been reported as a complex with a single guide RNA (sgRNA). For example, Liu et al., "C2 c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism”, Mol. Cel l, 2017 Jan. 19; 65(2):310-322, the entire contents of which are incorporated herein by reference. In addition, Alicyclobacillus acidoterrestris C2c1 bound to target DNA as a ternary complex For example, Yang et al., "PAM-dependent Target DN A Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease”, Cell, 2016 Dec. 15 167(7):1814-1828, the entire contents of which are incorporated herein by reference. Target DN The catalytically competent conformation of AacC2c1, along with both the A strand and the non-target DNA strand, is are independently trapped within the RuvC catalytic pocket, and Cas12b / C2c1-mediated cleavage occurs in a staggered fashion at the target DNA. The Cas12b / C2c1 ternary complex, previously identified as Cas9 and C2c1, produces a 7-nucleotide cleavage. Structural comparisons between pf1 counterparts demonstrate the diversity of mechanisms used by the CRISPR-Cas9 system. vinegar.

[0156] In some embodiments, the nucleic acid of any of the fusion proteins provided herein The programmable DNA-binding protein (napDNAbp) binds to the Cas12b / C2c1 or Cas12c / C2c3 proteins. In some embodiments, the napDNAbp can be a Cas12b / C2c1 protein. In some embodiments, the napDNAbp is a Cas12c / C2c3 protein. In some embodiments, the napDNAbp is a naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. At least 85%, at least 90%, at least 91%, at least 92%, or less At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 9 8%, at least 99%, or at least 99.5% identity. In some embodiments, the napDNAbp is a naturally occurring Cas12b / C2c1 or Cas12c / C2c3 tag. In some embodiments, the napDNAbp is a napD protein provided herein. At least 85%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , amino acid sequences with at least 98%, at least 99%, or at least 99.5% identity Cas12b / C2c1 or Cas12c / C2c3 from other bacterial species may also be used in accordance with the present disclosure. Please understand that this is possible.

[0157] Cas12b / C2c1((uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2| / C2C1_ALIAG CRISPR-assoc iated endonuclease C2c1 OS = Alicyclobacillus acido-terrestris (strain ATCC 4902 5 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1 SV=1) amino acid sequence is As follows: MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECDKTAEECKAELLERLRARQ VENGHRGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWE EEKEKAETRKSADRTADVLRALADFGLKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQ EYAKLVEQKNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDA EIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLNHAKMFATFTLPDATAHPIWTRFDKLGGN LHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAK IQCRRDQLAHMHRRRGARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGL LSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQR TLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVR RVWRHMGKQVRDWRKDVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREH IDHAKEDRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELI NQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADDLIPTGEGEIF VSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLRCDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYE RERGKKRRKVFAQEKLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMV NQRIEGYLVKQIRSRVPLQ DSACENTGDI

[0158] BhCas12b (Bacillus hisashii) NCBI Reference Sequence: WP_095142515

[0159] MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAE IQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRW YNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQAL ERFLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWL KMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPIN HPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKH AFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKE LTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGET LVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWV AFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHL NALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGE IYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDR KCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIK KGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSS KQSMKRPAATKKAGQAKKKK

[0160] In some embodiments, the Cas12b is BvCas12B, which is a variant of BhCas12b. It contains the following changes relative to BhCas12B: S893R, K846R, and E837G.

[0161] BvCas12b (Bacillus sp. V3-13) NCBI Reference Sequence: WP_101661451.1

[0162] MAIRSIKLKMKTNSGTDSIYLRKALWRTHQLINEGIAYYMNLLTLYRQEAIGDKTKEAYQAELINIIRNQQRNNGSSEEH GSDQEILALLRQLYELIIPSSIGESGDANQLGNKFLYPLVDPNSQSGKGTSNAGRKPRWKRLKEEGNPDWELEKKKDEER KAKDPTVKIFDNLNKYGLLPLFPLFTNIQKDIEWLPLGKRQSVRKWDKDMFIQAIERLLSWESWNRRVADEYKQLKEKTE SYYKEHLTGGEEWIEKIRKFEKERNMELEKNAFAPNDGYFITSRQIRGWDRVYEKWSKLPESAPEELWKVVAEQQNKMS EGFGDPKVFSFLANRENRDIWRGHSERIYHIAAYNGLQKKLSRTKEQATFTLPDAIEHPLWIRYESPGGTNLNLFKLEEK QKKNYYVTLSKIIWPSEEKWIEKENIEIPLAPSIQFNRQIKLKQHVKGKQEISFSDYSSRISLDGVLGGSRIQFNRKYIK NHKELLGEGDIGPVFFNLVVDVAPLQETRNGRLQSPIGKALKVISSDFSKVIDYKPKELMDWMNTGSASNSFGVASLLEG MRVMSIDMGQRTSASVSIFEVVKELPKDQEQKLFYSINDTELFAIHKRSFLLNLPGEVVTKNNKQQRQERRKKRQFVRSQ IRMLANVLRLETKKTPDERKKAIHKLMEIVQSYDSWTASQKEVWEKELNLLTNMAAFNDEIWKESLVELHHRIEPYVGQI VSKWRKGLSEGRKNLAGISMWNIDELEDTRRLLISWSKRSRTPGEANRIETDEPFGSSLLQHIQNVKDDRLKQMANLIIM TALGFKYDKEEKDRYKRWKETYPACQIILFENLNRYLFNLDRSRRRENSRLMKWAHRSIPRTVSMQGEMFGLQVGDVRSEY SSRFHAKTGAPGIRCHALTEEDLKAGSNTLKRLIEDGFINESELAYLKKGDIIPSQGGELFVTLSKRYKKDSDNNELTVI HADINAAQNLQKRFWQQNSEVYRVPCQLARMGEDKLYIPKSQTETIKKYFGKGSFVKNNTEQEVYKWEKSEKMKIKTDTT FDLQDLDGFEDISKTIELAQEQQKKYLTMFRDPSGYFFNNETWRPQKEYWSIVNNIIKSCLKKKILSNKVEL

[0163] Polynucleotide programmable nucleotide binding domains also bind to nuclear RNA. It is understood that the present invention may include an acid programmable protein. The polynucleotide-programmable nucleotide binding domain is The nucleotide-binding domain can be linked to a nucleic acid that guides the RNA. Other DNA-binding proteins are also within the scope of this disclosure, although they are not specifically listed in this disclosure. It has not been done.

[0164] The CAs proteins that can be used herein include class 1 and class 2. Non-limiting examples of proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5d, Cas5t, as5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also called Csn1 or Csx12), Cas10, Csy1, C sy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm 5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Cssx16, Cx16, Cx, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1 , Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, CARF, DinG, and their homologs Unmodified CRISPR enzymes, like Cas9, are composed of two It has functional endonuclease domains, RuvC and HNH, and can have DNA cleavage activity. CRISPR enzymes target sequences, such as within the target sequence and / or the complementary strand of the target sequence. For example, CRISPR enzymes can induce cleavage of one or both strands of a target sequence. Approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 nucleotides from the first or last nucleotide of the string Induce breaks in one or both strands at 50, 100, 200, 500 base pairs or more. It is possible.

[0165] Loss of ability to cleave one or both strands of a target polynucleotide containing the target sequence To achieve this, vectors were used to encode CRISPR enzymes that were mutated relative to the corresponding wild-type enzyme. Cas9 can be a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Cas9) and at least approximately 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93% %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology Cas9 can refer to a polypeptide having a wild-type exemplary Cas9 polypeptide ( For example, from S. pyogenes), at most, or at most, approximately, about 50%, 60%, 70% , 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or polypeptides having sequence homology. or deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any of these It can refer to modified forms of the Cas9 protein that may contain amino acid changes, such as combinations.

[0166] In some embodiments, the methods described herein involve recombinantly engineered The guide RNA (gRNA) is required for Cas binding. The required scaffold sequence and a user-defined approximately 20-base sequence that defines the genomic target to be modified. In some embodiments, the scaffold is a short synthetic RNA consisting of a GUUUU AGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUU The ability of a person skilled in the art to alter the genomic target of a Cas protein specificity is discussed below. In particular, how specific the gRNA targeting sequence is for the genomic target relative to other parts of the genome. It is determined by whether

[0167] Cas9 nuclease has two functional endonuclease domains, RuvC and HNH. Upon binding to the target DNA, Cas9 undergoes a second conformational change, which Cas9-mediated DNA cleavage is achieved by positioning the cleavage domain on the opposite strand of the target DNA. The end result is a double-strand break (DSB) in the target DNA (approximately 3–4 nucleotides upstream of the PAM sequence). The resulting DSBs are repaired by one of two general repair pathways: (1) efficient (2) the less efficient but more error-prone non-homologous end joining (NHEJ) pathway; or (3) the less efficient but more highly fidel Homologous directed repair (HDR) pathway.

[0168] The "efficiency" of non-homologous end joining (NHEJ) and / or homology-directed repair (HDR) is not known by any simple method. It can be calculated in a convenient way. For example, in some cases, the efficiency is For example, a test nuclease assay can be used to determine the percentage of cleavage product. The ratio of product to substrate can be used to calculate the percentage. For example, direct cleavage of DNA containing the newly integrated restriction sequence as a result of successful HDR. A nuclease enzyme can be used to measure the amount of substrate cleaved, which increases the rate of HDR. As an illustrative example, the HDR percentage (percentage) The percentage of cleavage can be calculated using the following formula: [(cleavage product) / (substrate + cleavage product)] (e.g., (b+c) / (a+b+c) where "a" is the band intensity of the DNA substrate, and "b" and and "c" are cleavage products.

[0169] In some cases, efficiency can be expressed as the success rate of NHEJ. For example, T7 endonuclease Cleavage products were generated using a cleavage enzyme I assay, and the ratio of product to substrate was used to determine the percentage of NHEJ. The conversion can be calculated using wild-type and mutant T7 endonuclease I. High levels of NHEJ (NHEJ generates small random insertions or deletions (indels) at the initial cleavage site) It cleaves the mismatched heteroduplex DNA resulting from hybridization. This indicates a high rate of NHEJ (high efficiency of NHEJ). (Percentage) is calculated using the formula (1-(1-(b+c) / (a+b+c)) 1 / 2 ) × 100 , where "a" is the band intensity of the DNA substrate, and "b" and "c" are the cleavage products ( Ran et. al., Cell. 2013 Sep. 12; 154(6):1380-9; and Ran et. al., Nat Protoc. 2013 Nov.;8(11):2281-2308).

[0170] The NHEJ repair pathway is the most active repair mechanism, resulting in small nucleotide insertions or deletions at DSB sites. The random nature of NHEJ-mediated DSB repair is due to the lack of Cas9 and gRNA. Alternatively, a population of cells expressing the guide polynucleotide may result in a diverse array of mutations. In most cases, NHEJ results in small indentations in the target DNA, which has important practical implications. This results in premature deletion of the target gene's open reading frame (ORF). Amino acid deletions, insertions, or frameshift mutations resulting in undesired stop codons occur. The ideal end result is a loss-of-function mutation in the target gene.

[0171] NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, but Homologous-directed repair (HDR) is a method for repairing single nucleotide changes, such as the addition of a fluorophore or tag. These can be used to generate specific nucleotide changes ranging from large insertions such as

[0172] To utilize HDR for gene editing, a DNA repair template containing the desired sequence is prepared using 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 regions immediately upstream and downstream of the target (called left and right homology arms). The length of each homologous arm determines the magnitude of the change introduced. The repair template may depend on the size of the insert, with larger inserts requiring longer homology arms. a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid The efficiency of HDR was measured in cells expressing Cas9, gRNA, and exogenous repair template. HDR occurs between the S and G2 phases of the cell cycle, so the risk of developing a mutation is generally low (less than 10% correcting alleles). The efficiency of HDR can be increased by synchronizing cells. Chemically or genetically inhibiting offspring can also increase HDR frequency.

[0173] In some embodiments, the Cas9 is a modified Cas9. There may be additional sites of partial homology throughout the genome. These targets must be considered when designing gRNAs, but they are often used to optimize gRNA design. In addition to this, the specificity of CRISPR can also be increased by modifying Cas9. Cas9 induces double-strand breaks (DS) through the combined activity of two nuclease domains, RuvC and HNH. B) Cas9 nickase, the D10A mutant of SpCas9, contains one nuclease domain This allows for the generation of DNA nicks rather than DSBs. Editing can also be combined with nickase.

[0174] In some embodiments, the modified Cas9 is a high-fidelity Cas9 enzyme. In this study, the high-fidelity Cas9 enzyme was selected from SpCas9(K855A), eSpCas9(1.1), SpCas9-HF1, or high-fidelity Cas9. The modified Cas9eSpCas9(1.1) is a precision Cas9 variant (HypaCas9) that binds the HNH / RuvC group and non- It contains alanine substitutions that weaken the interaction between the target DNA strand and the target DNA strand, preventing strand splitting at off-target sites. Similarly, SpCas9-HF1 disrupts the interaction of Cas9 with the DNA phosphate backbone, preventing cleavage and fragmentation. HypaCas9 reduces off-target editing through alanine substitutions that alter the proofreading of Cas9. and contains mutations in the REC3 domain that increase target discrimination (SpCas9 N692A / M694A / Q695A / H698 A). All three high-fidelity enzymes cause fewer off-target edits than wild-type Cas9. The amino acid sequence of a typical high-fidelity Cas9 is provided below. In this sequence, the amino acid sequence of the reference Cas9 is The high-fidelity Cas9 domain mutations 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.

[0175] In some cases, the Cas9 is a variant Cas9 protein. Variant Cas9 polypeptide differs by a single amino acid compared to the amino acid sequence of the wild-type Cas9 protein (e.g., In some instances, the amino acid sequence may be a sequence of a variant of the amino acid sequence, such as a nucleotide sequence of a nucleotide, a nucleotide sequence of ... The modified Cas9 polypeptide contains an amino acid sequence that reduces the nuclease activity of the Cas9 polypeptide. have alterations (e.g., deletions, insertions, or substitutions). For example, in some instances, The ant-Cas9 polypeptides exhibited less than 50% of the nuclease activity 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%. In this case, the variant Cas9 protein does not have substantial nuclease activity. If the protein is a variant Cas9 protein that does not have substantial nuclease activity, This may be referred to as "dCas9."

[0176] In some cases, the variant Cas9 protein has reduced nuclease activity. For example, A variant Cas9 protein may be a variant of 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 or less than about 0.1%.

[0177] In some cases, the variant Cas9 protein cleaves the complementary strand of the guide target sequence. but have a reduced ability to cleave the non-complementary strand of the double-stranded guide target sequence. Variant Cas9 proteins contain mutations (amino acid substitutions) that reduce the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant The Cas9 protein has D10A (aspartic acid to alanine at amino acid position 10). , thus, can cleave the complementary strand of the double-stranded guide target sequence, but This variant of Cas9 has a reduced ability to cleave the non-complementary strand of the target sequence (hence, When a protein cleaves a double-stranded target nucleic acid, it creates a single-strand break (S) instead of a double-strand break (DSB). (See, e.g., Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21) .

[0178] In some cases, the variant Cas9 protein cleaves the non-complementary strand of the double-stranded guide target sequence. can cleave the complementary strand of the guide target sequence, but with a reduced ability to cleave the complementary strand of the guide target sequence. Variant Cas9 proteins have mutations (amino acid substitutions) that reduce the function of the HNH domain. (RuvC / HNH / RuvC domain motif). Non-limiting examples include: In embodiments, the variant Cas9 protein is H840A (His at amino acid position 840). alanine to cysteine) mutation, thus inhibiting the non-complementary strand of the guide target sequence. can cleave the complementary strand of the guide target sequence, but has a reduced ability to cleave the complementary strand of the guide target sequence (Thus, this variant Cas9 protein cleaves the double-stranded guide target sequence.) (When the Cas9 protein is inserted into the guide target sequence, it generates an SSB instead of a DSB.) The ability to cleave the guide target sequence (e.g., single-stranded guide target sequence) is reduced, but the ability to cleave the guide target sequence (e.g., single-stranded guide target sequence) is reduced. The strand retains the ability to bind to the guide target sequence.

[0179] In some cases, the variant Cas9 protein is capable of cleaving complementary and non-complementary strands of a double-stranded target DNA. As a non-limiting example, in some cases, the variant The Cas9 protein has both the D10A and H840A mutations, resulting in a polypeptide These have a reduced ability to cleave both the complementary and non-complementary strands of double-stranded target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA). However, they retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0180] As another non-limiting example, in some cases, the variant Cas9 protein is 76A and W1126A mutations, such that the polypeptide binds to the target DNA (e.g., single-stranded target have reduced ability to cleave target DNA (e.g., single-stranded target DNA), but have reduced ability to bind to target DNA (e.g., single-stranded target DNA). The power is retained.

[0181] As another non-limiting example, in some cases, the variant Cas9 protein may be P4 75A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, resulting in Such Cas9 proteins have a reduced ability to cleave target DNA. Although the target DNA (e.g., single-stranded target DNA) has a reduced ability to cleave the target DNA (e.g., single-stranded target DNA), It retains the ability to bind to DNA.

[0182] As another non-limiting example, in some cases, the variant Cas9 protein is H8 40A, W476A, and W1126A mutations, so that the polypeptide binds to target DNA (e.g., The target DNA (e.g., single-stranded target DNA) has a reduced ability to cleave, but As another non-limiting example, in some cases, The antCas9 protein has H840A, D10A, W476A, and W1126A mutations, resulting in Such Cas9 proteins have a reduced ability to cleave target DNA. has a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but In some embodiments, the variant retains the ability to bind to single-stranded target DNA. This Cas9 has a restored catalytic His residue at position 840 of the Cas9 HNH domain (A840H). .

[0183] As another non-limiting example, in some cases, the variant Cas9 protein is H8 40A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, resulting in The polypeptide has reduced ability to cleave target DNA (e.g., single-stranded target DNA), but not target DNA. As another non-limiting example, some In some cases, the variant Cas9 protein is D10A, H840A, P475A, W476A, N477A, D1125A, W1126A, and D1127A mutations, such that the polypeptide binds to the target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded targets). have reduced ability to cleave target DNA (e.g., single-stranded target DNA), but have reduced ability to bind to target DNA (e.g., single-stranded target DNA). If the variant Cas9 protein has the W476A and W1126A mutations, or variant Cas9 proteins P475A, W476A, N477A, ​​D1125A, W1126A, and D1127 A mutation, the variant Cas9 protein does not bind efficiently to the PAM sequence. Therefore, in such cases, such variant Cas9 proteins are used in methods of conjugation. In other words, in some cases, such a barrier is not required. When a recombinant Cas9 protein is used in the method of attachment, the method may include a guide RNA. The method can be performed in the absence of a PAM sequence (thus, the specificity of binding depends on the guide RNA). (This is brought about by the target segment of A). To achieve the above effect, other residues were varied. Non-limiting examples include: Residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and and / or A987 can be altered (i.e., substituted). is also suitable.

[0184] In some embodiments, a variant Cas9 protein (e.g., Cas9) with reduced catalytic activity is provided. Proteins D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and and / or A987 mutations, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A , H983A, A984A, and / or D986A) that interacts with the guide RNA As long as it retains its ability to bind to target DNA in a site-specific manner (by guide RNA), (This is because the target DNA sequence is guided by the

[0185] As an alternative to S. pyogenes Cas9, the Cpf1 family exhibits cleavage activity in mammalian cells. RNA-guided endonucleases derived from Prevotella and Francisella 1 may be mentioned. The current CRISPR (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. 1 is a class II CRISPR / Cas RNA-guided endonuclease. This adaptive immune mechanism is involved in the Pre The Cpf1 gene is associated with the CRISPR locus and is found in the bacteria Vogelia and Francisella. It encodes an endonuclease that uses a guide RNA to find and cut viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, and therefore plays a key role in the regulation of the CRISPR / Cas9 system. Unlike Cas9 nuclease, Cpf1-mediated DNA cleavage results in , a double-strand break with a short 3' overhang. The staggered cleavage pattern of Cpf1 is consistent with the traditional restriction This opens up the possibility of directional gene transfer, similar to enzymatic cloning. Similar to the Cas9 variants and orthologues described above, Cpf1 limits the number of sites that CRISPR can target to A lacking the NGG PAM site preferred by SpCas9. It can also extend into T-rich regions or AT-rich regions of the genome. The Cpf1 locus is an α / β mixed dominance region. The main, RuvC-I, followed by the helical region, RuvC-II, and the zinc finger-like domain 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 does not contain a Ca The Cpf1 CRISPR-Cas domain organization does not have the α-helix recognition lobe of S9. showed that the CRISPR system is globally unique and classified as a Class 2, Type V CRISPR system. The Cpf1 locus encodes Cas1, Cas2, and Cas4 proteins that are more similar to type I and III than to type II systems. Functional Cpf1 requires trans-activating CRISPR RNA (tracrRNA). therefore, only CRISPR (crRNA) is required. Cpf1 is not only smaller than Cas9, but also more efficient. It has a smaller sgRNA molecule (about half the number of nucleotides of Cas9) that allows for genome editing. In contrast to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex targets the G-rich PAM motif. Cleavage of target DNA or RNA occurs through identification of the protospacer adjacent to 5'-YTN-3'. After the identification of the AM, Cpf1 binds sticky-end-like DNA fragments with 4- or 5-nucleotide overhangs. A double-strand break is introduced.

[0186] Some embodiments of the present disclosure act as nucleic acid programmable DNA binding proteins. Fusion proteins containing domains, which can be used to encode proteins such as base editors, are provided. can be used to direct proteins to specific nucleic acid (e.g., DNA or RNA) sequences. In embodiments, the fusion protein comprises a nucleic acid programmable DNA binding protein domain. DNA binding proteins include, but are not limited to, Cas9, Cas9A, Cas9B, Cas9C, Cas9D, Cas9E, Cas9F, Cas9H ... (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Ca These include s12e / CasX, Cas12g, Cas12h, and Cas12i. They have different PAM specificities than Cas9. Examples of nucleic acid programmable DNA binding proteins are those from Prevotella and Francisella Clustered Regularly Interspaced Short Palindromic Repeats from 1. Cas9 and Similarly, Cpf1 is a class 2 CRISPR effector. Cpf1 has distinct characteristics and robustness compared to Cas9. Cpf1 has been shown to mediate DNA interference in a single RNA-guided endonuclease that lacks tracrRNA. cleases, utilizing T-rich protospacer adjacent motifs (TTN, TTTN, or YTN) Furthermore, Cpf1 cleaves DNA at staggered double-strand breaks. Among proteins, two enzymes from Acidaminococcus and Lachnospiraceae are expressed in human cells. It has been shown that the Cpf1 protein has efficient genome editing activity. For example, Yamano et al., "Crystal structure of Cpf1 in complex with th guide RNA and target DNA.” Cell (165) 2016, pp. 949-962. The entire contents of which are incorporated herein by reference.

[0187] It can also be used as a guide nucleotide sequence-programmable DNA-binding protein domain. Nuclease-inactive Cpf1 (dCpf1) variants that can be used in the compositions and methods of the invention are also disclosed. The Cpf1 protein is useful in RuvC-like endonucleases, which are similar to the RuvC domain of Cas9. It has a cleavage domain but no HNH endonuclease domain, and is located at the N-terminus of Cpf1. does not have the α-helix recognition lobe of Cas9. Zetsche et al., Cell, 163, 759-771, 2 015 (hereby incorporated by reference) demonstrated that the RuvC-like domain of Cpf1 mediates cleavage of both DNA strands. showed that inactivation of the RuvC-like domain inactivates Cpf1 nuclease activity. For example, the mutations corresponding to D917A, E1006A, or D1255A in Francisella novicida Cpf1 The mutation inactivates Cpf1 nuclease activity. dCpf1 of D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or or mutations corresponding to D917A / E1006A / D1255A. Any mutation that can be used in accordance with the present disclosure, such as a substitution mutation, deletion, or insertion, can be used in accordance with the present disclosure. Please understand that.

[0188] In some embodiments, the nucleic acid of any of the fusion proteins provided herein The programmable DNA binding protein (napDNAbp) may be a Cpf1 protein. In some embodiments, the Cpf1 protein is Cpf1 nickase (nCpf1). In some cases, the Cpf1 protein is nuclease-inactive Cpf1 (dCpf1). In embodiments, Cpf1, nCpf1, or dCpf1 is selected from the group consisting of Cpf1 sequences disclosed herein. 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 In some embodiments, the amino acid sequence has at least 99%, or at least 99.5%, identity to the amino acid sequence. In some embodiments, dCpf1 has a sequence that is at least 85% identical to the Cpf1 sequence disclosed herein, at least At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, at least 99%, or at least 99 Contains amino acid sequences with 0.5% identity, D917A, E1006A, D1255A, D917A / E1006A, D917 containing mutations corresponding to A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that Cpf1 from bacterial species may also be used in accordance with the present disclosure.

[0189] The amino acid sequence of wild-type Francisella novicida Cpf1 is as follows: D917, E1006, and D1255 are in bold and underlined. MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIAANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI D RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFEFSFDYKNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN.

[0190] The amino acid sequence of Francisella novicida Cpf1 D917A is as follows: (A917, E1006 , and D1255 are underlined in bold.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEKELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGGEGYKKIVYKLLPGANKMLPKVFFSASKIFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIAANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI A RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFEFSFDYKNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN.

[0191] The amino acid sequence of Francisella novicida Cpf1 E1006A is as follows: (D917, A100 6, and D1255 are underlined in bold.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEKELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGGEGYKKIVYKLLPGANKMLPKVFFSASKIFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPCKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI D RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF ADLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFEFSFDYKNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN.

[0192] The amino acid sequence of Francisella novicida Cpf1 D1255A is as follows: (D917, E100 6 and A1255 mutation positions are underlined in bold.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEKELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGGEGYKKIVYKLLPGANKMLPKVFFSASKIFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPCKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI D RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFESFDYKNFGDKAAKKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN.

[0193] The amino acid sequence of Francisella novicida Cpf1 D917A / E1006A is as follows: (A917 , A1006, and D1255 are in bold and underlined.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIAANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI A RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF A DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFEFSFDYKNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA D ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN.

[0194] The amino acid sequence of Francisella novicida Cpf1 D917A / D1255A is as follows: (A917 , E1006, and A1255 are in bold and underlined.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIAANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI A RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFEFSFDYKNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN.

[0195] The amino acid sequence of Francisella novicida Cpf1 E1006A / D1255A is as follows: (D91 7, A1006, and A1255 are in bold and underlined.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEKELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGGEGYKKIVYKLLPGANKMLPKVFFSASKIFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPCKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI D RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF ADLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFEFSFDYKNFGDKAAKGKWTIASFFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN.

[0196] The amino acid sequence of Francisella novicida Cpf1 D917A / E1006A / D1255A is as follows: (A917, A1006, and A1255 are in bold and underlined.) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYS DVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDIT DIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEKELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNLLHKLKIFHISQSEDKANILDKDEH FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI FDDKAIKENKGGEGYKKIVYKLLPGANKMLPKVFFSASKIFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISEYIDSVVNQGKLYLFQIYNKDFSAYSKGR PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPCKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFF HCPITINFKSSGANKFNDEINLLLKEKANDVHILSI A RGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVF A DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVF KDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD KGYFESFDYKNFGDKAAKKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA A ANGAYHIGLKGLMLLGRIKNNQEGK KLNLVIKNEEYFEFVQNRNN.

[0197] In some embodiments, the variant Cas protein is spCas9, spCas9-VRQR, spCas9 -VRER, xCas9 (sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCa It can be s9-LRVSQL.

[0198] The amino acid sequence of an exemplary SaCas9 is as follows: KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTHS ELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKD GEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPGFGWKDIKEWYEMLMGHCTYFP EELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKP EFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELARE KNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPR SVSFDNSFNNKVLVKQEE N SKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDF INRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKL DKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIK YYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAE FIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEV KSKKHPQIIKKG. In this sequence, the bold and underlined residue N579 has been mutated (e.g., to A579). SaCas9 can produce nickase.

[0199] The amino acid sequence of an exemplary SaCas9n is as follows: KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTHS ELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKD GEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKWEYEMLMGHCTYFP EELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKP EFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIAIFNRKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIQUINAIIKKYGLPNDIIIELARE KNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPR SVSFDNSFNNKVLVKQEE A SKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTTKKEYLLEERDINRFSVQKDF INRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKKERNKGYKHHAEDALIIANADFIFKEWKKL DKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIK YYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAE FIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEV KSKKHPQIIKKG. In this sequence, residue A5 can be mutated from N579 to generate SaCas9 nickase. 79 is in bold and underlined.

[0200] The amino acid sequence of an exemplary SaKKH Cas9 is as follows: KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTHS ELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKD GEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPGFGWKDIKEWYEMLMGHCTYFP EELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKP EFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELARE KNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPR SVSFDNSFNNKVLVKQEE A SKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDF INRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKL DKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNR K LINDTLYSTRKDDKGNTLI VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIK YYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAE FIASFY K NDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPP H IIKTIASKTQSIKKYSTDILGNLYEV KSKKHPQIIKKG.

[0201] Residue A579, which can be mutated from N579 to generate SaCas9 nickase, is in bold. The underlined residues are E781, N967, and R1014, which can be mutated to generate SaKKH Cas9. Residues K781, K967, and H1014 above that can be identified are underlined in italics.

[0202] The polynucleotide-programmable nucleotide-binding domain of the base editor is The polynucleotide programmable vector itself can contain one or more domains. The nucleotide-binding domain capable of binding to the nuclease may include one or more nuclease domains. In some embodiments, the nucleic acid of a polynucleotide-programmable nucleotide binding domain The nuclease domain can comprise an endonuclease or an exonuclease. As used herein, the term "exonuclease" refers to an enzyme that liberates nucleic acids (e.g., RNA or DNA). The term "endonuclease" refers to a protein or polypeptide that can be digested from its termini. A "clease" is a nucleic acid that can catalyze (e.g., cleave) an internal region of a nucleic acid (e.g., DNA or RNA). In some embodiments, an endonuclease refers to a protein or polypeptide that It is capable of cleaving a single strand of double-stranded nucleic acid. Both strands of a double-stranded nucleic acid molecule can be cleaved. The programmable nucleotide binding domain can be a deoxyribonuclease. In some embodiments, the polynucleotide programmable nucleotide binding domain is a ribonucleotide. It may be a nuclease.

[0203] In one embodiment, the nucleotide of the polynucleotide-programmable nucleotide binding domain The cleavage domain can cleave zero, one, or two strands of a target polynucleotide. In some cases, the polynucleotide-programmable nucleotide binding domain can comprise a nickase domain. As used herein, the term "nickase" means A nuclease capable of cleaving only one strand of a double-stranded nucleic acid molecule (e.g., DNA) refers to a polynucleotide-programmable nucleotide-binding domain containing a nucleotidease domain In some embodiments, the nickase is an active polynucleotide programmable nucleotide. By introducing one or more mutations into the polypeptide binding domain, Derived from a fully catalytically active (e.g., native) form of a grammable nucleotide-binding domain For example, a polynucleotide programmable nucleotide binding domain can be When the nickase domain derived from Cas9 is included, the nickase domain derived from Cas9 is It may contain a D10A mutation and a histidine at position 840. In such cases, Residue H840 retains catalytic activity, thereby enabling single-strand cleavage of nucleic acid duplexes. In another example, the nickase domain from Cas9 can include an H840A mutation. , while the amino acid residue at position 10 remains D. The nickase may contain all or part of a nuclease domain that is not required for nickase activity. by removing the polynucleotide-programmable nucleotide binding domain It can be derived from a fully catalytically active (e.g., native) form. The programmable nucleotide-binding domain binds to the nickase domain derived from Cas9. When included, the nickase domain from Cas9 is the entire RuvC domain or HNH domain. It may contain a partial or partial deletion.

[0204] Thus, polynucleotides containing nickase domains can be used as programmable nucleotides. Base editors containing a base-binding domain can be used to target specific polynucleotide target sequences (e.g., binding domains). The DNA fragments are then ligated to the target nucleic acid, which then generates a single-stranded DNA break (a nick) at the target site (determined by the complementary sequence of the guide nucleic acid). In some embodiments, a nickase domain (e.g., a nickase domain derived from Cas9) can be used. Nucleic acid double-stranded target polynucleotides cleaved by base editors containing a base editor domain (a cleavage enzyme domain) The strand of the base sequence is the strand that is not edited by the base editor (i.e., (The strand cleaved by the cleavage is the opposite strand to the strand containing the base to be edited.) and base editors containing a nickase domain (e.g., a nickase domain derived from Cas9). can cut the strand of the DNA molecule targeted for editing. The non-target strand is not cleaved.

[0205] Catalytically dead (i.e., unable to cleave target polynucleotide sequence) polynucleotides Base editors comprising nucleotide-programmable nucleotide-binding domains are also described herein. As used herein, the terms "catalytically dead" and "dead nuclease" are used interchangeably. " refers to one or more mutations and / or mutations that result in the inability to cleave a strand of nucleic acid. Polynucleotides with deletions can be used to refer to programmable nucleotide binding domains. In some embodiments, catalytically dead polynucleotide proteases are used interchangeably. The programmable nucleotide-binding domain base editors bind to one or more nuclease domains. Nuclease activity can be lost as a result of specific point mutations in the For example, in the case of base editors containing the Cas9 domain, Cas9 is able to reverse the D10A mutation and the H840A mutation. Such mutations can render both nuclease domains inactive. In another embodiment, the catalytically dead polynuclease is Nucleotide-programmable nucleotide-binding domains can be used in catalytic domains (e.g., RuvC1 and The present invention may include one or more deletions of all or part of the HNH domain (e.g., the HNH domain and / or the HNH domain). In some embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain The inclusion of point mutations (e.g., D10A or H840A) as well as the entire or partial nuclease domain contains a partial deletion.

[0206] Also provided herein are the following polynucleotide-programmable nucleotide binding domains: Catalytically dead polynucleotide programmable nucleic acid from a previously functioning version Mutations that can generate peptide-binding domains are also contemplated. In the case of dead Cas9 ("dCas9"), mutations other than D10A and H840A are present, leading to the nuclease Variants that result in inactive Cas9 are provided. Such mutations include, for example, D10 and other amino acid substitutions at H840 or other substitutions within the nuclease domain of Cas9. (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain) Additional suitable nuclease-inactive dCas9 domains are described in this disclosure and in the art. Such modifications would be apparent to those skilled in the art based on their knowledge and are within the scope of the present disclosure. Exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to: Contains D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (e.g., Prashant et al., CAS9 transcriptional activators for target specific ficity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference. In some embodiments, the dCas9 domain is a nucleotide sequence similar to that provided herein. At least 60%, at least 65%, or at least 70% of any of the dCas9 domains , 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% In some embodiments, the Cas9 domain comprises an amino acid sequence having the identity: 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 compared to any of the amino acid sequences described herein 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 3 0, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 5 In some embodiments, the amino acid sequence includes an amino acid sequence having zero or more mutations. The Cas9 domain may have at least one amino acid sequence similar to any of the amino acid sequences described herein. At least 10, at least 15, 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 250, at least 300, at least 350, at least 400, a few At least 500, at least 600, at least 700, at least 800, at least 900, at least and each of the amino acid sequences has at least 1000, at least 1100, or at least 1200 identical consecutive amino acid residues. It contains an amino acid sequence.

[0207] Polynucleotide programmable nucleotides that can be incorporated into base editors Non-limiting examples of binding domains include domains derived from CRISPR proteins, restriction nucleases, and the like. Enzymes, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases In some cases, base editors are used to manipulate nucleic acids using CRISPR ( Clustered Regularly Interspaced Short Palindromic Repeats (CIR)-mediated modification natural or modified proteins that can bind to a nucleic acid sequence via a binding guide nucleic acid Polynucleotide-programmable nucleotide-binding domains containing proteins or portions thereof Such proteins are referred to herein as "CRISPR proteins." Thus, disclosed herein are polynucleotides containing all or part of a CRISPR protein. Nucleotide-programmable base editors containing nucleotide-binding domains (i.e. , a base editor containing all or part of a CRISPR protein as a domain (which is a base The CRISPR protein-derived domain of the base editor is also called the "CRISPR protein-derived domain." The CRISPR protein-derived domain integrated into the CRISPR protein is similar to that of the wild-type or naturally occurring CRISPR protein. For example, as described below, CRISPR protein-derived DNA can be modified compared to the target protein. The main one is a gene that contains one or more mutations or insertions compared to the wild-type or native CRISPR protein. , deletions, rearrangements and / or recombinations.

[0208] In some embodiments, the base editor comprises a CRISPR protein-derived domain. The main component is capable of binding to a target polynucleotide when combined with a binding guide nucleic acid. Endonucleases (e.g., deoxyribonucleases or ribonucleases) that can In some embodiments, the CRISPR protein incorporated into the base editor The protein-derived domain binds to the target polynucleotide when combined with the binding guide nucleic acid. In some embodiments, the base editor is a nickase that can bind to The CRISPR protein-derived domains integrated into the CRISPR protein bind to the CRISPR protein when combined with the guide nucleic acid. A catalytically dead domain is capable of binding to a target polynucleotide when In embodiments, a target polynucleotide that binds to a CRISPR protein-derived domain of a base editor is The nucleic acid is DNA, and in some embodiments, the nucleic acid comprises a CRISPR protein-derived domain of a base editor. The target polynucleotide that binds to the in is RNA.

[0209] In some embodiments, the CRISPR protein-derived domain of the base editor is Corynebacterium diphtheria (NCBI Refs: NC_015683.1, NC_017317.1); NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021 284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (N CBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella ba ltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1); St reptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP _472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningiti dis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus au The Cas9 fragment may comprise all or part of the Cas9 fragment derived from Cas9.

[0210] In some embodiments, the Cas9 domain is a Cas9 domain from Staphylococcus aureus (SaCa In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, nuclease The enzyme-inactive SaCas9 (SaCas9d), or SaCas9 nickase (SaCas9n). In embodiments, SaCas9 contains an N579A mutation, or an amino acid sequence provided herein. containing the corresponding mutation in either of the sequences.

[0211] In some embodiments, a SaCas9 domain, a SaCas9d domain, or a SaCas9n domain The nucleotides can bind to nucleic acid sequences with non-canonical PAMs, and in some embodiments The SaCas9 domain, SaCas9d domain, or SaCas9n domain is NNGRRT or NNNRRT In some embodiments, SaCa can bind to a nucleic acid sequence having a PAM sequence. The s9 domain may contain one or more of the E781X, N967X, and R1014X mutations, or any of the mutations provided herein. and corresponding mutations in any of the amino acid sequences described above, where X is any amino acid. In some embodiments, the SaCas9 domain is E781K, N967K, and R101. 4H mutations or in any of the amino acid sequences provided herein. In some embodiments, the SaCas9 domain comprises one or more corresponding mutations in the E781K, N967K, or R1014H mutation, or It includes corresponding mutations in either

[0212] The base editor comprises a domain derived from all or part of Cas9, a high-fidelity Cas9. In some embodiments, the high-fidelity Cas9 domain of the base editor can , between the Cas9 domain and the sugar-phosphate backbone of DNA compared to the corresponding wild-type Cas9 domain. An engineered Cas9 domain containing one or more mutations that reduce electrostatic interactions. High-fidelity Cas9 domains with reduced electrostatic interactions with the sugar-phosphate backbone of NAs exhibited fewer In some embodiments, the Cas9 domain may have off-target effects. (e.g., wild-type Cas9 domain) reduces the binding between the Cas9 domain and the sugar-phosphate backbone of DNA. In some embodiments, the Cas9 domain comprises one or more mutations that reduce Ca The binding between the s9 domain and the sugar-phosphate backbone of DNA is reduced by at least 1%, at least 2%, or at least At least 3%, 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 at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or more It contains one or more mutations that cause

[0213] [Guide polynucleotide] As used herein, the term "guide polynucleotide" refers to a polynucleotide that is specific to a target sequence. and polynucleotide-programmable nucleotide binding domain proteins (e.g., In one embodiment, the term "polynucleotide" refers to a polynucleotide that can form a complex with a gene encoding a gene encoding a gene encoding a gene (e.g., Cas9 or Cpf1). The guide polynucleotide is a guide RNA. As used herein, the term "guide RNA" "(gRNA)" and its grammatical equivalents are intended to mean a gene that can be specific for a target DNA and can act as a Cas protein. The RNA / Cas complex may represent an RNA that can form a complex with the target DNA. Cas9 / crRNA / tracrRNA can help guide the transcription of the target gene. Endonucleolytic cleavage of linear or circular dsDNA targets that are not complementary to the crRNA. The target strand is first cleaved by an endonuclease and then exonucleolytically cleaved in the 3'-5' direction. In nature, DNA binding and cleavage usually requires both a protein and RNA. However, it has been shown that both crRNA and tracrRNA aspects can be incorporated into a single RNA species. Alternatively, a single guide RNA ("sgRNA," or simply "gRNA") can be generated. For example, , Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are incorporated by reference. Cas9 targets short motifs (PAM or PAM) in the CRISPR repeats. protospacer adjacent motif) to help distinguish self from non-self.

[0214] In some embodiments, the guide polynucleotide comprises at least one single guide RNA ("s In some embodiments, the guide polynucleotide is at least In some embodiments, the guide polynucleotide is a polynucleotide. Inject a programmable DNA-binding domain (e.g., Cas9 or Cpf1) into the target nucleotide sequence. It does not require a PAM sequence to guide it.

[0215] The polynucleotide programmable nucleotide sequences of the base editors disclosed herein The guide polynucleotide is associated with a domain-binding domain (e.g., a CRISPR-derived domain). The target polynucleotide sequence can be recognized by the guide polynucleotide ( The target sequence of the polynucleotide is targeted by a gene encoding a gene (e.g., a gRNA), which is typically single-stranded and binds to the target sequence in a site-specific manner. can be programmed to bind (i.e., via complementary base pairing) and Thus, the base editor with the guide nucleic acid is guided to the target sequence. The guide polynucleotide may be DNA. The guide polynucleotide may be RNA. The oligonucleotides include natural nucleotides (e.g., adenosine). Polynucleotides may contain non-natural (or unnatural) nucleotides (e.g., peptide nucleic acids or nucleotides). In some cases, the targeting region of the guide nucleic acid sequence may be short in length. At least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 The targeting region of the guide nucleic acid can be between 10 and 30 nucleotides in length, or can be between 15 and 25 nucleotides in length, or between 15 and 20 nucleotides in length.

[0216] In some embodiments, the guide polynucleotide is a polynucleotide that is capable of, e.g., complementary base pairing. It comprises two or more individual polynucleotides that can interact with each other via a duplex. For example, guide polynucleotides include CRISPR RNA (crRNA) and and trans-activating CRISPR RNA (tracrRNA). For example, a guide polynucleotide The reticle can include one or more trans-activating CRISPR RNAs (tracrRNAs).

[0217] In Type II CRISPR systems, targeting of nucleic acids by CRISPR proteins (e.g., Cas9) is typically Specifically, the first RNA molecule (crRNA) contains a sequence that recognizes the target sequence, and the guide RNA-CRISPR tag. A second RNA molecule (trRNA) containing repeat sequences that form a scaffolding region that stabilizes the protein complex Such a dual guide RNA system requires complementary base pairing between the and a guide polynucleotide for directing a base editor to a target polynucleotide sequence, as disclosed in the document. It can be used as a protease inhibitor.

[0218] In some embodiments, the base editors provided herein comprise a single guidepo In some embodiments, the present invention utilizes a nucleic acid sequence (e.g., a gRNA) as provided herein. The base editors used are dual guide polynucleotides (e.g., dual guide polynucleotides). In some embodiments, the base editors provided herein utilize: Some embodiments utilize one or more guide polynucleotides (e.g., multiple gRNAs). In the method, a single guide polynucleotide is selected from the group consisting of a plurality of different base editors described herein. For example, a single guide polynucleotide can be synthesized using a cytidine base editor. and adenosine base editors.

[0219] In other embodiments, the guide polynucleotide comprises a polynucleotide targeting portion of a nucleic acid and Both the nucleic acid and the nucleic acid scaffold portion can be contained in a single molecule (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" refers to a base pair. any base editor that can interact with the base editor and guide the base editor to the target polynucleotide sequence Single, double or multi-molecule nucleic acids of the formula (I) are contemplated.

[0220] Typically, the guide polynucleotide (e.g., crRNA / trRNA complex or gRNA) is A "polynucleotide" refers to a sequence that can recognize and bind to a polynucleotide sequence. and a base editor for polynucleotide programmable nucleotide sequences. A "protein binding segment" that stabilizes the guide polynucleotide within the binding domain component. In some embodiments, the polynucleotide target segment of the guide polynucleotide The ment recognizes and binds to a DNA polynucleotide, thereby editing the bases in the DNA. In other embodiments, the polynucleotide target segment of the guide polynucleotide is The nucleotides recognize and bind to RNA polynucleotides, thereby facilitating the editing of bases in the RNA. Here, a "segment" refers to a portion or region of a molecule, such as a guide polynucleotide. A segment is a stretch of consecutive nucleotides in a nucleic acid. A segment can also represent a region / section, and thus can include regions of more than one molecule. For example, if the guide polynucleotide comprises multiple nucleic acid molecules, the protein binding segments may be The complement may be, for example, all or part of a plurality of separate molecules hybridized along regions of complementarity. In some embodiments, the targeting of the DNA-targeting RNA comprises two separate molecules. The protein-binding segment is: (i) a first RNA molecule that is 100 base pairs in length and spans base pairs 40-75 of the first RNA molecule; and (ii) 10 to 25 base pairs of a second RNA molecule that is 50 base pairs in length. The definition of "base pair" refers to a specific number of base pairs out of the total number of base pairs, unless otherwise defined in a particular context. is not limited to a specific number of base pairs from a given RNA molecule, but to distinct sequences within the complex. It is not limited to a particular number of molecules and can include regions of any full length RNA molecule, and can be used to identify regions of interest relative to other molecules. It may contain regions of complementarity.

[0221] A guide RNA or guide polynucleotide is a combination of two or more RNAs, such as CRISPR RNA (crRNA ) and trans-activating crRNA (tracrRNA). The polynucleotide sometimes comprises a fusion of portions (e.g., functional portions) of the crRNA and tracrRNA. The guide RNA or single-stranded RNA formed by the Alternatively, the guide polynucleotide may be a duplex RNA comprising a crRNA and a tracrRNA. Furthermore, the crRNA can hybridize with the target DNA.

[0222] As mentioned above, the guide RNA or guide polynucleotide can be an expression product. For example, the DNA encoding the guide RNA is a vector containing a sequence encoding the guide RNA. The guide RNA or guide polynucleotide is an isolated guide RNA or guide Transfect cells with plasmid DNA containing the RNA coding sequence and promoter The guide RNA or guide polynucleotide can be introduced into cells by The peptide can also be introduced into cells by other methods, such as using viral-mediated gene delivery.

[0223] The guide RNA or guide polynucleotide can be isolated. For example, the guide RNA The guide RNA can be transfected into a cell or organism in the form of isolated RNA. It can be prepared by in vitro transcription using any in vitro transcription system known in the art. The guide RNA can be delivered in the form of a plasmid containing the guide RNA coding sequence. , can be introduced into cells in the form of isolated RNA.

[0224] A guide RNA or guide polynucleotide may comprise the following three regions: a chromosomal sequence; a first region at the 5' end that may be complementary to a target site in the a second internal region that can be used to generate a fusion protein, and a third 3' region that can be single-stranded. The first region of each guide RNA can be different to guide the gene to a specific target site. Furthermore, the second and third regions of each guide RNA are identical in all guide RNAs. It could be.

[0225] The first region of the guide RNA or guide polynucleotide is It is complementary to a sequence at the target site in the chromosomal sequence so that it can form base pairs with the target site. In some cases, the first region of the guide RNA may be about 10 nucleotides to 25 nucleotides. nucleotides (i.e., from 10 nucleotides to nucleotides; or from about 10 nucleotides to about 25 nucleotides) or from 10 nucleotides to about 25 nucleotides; or about 10 nucleotides For example, the first nucleotide of a guide RNA may be 25 nucleotides or more. The region of base pairing between the region and the target site in the chromosomal sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25 or more nucleotides The first region of the guide RNA may be at or about the length of the first region of the guide RNA. may be 9, 20, or 21 nucleotides, or about 19, 20, or 21 nucleotides .

[0226] The guide RNA or guide polynucleotide may include a second region that forms a secondary structure. For example, the secondary structure formed by the guide RNA can be a stem (or hairpin ) and loops. The lengths of the loops and stems can vary. For example, For example, the loop length can range from (approximately) 3 to 10 nucleotides, and the stem length can range from (approximately) 6 to 10 nucleotides. The stem may range from 1 to 10 or about 10 nucleotides. The total length of the second region can range from about 16 to 60 nucleotides in length. For example, the loop can be (about) 4 nucleotides in length and the stem can be (about) 12 nucleotides. It may be a base pair.

[0227] The guide RNA or guide polynucleotide may be essentially single-stranded. A third region may also be included. For example, the third region may be any staining of the cells of interest. It may not be complementary to the target sequence or the rest of the guide RNA. Furthermore, the length of the third region can vary. The third region can be approximately 4 nucleotides in length. For example, the length of the third region can range from (about) 5 to 60 nucleotides in length. could be.

[0228] The guide RNA or guide polynucleotide can be inserted into any exon or intron of the gene target. In some cases, the guide targets exon 1 or 2 of the gene. in other cases, the guide targets exon 3 or 4 of the gene. The composition can contain multiple guide RNAs that all target the same exon, or In some cases, the gene can contain multiple guide RNAs that target different exons. Both exons and introns of a gene can be targeted.

[0229] The guide RNA or guide polynucleotide targets a nucleic acid sequence of (approximately) 20 nucleotides. The target nucleic acid may be less than (about) 20 nucleotides in length. 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, 16, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170 6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50 nucleotides, or 1 to 100 nucleotides The target nucleic acid sequence can be any length between the nucleotides. The guide RNA may target a nucleic acid sequence. The target nucleic acid may be: At least (approximately) 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, Or it may be 1 to 100 nucleotides.

[0230] The guide polynucleotide, e.g., guide RNA, is a nucleic acid sequence that encodes a nucleic acid sequence that is encoded by another nucleic acid, e.g., a nucleic acid sequence that is encoded by ... It can refer to a nucleic acid that can hybridize to a target nucleic acid or a protospacer. The guide polynucleotide can be RNA. The guide polynucleotide can be DNA. The polynucleotide is programmed or designed to bind site-specifically to a sequence of nucleic acid. The guide polynucleotide may comprise one polynucleotide strand, A guide polynucleotide may be a sequence of two polynucleotides. The guide RNA may comprise a double-stranded polynucleotide, which may be referred to as a dual-guide polynucleotide. For example, RNA molecules can be transcribed in vitro and / or synthesized. RNA can be synthesized chemically from synthetic DNA molecules, e.g., gBlocks® gene fragments. Guide RNAs can be introduced into cells or embryos as RNA molecules. Guide RNAs can also be It can be introduced into a cell or embryo in the form of a non-RNA nucleic acid molecule, such as a DNA molecule. For example, a guide RNA DNA encoding A is provided that encodes a promoter for expression of the guide RNA in the cells or embryo of interest. The RNA coding sequence may be operably linked to a regulatory sequence that encodes RNA polymerase III (Pol III). ) can be operably linked to a promoter sequence recognized by Plasmid vectors that can be used for this purpose are the px330 vector and the px333 vector. In some cases, a plasmid vector (e.g., px333 vector) may be used. The vector can comprise a DNA sequence encoding at least two guide RNAs.

[0231] Select and design guide polynucleotides (e.g., guide RNAs) and targeting sequences Methods for verifying and validating the identity of the gene are described herein and known to those skilled in the art. For example, Potential of deaminase domains (e.g., AID domains) in nucleobase editor systems To minimize the effects of potential substrate promiscuity, residues that may potentially be located on ssDNA within the target nucleic acid locus (e.g., off-target residues that may potentially be located on ssDNA within the target nucleic acid locus) Furthermore, software tools can be used to minimize the number of nucleotides (e.g., C residues). The gRNA corresponding to the target nucleic acid sequence can be optimized, e.g., by total offset across the genome. For example, targeting domains using S. pyogenes Cas9 can minimize targeting activity. For each possible in-choice, (preceding the selected PAM, e.g. NAG or NGG) Any of a number of mismatched base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) All off-target sequences can be identified across the genome. The primary regions of the gRNAs that are useful can be identified, and all primary regions (e.g., crRNAs) can be compared to their total predicted values. The off-target scores can be ranked according to the off-target score; The main one is likely to have the greatest on-target activity and the least off-target activity. Candidate targeting gRNAs can be prepared by methods known in the art and / or described herein. The method can be used to functionally assess.

[0232] As a non-limiting example, a target DNA fragment in a guide RNA crRNA for use with Cas9 can be used. Hybridizing sequences can be identified using DNA sequence searching algorithms. The total is from Bae S., Park J., & Kim J.-S. Cas-OFFinder: A fast and versatile algorithm. that searches for potential off-target sites of Cas9 RNA-guided endonucleases. B The public tool cas-offinder, as described in ioinformatics 30, 1473-1475 (2014), This can be done using custom gRNA design software based on this software. scores guides after calculating the genome-wide off-target propensity of the guide. Matches typically range from a perfect match to seven mismatches, with lengths ranging from 17 to 24. Once the off-target sites are computationally determined, An aggregate score is calculated for each guide and output in a tabular format using a web interface. In addition to identifying potential target sites adjacent to the PAM sequence, The software identifies all sequences that differ from the selected target site by 1, 2, 3, or more than 3 nucleotides. The PAM flanking sequences are also identified. The repeat elements were then identified using publicly available tools, such as the RepeatMasker program. RepeatMasker can identify repeats from an input DNA sequence. Finds repeats and low-complexity regions. Detailed annotation of repeats present in a given query sequence. The output is:

[0233] After identification, the first region of the guide RNA (e.g., crRNA) is identified based on its distance to the target site, orthogonality and close match to related PAM sequences. Presence of protease inhibitors (e.g., associated PAMs (e.g., NGG PAM for Streptococcus pyogenes, Close matches in the human genome containing NNGRRT or NNGRRV PAM for Staphylococcus aureus The term "genetic variants" can be ranked hierarchically based on the identification of the 5'G. Orthogonality, when used in this context, refers to the ability to generate sequences in the human genome that contain a minimal number of mismatches to the target sequence. "High level of orthogonality" or "good orthogonality" refers to, for example, the number of sequences that are orthogonal to the intended sequence. It has no identical sequences in the human genome other than the target and has one or two mutations in the target sequence. It can also refer to a 20-mer targeting domain that has no sequences containing a match. The targeting domain can be selected to minimize off-target DNA cleavage.

[0234] In some embodiments, detecting base editing activity and identifying candidate guide polynucleotides. A reporter system can be used to test for nucleotides. In the reporter system, base editing activity results in expression of a reporter gene, It can include reporter gene-based assays. For example, the reporter system can be , including an inactive start codon, e.g., a mutation of 3'-TAC-5' to 3'-CAC-5' on the template strand Upon successful deamination of the target C, the corresponding mRNA is converted to 5'-GUG- The reporter gene is transcribed as 5'-AUG-3' instead of 3', allowing translation of the reporter gene. Reporter genes will be apparent to those skilled in the art. Non-limiting examples of reporter genes include: Green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase, secreted alkaline phosphatase (SEAP) or any other gene whose expression is detectable and apparent to one skilled in the art. Reporter systems can be used to detect many different For example, different gRNAs can be tested, each of which residues in the target DNA sequence are The sgRNA that targets the non-template strand can be determined by the corresponding deaminase. also act as an off-target for certain base-editing proteins (e.g., Cas9 deaminase fusion proteins). In some embodiments, such a test can be performed to assess the effectiveness of the Such gRNAs can be designed so that the mutant start codon does not base pair with the gRNA. The guide polynucleotide may be any of a variety of ribonucleotides, including standard ribonucleotides, modified ribonucleotides (e.g., primers, pseudouridine), ribonucleotide isomers, and / or ribonucleotide analogs In some embodiments, the guide polynucleotide may comprise at least The detectable label may include a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluor, Halo Tag, or or suitable fluorescent dyes), detection tags (e.g., biotin, digoxigenin, etc.), quantum dots, Or it may be gold particles.

[0235] The guide polynucleotide can be chemically synthesized, enzymatically synthesized, or For example, the guide RNA may be a standard phosphoramidite. Alternatively, the guide RNA can be synthesized using a D-based solid-phase synthesis method. The RNA is operably linked to a promoter control sequence recognized by a phage RNA polymerase. By ligating the appropriate phage, guide RNA can be synthesized in vitro. Examples of promoter sequences include T7, T3, and SP6 promoter sequences, or variants thereof. In embodiments where the guide RNA comprises two separate molecules (e.g., crRNA and tracrRNA), , crRNA can be chemically synthesized and tracrRNA can be enzymatically synthesized.

[0236] In some embodiments, the base editor system comprises a plurality of guide polynucleotides, e.g., For example, the gRNA may comprise one or more target genes included in the base editor system. locus (e.g., at least 1 gRNA, at least 2 gRNAs, at least 5 gRNAs, at least 1 0 gRNAs, at least 20 gRNAs, at least 30 gRNAs, at least 50 gRNAs) The multiple gRNA sequences can be arranged in tandem, preferably separated by direct repeats. Be separated.

[0237] The DNA sequence encoding the guide RNA or guide polynucleotide is part of a vector. In addition, the vector may contain additional expression control sequences (e.g., enhancer sequences, Kozak sequences, etc.). sequences, polyadenylation sequences, transcription termination sequences, etc.), selectable marker sequences (e.g., GFP or or antibiotic resistance genes such as puromycin), a replication origin, etc. The DNA molecule encoding the guide RNA may be linear. The DNA molecule encoding the leucine may also be circular.

[0238] In some embodiments, one or more components of the base editor system are Such DNA sequences can be incorporated into an expression system (e.g., a cell) together with or encoded by the They can be introduced separately, e.g., polynucleotide programmable nucleotides. DNA sequences encoding the binding domain and guide RNA can be introduced into cells, and each DNA The sequence can be part of a separate molecule (e.g., a polynucleotide programmable nucleic acid). One vector containing the coding sequence for the nucleotide binding domain and the guide RNA coding sequence a second vector containing the polynucleotide), or both can be part of the same molecule (e.g., a polynucleotide The nucleotide-programmable nucleotide binding domain and the guide RNA are both coded for by the nucleotide sequence. one vector containing the gene (and regulatory) sequences).

[0239] The guide polynucleotide is used to provide nucleic acids with new or enhanced characteristics. The guide polynucleotide may include one or more of the following modifications: The guide polynucleotide may be a synthetic nucleotide, a synthetic nucleotide analog, or a The nucleic acid may include nucleotides, nucleotide derivatives, and / or modified nucleotides.

[0240] In some cases, the gRNA or guide polynucleotide may contain modifications. Modifications can be made at any position in the gRNA or guide polynucleotide. Alternatively, multiple modifications can be made to the guide polynucleotide. The polynucleotide may be subjected to quality control after modification. Controls can include PAGE, HPLC, MS, or any combination thereof.

[0241] 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.

[0242] The gRNA or guide polynucleotide also contains a 5' adenylate, 5' guanosine triphosphate ketone. cap, 5'N 7-methylguanosine triphosphate cap, 5' triphosphate cap, 3' phosphate , 3' thiophosphate, 5' phosphate, 5' thiophosphate, Cis-Syn thymidine dimer, trimer, C12 thrombin Pacer, C3 spacer, C6 spacer, d spacer, PC spacer, r spacer, Spacer 18, Spacer 9, 3'-3' modification, 5'-5' modification, abasic modification, acridine, azobe Biotin, Biotin BB, Biotin TEG, Cholesterol TEG, Desthiobiotin TE G, DNP TEG, DNP-X, DOTA, dT-biotin, double biotin, psoralen, psoralen C2, psoralen C6, TINA, 3'DABCYL, Black Hole Quencher 1, Black Hole Quencher 2 , DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, Carboxyluri Linker, thiol linker, 2'-deoxyribonucleoside analogue purine, 2'-deoxy Ribonucleoside analogues pyrimidines, ribonucleoside analogues, 2'-O-methylribonucleic acids Oxide analogs, sugar-modified analogs, wobble / universal bases, fluorescent dye labels, 2'-fluoro RNA, 2'-O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioester DNA, phosphorothioate RNA, UNA, pseudolysine-5'- triphosphate, 5'-methylcysteine-5'-triphosphate, or any combination thereof It can also be modified.

[0243] In some cases, the modification is permanent. In other cases, the modification is temporary. In this case, multiple modifications are made to the gRNA or guide polynucleotide. Modifications of guide polynucleotides can affect their conformation, polarity, hydrophobicity, and chemical reactivity. The physical and chemical properties of nucleotides, such as their affinity, base pairing interactions, or a combination thereof The properties can be changed.

[0244] The modification may also be a phosphorothioate substitution. In some cases, the natural phosphodiester The linkage is subject to rapid degradation by cellular nucleases, and phosphorothioate (PS) bonds Modification of internucleotide bonds with synthetic substitutes makes them more resistant to hydrolysis during cell lysis. The modifications can increase the stability of the gRNA or guide polynucleotide. Modifications can also enhance biological activity. In some cases, phosphorothioates Enhanced RNA gRNAs are resistant to RNase A, RNase T1, calf serum nuclease, or a combination thereof. These properties can be seen in the nuclear Enables the use of PS-RNA gRNA in applications where exposure to enzymes is likely For example, phosphorothioate (PS) bonds can be added to the terminal ends of the gRNA at the 5' or '6' ends. It can be introduced between 3 and 5 nucleotides of the In some cases, phosphorothioate linkages are added throughout the gRNA to protect it from endonucleases. It can reduce attacks.

[0245] [protospacer adjacent motif] The term "protospacer adjacent motif (PAM)" or PAM-like motif refers to the bacterial adaptation of CRISPR. A 2- to 6-base pair DNA sequence immediately following the DNA sequence targeted by Cas9 nuclease in the immune system In some embodiments, the PAM refers to a 5' PAM (i.e., a sequence at the 5' end of the protospacer). In other embodiments, the PAM may be a 3' PAM (i.e., located upstream of the protospacer). (located downstream of the 5' end of the

[0246] Protospacer adjacent motifs (PAMs) or PAM-like motifs mediate CRISPR-mediated bacterial adaptive immune responses This refers to the 2-6 base pair DNA sequence immediately following the DNA sequence targeted by Cas9 nuclease. In some embodiments, the PAM is a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (i.e., at the 5' end of the protospacer). The PAM sequence is essential for target binding, but the exact sequence is unknown. Depends on the type of protein. The base editors provided herein can be used to generate bases adjacent to canonical or non-canonical protospacers. CRISPR proteins that can bind to nucleotide sequences containing the PAM motif (PAM) sequence The PAM site may comprise a nucleotide sequence adjacent to the target polynucleotide sequence. Some aspects of the present disclosure provide CRISPR targets with different PAM specificities. For example, a base editor comprising all or part of a protein derived from S. pyogenes is provided. Cas9 proteins, such as Cas9 (spCas9), are typically engineered to bind to specific nucleic acid regions. Requires the standard NGG PAM sequence, where the "N" in "NGG" stands for adenine (A) or thymine (T). , guanine (G), or cytosine (C), where G is guanine. PAM is a CRISPR protein. Different base editors can be protein-specific and contain domains from different CRISPR proteins. The PAM can be located 5' or 3' of the target sequence. The PAM can be located upstream or The PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. In most cases, PAMs are 2-6 nucleotides in length.

[0247] In some embodiments, the Cas9 domain is a Cas9 domain from Streptococcus pyogenes. In some embodiments, the SpCas9 domain comprises a nuclease-active SpCas9, a nuclease-binding domain (SpCas9). SpCas9 is a nucleotide-binding protein that binds to the nucleotides in the nucleotide sequence of the target molecule, either ase-inactive SpCas9 (SpCas9d) or SpCas9 nickase (SpCas9n). In this embodiment, SpCas9 has a D9X mutation, or an amino acid sequence provided herein. and a corresponding mutation in any of the following sequences, where X is any amino acid other than D. In some embodiments, SpCas9 is a D9A mutation or a variant of any of the variants provided herein. In some embodiments, the SpCas9 domain contains a corresponding mutation in either of the amino acid sequences. Main, SpCas9d domain or SpCas9n domain binds to nucleic acid sequences with non-canonical PAM In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain The domain can bind to a nucleic acid sequence having an NGG, NGA, or NGCG PAM sequence. In some embodiments, the SpCas9 domain contains D1135X, R1335X, and T1337X mutations. or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, S The pCas9 domain may contain one or more of the D1135E, R1335Q, and T1337R mutations, or any of the mutations described herein. It includes corresponding mutations in any of the amino acid sequences provided. In this embodiment, the SpCas9 domain contains the D1135E, R1335Q, and T1337R mutations, or the mutations described herein. The amino acid sequences of the present invention may be modified in various ways, including corresponding mutations in any of the amino acid sequences provided in the publication. In embodiments, the SpCas9 domain contains one of a D1135X, R1335X, and T1337X mutation. or one or more corresponding hits in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 The domain may contain one or more of the D1135V, R1335Q, and T1337R mutations, or any of the mutations provided herein. In some embodiments, the amino acid sequence of the nucleotide ... In this case, the SpCas9 domain contains the D1135V, R1335Q, and T1337R mutations, or the mutations described herein. and corresponding mutations in any of the amino acid sequences provided. wherein the SpCas9 domain contains one or more of the following mutations: D1135X, G1218X, R1335X, and T1337X. or a corresponding mutation in any of the amino acid sequences provided herein. wherein X is any amino acid. In some embodiments, the SpCas9 domain is D one or more of the 1135V, G1218R, R1335Q, and T1337R mutations, or any of the mutations provided herein In some embodiments, the amino acid sequence of the nucleotide ... , the SpCas9 domain contains D1135V, G1218R, R1335Q, and T1337R mutations, or the mutations described herein. The amino acid sequences provided herein include corresponding mutations in any of the amino acid sequences provided herein.

[0248] In some embodiments, the Cas9 domain of any of the fusion proteins provided herein The main sequence is at least 60%, at least 6%, or 5%, 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 In some embodiments, The Cas9 domain of any of the fusion proteins provided herein may be any of the fusion proteins described herein. In some embodiments, the amino acid sequence of any Cas9 polypeptide described herein is The Cas9 domain of any of the fusion proteins provided herein may be any of the fusion proteins described herein. It consists of the amino acid sequence of the Cas9 polypeptide.

[0249] The amino acid sequences of exemplary SpCas9 proteins that can bind to PAM sequences are provided below: Provide:

[0250] The amino acid sequence of an exemplary PAM-bound SpCas9 is as follows: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD.

[0251] The amino acid sequence of an exemplary PAM-bound SpCas9n is as follows: MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD.

[0252] The amino acid sequence of an exemplary PAM-bound SpEQR Cas9 is as follows: MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGF E SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRI In this sequence, D1135, R1335, and T1337 were mutated to form SpEQR Cas9. Possible residues E1135, Q1335, and R1337 are in bold and underlined.

[0253] The amino acid sequence of an exemplary PAM-linked SpVQR Cas9 is as follows: MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGF V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRI In this sequence, D1135, R1335, and T1337 were mutated to form SpVQR Cas9. Possible residues V1135, Q1335, and R1337 are in bold and underlined.

[0254] The amino acid sequence of an exemplary PAM-linked SpVRER Cas9 is as follows: MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGF V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASA R ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK E Y R STKEVLDATLIHQSITGLYETRI DLSQLGGD.

[0255] The amino acid sequence of an exemplary PAM-linked SpVRQR Cas9 is as follows: MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGF V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASA R ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRI DLSQLGGD. It can be mutated from D1135, G1218, R1335, and T1337 to yield SpVRQR Cas9. Residues V1135, R1218, Q1335, and R1337 are bold and underlined.

[0256] In some embodiments, the Cas9 domain is a recombinant Cas9 domain. The recombinant Cas9 domain is a SpyMacCas9 domain. The acCas9 domain is used to generate nuclease-active SpyMacCas9 and nuclease-inactive SpyMacCas9 (SpyM acCas9d), or SpyMacCas9 nickase (SpyMacCas9n). In this case, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain has a non-standard PAM. In some embodiments, the SpyMacCas9 domain can bind to a nucleic acid sequence that encodes the SpyMacCas9 domain. The SpCas9d domain or the SpCas9n domain binds to a nucleic acid sequence having a NAA PAM sequence. It is possible. Exemplary SpyMacCas9 MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFGSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGHSL HEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEIQTVGQNGGLFDDNPKSPLEVT PSKLVPLKKELNPKKYGGYQKPTTAYPVLLITDTKQLIPISVMNKKQFEQNPVKFLRDRGYQQVGKNDFIKLPKYTLVDI GDGIKRLWASSKEIHKGNQLVVSKKSQILLYHAHHLDSDLSNDYLQNHNQQFDVLFNEIISFSKKCKLGKEHIQKIENVY SNKKNSASIEELAESFIKLLGFTQLGATSPFNFLGVKLNQKQYKGKKDYILPCTEGTLIRQSITGLYETRVDLSKIGED.

[0257] [High-fidelity Cas9 domain] Some aspects of the present disclosure provide high-fidelity Cas9 domains. In this study, high-fidelity Cas9 domains were characterized by a Cas9 domain with a higher fidelity than the corresponding wild-type Cas9 domain. those containing one or more mutations that reduce the electrostatic interactions between the phosphate and the sugar-phosphate backbone of DNA Without wishing to be bound by any particular theory, it is believed that the sugar-phosphate backbone of DNA High-fidelity Cas9 domains with reduced electrostatic interactions have fewer off-target effects In some embodiments, the Cas9 domain (e.g., a wild-type Cas9 domain) may have a It contains one or more mutations that reduce the bond between the phosphate and the sugar-phosphate backbone of DNA. In this manner, the Cas9 domain reduces the bond between the Cas9 domain and the sugar-phosphate backbone of DNA. At least 1%, at least 2%, at least 3%, 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 at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least The gene may also contain one or more mutations that reduce the gene expression level by 65%, or at least 70%.

[0258] In some embodiments, any of the Cas9 fusion proteins provided herein , N497X, R661X, Q695X, and / or Q926X mutations, or any of the mutations provided herein. and corresponding mutations in any of the amino acid sequences described above, where X is any amino acid. In some embodiments, any of the Cas9 fusion proteins provided herein is an acid. Any of the mutations may contain one or more of the N497A, R661A, Q695A, and / or Q926A mutations, or any of the mutations described herein. In some embodiments, the amino acid sequence of the present invention may be a mutation in the corresponding amino acid sequence of the present invention. In some embodiments, the Cas9 domain may comprise a D10A mutation, or the amino acid sequence provided herein. The Cas9 domains with high fidelity are well known in the art and contain corresponding mutations in either of the sequences. These techniques are well known in the art and will be apparent to those skilled in the art. For example, Cas9 domains with high fidelity can be used. The article is based on Kleinstiver, B.P., et al. "High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495 (2016); and S laymaker, IM, et al. “Rationally engineered Cas9 nucleases with improved spec ificity.” Science 351, 84-88 (2015), the full contents of which are hereby incorporated by reference. and is hereby incorporated by reference. In Figure 1, high-fidelity Cas9 domain mutations compared to Cas9 are shown in bold and underlined. MDKKYSIGL A IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEE VVDKGASAQSFIERMT A FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWG A LSRKLINGIRDKQSGKTILDFLKSDGFANRNFM A LIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR A ITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD.

[0259] In some cases, the variant Cas9 protein is H840A, P475A, W476A, N477A, ​​D1125A, W1 It has mutations D126A and D1127A, resulting in a reduced ability to cleave target DNA or RNA. Such Cas9 proteins have the ability to cleave target DNA (e.g., single-stranded target DNA). Although the binding capacity is reduced, it still retains the ability to bind to target DNA (e.g., single-stranded target DNA). By way of non-limiting example, in some cases, the variant Cas9 protein may be D10A, H8 40A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, resulting in The polypeptide has a reduced ability to cleave target DNA (e.g., single-stranded target DNA). Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA). The variant Cas9 protein retains the ability to bind to target DNA (e.g., single-stranded target DNA). If the protein has the W476A and W1126A mutations, or if the variant Cas9 protein has P475A, Variant Cas9 proteins with W476A, N477A, ​​D1125A, W1126A, and D1127A mutations Proteins do not bind efficiently to PAM sequences. When a variant Cas9 protein is used in the conjugation method, the method does not require a PAM sequence. In other words, in some cases, such variant Cas9 proteins are used in methods of conjugation. In some cases, the method may include a guide RNA, but the method may be performed in the absence of a PAM sequence. (Thus, binding specificity is provided by the target segment of the guide RNA) Other residues may be mutated (i.e., one or the other nucleoside) to achieve the above effect. (This inactivates the cleavage portion.) Non-limiting examples include residues D10, G12, G17, E762, H840 , modify (i.e., replace) N854, N863, H982, H983, A984, D986, and / or A987 Mutations other than alanine substitution are also suitable.

[0260] In some embodiments, the CRISPR protein-derived domain of the base editor is a canonical PAM sequence. In other embodiments, the Cas9 protein may comprise all or a portion of the Cas9 protein having the base sequence (NGG). The Cas9-derived domain of the editor can use non-canonical PAM sequences. Sequences are described in the art and will be apparent to those skilled in the art. For example, non-standard PAM sequences may be used. The Cas9 domains that bind to the array are described in Kleinstiver, B.P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and K leinstiver, BP, et al., “Broadening the targeting range of Staphylococcus au reus CRISPR-Cas9 by modifying PAM recognition”Nature Biotechnology, 33, 1293-12 98 (2015), the entire contents of each of which are incorporated herein by reference.

[0261] In some examples, the base editors disclosed herein are derived from CRISPR proteins. The PAM recognized by the domain is inserted into an insert (e.g., AAV The insert may be provided to the cell on a separate oligonucleotide. Providing the PAM on a separate oligonucleotide allows for the PAM to be isolated from an otherwise identical polynucleotide to the target sequence. Cleavage of a target sequence that cannot be cleaved due to the absence of an adjacent PAM on the nucleotide This makes it possible.

[0262] In one embodiment, S. pyogenes Cas9 (SpCas9) is used as a CRISPR enzyme for genome engineering. It can be used as a nuclease, although others can be used. Therefore, different endonucleases can be used to target specific genomic targets. In some cases, synthetic SpCas9-derived variants with non-NGG PAM sequences may be used. In addition, other Cas9 orthologs from various species have been identified, and these Non-SpCas9s can bind to a variety of PAM sequences that may also be useful in the present disclosure. For example, The large size of SpCas9 (approximately 4 kb coding sequence) allows efficient expression in cells. Conversely, the SpCas9 cDNA plasmid may not be effective against Staphylococcus aureus C. The coding sequence of as9 (SaCas9) is approximately 1 kilobase shorter than that of SpCas9, and therefore it is expressed efficiently in cells. Like SpCas9, the SaCas9 endonuclease can be expressed in mammalian cells in vitro and in vivo. They have the ability to modify target genes in vivo in mice. In some cases, Cas proteins Proteins can target different PAM sequences. In some cases, the target gene The Cas9 ortholog may be flanked by a Cas9 PAM, e.g., 5'-NGG. In other cases, other Cas9 orthologs may be different. For example, PAMs such as those of S. thermophilus (in the case of CRISPR1) may have different PAM requirements. 5'-NNAGAA for CRISPR3 and 5'-NGGNG for CRISPR3) and those of Neisseria meningiditis (5' Other PAMs, such as -NNNNGATT, can also be found adjacent to target genes.

[0263] In some embodiments, for S. pyogenes systems, the target gene sequence is 5'-NGG P The AM may be preceded by (i.e., 5' to) a 20 nt guide RNA sequence that base pairs with the opposite strand. The PAM can be formed to mediate Cas9 cleavage adjacent to the PAM. In some cases, the adjacent cleavage , (approximately) 3 base pairs upstream of the PAM. In some cases, the adjacent cleavage occurs (approximately) 10 base pairs upstream of the PAM. In some cases, the adjacent cleavage may be (approximately) 0 to 20 base pairs upstream of the PAM. For example, adjacent cleavages occur at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 sites upstream of the PAM. , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs away The adjacent cleavage can also be 1 to 30 base pairs downstream of the PAM.

[0264] [Fusion protein containing a nuclear localization sequence (NLS)] Vectors encoding CRISPR enzymes containing one or more nuclear localization sequences (NLS) can be used. For example, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs can be used. ISPR enzymes have approximately one NLS at or near the amino terminus, one at or near the carboxy terminus, and 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs or Any combination (e.g., one or more NLSs at the amino terminus and one at the carboxy terminus) If there are multiple NLSs, each NLS can be selected independently of the others. Therefore, one NLS can exist in more than one copy, and / or or in combination with one or more other NLSs present in one or more copies. do.

[0265] The CRISPR enzyme used in the method may contain approximately six NLSs. The amino acid is within about 50 amino acids (e.g., 1, 2, 3) along the polypeptide chain from the N- or C-terminus. , 4, 5, 10, 15, 20, 25, 30, 40, or 50 amino acids), the NLS is N- or is thought to be near the C-terminus.

[0266] In some embodiments, the NLS has the amino acid sequence PKKKRKVEGADKRTADGSEFES PKKKRKV, KRTADGSEF ESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVV Contains KRPRKPKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0267] In some embodiments, the NLS is present in a linker, or the NLS is present in a linker, For example, the linkers are flanked by linkers described herein. The N-terminal or C-terminal NLS is a bipartite NLS. A bipartite NLS is a NLS that is bound to a relatively short spacer sequence. It contains two basic amino acid clusters separated by a The NLS of nucleoplasmin is called KR[PAATK KAGQA]KKKK is a prototype of a ubiquitous bipartite signal, which binds two classes of basic amino acids. The stars are separated by a spacer of about 10 amino acids. The sequence is PKKKRKVEGADKRTADGSEFES PKKKRKV.

[0268] In some embodiments, the fusion proteins of the present invention do not include a linker sequence. In embodiments, there is a linker sequence between one or more domains or proteins.

[0269] The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include the following: Includes, but is not limited to: NGG, NGA, NGC, NGN, NGT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNG ATT, NNAGAAW, or NAAAAC, where Y is a pyrimidine and N is any nucleotide base. and W is A or T.

[0270] [Nucleobase editing domain] Polynucleotide-programmable nucleotide-binding domains and nucleobase-editing domains The present disclosure provides base editors that include fusion proteins containing a deaminase domain. The base editor is a guide polynucleotide that can recognize a target sequence. The target polynucleotide sequence is then edited by interacting with the target base. Once the target sequence is recognized, editing takes place. The base editor is immobilized on a polynucleotide to be synthesized, and then the base editor's deamina The enzyme domain component can edit the target base.

[0271] In some embodiments, the nucleobase editing domain is a deaminase domain. In other words, the deaminase domain is a cytosine deaminase or a cytidine deaminase. In some embodiments, "cytosine deaminase" and "cytidine deaminase" may be used. The terms "deaminase" and "deaminase" can be used interchangeably. The enzyme can be adenine deaminase or adenosine deaminase. In the form, the terms "adenine deaminase" and "adenosine deaminase" are Further details of nucleobase-editing proteins can be found in International PCT Application No. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632) , each of which is incorporated herein by reference in its entirety. Komor, AC, et al. ., “Programmable editing of a target base in genomic DNA without double-strande d DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programma ble base editing of A·T to G·C in genomic DNA without DNA cleavage”Nature 55 1, 464-471 (2017); and Komor, AC, et al., “Improved base excision repair i nhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with h See also "Higher efficiency and product purity" Science Advances 3:eaao4774 (2017) the entire co...

Claims

1. SER including single nucleotide polymorphisms (SNPs) associated with alpha-1 antitrypsin deficiency (A1AD) A method for editing a PINA1 polypeptide, comprising: The SERPINA1 polypeptide is complexed with one or more guide polynucleotides. This includes contacting the editor. The base editor comprises a polynucleotide programmable DNA binding domain and a cytogenetic and a base deaminase domain, wherein the one or more guide polynucleotides comprise the base endonucleases. Targeting the regulator to alter single nucleotide polymorphisms (SNPs) associated with A1AD. method.

2. The contacting can be in a cell, in a eukaryotic cell, in a mammalian cell, or in a human cell. The method of claim 1 .

3. The method of claim 1 or 2, wherein the cell is in vivo or ex vivo.

4. The base editor deaminates the cytidine at position 1455 of the SERPINA1 polynucleotide , thereby at amino acid position 374 of the alpha-1 antitrypsin (A1AT) protein. The method according to any one of claims 1 to 3, which induces a mutation from methionine to isoleucine in The method described.

5. The A1AT polypeptide according to any one of claims 1 to 4, wherein the A1AT polypeptide comprises a lysine at amino acid position 342. The method according to any one of claims 1 to 5.

6. The A1AT polypeptide according to any one of claims 1 to 4, wherein the A1AT polypeptide comprises a lysine at amino acid position 376. The method according to any one of claims 1 to 5.

7. The polynucleotide programmable DNA binding domain is selected from the group consisting of Streptococcus pyogenes The method according to any one of claims 1 to 4, wherein the target polypeptide is Cas9 (SpCas9) or a variant thereof. 。

8. The SpCas9 has specificity for a PAM sequence selected from 5'-NGG-3' or 5'-GGG-3'. The method of claim 7, wherein the

9. The polynucleotide programmable DNA binding domain comprises a nuclease inactive valence The method according to any one of claims 1 to 8, wherein the antibody is an antagonism or a nickase variant.

10. The nickase variant comprises the amino acid substitution D10A or a corresponding amino acid substitution. The method of claim 9 , comprising:

11. The cytidine deaminase domain deactivates cytidine in deoxyribonucleic acid (DNA). The method according to any one of claims 1 to 10, wherein the compound can be amine-modified.

12. The cytidine deaminase domain is a modified cytidine deaminase domain that does not occur in nature. The method of claim 11, which is a kinase domain.

13. 12. The method of claim 11, wherein the cytidine deaminase domain is an APOBEC deaminase domain. Or the method according to claim 12.

14. 14. The method of any one of claims 11-13, wherein the base editor is BE4.

15. The one or more guide polynucleotides are transduced with CRISPR RNA (crRNA). and a small RNA (tracrRNA), the crRNA comprising a SERPINA1 nucleic acid sequence comprising a SNP associated with A1AD. The method of any one of claims 1 to 14, comprising a nucleic acid sequence complementary to:

16. The base editor is complementary to a SERPINA1 nucleic acid sequence encoding methionine 374.

16. The method according to claim 1 , wherein the method is complexed with a single guide RNA (sgRNA) comprising a nucleic acid sequence having a specific structure. The method according to claim 5.

17. The cells or their precursor cells, Polynucleotide-programmable DNA-binding domains and cytidine deaminase domains an editor comprising the gene, a polynucleotide encoding the same, targeting said base editor to nucleic acid position 1455 of the SERPINA1 polynucleotide; one or more guide polynucleotides that deaminate cytidines in A cell produced by introducing

18. The cell of claim 17 , wherein the cell produced is a hepatocyte.

19. The cell or its precursor cell is an induced pluripotent stem cell or a hepatocyte.

19. The cell according to claim 18.

20. The cell of claim 18 or 19, wherein the hepatic cell expresses an A1AT polypeptide.

21. The cell of any one of claims 17 to 20, which is from a subject with A1AD.

22. The cell according to any one of claims 17 to 21, which is a mammalian cell or a human cell.

23. The modification at cytidine replaces the methionine at position 374 of the A1AT polypeptide with an isoleucine. The cell according to any one of claims 17 to 22, wherein the cell is transformed into a

24. The deamination of the cytidine results in the formation of an A1AT polypeptide having an isoleucine at amino acid position 374. A cell according to any one of claims 17 to 23, which results in expression of a polypeptide.

25. The SNP associated with A1AD substitutes lysine for glutamic acid at amino acid position 342, claims 25. The cell according to any one of items 17 to 24.

26. The cell comprises a cytidine deamination site at nucleic acid position 1455 of the SERPINA1 polynucleotide. The cell of any one of claims 17 to 25, which is selected for:

27. The polynucleotide programmable DNA binding domain is selected from the group consisting of Streptococcus pyogenes The method according to any one of claims 17 to 26, which is Cas9 (SpCas9) or a variant thereof. cell.

28. The SpCas9 has specificity for a PAM sequence selected from 5'-NGG-3' or 5'-GGG-3'. The cell of claim 27 .

29. The polynucleotide programmable DNA binding domain comprises a nuclease inactive valence The method according to any one of claims 17 to 28, which is an antagonism or a nickase variant. Cells on the plate.

30. The nickase variant comprises the amino acid substitution D10A or a corresponding amino acid substitution. The cell of claim 29.

31. The cytidine deaminase domain removes cytidine from deoxyribonucleic acid (DNA). The cell according to any one of claims 17 to 30, which is capable of aminating.

32. The cytidine deaminase domain is a modified cytidine deaminase domain that does not occur in nature. The cell of claim 31 , which is a kinase domain.

33. 32. The method of claim 31, wherein the cytidine deaminase domain is an APOBEC deaminase domain. Or the cell described in 32.

34. 34. The cell of any one of claims 31-33, wherein the base editor is BE4.

35. The one or more guide polynucleotides comprise a CRISPR RNA (crRNA) and a transactivating oligonucleotide. (tracrRNA), wherein the crRNA comprises a sequence identical to that at nucleic acid position 1455 of the SERPINA1 polynucleotide. The cell according to any one of claims 17 to 34, comprising a nucleic acid sequence complementary to cytosine. 。

36. A complex between the base editor and the one or more guide polynucleotides in the cell. The cell according to any one of claims 17 to 35, which forms

37. The base editor modifies the cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide.

37. The method of claim 36, wherein the method is complexed with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to Cells on the plate.

38. A method for treating A1AD in a subject, comprising administering to a subject a compound according to any one of claims 17 to 37. administering a cell to said subject.

39. 39. The method of claim 38, wherein the cells are autologous or allogeneic to the subject.

40. An isolated cell or a cell culture medium, grown or expanded from a cell according to any one of claims 17 to 37. or cell population.

41. 1. A method of treating A1AD in a subject, comprising: Polynucleotide-programmable DNA-binding domains and cytidine deaminase domains a base editor or a polynucleotide encoding same, comprising: targeting said base editor to nucleic acid position 1455 of the SERPINA1 polynucleotide; one or more guide polynucleotides that result in cytidine modifications in to a subject in need thereof. method.

42. 42. The method of claim 41, wherein the subject is a mammal or a human.

43. The base editor or a polynucleotide encoding same and the one or more guides 43. The method of claim 41 or 42, comprising delivering a polypeptide to a cell of the subject. The method described.

44. 44. The method of claim 43, wherein the cells are hepatocytes or hepatocyte progenitor cells. 。

45. The method of claim 44, wherein the hepatocytes express A1AT protein.

46. The deamination of the cytidine converts the methionine to an isoleucine in the A1AT polynucleotide. The method according to any one of claims 41 to 45, wherein the

47. 47. The method of claim 45 or 46, wherein the A1AT protein comprises a SNP associated with A1AD. 。

48. The SNP associated with A1AD is a substitution of glutamic acid for lysine at amino acid position 342. The method according to any one of claims 41 to 47.

49. The polynucleotide programmable DNA binding domain is selected from the group consisting of Streptococcus pyogenes The method according to any one of claims 41 to 48, comprising Cas9 (SpCas9) or a variant thereof. method.

50. The specificity of the SpCas9 for a PAM sequence selected from 5'-NGG-3' or 5'-GGG-3'.

50. The method of claim 49, having the following structure:

51. The polynucleotide programmable DNA binding domain comprises a nuclease inactive valence The method according to any one of claims 41 to 50, which is an antagonism or a nickase variant. Law.

52. The nickase variant has the amino acid substitution D10A, or a corresponding amino acid substitution 52. The method of claim 51 , comprising:

53. The cytidine deaminase domain removes cytidine from deoxyribonucleic acid (DNA).

43. The method according to claim 41 or 42, wherein the compound can be aminated.

54. The cytidine deaminase domain is a modified cytidine deaminase domain that does not occur in nature. The method of claim 53, which is a kinase domain.

55. 53. The method of claim 52, wherein the cytidine deaminase domain is an APOBEC deaminase domain. Or the method described in 54.

56. 56. The method of Claim 54 or 55, wherein the base editor is BE4.

57. The one or more guide polynucleotides are CRISPR RNA (crRNA) and transcoding The crRNA comprises a small RNA (tracrRNA) that has a nucleic acid sequence complementary to a SERPINA1 nucleic acid sequence. The method of any one of claims 41 to 56, comprising:

58. The base editor comprises a nucleic acid complementary to a cytidine at nucleic acid position 1455 of SERPINA1.

58. The method of claim 41, wherein the nucleic acid sequence is a single guide RNA (sgRNA). The method according to

59. 1. A method for producing hepatocytes or their progenitor cells, comprising: (a) Hepatic progenitor cells containing an A1AD-associated SNP were Polynucleotide programmable nucleotide binding domains and cytidine deamina a base editor comprising an ELISA domain, or a polynucleotide encoding the same; targeting said base editor to nucleic acid position 1455 of the SERPINA1 polynucleotide; one or more guide polynucleotides that result in cytidine deamination at cytidine in Chid and Introducing (b) differentiating the hepatic progenitor cells into hepatocytes. A method comprising:

60. The method of claim 59, wherein the hepatic progenitor cells express an A1AT polypeptide.

61. 60. The method of claim 59, wherein the hepatic progenitor cells are obtained from a subject with A1AD. The method according to

62. 62. The method according to claim 59, wherein the hepatic progenitor cells are mammalian or human cells. The method according to

63. The polynucleotide programmable DNA binding domain is selected from the group consisting of Streptococcus pyogenes The method according to any one of claims 59 to 62, comprising Cas9 (SpCas9) or a variant thereof. method.

64. The SpCas9 has specificity for a PAM sequence selected from 5'-NGG-3' or 5'-GGG-3'. The method of claim 63, having the following properties:

65. The polynucleotide programmable DNA binding domain comprises a nuclease inactive valence The method according to any one of claims 59 to 64, which is an antagonism or a nickase variant. Law.

66. The nickase variant comprises the amino acid substitution D10A or a corresponding amino acid substitution. The method of claim 65.

67. The cytidine deaminase domain binds cytidine to deoxyribonucleic acid (DNA).

67. The method of any one of claims 59 to 66, wherein the compound can be deaminated.

68. The cytidine deaminase domain is a modified cytidine deaminase domain that does not occur in nature. The method of claim 57, which is a kinase domain.

69. 6. The method of claim 5, wherein the cytidine deaminase domain is an APOBEC deaminase domain. 7 or 58. The method according to claim 7 or 58.

70. 70. The method of any one of claims 67-69, wherein the base editor is BE4.

71. The one or more guide polynucleotides are CRISPR RNA (crRNA) and transcoding The crRNA contains a small RNA that encodes a cytidine at nucleic acid position 1455 of SERPINA1.

70. The method of any one of claims 57 to 69, comprising a nucleic acid sequence complementary to

72. The base editor and one or more guide polynucleotides are complexed in the cell. The method of any one of claims 57 to 71, further comprising forming

73. The base editor comprises a nucleic acid complementary to a cytidine at nucleic acid position 1455 of SERPINA1.

73. The method of claim 72, wherein the nucleic acid sequence is complexed with a single guide RNA (sgRNA) comprising an amino acid sequence.

74. 5'-CAAUCAUUAAGAAGACAAAGGGUUU-3' 5'-UCAAUCAUUAAGAAGACAAAGGGUUU-3' 5'-UUCAAUCAUUAAGAAGACAAAGGGUUU-3' 5'-GUUCAAUCAUUAAGAAGACAAAGGGUUU-3' 5'-UGUUCAAUCAUUAAGAAGACAAAGGGUUU-3' 5'-UUGUUCAAUCAUUAAGAAGACAAAGGGUU-3' 5'-UUCAAUCAUUAAGAAGACAAAG-3' 5'-UUCAAUCAUUAAGAAGACAAAGG-3' 5'-UCAAUCAUUAAGAAGACAAAGGG-3' 5'-AAUCAUUAAGAAGACAAAGGGU-3' comprising a nucleic acid sequence selected from Guide RNA.

75. 75. A guide RNA comprising 18, 19, 20, 21, or 22 nucleotides of the guide RNA of claim 74.

76. 75. A protein-nucleic acid complex comprising a base editor and a guide RNA of claim 74.

77. 1. A method of treating a genetic disorder in a subject, comprising: A polynucleotide having a programmable nucleotide binding domain and a deoxyribonucleic acid domain is provided to a subject of interest. and a base editor comprising an aminase domain, or a polynucleotide encoding the same. administering a guidepoietin that targets the base editor to a target nucleotide sequence of a subject; administering a oligonucleotide to said subject; and Upon targeting of the base editor to the target nucleotide sequence, a nucleobase and deaminating the nucleic acid base of the target nucleotide sequence, thereby Treating said genetic disorder by altering said nucleobase to another nucleobase. Including, The nucleobase is in a protein coding region of a polynucleotide, It is not the cause of genetic disorders. method.

78. A method for producing a cell, tissue, or organ for treating a genetic disorder in a subject. So, Polynucleotide programmable nucleotide binding domains and deaminase domains or a polynucleotide encoding the same, in a cell, tissue, or contact with any part or organ; targeting said base editor to a target nucleotide sequence in said cell, tissue, or organ contacting said cell, tissue, or organ with a guide polynucleotide that binds to said cell, tissue, or organ; and Upon targeting of the base editor to the target nucleotide sequence, a nucleobase and deaminating the nucleic acid base of the target nucleotide sequence, thereby A method for treating said genetic disorder by changing said nucleobase to another nucleobase. To produce cells, tissues, or organs Including, The nucleobase is in a protein coding region of a polynucleotide, It is not the cause of genetic disorders. method.

79. The method of claim 1, further comprising administering the cell, tissue, or organ to a subject in need thereof.

78. The method according to claim 78.

80. The cells, tissues, or organs may be autologous, allogeneic, or xenogeneic to the subject.

80. The method of claim 78 or 79,

81. The change of the nucleobase to another nucleobase is The method according to any one of claims 77 to 80, which results in an increase in the activity of the protein to be treated. 。

82. The change of the nucleobase to another nucleobase is 77 to 79, which result in improved folding and / or increased stability of the protein to be treated.

80. The method of any one of claims 80 to 80.

83. The change of the nucleobase to another nucleobase is The method according to any one of claims 77 to 80, which results in an increased expression of a protein to be expressed. 。

84. The increase in expression of the protein Improved translation rate of said protein, or organelles or increases the release rate from the cellular compartment; Improved processing rate of the signal peptide of said protein; or Altering the interaction of said protein with another protein 84. The method of claim 83, wherein the method results from one or more of:

85. The nucleic acid base is located in a gene responsible for the genetic disorder according to any one of claims 77 to 84.

2. The method according to any one of claims 1 to 11.

86. The editing comprises editing a plurality of nucleobases located in the gene, 86. The method of claim 85, wherein the plurality of nucleobases does not cause the genetic disorder.

87. The editing step comprises: editing one or more additional nucleic acid salts located in at least one additional gene.

87. The method of claim 85 or 86, further comprising editing the group.

88. The gene and the at least one other gene may encode one or more subunits of a protein.

88. The method of claim 87, wherein the nucleic acid sequence encodes a nucleic acid sequence that is a nucleic acid fragment of the nucleic acid sequence.

89. The nucleobase is in a gene listed in Table 3A or Table 3B, and the editing is an amino acid sequence in a protein encoded by a gene shown in Table 3A or Table 3B.

89. The method of any one of claims 77 to 88, which results in an acid change.

90. The genetic disorders include retinitis pigmentosa, Usher syndrome, sickle cell disease, beta-thalassemia, MIA, Alpha-1 Antitrypsin Deficiency (A1AD), Hepatic Porphyria, Medium Chain Acyl CoA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenyl Ketonuria, hemochromatosis, von Gierke disease, Pompe disease, Gaucher disease, Hermann disease 90. The method according to any one of claims 77 to 89, wherein the treatment is selected from the group consisting of Lehr syndrome, cystic fibrosis, and chronic pain. The method described.

91. 77 to 79, wherein the genetic disorder is alpha-1 antitrypsin deficiency (A1AD).

90. The method of any one of claims 90 to 94.

92. The edits consist of F51L, M374I, A348V, A347V, K387R, T59A, and T68A. an amino acid in the alpha-1 antitrypsin (A1AT) protein selected from the group 92. The method of claim 91, which produces a change.

93. The editing results in an M374I amino acid change in the A1AT protein.

92. The method according to claim 92.

94. 91. The method of any one of claims 77 to 90, wherein the genetic disorder is sickle cell disease. 。

95. The editing results in amino acid changes that reduce the polymerization ability of the HbA / HbS tetramer. The method of claim 94.

96. The nucleobase is located in the HBB gene, which encodes the beta subunit of hemoglobin (HbB). The method of claim 94 or 9595, wherein the

97. 97. The method of claim 96, wherein the HBB gene is the sickle cell hemoglobin allele (HbS). method.

98. The editing results in an amino acid change in the beta subunit of hemoglobin. The method of claim 96 or 97.

99. The amino acid changes in the beta subunit of hemoglobin are A70T, A70V, L88P, 99. The method of claim 98, comprising administering to said patient an effective amount of at least one of the following: F85L, F85P, E22G, G16D, G16N, or any combination thereof. The method described.

100. The nucleobase is HBA1, which encodes the alpha subunit of hemoglobin (HbA), or The method of claim 94 or 95, wherein said gene is located in the HBA2 gene.

101. The editing also includes amino acid changes in the alpha subunit of hemoglobin. The method of claim 100, further comprising:

102. The amino acid change in the alpha subunit is the same as that in sickle cell hemoglobin. The method of claim 101, wherein the β-subunit is located at the polymerization interface between the β-subunit and the β-subunit.

103. The amino acid changes in the alpha subunit of hemoglobin are K11E, D47G, Q54R , N68D, E116K, H20Y, H50Y, or any combination thereof.

102. The method according to claim 102.

104. and a second edit of additional nucleobases, said additional nucleobases being an inhibitor of said genetic disorder. or the additional nucleic acid base is the cause of the genetic disorder.

103. The method of any one of claims 103.

105. The deaminase domain is a cytidine deaminase domain or an adenosine deaminase domain. The method of any one of claims 77 to 104, wherein the protein is a kinase domain.

106. The adenosine deaminase domain removes adenine from deoxyribonucleic acid (DNA). The method of claim 105, wherein the compound can be aminated.

107. The guide polynucleotide may comprise ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The method according to any one of claims 77 to 106.

108. The guide polynucleotide may be a CRISPR RNA (crRNA) sequence, a transactivating CRISPR RNA (crRNA) sequence, (tracrRNA) sequences, or combinations thereof, according to any one of claims 77 to 107. The method described.

109. The method according to any one of claims 77 to 108, further comprising a second guide polynucleotide. Method of posting.

110. The second guide polynucleotide may be a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA 110. The method of claim 109, comprising:

111. The second guide polynucleotide may comprise a CRISPR RNA (crRNA) sequence, a transactivating CRIS 110. The method of claim 109, comprising a PR RNA (tracrRNA) sequence, or a combination thereof.

112. The second guide polynucleotide directs the base editor to a second target nucleotide sequence.

112. The method of any one of claims 109 to 111, targeting to a column.

113. The polynucleotide programmable DNA binding domain may be a Cas9 domain, a Cpf1 domain, or a combination thereof. In, CasX domain, CasY domain, Cas12b / C2c1 domain, or Cas12c / C2c3 domain The method of any one of claims 77 to 112, comprising:

114. The polynucleotide programmable DNA binding domain is nuclease inactive The method according to any one of claims 77 to 113.

115. The polynucleotide programmable DNA binding domain is a nickase. 77-113. A method according to any one of claims 77 to 113.

116. The polynucleotide programmable DNA binding domain is a nuclease-inactivated Cas9 (d Cas9 domain selected from Cas9 (Cas9), Cas9 nickase (nCas9), or nuclease-active Cas9 The method of any one of claims 77 to 115, comprising in.

117. 117. The method of Claim 116, wherein the Cas9 domain comprises a Cas9 nickase.

118. The polynucleotide programmable DNA binding domain may be an engineered or modified polynucleotide. Any of claims 77 to 117, which is a oligonucleotide programmable DNA binding domain.

3. The method according to claim 1 .

119. The method of any one of claims 77 to 118, further comprising a second base editor. 。

120. The second base editor comprises a deaminase domain that is different from the base editor.

120. The method of claim 119, comprising:

121. Any of claims 77 to 120, wherein the editing results in less than 20% indel formation. The method according to any one of claims 1 to 5.

122. The method according to any one of claims 77 to 121, wherein the editing does not result in a translocation. method.

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