Methods for suppressing pathogenic mutations using programmable base editor systems

Abstract

Provided herein are compositions comprising a polynucleotide programmable nucleotide binding domain and a nucleoside base editing domain together with a guide polynucleotide base editor, and methods of using the base editor. Also provided herein are base editor systems for editing nucleoside bases of a target nucleotide sequence.

Description

[0001] Related applications

[0002] This application claims the benefits of U.S. Provisional Application No. 62 / 670,498, filed May 11, 2018, and U.S. Serial No. 62 / 780,864, filed December 17, 2018, the full disclosures of which are incorporated herein by reference. Background Technology

[0003] For the most well-known genetic diseases, the aim is to correct point mutations at target loci, rather than randomly disrupting genes, in order to investigate or resolve the underlying causes of the disease. Currently, gene editing techniques using the CRISPR system (Clustered Regularly Interspaced Short Palindromic Repeats) introduce double-stranded DNA breaks at target loci as the first step in gene correction. In response to double-stranded DNA breaks, cellular DNA repair processes largely involve non-homologous end junctions, resulting in random insertions or deletions (indels) at the DNA cleavage site. Although most genetic diseases originate from point mutations, current point mutation correction methods are ineffective, typically inducing a rich influx of random insertions or deletions (indels) at the target site due to cellular responses to dsDNA breaks. Therefore, there is a need for improved gene editing techniques that are more efficient and produce far fewer undesirable products, such as random insertions or deletions (indels) or translocations.

[0004] Alpha-1 antitrypsin deficiency (A1AD) is a genetic disorder caused by a pathogenic mutation in the SERPINA1 gene, which encodes the alpha-1 antitrypsin (A1AT) protein. This mutation results in reduced protein production in individuals with the disease. A1AT is an excellent inhibitor of neutrophil elastase and protects tissues and organs, such as the lungs, from elastase degradation. As a result, elastin in the lungs of patients with A1AD is more easily degraded by neutrophil elastase, and over time, the loss of lung elasticity develops into chronic obstructive pulmonary disease (COPD). In healthy individuals, A1AT is produced by hepatocytes in the liver and secreted into the systemic circulation, where it acts as an inhibitor of proteases.

[0005] The most common pathogenic A1AT variant is the guanine-to-adenine (G→A) mutation in the SERPINA1 gene, which results in a glutamic-to-lysine substitution at amino acid position 342 of the A1AT protein. This substitution causes misfolding of the protein and aggregation within hepatocytes, ultimately leading to toxic accumulations that cause liver damage and cirrhosis. While hepatotoxicity can be addressed with gene knockout (CRISPR / ZFN / TALEN) or gene knock-out (siRNA), these methods fail to resolve the lung pathology. Although lung pathology can be addressed with protein supplementation therapy, this therapy does not resolve hepatotoxicity. Gene therapy is also insufficient to address A1AT genetic defects. Because the livers of patients with A1AD are already under a severe disease burden due to endogenous A1AT accumulation, gene therapy that increases hepatic A1AT will be counterproductive. Therefore, a treatment for patients with A1AD is needed that addresses both the accompanying lung pathology and hepatotoxicity.

[0006] Included by citation

[0007] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety as if each publication, patent, and patent application were specifically and individually identified as incorporated herein by reference. Unless otherwise indicated, all publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety. Summary of the Invention

[0008] Invention Summary

[0009] This article provides a method for treating a genetic disease in an individual, wherein the method comprises administering a base editor or a polynucleotide encoding the base editor to the individual in need, wherein the base editor comprises a polynucleotide programmable DNA-binding domain and a cytidine deaminase domain; administering a guide polynucleotide to the individual, wherein the guide polynucleotide targets the base editor to a target nucleotide sequence of the individual; and when the base editor targets the target nucleotide sequence, editing the nucleoside base by deaminating a nucleoside base of the target nucleotide sequence, thereby treating the genetic disease by changing the nucleoside base to another nucleoside base; wherein the nucleoside base is in a protein coding region of the polynucleotide; and wherein the nucleoside base is not the cause of the genetic disease (i.e., the nucleoside base is not coding for a mutation that causes the genetic disease).

[0010] This disclosure also provides a method for manufacturing cells, tissues, or organs for treating a genetic condition in an individual, wherein the method includes contacting the cell, tissue, or organ with a base editor or a polynucleotide encoding the base editor, wherein the base editor includes a polynucleotide programmable DNA-binding domain and a deaminase domain; contacting the cell, tissue, or organ with a guide polynucleotide, wherein the guide polynucleotide targets the base editor to a target nucleotide sequence of the cell, tissue, or organ; and when the base editor targets the target nucleotide sequence, editing the nucleoside base by deaminating a nucleoside base of the target nucleotide sequence, thereby manufacturing the cell, tissue, or organ for treating the genetic condition by altering the nucleoside base to another nucleoside base; wherein the nucleoside base is in a protein coding region of the polynucleotide; and wherein the nucleoside base is not the cause of the genetic condition. In several embodiments, the method further includes delivering the cell, tissue, or organ to the individual. In several embodiments, the cell, tissue, or organ is autologous to the individual. In some embodiments, the cell, tissue, or organ is allogeneic to the individual. In some embodiments, the cell, tissue, or organ is xenogeneic to the individual.

[0011] In several embodiments, the change of one nucleoside base to another results in increased activity of the protein encoded by the polynucleotide. In several embodiments, the change of one nucleoside base to another results in improved folding and / or increased stability of the protein encoded by the polynucleotide. In several embodiments, the change of one nucleoside base to another results in increased expression of the protein encoded by the polynucleotide. In several embodiments, the increased protein expression is due to improved protein translation rate. In several embodiments, the increased protein expression is due to increased release rate from organelles or cellular compartments containing the protein. In several embodiments, the increased protein expression is due to improved processing rate of the protein's signal peptide. In several embodiments, the increased protein expression is due to altered interaction between the protein and another protein.

[0012] In several embodiments, the nucleoside base is located in a gene that causes the genetic condition. In several embodiments, the editing comprises editing a plurality of nucleoside bases located in the gene, wherein the plurality of nucleoside bases are not the cause of the genetic condition. In several embodiments, the editing further comprises editing one or more additional nucleoside bases located in at least one other gene. In several embodiments, the gene and the at least one other gene encode one or more subunits of the protein. In several embodiments, the nucleoside base is among the genes listed in Table 3A or Table 3B, and the editing results in an amino acid change in the protein encoded by the gene indicated in Table 3A or Table 3B.

[0013] In several embodiments, the genetic disorder is retinitis pigmentosa, Ussell's syndrome, sickle cell disease, β-thalassemia, α-1 antitrypsin deficiency (A1AD), hepatic pyrroloporosis, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosome acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, von Kilke's syndrome, Pompe syndrome, Gaucher's syndrome, Hollerithia syndrome, cystic fibrosis, or chronic pain. In several embodiments, the genetic disorder is α-1 antitrypsin deficiency (A1AD). In several embodiments, the editing results in an amino acid alteration in the α-1 antitrypsin (A1AT) protein selected from the group consisting of F51L, M374I, A348V, A347V, K387R, T59A, and T68A. In several implementations, this editing results in a change in the M374I amino acid in the A1AT protein.

[0014] In several embodiments, the genetic condition is sickle cell disease. In several embodiments, the editing results in an amino acid alteration that reduces the polymerization potential of the HbA / HbS tetramer. In several embodiments, the nucleoside base is the HBB gene encoding the β subunit (HbB) of hemoglobin. In several embodiments, the HBB gene is the sickle hemoglobin allele (HbS). In several embodiments, the editing results in an amino acid alteration in the β subunit of hemoglobin. In several embodiments, the amino acid alteration in the β subunit of hemoglobin comprises A70T, A70V, L88P, F85L, F85P, E22G, G16D, G16N, or any combination thereof. In several embodiments, the nucleoside base is the HBA1 or HBA2 gene encoding the α subunit (HbA) of hemoglobin. In several embodiments, the editing results in an amino acid alteration in the α subunit of hemoglobin. In several embodiments, the amino acid change in the α subunit is located at the polymerization interface between the α and β subunits of the sickle-shaped hemoglobin. In several embodiments, the amino acid change in the α subunit of the hemoglobin includes K11E, D47G, Q54R, N68D, E116K, H20Y, H50Y, or any combination thereof.

[0015] In one embodiment, compositions and methods for suppressing pathogenic mutations using a programmable nucleoside base editor are provided. The present invention provides a method for treating A1AD by using a base editor (e.g., BE4) to induce alterations in the endogenous SERPINA1 gene. The altered SERPINA1 gene encodes an M374I mutation that stabilizes E342K in the α-1 antitrypsin protein. Introducing M374I using BE4 simultaneously improves hepatotoxicity and increases A1AT circulation to the lungs, thereby compensating for the presence of the harmful M374I mutation. This strategy simultaneously reduces the pathogenic protein load on the liver and restores functional proteins to the lungs.

[0016] In another embodiment, the present invention provides a method for editing a SERPINA1 polynucleotide comprising a single nucleotide polymorphism (SNP) associated with α-1 antitrypsin deficiency (A1AD). The method involves contacting the SERPINA1 polynucleotide with a base editor that complexes with one or more guide polynucleotides, wherein the base editor comprises a polynucleotide-programmable DNA-binding domain and a cytidine deaminase domain, and wherein the one or more guide polynucleotides target the base editor to achieve a change in the A1AD-associated SNP. In one embodiment, the contact is in a cell, eukaryotic cell, mammalian cell, or human cell. In another embodiment, the cell is in vivo or in vitro.

[0017] In another embodiment, the present invention provides a cell, or a progenitor cell thereof, manufactured by introducing a base editor, or a polynucleotide encoding the base editor, into a cell, wherein the base editor comprises a polynucleotide-programmable DNA-binding domain and a cytidine deaminase domain; and one or more guide polynucleotides of the base editor are targeted to deaminate cytidine at nucleotide position 1455 of the SERPINA1 polynucleotide. In one embodiment, the manufactured cell is a hepatocyte. In another embodiment, the cell or its progenitor cell is a germ cell, an induced pluripotent stem cell, or a hepatocyte. In another embodiment, the hepatocyte expresses the A1AT polypeptide. In another embodiment, the cell is derived from an individual suffering from A1AD. In another embodiment, the cell is a mammalian cell or a human cell.

[0018] In another embodiment, the present invention provides a method for treating A1AD in an individual, comprising administering cells of any prior form to the individual. In one embodiment, the cells are autologous to the individual. In another embodiment, the cells are allogeneic to the individual.

[0019] In another state, the present invention provides isolated cells or cell populations that have proliferated or expanded from cells of any previous state.

[0020] In another embodiment, the present invention provides a method for treating A1AD in an individual, wherein the method comprises administering to the individual:

[0021] A base editor, or a polynucleotide encoding the base editor, wherein the base editor contains a polynucleotide programmable DNA-binding domain and a cytidine deaminase domain; and

[0022] One or more guide polynucleotides of the base editor are targeted to achieve a change in cytidine at position 1455 of the SERPINA1 polynucleotide.

[0023] In one embodiment described above, the individual is a mammal or a human. In another embodiment, the method involves delivering the base editor, or the polynucleotide encoding the base editor, and one or more guide polynucleotides, to the cells of the individual. In another embodiment, the cell is a hepatocyte. In another embodiment, the cell is a progenitor cell of a hepatocyte. In another embodiment, the hepatocyte expresses the A1AT protein.

[0024] In another embodiment, a method for producing hepatocytes or their progenitor cells includes:

[0025] Introducing a base editor, or a polynucleotide encoding the base editor, into hepatocyte progenitor cells containing an SNP associated with A1AD, wherein the base editor comprises a polynucleotide-programmable DNA-binding domain and a cytidine deaminase domain; and one or more guide polynucleotides, wherein the one or more guide polynucleotides target the base editor to deaminate cytidine at nucleotide position 1455 of the SERPINA1 polynucleotide, and

[0026] (b) Differentiating the hepatocyte progenitor cell into hepatocytes. In one embodiment, the method involves differentiating the hepatocyte progenitor cell into hepatocytes. In another embodiment, the hepatocyte progenitor cell expresses the A1AT polypeptide. In another embodiment, the hepatocyte progenitor cell is derived from an individual suffering from A1AD. In another embodiment, the hepatocyte progenitor cell is a mammalian cell or a human cell.

[0027] In another embodiment, the present invention provides a guide RNA containing a nucleic acid sequence selected from the following:

[0028] 5'-CAAUCAUUAAGAAGACAAAGGGUUU-3'

[0029] 5'-UCAAUCAUUAAGAAGACAAAGGGUUU-3'

[0030] 5'-UUCAAUCAUUAAGAAGACAAAGGGUUU-3'

[0031] 5'-GUUCAAUCAUUAAGAAGACAAAGGGUUU-3'

[0032] 5'-UGUUCAAUCAUUAAGAAGACAAAGGGUUU-3'

[0033] 5'-UUGUUCAAUCAUUAAGAAGACAAAGGGUU-3'

[0034] 5'-UUCAAUCAUUAAGAAGACAAAG-3'

[0035] 5'-UUCAAUCAUUAAGAAGACAAAGG-3'

[0036] 5'-UCAAUCAUUAAGAAGACAAAGGG-3'

[0037] 5'-AAUCAUUAAGAAGACAAAGGGU-3'.

[0038] In another embodiment, the present invention provides a guide RNA containing 18, 19, 20, 21, or 22 nucleotides of a guide RNA of the embodiment described herein or otherwise.

[0039] In another embodiment, the present invention provides a protein-nucleic acid complex containing a base editor of the embodiment elucidated herein and a guide RNA as described herein.

[0040] In any of the above-described embodiments or any other embodiments of the invention as disclosed herein, the base editor deamination of the SERPINA1 polynucleotide cytidine at position 1455, thereby inducing a methionine-to-isoleucine mutation at amino acid position 374 of the A1AT protein. In any of the above-described embodiments or any other embodiments of the invention as disclosed herein, the A1AT polypeptide contains lysine at amino acid position 342 and / or contains lysine at amino acid position 376. In any of the above-described embodiments or any other embodiments of the invention as disclosed herein, the polynucleotide programmable DNA binding domain is Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof. In any of the above-described embodiments or any other embodiments of the invention as disclosed herein, the SpCas9 has specificity for PAM sequences selected from 5'-NGG-3' or 5'-GGG-3'.

[0041] In any of the above-described embodiments or any other embodiments of the invention as described in this disclosure, the polynucleotide programmable DNA binding domain is a nuclease inactivation or nicking enzyme variant. In any of the above-described embodiments or any other embodiments of the invention as described in this disclosure, the nicking enzyme variant contains an amino acid substitution D10A or its corresponding amino acid substitution. In any of the above-described embodiments or any other embodiments of the invention as described in this disclosure, the cytidine deaminase domain is capable of deaminating cytidine in deoxyribonucleic acid (DNA). In any of the above-described embodiments or any other embodiments of the invention as described in this disclosure, the cytidine deaminase domain is a modified cytidine deaminase domain that does not occur naturally. In any of the above-described embodiments or any other embodiments of the invention as described in this disclosure, the cytidine deaminase domain is an APOBEC deaminase domain. In any of the above-described embodiments or any other embodiments of the invention as described in this disclosure, the base editor is BE4. In any of the above-described or any other embodiments of the invention as disclosed herein, the one or more guide polynucleotides contain CRISPR RNA (crRNA) and trans small RNA (tracrRNA), wherein the crRNA contains a nucleic acid sequence complementary to the SERPINA1 nucleic acid sequence, the SERPINA1 nucleic acid sequence containing an SNP associated with A1AD. In any of the above-described or any other embodiments of the invention as disclosed herein, the base editor is complexed with a single guide RNA (sgRNA) containing a nucleic acid sequence complementary to the SERPINA1 nucleic acid sequence encoding methionine 374.

[0042] In several embodiments, any method provided herein further includes a second editing of an additional nucleoside base. In some cases, this additional nucleoside base is not the cause of the genetic condition. In other cases, this additional nucleoside base is the cause of the genetic condition.

[0043] In several embodiments, the deaminase domain is a cytidine deaminase domain or an adenosine deaminase domain. In several embodiments, the deaminase domain is a cytidine deaminase domain. In several embodiments, the deaminase domain is an adenosine deaminase domain. In several embodiments, the adenosine deaminase domain is capable of deaminating adenine in deoxyribonucleic acid (DNA). In several embodiments, the guide polynucleotide comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In several embodiments, the guide polynucleotide comprises a CRISPR RNA (crRNA) sequence, a trans-activated RNA (tracrRNA) sequence, or a combination thereof.

[0044] In several embodiments, any method provided herein further comprises a second guide polynucleotide. In several embodiments, the second guide polynucleotide comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In several embodiments, the second guide polynucleotide comprises a CRISPR RNA (crRNA) sequence, a trans-activated RNA (tracrRNA) sequence, or a combination thereof. In several embodiments, the second guide polynucleotide targets the base editor to a second target nucleotide sequence. In several embodiments, the polynucleotide-programmable DNA binding domain comprises a Cas9 domain, a Cpf1 domain, a CasX domain, a CasY domain, a Cas12b / C2c1 domain, or a Cas12c / C2c3 domain. In several embodiments, the polynucleotide-programmable DNA binding domain is a nuclease death domain. In several embodiments, the polynucleotide-programmable DNA binding domain is a nickase. In several embodiments, the polynucleotide-programmable DNA binding domain comprises a Cas9 domain. In several embodiments, the Cas9 domain comprises a dead nuclease Cas9 (dCas9), a Cas9 nicking enzyme (nCas9), or an active nuclease Cas9. In several embodiments, the Cas9 domain comprises a Cas9 nicking enzyme. In several embodiments, the polynucleotide programmable DNA binding domain is an engineered or modified polynucleotide programmable DNA binding domain.

[0045] In several embodiments, any method provided herein further includes a second base editor. In several embodiments, the second base editor includes a deaminase domain different from that of the base editor.

[0046] In several embodiments, the editing results in less than 20% of insertions or deletions. In several embodiments, the editing results in less than 15% of insertions or deletions. In several embodiments, the editing results in less than 10% of insertions or deletions. In several embodiments, the editing results in less than 5% of insertions or deletions. In several embodiments, the editing results in less than 4% of insertions or deletions. In several embodiments, the editing results in less than 3% of insertions or deletions. In several embodiments, the editing results in less than 2% of insertions or deletions. In several embodiments, the editing results in less than 1% of insertions or deletions. In several embodiments, the editing results in less than 0.5% of insertions or deletions. In several embodiments, the editing results in less than 0.1% of insertions or deletions. In several embodiments, the editing does not result in transposition. Attached Figure Description

[0047] The features of this disclosure are set forth in specific detail in the appended claims. Further understanding of the features and advantages of this disclosure will be gained by referring to the following detailed description, which sets forth specific embodiments in which the principles of this disclosure are applied, and clarifies the accompanying drawings:

[0048] Figure 1 This diagram illustrates a comparison between healthy individuals and patients 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, there is a deficiency of normally functioning A1AT protein, leading to lung tissue damage. Furthermore, abnormal A1AT accumulates in hepatocytes, resulting in cirrhosis.

[0049] Figure 2 For illustration, it shows typical ranges of serum α-1 antitrypsin (A1AT) levels for different genotypes (normal (MM), heterozygous vectors of α-1 antitrypsin deficiency (MZ, SZ), and homozygous deficiency (SS, ZZ)). Serum α-1 antitrypsin (AAT) concentrations are expressed in μM on the left Y-axis, as commonly found in the references. The right Y-axis shows the approximate conversion of serum AAT concentrations to mg / dL, as commonly reported in clinical laboratories and using various measurement techniques (turbidity assays or radioimmunodiffusion).

[0050] Figure 3 The target site sequences for introducing the repressor mutation M374I into SERPINA1 are depicted. The highlighted portion is the canonical spCas9 NGG PAM, and the target C, whose editing will result in the desired codon change M374I. Also highlighted is the non-target C, whose editing would result in the undesired codon change E376K.

[0051] Figure 4This is a bar chart showing the amount of secreted protein in the culture supernatant of HEK293T cells transiently transfected with plasmids encoding different variants of the A1AT protein. A1AT concentration was determined by ELISA, as disclosed in Borel, Florie & Mueller, Christian. (2017). Alpha-1 Antitrypsin Deficiency: Methods and Protocols. 10.1007 / 978-1-4939-7163-3, the full text of which is incorporated herein by reference. The two most common variants of A1AT (e.g., pathogenic mutations) are E264V (PiS allele) and E342K (PiZ allele). PiS and PiZ proteins are produced at lower abundances than wild-type proteins. Adding the M374I repressor mutation (… Figure 4 The mutation (designated as a "compensatory mutation") clearly increases the secretion of PiS and PiZ A1AT proteins. Therefore, we hypothesize that introducing the M374I mutation using the base editor and base editing methods described herein can increase A1AT secretion from hepatocytes, while simultaneously reducing hepatotoxicity and increasing A1AT circulation to the lungs. A1AT: α-1 antitrypsin; A1AD: α-1 antitrypsin deficiency; "Z mutation" is an E342K (PiZ allele) mutation; "S mutation" is an E264V (PiS allele) mutation.

[0052] Figure 5 The bar chart shows the base editing efficiency of the M374I mutation in HEK293T. Using bpNLS is superior to the SV40 nuclear localization signal. Comparing starting codon usage, codon optimization 2 achieves higher editing efficiency when delivered via plasmids and both mRNA and gRNA.

[0053] Figure 6 This diagram illustrates the evolutionary strategy of DNA deoxyadenosine deaminase, originating from TadA. The *E. coli* library contains a plastid library of the mutant ecTadA (TadA*) gene fused to dCas9, and plastids targeting A·T to G·C mutations to repair antibiotic resistance genes. Mutations from surviving TadA* variants enter the human ABE base editing architecture.

[0054] Figure 7The graphs present a prediction of the functional elastase activity of base-edited A1AT variants. The graphs show the percentage of elastase activity of the following A1AT variants: those with the E342K (PiZ) mutation; those with the E342K (PiZ) mutation; those with the E342K mutation and compensated M374I mutation; those with the E342K (PiZ) mutation; those with the E264V (PiS) mutation; and those with the E264V mutation and compensated M374I mutation, compared to wild-type (WT) A1AT.

[0055] Figures 8A to 8C Three images are provided, showing the cells in HEK293. Figure 8A ) and induced pluripotent stem cells (iPSCs) Figure 8B The observed percentages of base editing were all obtained by transfection with the base editor BE4. Figure 8C This shows the percentage of edits achieved when wild-type primary hepatocytes were transfected.

[0056] Figure 9 The percentage of base editing achieved and A1AT secretion are shown in BE4-edited IPSC-derived hepatocytes. Detailed Implementation

[0057] [Detailed Description of the Preferred Embodiment]

[0058] The following description and embodiments illustrate the implementation of this disclosure in detail. It should be understood that this disclosure is not limited to the specific embodiments described herein and therefore can be modified. Those skilled in the art will understand that numerous variations and modifications are possible and are covered within the scope of this invention.

[0059] It should be understood that all terms are intended as understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same definitions as generally understood by those skilled in the art in connection with this disclosure.

[0060] Unless otherwise indicated, some of the embodiments disclosed herein employ known techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA that fall within the skill range of the industry. For example, see Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (FMAusubel, et al. eds.); the series Methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, B.D. Hames and G.T. Taylor eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (RIFreshney, ed. (2010)).

[0061] The section markers used in the text are for organizational purposes only, and are not to be interpreted as limiting the main points of the description.

[0062] While the features of this disclosure may be described in the text of a single embodiment, such features may also be provided separately or in any suitable combination. Conversely, although this disclosure is described in the text of separate embodiments for clarity, this disclosure may also be implemented in a single embodiment.

[0063] [definition]

[0064] The following definitions supplement industry definitions and are specific to this case, but do not refer to any related or unrelated cases, such as any commonly owned patent cases or patent applications. While any similar or equivalent methods and materials described herein may be used in the testing practices disclosed herein, preferred materials and methods are described herein. Therefore, the terminology used herein is for describing particular embodiments only and is not intended to be restrictive.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same definitions as commonly understood by one of ordinary skill in the art to which this invention pertains. The following references provide common definitions for many of the terms used in this invention for those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0066] Unless otherwise specifically stated, the use of the singular includes the plural in this case. It should be noted that, unless the context clearly requires otherwise, the singular forms "a," "an," and "the" include the plural forms as used in the description. Furthermore, the use of the term "including" and other forms such as "include," "includes," and "included" is not restrictive.

[0067] As used in the specification and claims, the terms "comprising" (and any forms of inclusion, such as "comprise" and "comprises"), "having" (and any forms of having, such as "have" and "has"), "including" (and any forms of inclusion, such as "includes" and "include"), or "containing" (and any forms of containing, such as "contains" and "contains") are inclusive or open-ended and do not exclude additional unreferenced elements or method steps. It is anticipated that any embodiments discussed in this specification can be practiced with respect to any method or composition disclosed herein, and vice versa. Furthermore, the compositions disclosed herein can be used to achieve the methods disclosed herein.

[0068] The terms "about" or "approximately" indicate that, as determined by a person skilled in the art, a particular value falls within an acceptable range of error, which depends in part on the method of measurement or determination of that value, i.e., the limits of the measurement system. For example, according to industry practice, "about" may mean falling within 1 or greater than 1 standard deviation. Additionally, "about" may mean up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Furthermore, particularly in the context of biological systems or processes, the term may mean falling within an order of magnitude of a value, preferably within 5 times, and more preferably within 2 times. When a particular value is described in the specification and claims, unless otherwise stated, the term "about" is presumed to mean falling within an acceptable range of error for that particular value.

[0069] The use of terms such as "several embodiments," "an embodiment," "one embodiment," or "other embodiments" in the specification indicates that a particular feature, structure, or characteristic described in connection with that embodiment is encompassed in at least several embodiments disclosed herein, but not necessarily all embodiments.

[0070] In this text, "administer" means providing one or more of the compositions described herein to a patient or individual. For example, but not limitingly, the composition may be administered (e.g., by injection) via intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im). One or more of these routes may be used. Parenteral administration may be by large-dose injection or by slow infusion over time. Additionally or simultaneously, administration may be by oral route.

[0071] "Adenosine deaminase" refers to a deaminase that catalyzes the hydrolytic deamination of adenine (A) to inosine (I). In several embodiments, the deaminase or deaminase domain is adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In several embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., genetically modified adenosine deaminases, evolved adenosine deaminases) can be derived from any organism, such as bacteria. In several embodiments, the adenosine deaminase is derived from bacteria, such as Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, or C. crescentus. In several embodiments, the adenosine deaminase is a TadA deaminase. In several embodiments, the TadA deaminase is Escherichia coli TadA (ecTadA) deaminase or a fragment thereof.

[0072] For example, a truncated ecTadA may lack one or more N-terminal amino acids compared to full-length ecTadA. In some embodiments, a truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues compared to full-length ecTadA. In some embodiments, a truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues compared to full-length ecTadA. In some embodiments, the ecTadA deaminase does not contain an N-terminal methionine. In some embodiments, the ecTadA deaminase is an N-terminal truncated TadA. In a particular implementation, TadA may be any of the TadA described in PCT / US2017 / 045381, the entire contents of which are incorporated herein by reference.

[0073] "Active agent" refers to any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0074] "Improvement" means to reduce, stop, weaken, shrink, stop, or stabilize the development or progression of a disease.

[0075] "Change" means a change (increase or decrease) in the expression level or activity of a gene or peptide as detected by standard, industry-known methods (such as those described herein). As used in this disclosure, a change includes a 10% change in expression level, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression level.

[0076] "Analogous" refers to molecules that are not identical but have similar functional or structural features. For example, peptide analogs retain the biological activity of the corresponding natural peptide while having certain biochemical modifications that enhance the function of the analog compared to the natural peptide. These biochemical modifications can increase the protease resistance, membrane permeability, or half-life of the analog without altering, for example, ligand binding. Analogs may include non-natural amino acids.

[0077] "α-1 antitrypsin (A1AT) protein" refers to a polypeptide or fragment thereof having at least about 95% amino acid sequence identity with UniProt Accession No. P01009. In a particular embodiment, the A1AT protein includes one or more modifications compared to the reference sequences below. In one particular embodiment, the A1AT protein associated with A1AD includes an E342K mutation. Examples of A1AT amino acid sequences are provided below.

[0078]

[0079] The term "base editor (BE)" refers to an agent comprising a polypeptide capable of modifying nucleoside bases (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is a fusion protein comprising a polynucleotide-programmable nucleotide-binding domain and a nucleoside base editing domain (e.g., a cytidine deaminase domain or an adenosine deaminase domain) along with a guide polynucleotide (e.g., guide RNA). In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the polynucleotide-programmable DNA-binding domain is fused to or linked to a deaminase domain. In some embodiments, the base editor comprises a polynucleotide-programmable DNA-binding domain and a deaminase domain along with a guide polynucleotide (e.g., guide RNA). In some embodiments, the polynucleotide-programmable DNA-binding domain is a CRISPR-associated enzyme (e.g., Cas or Cpf1). In several embodiments, the base editor is a Cas9 protein fused to a deaminase domain (e.g., adenosine deaminase or cytidine deaminase). In several embodiments, the base editor is a catalytic death Cas9 (dCas9) fused to a deaminase domain. In several embodiments, the base editor is a Cas9 cleavage enzyme (nCas9) fused to a deaminase domain. In several embodiments, the base editor is fused to a base excision repair (BER) inhibitor. In several embodiments, the base excision repair inhibitor is a uracil DNA glycosylation inhibitor (UGI). In several embodiments, the base excision repair inhibitor is an inosine base excision repair inhibitor. In several embodiments, the base editor is capable of deaminating bases within nucleic acids. In several embodiments, the base editor is capable of deaminating bases within DNA molecules. In several embodiments, the base editor is capable of deaminating bases within RNA molecules. In several embodiments, the base editor is capable of deaminating adenine (A). In several embodiments, the adenosine deaminase is evolved from TadA. In some embodiments, the base editor is capable of deaminating guanine (G). In some embodiments, the base editor is capable of deaminating adenine (A). In some embodiments, the base editor is capable of deaminating cytosine (C). Details of the base editor are described in International PCT Applications Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), the full text of which is incorporated herein by reference.Please also refer to Komor, AC, et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage" (Nature 533, 420-424 (2016); Gaudelli, NM, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage" (Nature 551, 464-471 (2017); and Komor, AC, et al., "Improved base excision repair inhibition andbacteriophage Mu Gam protein yields C:G-to-T:A base". The full text of the article “Editors with higher efficiency and product purity” (Science Advances 3:eaao4774 (2017) is incorporated herein by reference.)

[0080] In several embodiments, the cytidine base editor BE4 used in the base editing compositions, systems, and methods described herein has the following nucleic acid sequence (8877 base pairs), provided below (Addgene, Watertown, MA.; Komor AC, et al., 2017, Sci Adv., 30; 3(8):eaao4774.doi:10.1126 / sciadv.aao4774). Polynucleotide sequences having at least 95% or more identity with the BE4 nucleic acid sequence are also included.

[0081]

[0082]

[0083]

[0084]

[0085] In several embodiments, the cytidine base editor has the following sequence:

[0086]

[0087]

[0088]

[0089] In several embodiments, the cytidine base editor has the following sequence:

[0090]

[0091]

[0092]

[0093]

[0094] "Base editing activity" refers to the action of chemically altering more bases within a nucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, for example, converting a target C·G to T·A. In another embodiment, the base editing activity is adenosine deaminase activity, for example, converting a target A·T to G·C.

[0095] The term "base editor system" refers to a system for editing nucleoside bases of a target nucleotide sequence. In some embodiments, the base editor system comprises (1) a base editor (BE) including a polynucleotide programmable nucleotide binding domain and a deaminase domain for deaminating nucleoside bases; and (2) a guide polynucleotide (e.g., guide RNA) together with the polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE).

[0096] In several embodiments, the nucleoside base editor system may include more than one base editing component. For example, the nucleoside base editor system may include more than one deaminase. In several embodiments, the nuclease base editor system may include one or more cytidine deaminases and / or one or more adenosine deaminases. In several embodiments, a single guide polynucleotide may be used to target different deaminases to the target nucleic acid sequence. In several embodiments, a single pair of guide polynucleotides may be used to target different deaminases to the target nucleic acid sequence.

[0097] The nucleoside base components and polynucleotide programmable nucleotide binding components of the base editor system can be covalently or non-covalently or covalently associated with each other. For example, in some embodiments, a deaminase domain can be targeted to a target nucleotide sequence via a polynucleotide programmable nucleotide binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused to or linked to the deaminase domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the deaminase domain to a target nucleotide sequence through non-covalent interaction or association with the deaminase domain. For example, in some embodiments, the nucleoside base editing component, such as the deaminase component, may include additional heterologous portions or domains that can interact, associate, or form complexes with additional heterologous portions or domains that are part of the polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous portion can bind to, interact with, associate with, or form a complex with a polypeptide. In some embodiments, the additional heterologous portion can bind to, interact with, associate with, or form a complex with a polynucleotide. In several embodiments, the additional heterologous portion can bind to a guide polynucleotide. In several embodiments, the additional heterologous portion can bind to a polypeptide linker. In several embodiments, the additional heterologous portion can bind to a polynucleotide linker. The additional heterologous portion may be a protein domain. In several embodiments, the additional heterologous portion may be a K homology (KH) domain, an MS2 shell protein domain, a PP7 shell protein domain, an SfMuCom shell protein domain, a sterile α motif, a telomerase Ku-binding motif with Ku protein, a telomerase Sm7-binding motif with Sm7 protein, or an RNA recognition motif.

[0098] The base editor system may further include a guiding polynucleotide component. It should be understood that the components of the base editor system can be associated with each other through covalent bonds, non-covalent interactions, or any combination of their associations and interactions. In several embodiments, the deaminase domain can target a target nucleotide sequence via a guiding polynucleotide. For example, in several embodiments, the nucleoside base editing component of the base editor system, such as the deaminase component, may include additional heterologous portions or domains (e.g., polynucleotide-binding domains, such as RNA or DNA-binding proteins) that can interact with, associate with, or form a complex with a portion or segment (e.g., a polynucleotide motif) of the guiding polynucleotide. In several embodiments, the additional heterologous portions or domains (e.g., polynucleotide-binding domains, such as RNA or DNA-binding proteins) can be fused to or linked to the deaminase domain. In several embodiments, the additional heterologous portions can bind to, interact with, associate with, or form a complex with a polypeptide. In several embodiments, the additional heterologous portions can bind to, interact with, associate with, or form a complex with a polynucleotide. In several embodiments, the additional heterologous portion can bind to a guide polynucleotide. In several embodiments, the additional heterologous portion can bind to a polypeptide linker. In several embodiments, the additional heterologous portion can bind to a polynucleotide linker. The additional heterologous portion may be a protein domain. In several embodiments, the additional heterologous portion may be a K homology (KH) domain, an MS2 shell protein domain, a PP7 shell protein domain, an SfMuCom shell protein domain, a sterile α motif, a telomerase Ku-binding motif with Ku protein, a telomerase Sm7-binding motif with Sm7 protein, or an RNA recognition motif.

[0099] In several embodiments, the base editor system may further include a base excision repair (BER) inhibitor component. It should be understood that components of the base editor system can be associated with each other through covalent bonds, non-covalent interactions, or any combination of associations and interactions. The BER inhibitor component may include a base excision repair inhibitor. In several embodiments, the base excision repair inhibitor may be a uracil DNA glycosylation inhibitor (UGI). In several embodiments, the base excision repair inhibitor may be an inosine base excision repair inhibitor. In several embodiments, the base excision repair inhibitor can be targeted to a target nucleotide sequence via a polynucleotide-programmable nucleotide binding domain. In several embodiments, the polynucleotide-programmable nucleotide binding domain can be fused to or linked to the base excision repair inhibitor. In several embodiments, the polynucleotide-programmable nucleotide binding domain can be fused to or linked to a deaminase domain and a base excision repair inhibitor. In several embodiments, the polynucleotide-programmable nucleotide binding domain can target the base excision repair inhibitor to a target nucleotide sequence through non-covalent interaction or association with the base excision repair inhibitor. For example, in several embodiments, a base excision repair inhibitor may include an additional heterologous portion or domain that can interact, associate with, or form a complex with an additional heterologous portion or domain that is part of a polynucleotide programmable nucleotide binding domain. In several embodiments, the base excision repair inhibitor can target a target nucleotide sequence via a guide polynucleotide. For example, in several embodiments, the base excision repair inhibitor may include an additional heterologous portion or domain (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) that can interact, associate with, or form a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide. In several embodiments, the additional heterologous portion or domain of the guide polynucleotide (e.g., a polynucleotide binding domain, such as an RNA or DNA binding protein) can be fused to or linked to the base excision repair inhibitor. In several embodiments, the additional heterologous portion can bind to, interact with, associate with, or form a complex with the polynucleotide. In several embodiments, the additional heterologous portion can bind to, interact with, associate with, or form a complex with the guide polynucleotide. In several embodiments, the additional heterologous portion can bind to a guide polynucleotide. In several embodiments, the additional heterologous portion can bind to a polypeptide linker. In several embodiments, the additional heterologous portion can bind to a polynucleotide linker. The additional heterologous portion may be a protein domain. In several embodiments, the additional heterologous portion may be a K homology (KH) domain, an MS2 shell protein domain, a PP7 shell protein domain, an SfMuCom shell protein domain, a sterile α motif, a telomerase Ku-binding motif with Ku protein, a telomerase Sm7-binding motif with Sm7 protein, or an RNA recognition motif.

[0100] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease containing the Cas9 protein or a fragment thereof (e.g., a protein containing an active, inactive, or partially active DNA-cleaving domain of Cas9, and / or a gRNA-binding domain of Cas9). Cas9 nucleases are occasionally also referred to as casnl nucleases or CRISPR (clustered regularly spaced short palindromic repeats)-associated nucleases. An example of Cas9 is *Streptococcus pyogenes* Cas9, whose amino acid sequence is provided below.

[0101]

[0102]

[0103] (Single underline: HNH domain; Double underline: RuvC domain).

[0104] The term "conserved amino acid substitution" or "conserved mutation" refers to the substitution of one amino acid for another that shares a common property. A functional way to define the common property between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on such analyses, groups of amino acids can be defined, within which amino acids within a group tend to exchange with each other, thus having the most similar effects on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conserved mutations include amino acid substitutions, for example, lysine replacing arginine, and vice versa, to maintain a positive charge; glutamic acid replacing aspartic acid, and vice versa, to maintain a negative charge; serine replacing threonine to maintain a free -OH state; and glutamylamine replacing asparagine to maintain a free -NH2 state.

[0105] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease containing the Cas9 protein or a fragment thereof (e.g., a protein containing an active, inactive, or partially active DNA splitting domain of Cas9, and / or a gRNA-binding domain of Cas9). Cas9 nucleases are occasionally also referred to as casnl nucleases or CRISPR (clustered regularly spaced short palindromic repeats)-associated nucleases. An example of Cas9 is *Streptococcus pyogenes* Cas9, whose amino acid sequence is provided below.

[0106]

[0107] (Single underline: HNH domain; Double underline: RuvC domain).

[0108] The terms "code-writing sequence" or "protein code-writing sequence" are used interchangeably in this disclosure and refer to a polynucleotide segment that encodes a protein. The boundaries of the region or sequence are closer to the 5' end at the start codon and closer to the 3' end at the stop codon. Code-writing sequences are also known as open reading frames.

[0109] The term "conserved amino acid substitution" or "conserved mutation" refers to the substitution of one amino acid for another that shares a common property. A functional way to define the common property between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on such analyses, groups of amino acids can be defined, within which amino acids within a group tend to exchange with each other, thus having the most similar effects on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conserved mutations include amino acid substitutions, for example, lysine replacing arginine, and vice versa, to maintain a positive charge; glutamic acid replacing aspartic acid, and vice versa, to maintain a negative charge; serine replacing threonine to maintain a free -OH state; and glutamylamine replacing asparagine to maintain a free -NH2 state.

[0110] "Cytidine deaminase" refers to a polypeptide or fragment thereof that catalyzes a deamination reaction to convert an amino group to a carbonyl group. In one embodiment, cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. PmCDA1, which is derived from the hagfish (Petromyzon marinus) (hagfish cytidine deaminase 1, "PmCDA1"); AID (activation-inducible cytidine deaminase, AICDA), which is derived from mammals (e.g., humans, pigs, cattle, horses, monkeys, etc.); and APOBEC are examples of cytidine deaminases.

[0111] As used herein, the terms "deaminase" or "deaminase domain" refer to a protein or enzyme that catalyzes a deamination reaction. In several embodiments, the deaminase or deaminase domain is a cytidine deaminase, which catalyzes the hydrolysis and deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In several embodiments, the deaminase or deaminase domain is a cytidine deaminase, which catalyzes the hydrolysis and deamination of cytosine to uracil. In several embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolysis and deamination of adenine to hypoxanthine.

[0112] In several embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase derived from an organism, such as a human, chimpanzee, orangutan, monkey, cow, dog, rat, or mouse. In several embodiments, the deaminase or deaminase domain does not occur in nature. For example, in several embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 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%, 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 99.9%. For example, the deaminase domain is described in international PCT applications Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), the full text of which is incorporated herein by reference. Please also refer to Komor, AC, et al., "Programmable editing of target bases in genomic DNA without double-stranded DNA division," Nature 533, 420-424 (2016); Gaudelli, NM, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA division," Nature 551, 464-471 (2017); and Komor, AC, et al., "Improved base excision repair inhibition and C:G-to-T:A base editor obtained from phage μGam protein with higher efficiency and product purity," Science Advances 3: eaao4774 (2017); and Rees, HA, et al., "Base editing: precision chemistry on the genome and transcriptome of living cells." Nat RevGenet.2018Dec;19(12):770-788.doi:10.1038 / s41576-018-0059-1, the full text of which is incorporated herein by reference.

[0113] "Detectable label" refers to a composition that, when linked to a molecule of interest, enables the latter to be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Examples of useful labels include radioactive isotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxigenin, or haptens.

[0114] "Disease" refers to any condition or symptom that impairs or interferes with the normal function of cells, tissues, or organs. Examples of diseases include retinitis pigmentosa, Ussell's syndrome, sickle cell disease, β-thalassemia, α-1 antitrypsin deficiency (A1AD), hepatic pyrrole porphyria, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosome acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, von Kilke's syndrome, Pompe syndrome, Gaucher's syndrome, Holler's syndrome, cystic fibrosis, or chronic pain. In a particular embodiment, the disease is A1AD.

[0115] "Effective dose" refers to the amount of agent or active compound (e.g., a base editor as described herein) required to improve disease symptoms relative to an untreated patient. The effective dose of an active compound used in the practice of this invention to therapeutically treat disease varies depending on the administration method, individual age, weight, and general health condition. Ultimately, a clinician or veterinarian will determine the appropriate dosage. This dosage is referred to as the "effective" dose. In one embodiment, the effective dose is the amount of the base editor of this invention sufficient to introduce gene changes into cells (e.g., in vitro or in vivo). In one implementation, the effective amount is the amount of base editor required to achieve the therapeutic effect (e.g., to alleviate or control retinitis pigmentosa, Ussell's syndrome, sickle cell disease, β-thalassemia, α-1 antitrypsin deficiency (A1AD), hepatic pyrroloporosis, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosome acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, von Kilke's syndrome, Pompe syndrome, Gaucher's syndrome, Holler's syndrome, cystic fibrosis, or chronic pain). This therapeutic effect does not require sufficient alteration of the pathogenic gene in all cells of an individual, tissue, or organ, but only alteration of the pathogenic gene present in approximately 1%, 5%, 10%, 25%, 50%, 75%, or more of the cells of an individual, tissue, or organ. In one embodiment, the effective amount is sufficient to improve one or more symptoms of a disease (e.g., retinitis pigmentosa, Ussell's syndrome, sickle cell disease, β-thalassemia, α-1 antitrypsin deficiency (A1AD), hepatic pyrrole porphyria, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosome acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, von Kilke's syndrome, Pompe syndrome, Gaucher's syndrome, Holler's syndrome, cystic fibrosis, or chronic pain).

[0116] A "fragment" refers to a portion of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of a preferred reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0117] "Hybridization" refers to the hydrogen bond between complementary nucleoside bases, which can be a Watson-Crick, Husstein, or anti-Husstein hydrogen bond. For example, adenine and thymine are complementary nucleoside bases that pair up through hydrogen bonding.

[0118] The terms "base repair inhibitor," "base repair inhibitor," or their grammatical synonyms refer to proteins that inhibit the activity of nucleic acid repair enzymes (e.g., base excision repair enzymes). Non-limiting examples of base repair inhibitors include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4PDG, UDG, hSMUGl, and hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the base repair inhibitor is catalytically inactive Endo V or catalytically inactive hAAG. In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein that inhibits uracil DNA glycosylase base excision repair enzymes. In some embodiments, the UGI domain comprises wild-type UGI or a wild-type UGI fragment. In some embodiments, the UGI proteins provided herein comprise UGI fragments and proteins homologous to UGI fragments. In some embodiments, the base repair inhibitor is an inosine base excision repair inhibitor. In some embodiments, the base repair inhibitor is a "catalytically inactive inosine-specific nuclease" or a "dead inosine-specific nuclease".

[0119] Without being confined to any particular theory, catalytically inactive inosine glycosylation enzymes (e.g., alkyladenine glycosylation enzymes (AAG)) can bind inosine but cannot generate a basesettling site or remove inosine, thereby stereoblocking the newly generated inosine portion from DNA damage / repair mechanisms. In some embodiments, catalytically inactive inosine-specific nucleases can bind inosine in nucleic acids but do not cleave the nucleic acids. Non-limiting examples of catalytically inactive inosine-specific nucleases include, for example, catalytically inactive alkyladenine glycosylation enzymes (AAG nucleases) from humans, and catalytically inactive endonuclease V (EndoV nucleases) from E. coli. In some embodiments, catalytically inactive AAG nucleases contain an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0120] The terms "separated," "purified," or "biopure" refer to materials free from components normally present in their natural state to varying degrees. "Separated" indicates the degree of separation from its original source or surrounding environment. "Purified" indicates a higher degree of separation than separation. "Purified" or "biopure" proteins are sufficiently free of other materials such that any impurities do not substantially affect the protein's biological properties or cause other adverse consequences. In other words, if the nucleic acids or peptides of the present invention, when produced using recombinant DNA technology, are substantially free of cellular material, viral material, or culture medium, or when chemically synthesized, are substantially free of chemical precursors or other chemicals, then they have been purified. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" indicates that the nucleic acid or protein essentially forms a band in the electrophoretic gel. For proteins that can be modified, such as phosphorylation or glycosylation, different modifications can produce different separated proteins that can be purified.

[0121] "Separated polynucleotide" refers to a nucleic acid (e.g., DNA) that, in the natural genome of the organism from which the nucleic acid molecule of the present invention is derived, does not contain any genes that are extrinsic from that gene. Therefore, this term includes, for example, recombinant DNA, which is incorporated into a vector; incorporated into a self-replicating plasmid or virus; or incorporated into a prokaryotic or eukaryotic genome DNA; or exists as a separate molecule independent of other sequences (e.g., cDNA or genome or cDNA fragment produced by PCR or restriction endonuclease digestion). Furthermore, this term includes RNA molecules transcribed from DNA molecules, and recombinant DNA that is part of a fusion gene encoding an additional polypeptide sequence.

[0122] "Isolated polypeptide" refers to the polypeptide of the present invention that has been separated from its naturally occurring accompanying components. Typically, a polypeptide is considered isolated when it contains at least 60% by weight of its naturally associated proteins and naturally occurring organic molecules. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the polypeptide of the present invention. The isolated polypeptide of the present invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding the polypeptide, or by chemical synthesis of the protein. Purity can be measured by any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0123] As used in this disclosure, the term "linker" can refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule that links two molecules or portions, such as two components of a protein complex or ribonucleoside complex, or two domains of a fusion protein, such as a polynucleotide-programmable DNA-binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine deaminase or cytidine deaminase). Linkers can bind different components or different portions of a base editor system. For example, in some embodiments, a linker can bind a guiding polynucleotide-binding domain of a polynucleotide-programmable nucleotide-binding domain to a catalytic domain of a deaminase. In some embodiments, a linker can bind a CRISPR peptide to a deaminase. In some embodiments, a linker can bind Cas9 to a deaminase. In some embodiments, a linker can bind dCas9 to a deaminase. In some embodiments, a linker can bind nCas9 to a deaminase. In some embodiments, a linker can bind a deamination component of a base editor system to a polynucleotide-programmable nucleotide-binding component. In several embodiments, the linker can bind to the RNA-binding portion of the deamination component of the base editor system and the RNA-binding portion of the polynucleotide programmable nucleotide binding component. The linker may be located between, or adjacent to, two groups, molecules, or other portions, and linked to each other by covalent or non-covalent interactions, thus linking them. In several embodiments, the linker may be an organic molecule, group, polymer, or chemical portion. In several embodiments, the linker may be a polynucleotide. In several embodiments, the linker may be a DNA linker. In several embodiments, the linker may be an RNA linker. In several embodiments, the linker may contain a nucleic acid aptamer capable of binding to a ligand. In several embodiments, the linker may be a carbohydrate, peptide, protein, or nucleic acid. In several embodiments, the linker may contain a nucleic acid aptamer derived from a riboswitch. The riboswitch derived from the nucleic acid aptamer may be selected from: theophylline riboswitch, thiamine pyrophosphate (TPP) riboswitch, adenosylcobalamin (AdoCbl) riboswitch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch, riboflavin mononucleotide (FMN) riboswitch, tetrahydrofolate riboswitch, lysine riboswitch, glycine riboswitch, purine riboswitch, GlmS riboswitch, or pre-Q nucleoside 1 (PreQ1) riboswitch. In some embodiments, the linker may comprise a nucleic acid aptamer capable of binding to a polypeptide or protein domain, such as a polypeptide linker.In several embodiments, the linker may be a K homology (KH) domain, an MS2 shell protein domain, a PP7 shell protein domain, an SfMuCom shell protein domain, a sterile α motif, a telomerase Ku-binding motif and Ku protein, a telomerase Sm7-binding motif and Sm7 protein, or an RNA recognition motif. In several embodiments, the peptide linker may be part of a base editor system component. For example, a nucleoside base editing component may include a deaminase domain and an RNA recognition motif.

[0124] In several embodiments, the linker may be an amino acid or a plurality of amino acids (e.g., a peptide or a protein). In several embodiments, the linker may be about 5-100 amino acids in length, for example, about 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 length. In several embodiments, the linker may be about 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 amino acids in length. Longer or shorter linkers are also contemplated.

[0125] In several embodiments, the linker conjugates the gRNA-binding domain (including the Cas9 nuclease domain) of an RNA-programmable nuclease to the catalytic domain of a nucleic acid editing protein (e.g., cytidine deaminase or adenosine deaminase). In several embodiments, the linker conjugates dCas9 to the nucleic acid editing protein. For example, the linker is located between, or adjacent to, two groups, molecules, or other parts, and is covalently linked to each other, thus linking the two. In several embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In several embodiments, the linker is an organic molecule, group, polymer, or chemical part. In several embodiments, the linker is 5-200 amino acids long, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190, or 200 amino acids long. Longer or shorter linkers are also anticipated. In several embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In several embodiments, the linker comprises the amino acid sequence SGGS. In several embodiments, the linker comprises (SGGS). n (GGGS) n (GGGGS) n (G) n (EAAAK) n (GGS) n , SGSETPGTSESATPES, or (XP) n The motif, or any combination thereof, wherein n is independently an integer from 1 to 30, and wherein X is any amino acid. In several embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In several embodiments, the linker comprises a plurality of proline residues and 5-21, 5-14, 5-9, or 5-7 amino acids, for example, PAPAP, PAPAPA, PAPAPAP, PAPAPAPA, P(AP)4, P(AP)7, P(AP) 10 These proline-rich linker groups are also known as "rigid" linker groups.

[0126] In several implementations, the fields of the base editor are fused via linker bases, which include SGGSSGSETPGTSESATPESSGGS,

[0127] SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or

[0128] The amino acid sequence is GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS. In some embodiments, the domains of the base editor are fused via a linker containing the amino acid sequence SGSETPGTSESATPES, also known as an XTEN linker. In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker contains the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids long. In some embodiments, the linker contains the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids long. In some embodiments, the linker contains the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS. In some embodiments, the linker is 92 amino acids long. In some embodiments, the linker contains an amino acid sequence.

[0129]

[0130] As used herein, the term "mutation" refers to the substitution of one residue within a sequence, such as a nucleic acid sequence or an amino acid sequence, by another residue, or the deletion or insertion of one or more residues within a sequence. Typically, mutations in this disclosure are described by identifying the original residue, then the position of the residue within the sequence, and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) described herein are well known in the art and are provided, for example, by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). In several embodiments, the base editor of this disclosure can efficiently generate "planned mutations," such as point mutations, in nucleic acids (e.g., nucleic acids within an individual's genome) without generating a significant number of unplanned mutations, such as unplanned point mutations. In some embodiments, the planned mutation is a mutation generated by a specific base editor (e.g., a cytidine base editor or an adenosine base editor) that binds to a guide polynucleotide (e.g., gRNA) (which is specifically designed to generate the planned mutation). Typically, mutations created or identified in a sequence (e.g., an amino acid sequence as described herein) are numbered relative to a reference (or wild-type) sequence (i.e., a sequence without the mutation). Those skilled in the art readily understand how to determine the location of mutations in amino acid and nucleic acid sequences relative to a reference sequence.

[0131] The term "non-conservative mutation" involves the substitution of amino acids in different groups, such as lysine replacing tryptophan, or phenylalanine replacing serine. In such cases, non-conservative amino acid substitutions are preferred to avoid interfering with or inhibiting the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, resulting in increased biological activity compared to the wild-type protein.

[0132] The terms "nuclear localization sequence," "nuclear localization signal," or "NLS" refer to amino acid sequences that facilitate protein importation into the cell nucleus. Nuclear localization sequences are known in the art and are described, for example, in Plank et al.'s international PCT application PCT / EP2000 / 011690, filed November 23, 2000, and published as WO / 2001 / 038547 on May 31, 2001, the contents of which are incorporated herein by reference for disclosed embodiments of nuclear localization sequences. In other embodiments, the NLS is, for example, an optimized NLS described by Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In several embodiments, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0133] As used in this disclosure, the terms "nucleic acid" and "nucleic acid molecule" refer to compounds containing nucleoside bases and acidic moieties, such as nucleosides, nucleotides, or polymers of nucleotides. Typically, polymeric nucleic acids, for example, nucleic acid molecules containing three or more nucleotides, are linear molecules in which adjacent nucleotides are linked together by phosphodiester linkages. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain containing three or more individual nucleotide residues. As used in this disclosure, the terms "oligonucleotide," "polynucleotide," and "polynucleic acid" are used interchangeably to refer to polymers of nucleotides (e.g., a chain of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA as well as single-stranded DNA and / or double-stranded DNA. Nucleic acids can occur naturally in, for example, genomic bodies, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plastids, cohesive plastids, chromosomes, chromatids, or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be non-natural molecules, such as recombinant DNA or RNA, artificial chromosomes, genetically modified genomes, fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or including non-natural nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, such as analogs having a non-phosphodiester backbone. Nucleic acids can be purified from natural sources, manufactured using recombinant expression systems, and optionally purified, chemically synthesized, etc. When appropriate, for example, as chemically synthesized molecules, nucleic acids may contain nucleoside analogs, such as analogs having chemically modified bases or sugars and backbone modifications. Unless otherwise indicated, nucleic acid molecules are presented in a 5' to 3' orientation. In several embodiments, the nucleic acid is or comprises a natural nucleoside (e.g., adenosine, thymosine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymosine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiathymosine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deadenosine, 7-deadenosine, 8-sideoxyadenosine, 8-sideoxyguanosine, O... 6-Methylguanine and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); embedded bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., thiophosphates and 5'-N-phosphorylamine links). In some embodiments, the RNA is RNA associated with the Cas9 system. For example, the RNA may be CRISPR RNA (crRNA), trans small RNA (tracrRNA), single guide RNA (sgRNA), or guide RNA (gRNA).

[0134] The terms "nucleoside base," "nitrogenous base," or "base" are used interchangeably in this disclosure to refer to nitrogenous biological compounds that form nucleosides, which in turn become nucleotides. Nucleoside bases are capable of forming base pairs and stacking on each other, directly resulting in long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleoside bases, namely adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), are referred to as primary or canonical. Adenine and guanine are derived from purines; cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA may also contain other (non-initial) modified bases. Non-limiting examples of modified bases include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytidine (m5C), and 5-hydromethylcytidine. Hypoxanthine and xanthine can be generated in the presence of a mutagen, both through deamination (replacing an amino group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be obtained from the deamination of cytosine. A nucleoside is composed of a nucleoside base and a pentose sugar (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleoside bases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A nucleotide is composed of a nucleoside base, a pentose sugar (ribose or deoxyribose), and at least one phosphate group.

[0135] The terms "nucleic acid programmable DNA-binding protein" or "napDNAbp" are used interchangeably with "polynucleotide programmable nucleotide binding domain" to refer to proteins associated with nucleic acids (e.g., DNA or RNA), such as guide nucleic acids that guide napDNAbp to a specific nucleic acid sequence. For example, a Cas9 protein may be associated with guide RNA, which guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, napDNAbp is a Cas9 domain, such as an active nuclease Cas9, a Cas9 nickase (nCas9), or an inactive nuclease Cas9 (dCas9). Examples of nucleic acid programmable DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Other nucleic acid-programmable DNA-binding proteins also fall within the scope of this disclosure, but may not be specifically listed herein. For example, see Makarova 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, the full text of which is incorporated herein by reference.

[0136] As used herein, the terms "nucleobase editing domain" or "nucleobase editing protein" refer to a protein or enzyme capable of catalyzing nucleobase modifications in RNA or DNA, such as deamination from cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine), or from adenine (or adenosine) to hypoxanthine (or inosine), as well as non-template nucleotide addition and insertion. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., cytidine deaminase, cytosine deaminase, adenine deaminase, or adenosine deaminase). In some embodiments, the nucleobase editing domain may be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain may be a nucleobase editing domain genetically modified or evolved from a naturally occurring nucleobase editing domain. The nucleobase editing domain may be derived from any organism, such as bacteria, humans, chimpanzees, orangutans, monkeys, cattle, dogs, rats, or mice. For example, nucleoside base editing proteins are described in international PCT applications Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), the full text of which is incorporated herein by reference. Please also refer to Komor, AC, et al., "Programmable editing of target bases in genomic DNA without double-strand DNA division" Nature 533, 420-424 (2016); Gaudelli, NM, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA division" Nature 551, 464-471 (2017); and Komor, AC, et al., "Improved base excision repair inhibition and C:G-to-T:A base editor from phage μGam protein with higher efficiency and product purity" Science Advances 3:eaao4774 (2017), the full contents of which are incorporated herein by reference.

[0137] As used in this disclosure, “obtain” in “obtaining the agent” includes synthesizing, purchasing, or otherwise obtaining the agent.

[0138] As used in this disclosure, "patient" or "individual" means a mammalian individual or person who is diagnosed with, at risk of developing, or suspected of developing a disease or condition. In some embodiments, the term "patient" refers to a mammalian individual with a higher probability than the average probability of developing a disease or condition. Examples of patients may include humans, non-human primates, cats, dogs, pigs, cattle, horses, camels, alpacas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other mammals that may benefit from the therapies disclosed herein. Examples of human patients may include men and / or women.

[0139] "Patients in need" or "individuals in need" in this disclosure means patients who have been diagnosed with or suspected of having a disease or condition, such as, but not limited to, alpha-1 antitrypsin deficiency (A1AD).

[0140] The terms "pathogenic mutation," "pathogenic variant," "pathogenic mutation," "pathogenic variant," "harmful mutation," or "susceptibility mutation" refer to a genetic alteration or mutation that increases an individual's susceptibility to or predisposition to a disease or condition. In some embodiments, the pathogenic mutation is contained in a protein encoded by a gene, wherein at least one wild-type amino acid is replaced by at least one pathogenic amino acid.

[0141] The terms "peptide," "polypeptide," "protein," and their grammatical synonyms are used interchangeably throughout this disclosure to refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The term refers to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide may be modified, for example, by adding chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linking groups for conjugation, functionalization, or other modifications. A protein, peptide, or polypeptide may be a single molecule or a multi-molecule complex. A protein, peptide, or polypeptide may simply be a fragment of a natural protein or peptide. A protein, peptide, or polypeptide may be natural, recombinant, synthetic, or any combination thereof. As used in this disclosure, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein may be located at the N-terminal (N-terminus) or C-terminal (C-terminus) portion of a fusion protein, thus generating an N-terminal fusion protein or a C-terminal fusion protein, respectively. The protein may contain various domains, such as a nucleic acid binding domain (e.g., a gRNA binding domain of Cas9 guiding the protein to a target site) and a nucleic acid cleavage domain, or a catalytic domain of a nucleic acid editing protein. In several embodiments, the protein comprises a protein moiety, such as an amino acid sequence constituting the nucleic acid binding domain, and an organic compound, such as a compound capable of acting as a nucleic acid cleavage agent. In several embodiments, the protein is complexed with or associated with a nucleic acid, such as RNA or DNA. Any protein provided in this disclosure can be manufactured by any method known in the art. For example, the proteins provided in this disclosure can be manufactured by recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkages. Methods for the expression and purification of recombinant proteins are well known, including those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), which are incorporated herein by reference in their entirety.

[0142] The polypeptides and proteins (including their functional proteins and functional variants) disclosed herein may contain synthetic amino acids that replace one or more natural amino acids. These amino acids are known in the art and include homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, and 1,2,3,4-tetrahydroisoquinone. Phospho-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoacylamine, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornene)carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tertiary butylglycine. Peptides and proteins can be associated with post-translational modifications of one or more amino acids in the polypeptide construct. Non-limiting embodiments of post-translation modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-linking and O-linking), acylation, hydroxylation, alkylation including methylation and ethylation, ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristylation, palmitoylation, isoprenylation, farnesylation, geraniylation, glycosylphosphatidylinositolization, esterification, and iodination.

[0143] The term "polynucleotide-programmable nucleotide binding domain" refers to a protein associated with a nucleic acid (e.g., DNA or RNA), such as a guiding polynucleotide (e.g., guiding RNA), which guides a polynucleotide-programmable DNA binding domain to a specific nucleic acid sequence. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a polynucleotide-programmable DNA binding domain. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a polynucleotide-programmable RNA binding domain. In some embodiments, the polynucleotide-programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein can be associated with guiding RNA, which guides the Cas9 protein to a specific DNA sequence having a complement to the guiding RNA. In some embodiments, the polynucleotide-programmable nucleotide binding domain has a Cas9 domain, such as an active nuclease Cas9, a Cas9 nickase (nCas9), or an inactive nuclease Cas9 (dCas9). Non-limiting embodiments of nucleic acid programmable DNA-binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Non-limiting embodiments of Cas include 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, Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, and Csa. 5. Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, 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, their equivalents, or modified or engineered versions thereof.Other nucleic acid programmable DNA-binding proteins also fall within the scope of this disclosure, but are not specifically listed herein.

[0144] As used in the context of proteins or nucleic acids in this disclosure, the term "recombinant" refers to a protein or nucleic acid that does not occur in nature but is a product of human engineering. For example, in some embodiments, the recombinant protein or nucleic acid molecule comprises, compared to any natural sequence, at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutated amino acid or nucleotide sequences.

[0145] "Reduction" means a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0146] "Reference" indicates a standard or control condition. In one implementation, the reference is wild-type or healthy cells.

[0147] A "reference sequence" refers to a defined sequence used as the basis for sequence comparison. A reference sequence can be a subset or the entirety of a specific sequence, such as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For peptides, the reference peptide sequence is typically at least about 16 amino acids in length, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the reference nucleic acid sequence is typically at least about 50 nucleotides in length, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides, or about 300 nucleotides, or any integer in length surrounding or within the specified range.

[0148] The terms “RNA-programmable nuclease” and “RNA-guided nuclease” are used in conjunction with one or more RNAs that are not targets for splitting (e.g., binding or associating). In some embodiments, an RNA-programmable nuclease, when complexed with RNA, may be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as guide RNA (gRNA). gRNA may exist as a complex of two or more RNAs or as a single RNA molecule. gRNA existing as a single RNA molecule may be referred to as a single guide RNA (RNA), but “gRNA” is used interchangeably to refer to guide RNA existing as a single molecule or as a complex of two or more RNAs. Typically, gRNA existing as a single RNA contains two domains: (1) a domain that shares homology with the target nucleic acid (e.g., and guides the Cas9 complex to bind to the target); and (2) a domain that binds the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence called tracrRNA and includes a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to tracrRNA, as provided in Jinek et ah, Science 337:816-821 (2012), the entire disclosure of which is incorporated herein by reference. Other embodiments of gRNA (e.g., including domain 2) are described in reference to U.S. Provisional Patent Application USSN 61 / 874,682, filed September 6, 2013, entitled "Switchable Cas9 Nucleases And Uses Thereof," and U.S. Provisional Patent Application USSN 61 / 874,746, filed September 6, 2013, entitled "Delivery System For Functional Nucleases," the entire disclosure of which is incorporated herein by reference. In some embodiments, gRNA comprising two or more of domains (1) and (2) may be referred to as "extended gRNA." For example, as described herein, the extended gRNA will, for instance, bind two or more Cas9 proteins and target nucleic acids in two or more separate regions. The gRNA contains a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity of the nuclease:RNA complex.In several embodiments, the RNA-programmable nuclease is a CRISPR-associated system (CRISPR-associated system) Cas9 endonuclease, such as Cas9 (Csnl) derived from Streptococcus pyogenes (e.g., refer to "Complete genome sequence of an Ml strain of Streptococcus pyogenes."). Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin R.E., Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded smallRNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM., Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011)).

[0149] "SERPINA1 polynucleotide" refers to a nucleic acid molecule encoding the A1AT protein or a fragment thereof. An example sequence of the SERPINA1 polynucleotide, available under NCBI Accession NO. NM_000295, is provided below:

[0150]

[0151]

[0152]

[0153] The positions of bases complementary to the PAM sequence are shown in italics with double underlines. The G at position 1455 (which is complementary to the target C at position 1455) is indicated in bold with an underline.

[0154] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide variation that occurs at a specific location in the genome, where each variation is present to a perceptible degree within a population (e.g., >1%). For example, at a specific base position in the human genome, the C nucleotide may be present in most individuals, but in a minority of individuals, that position is occupied by A. This indicates that there is an SNP at that specific location, with two possible nucleotide variations, C or A, representing the allele at that location. SNPs constitute the basis for susceptibility to diseases (a broad range of human diseases). Disease severity and the body's response to treatment are also characteristics of gene variations. SNPs can fall into gene code regions, gene non-code regions, or intergenic regions (intergenic regions). In some embodiments, SNPs within the code region do not necessarily alter the amino acid sequence of a protein, which is produced due to the degeneracy of the genetic code. SNPs in the code region are divided into two types: synonymous SNPs and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs alter the amino acid sequence of a protein. Nonsynonymous SNPs are divided into two types: missense and nonsense. SNPs not located in protein coding regions can still affect gene splicing, transcription factor binding, signaling RNA degradation, or the sequence of encoded RNA. Gene expression affected by this type of SNP is called an eSNP (expressed SNP), which can occur upstream or downstream of the gene. Single nucleotide variants (SNVs) are variations in a single nucleotide without any frequency limit and can occur in somatic cells. Somatic single nucleotide variants (e.g., those caused by cancer) are also called single nucleotide alterations.

[0155] "Specific binding" means that a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid programmable DNA binding domain and a guiding nucleic acid), compound, or molecule recognizes and binds to the polypeptide and / or nucleic acid molecules of the present invention, but does not actually recognize and bind to other molecules in a sample (e.g., a biological sample).

[0156] The nucleic acid molecules useful in the methods of this invention include nucleic acid molecules encoding the polypeptides or fragments thereof of the invention. These nucleic acid molecules need not be 100% identical to endogenous nucleic acid sequences, but will typically have substantial similarity. Polynucleotides having "substantial similarity" to endogenous sequences are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridization" means pairing to generate a double-stranded molecule, or a portion thereof, between complementary polynucleotide sequences (e.g., the genes described herein) under various stringent conditions (e.g., see Wahl, GM and SLBerger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0157] For example, harsh salt concentrations are typically below about 750 mM NaCl and 75 mM trisodium citrate, preferably below about 500 mM NaCl and 50 mM trisodium citrate, and even more preferably below about 250 mM NaCl and 25 mM trisodium citrate. Low-hardness hybridization can be obtained in the absence of organic solvents (e.g., formamide), while high-hardness hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Harsh temperature conditions typically include temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergent (e.g., sodium dodecyl sulfate (SDS)), and inclusion or exclusion of vector DNA, are well known to those skilled in the art. Different degrees of harshness are achieved as needed by combining these various conditions. In a preferred embodiment, hybridization will occur at 30°C with 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C with 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm (ssDNA). In the most preferred embodiment, hybridization will occur at 42°C with 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to those skilled in the art.

[0158] The severity of the washing steps after hybridization will vary for most applications. Washing severity conditions can be defined by salt concentration and temperature. As mentioned above, washing severity can be increased by reducing salt concentration and increasing temperature. For example, the severe salt concentration for the washing step is preferably below about 30 mM NaCl and 3 mM trisodium citrate; and most preferably below about 15 mM NaCl and 1.5 mM trisodium citrate. The severe temperature for the washing step typically includes a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the washing step will occur at 25°C with 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step will occur at 42°C with 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a preferred embodiment, the washing step will occur at 68°C with 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations in these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well-known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology), Wiley Interscience, New York, 2001; Berger and Kimmel (Guide to Molecular Cloning Techniques), 1987, Academic Press, New York); and Sambrook et al. (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor Laboratory Press, New York.

[0159] "Individual" means mammal, including but not limited to humans or non-human mammals, such as cows, horses, dogs, sheep, or cats.

[0160] "Substantially identical" means a polypeptide or nucleic acid molecule that has at least 50% identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and even more preferably 90%, 95%, or even 99% identical to the sequence used for comparison at the amino acid level or nucleic acid level.

[0161] Sequence identity is typically determined using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin-Madison, 1710 University Avenue, Madison, Wisconsin 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programming). This software pairs identical or similar sequences by specifying the degree of homology for each substitution, deletion, and / or other modification. Conserved substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamyl; asparagine, glutamic acid; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In the methodological examples for identity determination, BLAST programming with e -3 to e -100 The probability fractions between them indicate closely related sequences.

[0162] The term "target site" refers to a sequence within a nucleic acid molecule modified by a nucleoside base editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein containing a deaminase (e.g., cytidine deaminase or adenosine deaminase).

[0163] Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, in principle, these proteins can be targeted to any sequence specified by the guide RNA. The use of RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify genomes) is well-known in the industry (see, for example, Cong, L. et ah, Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et ah, RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W. et ah, Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et ah, RNA-programmed genome editing in human cells. eLife). 2,e00471 (2013); Dicarlo, JEet ah, Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas system. Nucleic acids research (2013); Jiang, W. et ah, RNA-guided editing of bacterial genomes using CRISPR-Cas system. Nature biotechnology 31, 233-239 (2013); their full texts are incorporated herein by reference.

[0164] As used herein, the terms "therapy," "treatment," or their grammatical synonyms refer to achieving the desired pharmacological and / or physiological effects. In some embodiments, the effect is therapeutic, meaning that the effect partially or completely cures the disease and / or adverse symptoms attributable to the disease. In some embodiments, the effect is preventative, meaning that the effect prevents the occurrence or recurrence of the disease or condition. To achieve this purpose, the method disclosed herein comprises administering a therapeutically effective amount of the composition as described herein.

[0165] "Uracil glycosylation enzyme inhibitor" refers to an inhibitor of the uracil excision repair system. In one embodiment, the inhibitor is a protein or fragment thereof that binds to host uracil-DNA glycosylation enzymes and prevents the removal of uracil residues from DNA.

[0166] It should be understood that the ranges provided in this disclosure are a simplified representation of all values ​​within that range. For example, it should be understood that the range 1 to 50 includes any number, combination of numbers, or subrange derived from the group consisting of 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.

[0167] In any definition of a variable in this disclosure, the enumeration of each chemical group in the list includes the definition of the variable as any single group or combination of the enumerated groups. References to embodiments of a variable or state in this disclosure include the embodiment as a single embodiment or in combination with any other embodiment or part thereof.

[0168] Any composition or method provided in this disclosure may be combined with one or more of any other compositions or methods provided in this disclosure.

[0169] DNA editing has become a viable method for modifying disease states by correcting pathogenic mutations at the gene level. Until recently, all DNA editing platforms functioned by inducing DNA double-strand breaks (DSBs) at specific genomic sites and relying on endogenous DNA repair pathways to determine product outcomes in a semi-random manner, resulting in a complex population of gene products. While homology-guided repair (HDR) can achieve precise, user-defined repair results, several challenges prevent the use of HDR for effective repair in therapeutically relevant cell types. In fact, this pathway is less efficient than the competing, error-prone non-homologous end-binding pathway. Furthermore, HDR is highly restricted to the G1 and S phases of the cell cycle, hindering precise repair of DSBs in post-mitotic cells. Consequently, it has been demonstrated that in these populations, it is difficult or impossible to alter the genomic sequence in a user-defined, programmable manner.

[0170] Nucleoside base editor

[0171] The present invention discloses a base editor or nucleoside base editor for editing, modifying, or changing target nucleotide sequences. The present invention describes a nucleoside base editor or base editor comprising a polynucleotide programmable nucleotide binding domain or a nucleoside base editing domain. The polynucleotide programmable nucleotide binding domain, when in conjunction with a bound guide polynucleotide (e.g., gRNA), specifically binds to the target polynucleotide sequence (in other words, through complementary base pairing between the bases of the bound guide nucleic acid and the bases of the target polynucleotide sequence), thus positioning the base editor to the target nucleic acid sequence to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA fusion.

[0172] Multinucleotide programmable nucleotide binding domain

[0173] The term "multinucleotide programmable nucleotide binding domain" refers to a protein associated with a nucleic acid (e.g., DNA or RNA), such as a guiding polynucleotide (e.g., guiding RNA), which directs the multinucleotide programmable nucleotide binding domain to a specific nucleic acid sequence. In some embodiments, the multinucleotide programmable nucleotide binding domain is a multinucleotide programmable DNA binding domain. In some embodiments, the multinucleotide programmable nucleotide binding domain is a multinucleotide programmable RNA binding domain. In some embodiments, the multinucleotide programmable nucleotide binding domain is a Cas9 protein. In some embodiments, the multinucleotide programmable nucleotide binding domain is a Cpf1 protein.

[0174] CRISPR is an adaptive immune system that provides protection against mobile genetic entities (viruses, substitutional entities, and conjugates). CRISPR clusters contain a spacer, a complementary sequence to the ancestral mobile genetic entity, and a target invading nucleic acid. These clusters are transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, proper processing of pre-crRNA requires transcoding of a small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. tracrRNA acts as a guide for ribonuclease 3-assisted processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endo-cleaves the linear or circular dsDNA target complementary to the spacer. Target strands not complementary to crRNA are first endo-cleaved and then exogenously cleaved at 3'-5'. In nature, DNA binding and DNA cleavage typically require both proteins and two types of RNA. However, single-guide RNA ("sgRNA" or simply "gRNA") can be genetically modified to combine the states of both crRNA and tracrRNA into a single RNA. For example, see Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the full disclosure of which is incorporated herein by reference. Cas9 identifies short motifs (PAM or protospacer adjacent motifs) in CRISPR repeat sequences to help distinguish itself from non-self motifs.

[0175] The Cas9 nuclease sequence and structure are well-known to industry professionals (for example, see "Complete Genome Sequence of Streptococcus pyogenes M1" Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA Maturation via Encoding Small RNA and Host Factor RNase III" Deltcheva E., Chylinski K., Sharma CM., Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity” (Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), each incorporated herein by reference in its entirety). The Cas9 homolog has been described in various species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus.Based on the disclosures herein, additional suitable Cas9 nucleases and sequences are readily apparent to those skilled in the art. These Cas9 nucleases and sequences include Cas9 sequences and loci derived from organisms, as disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737, the disclosure of which is incorporated herein by reference in its entirety.

[0176] In several embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) is a Cas9 domain. Non-limiting embodiments of the Cas9 domain are provided in this disclosure. The Cas9 domain can be a nuclease-active Cas9 domain, a nuclease-inactivating Cas9 domain, or a Cas9 cleavage enzyme. In several embodiments, the Cas9 domain is a nuclease-active domain. For example, the Cas9 domain can be a Cas9 domain that cleaves both strands of a diploid nucleic acid (both strands of a diploid DNA molecule). In several embodiments, the Cas9 domain comprises any of the amino acid sequences as described in this disclosure. In several embodiments, the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the amino acid sequences as described in this disclosure. In several embodiments, the Cas9 domain comprises an amino acid sequence having, compared to any of the amino acid sequences described herein, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations. In several embodiments, the Cas9 domain comprises an amino acid sequence having 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, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical adjacent amino acid residues.

[0177] In several embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA splitting domain; in other words, Cas9 is a nicking enzyme. The inactivated Cas9 protein is interchangeably referred to as "dCas9" protein (for Cas9 nuclease death). Methods for generating Cas9 protein (or fragments thereof) with an inactive DNA splitting domain are known (e.g., see 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, each incorporated herein by reference in its entirety). For example, the DNA splitting domain of Cas9 is known to include two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain splits the complementary strand of the gRNA, while the RuvC1 subdomain splits the non-complementary strand. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, mutations in D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al, Science. 337:816-821 (2012); Qi et al, Cell. 28; 152(5):1173-83 (2013)). In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA splitting domain; in other words, Cas9 is a nicking enzyme, referred to as the "nCas9" protein (for "nicking enzyme" Cas9). In some embodiments, a protein comprising a fragment of Cas9 is provided. For example, in some embodiments, the protein comprises one of two Cas9 domains: (1) a gRNA-binding domain of Cas9; or (2) a DNA splitting domain of Cas9. In some embodiments, a protein containing Cas9 or a fragment thereof is referred to as a "Cas9 variant". Cas9 variants share homology with Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identical to wild-type Cas9.In several embodiments, compared to wild-type Cas9, Cas9 variants may have 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 acid alterations. In several embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA-binding domain or a DNA splitting domain) such that the fragment is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In several embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the corresponding wild-type Cas9. In several embodiments, the fragment is at least 100 amino acids long. In several embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids long.

[0178] In several embodiments, wild-type Cas9 corresponds to Cas9 derived from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences are as follows).

[0179]

[0180]

[0181]

[0182]

[0183] (Single underline: HNH domain; Double underline: RuvC domain)

[0184] In several embodiments, wild-type Cas9 corresponds to or includes the following nucleotide and / or amino acid sequences:

[0185]

[0186]

[0187]

[0188] (Single underline: HNH domain; Double underline: RuvC domain)

[0189] In several embodiments, wild-type Cas9 corresponds to Cas9 derived from Streptococcus pyogenes (NCBI reference sequence: NC_002737.2 (nucleotide sequence below); and Uniprot reference sequence: Q99ZW2 (amino acid sequence below).

[0190]

[0191]

[0192]

[0193] (Single underline: HNH domain; Double underline: RuvC domain)

[0194] In several embodiments, Cas9 refers to Cas9 derived from the following: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021846.1); Ref:NC_021314.1); Belliella baltica (NCBI Ref:NC_018010.1); Psychroflexus torquis I (NCBI Ref:NC_018721.1); Streptococcus thermophilus (NCBI Ref:YP_820832.1); Listeria innocua (NCBI Ref:NP_472073.1); Campylobacter jejuni (NCBI Ref:YP_002344900.1); or Neisseria meningitidis (NCBI Ref:YP_002342100.1), or Cas9 derived from any other organism.

[0195] In several embodiments, dCas9 corresponds to, or partially or wholly comprises, a Cas9 amino acid sequence with one or more mutations that inactivate Cas9 nuclease activity. Unless otherwise stated, Cas9 is identified relative to a wild-type reference sequence. For example, in several embodiments, the dCas9 domain comprises the D10A and H840A mutations, or corresponding mutations in another Cas9. In several embodiments, the dCas9 domain comprises the amino acid sequence of dCas9 (D10A and H840A).

[0196]

[0197] (Single underline: HNH domain; Double underline: RuvC domain)

[0198] In several embodiments, the Cas9 domain contains a D10A mutation, while the residue at position 840 remains at the corresponding position in the amino acid sequence provided above, or in any of the amino acid sequences provided in this disclosure.

[0199] In other embodiments, dCas9 variants with mutations other than D10A and H840A are provided, such as Cas9 (dCas9) that leads to nuclease inactivation. For example, such mutations include other amino acid substitutions in D10A and H840A, or other substitutions in the Cas9 nuclease domain (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In several embodiments, variants or equivalents of dCas9 are provided that are 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 about 99.9% identical. In several embodiments, dCas9 variants are provided having shorter or longer amino acid sequences of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, or about 100 amino acids.

[0200] In some embodiments, the Cas9 fusion protein provided in this disclosure comprises the full-length amino acid sequence of the Cas9 protein, for example, one of the Cas9 sequences provided in this disclosure. However, in other embodiments, the fusion protein provided in this disclosure does not comprise the full-length Cas9 sequence, but instead contains only one or more Cas9 fragments. Suitable amino acid sequence examples of Cas9 domains and Cas9 fragments are provided in this disclosure; other suitable sequences of Cas9 domains and Cas9 fragments will be readily apparent to those skilled in the art.

[0201] The Cas9 protein can be associated with a guide RNA, which guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In several embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 domain, such as an active nuclease Cas9, a Cas9 nickase (nCas9), or a nuclease inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2c1, and Cas12c / C2c3.

[0202] The nuclease-inactivated Cas9 protein is interchangeably referred to as "dCas9" protein (for nuclease-"dead" Cas9) or catalytically inactivated Cas9. Methods for generating Cas9 proteins (or fragments thereof) with an inactivated DNA splitting domain are known (e.g., see Jinek et al, Science. 337:816-821 (2012); Qi et al, "Reusing CRISPR as an RNA-guided platform for specific sequence control of gene expression" (2013) Cell. 28; 152(5):1173-83, each incorporated herein by reference in its entirety). For example, the DNA splitting domain of Cas9 is known to comprise two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain splits the complementary strand of the gRNA, while the RuvC1 subdomain splits the non-complementary strand. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, mutants D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al, Science. 337:816-821 (2012); Qi et al, Cell. 28; 152(5):1173-83 (2013)). As an example, the nuclease-inactivated Cas9 domain contains the amino acid sequence stated in the cloning vector pPlatTET-gRNA2 (Accession No. BAV54124).

[0203] The amino acid sequence of the catalytically deactivated Cas9 (dCas9) example is as follows:

[0204]

[0205] The amino acid sequence of the embodiment catalyzing Cas9 nickase (nCas9) is as follows:

[0206]

[0207]

[0208] The amino acid sequence of the example of catalytic activation of Cas9 is as follows:

[0209]

[0210]

[0211] In some embodiments, Cas9 refers to Cas9 derived from archaea (e.g., nanoarchaea), which constitute the domain and kingdom of single-celled prokaryotic microorganisms. In some embodiments, the programmable nucleotide-binding protein may be a CasX or CasY protein, which has been described, for example, in Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi:10.1038 / cr.2017.21, the disclosure of which is incorporated herein by reference in its entirety. Using metagenomically resolved genomics, multiple CRISPR-Cas systems have been identified, including Cas9, which was first reported in the archaeal life domain. This divergent Cas9 protein appears in rarely studied nanoarchaea as part of a CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, are explored, representing some of the most densely packed systems yet to be explored. In some embodiments, within the base editor system described herein, Cas9 is replaced by CasX or a variant of CasX. In some embodiments, within the base editor system described herein, Cas9 is replaced by CasY or a variant of CasY. It should be understood that other RNA-guided DNA-binding proteins may be used as nucleic acid-programmable DNA-binding proteins (napDNAbp) and fall within the scope of this disclosure.

[0212] In several embodiments, the programmable nucleotide-binding protein, also referred to herein as a programmable DNA-binding protein (napDNAbp), is a CasX protein. In several embodiments, the programmable nucleotide-binding protein is a CasY protein. In several embodiments, the programmable nucleotide-binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to that of a natural CasX or CasY protein. In several embodiments, the programmable nucleotide-binding protein is a natural CasX or CasY protein. In several embodiments, the programmable nucleotide-binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to that of the CasX or CasY protein described herein. It should be understood that, as disclosed in this article, CAX and CasY derived from other strains can also be used.

[0213] An example of the amino acid sequence of the CasX protein OS = Sulfolobus islandicus (species HVE10 / 4) GN = SiH_0402PE = 4SV = 1 is as follows:

[0214]

[0215] An example of the amino acid sequence of CasX(>tr|F0NH53|F0NH53_SULIR CRISPR-associated protein, Casx OS = Sulfolobus islandicus (species REY15A) GN = SiRe_0771 PE = 4 SV = 1) is as follows:

[0216]

[0217] Deltaproteobacteria (CasX)

[0218]

[0219]

[0220] An example of the amino acid sequence of CasY ((ncbi.nlm.nih.gov / protein / APG80656.1)>APG80656.1 CRISPR-associated protein CasY [wild-type Parcubacteria]) is as follows:

[0221]

[0222] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) is a single effector of the microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have a single protein effector. For example, Cas9 and Cpf1 belong to Class 2 systems. Besides Cas9 and Cpf1, three separate class 2 CRISPR-Cas systems (Cas12b / C2c1 and Cas12c / C2c3) have been described in Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems", Mol. Cell, 2015 Nov. 5; 60(3):385-397, the full text of which is incorporated herein by reference. The effectors of two of these systems, Cas12b / C2c1 and Cas12c / C2c3, contain a Cpf1-associated RuvC-shaped endonuclease domain. The third system contains an effector with two predicted HEPN RNase domains. The production of mature CRISPR RNA is independent of tracrRNA production, unlike the production of CRISPR RNA via Cas12b / C2c1. Cas12b / C2c1 relies on both CRISPR RNA and tracrRNA for DNA division.

[0223] The crystal structure of the complex chimeric single-molecule guide RNA (sgRNA) of *Alicyclobaccillus acidoterrastris* Cas12b / C2c1 (AacC2c1) has been reported. For example, see Liu et al., "C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism", Mol. Cell, 2017 Jan. 19; 65(2):310-322, the disclosure of which is incorporated herein by reference in its entirety. The crystal structure of *Alicyclobaccillus acidoterrastris* C2c1 has also been reported to bind to target DNA in a ternary complex. For example, see Yang et al., “PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease”, Cell, 2016 Dec. 15; 167(7): 1814-1828, the full text of which is incorporated herein by reference. The catalytically competent conformation of AacC2c1, containing both target and non-target DNA strands, is known to be independently located within a single RuvC catalytic pocket. Cleavage mediated by Cas12b / C2c1 results in staggered 7-nucleotide breaks of the target DNA. Structural comparisons between the Cas12b / C2c1 ternary complex and previously identified Cas9 and Cpf1 dual moieties validate the diversity of mechanisms utilized in the CRISPR-Cas system.

[0224] In several embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) of any fusion protein provided in this disclosure may be a Cas12b / C2c1 protein or a Cas12c / C2c3 protein. In several embodiments, napDNAbp is a Cas12b / C2c1 protein. In several embodiments, napDNAbp is a Cas12c / C2c3 protein. In several embodiments, napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the native Cas12b / C2c1 or Cas12c / C2c3 protein. In several embodiments, napDNAbp is a native Cas12b / C2c1 or Cas12c / C2c3 protein. In several embodiments, the napDNAbp contains an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the napDNAbp sequences provided herein. It should be understood that Cas12b / C2c1 or Cas12c / C2c3 derived from other bacteria may also be used according to the disclosure herein.

[0225] An example of the amino acid sequence Cas12b / C2c1((uniprot.org / uniprot / T0D7A2#2)sp|T0D7A2|C2C1_AL IAGCRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acido-terrestris)(strain ATCC 49025 / DSM 3922 / CIP 106132 / NCIMB 13137 / GD3B)GN=c2c1PE=1SV=1) is as follows:

[0226]

[0227] BhCas12b Bacillus hisashii NCBI reference sequence: WP_095142515

[0228]

[0229] In some embodiments, Cas12b is BvCas12B, which is a variant of BhCas12b, and includes the following variations related to BhCas12B: S893R, K846R, and E837G.

[0230] BvCas12b (Bacillus sp.) V3-13 NCBI reference sequence: WP_101661451.1

[0231]

[0232]

[0233] It should be understood that polynucleotide-programmable nucleotide-binding domains can also include nucleic acid-programmable proteins that bind to RNA. For example, a polynucleotide-programmable nucleotide-binding domain can be associated with a nucleic acid that guides the polynucleotide-programmable nucleotide-binding domain to RNA. Other nucleic acid-programmable DNA-binding proteins also fall within the scope of this disclosure, but are not specifically listed herein.

[0234] The Cas proteins that can be used in this disclosure include those of classes 1 and 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cm r4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i, CARF, DinG, their equivalents, or modified versions thereof. Unmodified CRISPR enzymes, such as Cas9, possess DNA-splitting activity and have two functional endonuclease domains: RuvC and HNH. CRISPR enzymes can direct one or two splits within the target sequence, such as within the target sequence and / or within the complement region of the target sequence. For example, CRISPR enzymes can direct one or two splits within approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0235] A vector encoding a CRISPR enzyme may be used, which is mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or two strands of a target polynucleotide containing the target sequence. Cas9 may refer to a polypeptide having at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology with a wild-type Cas9 polypeptide example (e.g., Cas9 derived from Streptococcus pyogenes). Cas9 can refer to a polypeptide having at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology with wild-type Cas9 polypeptide examples (e.g., Cas9 derived from Streptococcus pyogenes). Cas9 can refer to wild-type or modified Cas9 proteins, which can include amino acid variations such as deletions, insertions, substitutions, mutations, fusions, chimeras, or any combination thereof.

[0236] In several embodiments, the methods described herein may utilize genetically engineered Cas proteins. The guide RNA (gRNA) is a short synthetic RNA consisting of a Cas binding backbone sequence and a user-defined spacer of approximately 20 nucleotides (which defines the genosome target to be modified). In several embodiments, the backbone comprises GUUUUAGAGC UAGAAAUAGCAAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU. Whether a person skilled in the art can modify the Cas protein-specific genosome target is determined in part by comparing the specificity of the gRNA targeting sequence to the genosome target against the rest of the genosome.

[0237] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Upon target binding, it positions these domains to cleave the opposite strand of the target DNA, resulting in a second conformational change. The final result of Cas9-mediated DNA splitting is a double-strand break (DSB) within the target DNA (approximately 3-4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired via one of two common repair pathways: (1) the efficient but error-prone non-homologous terminal conjugation (NHEJ) pathway, or (2) the less efficient but highly faithful homology-directed repair (HDR) pathway.

[0238] The "efficiency" of non-homologous end joining (NHEJ) and / or homologous directed repair (HDR) can be determined by any convenient method. For example, in some cases, efficiency can be expressed as a percentage of successful HDR. For example, a DNase assay can be used to generate cleavage products, and the ratio of products to enzyme matrix can be used to calculate the percentage. For example, a DNase assay can be used to directly cleave DNA containing newly integrated restricted-split sequences due to a successful HDR result. More cleaved enzyme matrix indicates a larger percentage of HDR (higher HDR efficiency). As in a specific embodiment, a component (percentage) of HDR can be calculated using the following programming: [(cleavage product) / (enzyme matrix + cleavage product)] (e.g., (b+c) / (a+b+c), where a is the band intensity of the DNase matrix, and b and c are the cleavage products).

[0239] In some cases, efficiency can be expressed as a percentage of NHEJ. For example, the T7 endonuclease I assay can be used to generate cleavage products, and the product-to-enzyme matrix ratio can be used to calculate the percentage of NHEJ. For example, T7 endonuclease I cleaves mispaired diploid DNA, which is derived from a hybridization of wild-type and mutant DNA strands (NHEJ generates small random insertions or deletions at the original break sites). More cleavage indicates a larger percentage of NHEJ (higher NHEJ efficiency). In a specific embodiment, a component (percentage) of NHEJ can be calculated using the following programming: (1 - (1 - (b+c) / (a+b+c))) 1 / 2 )×100, where a is the band intensity of the DNA enzyme matrix, and b and c are the splitting products (Ran et al., Cell. 2013 Sep. 12; 154(6): 1380-9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11): 2281–2308).

[0240] NHEJ repair is the most active repair mechanism, often resulting in small nucleotide insertions or deletions (indels) at DSB sites. The randomness of NHEJ-mediated DSB repair has significant practical implications, as cell populations expressing Cas9 and gRNA or guide polynucleotides may lead to a host of divergent mutations. In most cases, NHEJ produces small insertions or deletions in the target DNA, resulting in amino acid deletions, insertions, or frameshift mutations, leading to the generation of immature stop codons within the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation within the target gene.

[0241] Although NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, similar to adding fluorescent groups or tags, homology-directed repair (HDR) can be used to generate specific nucleotide changes ranging from single nucleotide alterations to large insertions.

[0242] To use HDR for gene editing, DNA repair templates containing the desired sequence are delivered into the cell type of interest using gRNA and Cas9 or Cas9 nickase. The repair template may contain the desired edit, along with additional homologous sequences (named left and right homologous arms) immediately upstream and downstream of the target. The length of each homologous arm depends on the magnitude of the inserted change; larger insertions require longer homologous arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. HDR efficiency is typically low (less than 10% of modified alleles), even in cells expressing Cas9, gRNA, and exogenous repair templates. HDR efficiency can be improved by synchronizing the cells, as HDR occurs during the S and G2 phases of the cell cycle. Chemically or genetically repressive genes involved in NHEJ also enhance HDR efficiency.

[0243] In several implementations, Cas9 is a modified Cas9. Additional sites may be present throughout the genome (where some homologs exist) designated as gRNA target sequences. These sites are called off-target sites and must be considered when designing gRNAs. In addition to optimizing gRNA design, CRISPR specificity can also be enhanced by modifying Cas9. Cas9 generates double-stranded breaks (DSBs) through the combined activity of its two nuclease domains, RuvC and HNH. The Cas9 nickase, specifically the D10A mutant of SpCas9, retains one nuclease domain that generates DNA nicks instead of DSBs. Nickase systems can also be combined with HDR-mediated gene editing targeting specific gene editing.

[0244] In several embodiments, the modified Cas9 is a high-fidelity Cas9 enzyme. In several embodiments, the high-fidelity Cas9 enzyme is SpCas9 (K855A), eSpCas9 (1.1), SpCas9-HF1, or a highly accurate Cas9 variant (HypaCas9). The modified Cas9 eSpCas9 (1.1) contains an alanine substitution, which weakens the interaction between the HNH / RuvC groove and the non-target DNA strand, preventing strand dissociation and off-target cleavage. Similarly, SpCas9-HF1, through an alanine substitution, disrupts the interaction between Cas9 and the DNA phosphate backbone, thereby reducing off-target editing. HypaCas9 contains mutations in the REC3 domain (SpCas9 N692A / M694A / Q695A / H698A), which increases Cas9 correction and target selection. All three high-fidelity enzymes produce less off-target editing than wild-type Cas9. The amino acid sequences of the high-fidelity Cas9 examples are provided below. In this sequence, high-fidelity Cas9 domain mutations relative to the reference Cas9 are shown in bold and underlined:

[0245]

[0246]

[0247] In some cases, Cas9 is a variant Cas9 protein. When compared to the amino acid sequence of the wild-type Cas9 protein, the variant Cas9 polypeptide has an amino acid sequence that differs by at least one amino acid (e.g., deletion, insertion, substitution, or fusion). In some cases, the variant Cas9 polypeptide has amino acid changes that reduce the nuclease activity of the Cas9 polypeptide (e.g., deletion, insertion, or substitution). For example, in some cases, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity compared to the corresponding wild-type Cas9 protein. In some cases, the variant Cas9 protein is essentially non-nuclease-active. When the primary Cas9 protein is a variant Cas9 protein that is essentially non-nuclease-active, it can be called "dCas9".

[0248] In some cases, the mutant Cas9 protein has reduced nuclease activity. For example, the mutant Cas9 protein has less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, less than about 0.1%, or less than the corresponding wild-type Cas9 protein.

[0249] In some cases, the mutant Cas9 protein can cleave the complementary strand of the target sequence, but its ability to cleave the non-complementary strand of the double-stranded target sequence is reduced. For example, the mutant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As for non-limiting embodiments, in some embodiments, the mutant Cas9 protein has D10A (aspartic acid at amino acid position 10 is replaced by alanine), and thus can cleave the complementary strand of the double-stranded target sequence, but its ability to cleave the non-complementary strand of the double-stranded target sequence is reduced (so when the mutant Cas9 protein cleaves the double-stranded target nucleic acid, the result is a single-strand break (SSB) rather than a double-strand break (DSB)) (e.g., see Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21).

[0250] In some cases, a mutant Cas9 protein can cleave the non-complementary strand of a lead target sequence, but its ability to cleave the complementary strand of a double-stranded lead target sequence is reduced. For example, a mutant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As for non-limiting embodiments, in some embodiments, a mutant Cas9 protein has an H840A mutation (histidine at amino acid position 840 is replaced with alanine), thus enabling it to cleave the non-complementary strand of a lead target sequence, but reducing its ability to cleave the complementary strand of a double-stranded lead target sequence (so that when the mutant Cas9 protein cleaves a double-stranded target nucleic acid, the result is a single-strand break (SSB) instead of a double-strand break (DSB)). Such a Cas9 protein has a reduced ability to cleave a lead target sequence (e.g., a single lead target sequence), but retains the ability to bind to a lead target sequence (e.g., a single lead target sequence).

[0251] In some cases, the mutant Cas9 protein has a reduced ability to cleave both the complementary and non-complementary strands of a double-stranded target DNA. As for non-limiting embodiments, in several embodiments, the mutant Cas9 protein has both D10A and H840A mutations, thus reducing the ability of the polypeptide to cleave both the complementary and non-complementary strands of a double-stranded target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0252] In another non-limiting embodiment, in several cases, the mutant Cas9 protein has mutations in W476A and W1126A, thereby reducing the ability of the polypeptide to cleave target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0253] In another non-limiting embodiment, in several cases, the mutant Cas9 protein has mutations of P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, thereby reducing the peptide's ability to cleave target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0254] In another non-limiting embodiment, in several cases, the mutant Cas9 protein has mutations of H840A, W476A, and W1126A, thus reducing the polypeptide's ability to cleave target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind target DNA (e.g., single-stranded target DNA). In several embodiments, the mutant Cas9 has a repaired catalytic His residue at position 840 (A840H) in the Cas9 HNH domain.

[0255] In another non-limiting embodiment, in several cases, the mutant Cas9 protein has mutations of H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, thus reducing the polypeptide's ability to cleave target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind target DNA (e.g., single-stranded target DNA). In another non-limiting embodiment, in several cases, the mutant Cas9 protein has mutations of D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, thus reducing the polypeptide's ability to cleave target DNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind target DNA (e.g., single-stranded target DNA). In some cases, when the mutant Cas9 protein has mutations such as W476A and W1126A, or mutations such as P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, the mutant Cas9 protein cannot effectively bind to the PAM sequence. Therefore, in some cases, when this mutant Cas9 protein is used in a binding method, the method does not require the PAM sequence. In other words, in some cases, when this mutant Cas9 protein is used in a binding method, the method may include guide RNA (gRNA), but the method can be performed without the PAM sequence (and therefore, the binding specificity is provided by the target segment of the guide RNA). Other residues can be mutated to achieve the above effect (i.e., inactivating one or another nuclease portion). As for non-limiting embodiments, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 may be modified (i.e., substituted). Furthermore, mutations other than alanine substitution are also suitable.

[0256] In several embodiments, a variant Cas9 protein with reduced catalytic activity (e.g., when the Cas9 protein has mutations such as D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987, for example, D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A) can still bind to the target DNA in a site-specific manner (because it is still guided to the target DNA sequence by guide RNA), as long as it retains the ability to interact with the guide RNA.

[0257] An alternative to Streptococcus pyogenes, Cas9 can include RNA-guided endonucleases from the Cpf1 family, which exhibit cleavage activity in mammalian cells. CRISPR (CRISPR / Cpf1) from Prevotella and Francisella 1 is a CRISPR / Cas-like DNA editing technology. Cpf1 is a class II CRISPR / Cas RNA-guided endonuclease. This acquired immune mechanism is present in Prevotella and Francisella. The Cpf1 gene is associated with a CRISPR locus and encodes an endonuclease that uses guide RNA to locate and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming several limitations of the CRISPR / Cas system. Unlike the Cas9 nuclease, Cpf1-mediated DNA cleavage results in double-stranded breaks with short 3' protrusions. The staggered splitting pattern of Cpf1 enables targeted gene transfer (similar to traditional restriction enzyme cloning), which improves gene editing efficiency. Similar to the aforementioned Cas9 variants and homologs, Cpf1 can also amplify the number of sites targeted by CRISPR to AT-rich regions or AT-rich gene bodies (lacking the NGGPAM site preferred by SpCas9). The Cpf1 locus contains a fixed α / β domain, RuvC-I, followed by a helical region, RuvC-II, and a zinc finger domain. The Cpf1 protein possesses a RuvC-shaped endonuclease domain, which is similar to the RuvC domain of Cas9. However, Cpf1 lacks an HNH endonuclease domain, and its N-terminus lacks the α-helical recognition leaflet of Cas9. The Cpf1 CRISPR-Cas domain architecture demonstrates that Cpf1 is functionally unique and is classified as a type 2, type V CRISPR system. The Cpf1 locus encodes Cas1, Cas2, and Cas4 proteins that are more similar to those of types I and III than those of type II systems. Functional Cpf1 does not require trans-activation of CRISPR RNA (tracrRNA), thus only CRISPR (crRNA) is needed. This facilitates genome editing because Cpf1 is not only smaller than Cas9 but also has a smaller sgRNA molecule (approximately half the number of nucleotides as Cas9). The Cpf1-crRNA complex cleaves target DNA or RNA by recognizing the adjacent 5'-YTN-3' protospacer motif (as opposed to G-rich PAMs targeted by Cas9). Upon PAM recognition, Cpf1 introduces a sticky double-stranded DNA break at a 4 or 5 nucleotide overhang.

[0258] This disclosure provides certain forms of fusion proteins that include a domain acting as a nucleic acid-programmable DNA-binding protein, which can be used to guide proteins (such as base editors) to specific nucleic acid (e.g., DNA or RNA) sequences. In certain embodiments, the fusion protein includes a nucleic acid-programmable DNA-binding protein domain and a deaminase domain. DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. One embodiment of a nucleic acid-programmable DNA-binding protein with PAM specificity different from Cas9 is a clustered, regularly spaced short palindromic repeat (CRISPR) sequence derived from *Prevotella* and *Francisella 1* (Cpf1). Similar to Cas9, Cpf1 is also a class 2 CRISPR effector. Cpf1 has been shown to mediate robust DNA interference with properties different from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, which utilizes T-rich protospacer adjacent motifs (TTN, TTTN, or YTN). Furthermore, Cpf1 cleaves DNA through staggered double-strand breaks. Of the 16 Cpf1 family proteins, two enzymes derived from *Acidaminococcus* and *Lachnospiraceae* have shown effective genome editing activity in human cells. Cpf1 proteins are known in the industry and have been previously described, for example, Yamano et al., "Crystal structure of Cpf1 in complex with guide RNA and target DNA." Cell (165) 2016, pp. 949-962; the full disclosure of which is incorporated herein by reference.

[0259] The nuclease-inactive Cpf1 (dCpf1) variant is also used in this composition and method, which can be used as a programmable DNA-binding protein domain for guiding nucleotide sequences. The Cpf1 protein has a RuvC-like endonuclease domain, which is similar to the RuvC domain of Cas9, but lacks the HNH endonuclease domain, and the N-terminus of Cpf1 does not have the α-helix recognition leaf of Cas9. As shown in Zetsche et al., Cell, 163, 759-771, 2015 (disclosure incorporated herein by reference), the RuvC-like domain of Cpf1 is responsible for splitting the DNA strands, and inactivation of the RuvC-like domain inactivates the Cpf1 nuclease activity. For example, mutations in D917A, E1006A, or D1255A corresponding to Cpf1 in *Francisella novicida* inactivate the Cpf1 nuclease activity. In several embodiments, the dCpf1 disclosed herein includes mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that any mutation in the RuvC domain of inactivated Cpf1, such as substitution, deletion, or insertion, may be used in accordance with the disclosure herein.

[0260] In several embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) of any fusion protein provided herein may be a Cpf1 protein. In several embodiments, the Cpf1 protein is a Cpf1 cleavage enzyme (nCpf1). In several embodiments, the Cpf1 protein is a nuclease-inactivated Cpf1 (dCpf1). In several embodiments, Cpf1, nCpf1, or dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cpf1 sequence disclosed herein. In several embodiments, dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cpf1 sequence disclosed herein, and includes mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that Cpf1 derived from other bacterial species may also be used according to the disclosure herein.

[0261] The amino acid sequence of wild-type *Neofragmites aeruginosa* Cpf1 is as follows. D917, E1006, and D1255 are in bold and underlined.

[0262]

[0263] The amino acid sequence of *Neofrancella* Cpf1 D917A is as follows. (A917, E1006, and D1255 are in bold and underlined).

[0264]

[0265] The amino acid sequence of *Neofrancella* Cpf1 E1006A is as follows. (D917, A1006, and D1255 are in bold and underlined).

[0266]

[0267] The amino acid sequence of *Neofrancella* Cpf1 D1255A is as follows. (The mutation positions D917, E1006, and A1255 are in bold and underlined).

[0268]

[0269] The amino acid sequence of *Neofrancella* Cpf1 D917A / E1006A is as follows. (A917, A1006, and D1255 are in bold and underlined).

[0270]

[0271]

[0272] The amino acid sequence of *Neofrancella* Cpf1 D917A / D1255A is as follows. (A917, E1006, and A1255 are in bold and underlined).

[0273]

[0274] The amino acid sequence of *Neofrancella* Cpf1 E1006A / D1255A is as follows. (D917, A1006, and A1255 are in bold and underlined).

[0275]

[0276] The amino acid sequence of *Neofrancella* Cpf1 D917A / E1006A / D1255A is as follows. (A917, A1006, and A1255 are in bold and underlined).

[0277]

[0278]

[0279] In several embodiments, the variant Cas protein may be spCas9, spCas9-VRQR, spCas9-VRER, xCas9(sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.

[0280] The amino acid sequence of the SaCas9 example is as follows:

[0281]

[0282]

[0283] In this sequence, the underlined and bold residue N579 can be mutated (e.g., to A579) to obtain the SaCas9 nickase.

[0284] The amino acid sequence of the SaCas9n example is as follows:

[0285]

[0286] In this sequence, residue A579 (which can be mutated from N579 to obtain the SaCas9 nickase) is underlined and in bold. The amino acid sequence of the SaKKH Cas9 example is as follows:

[0287]

[0288]

[0289] The residue A579 above (which can be mutated from N579 to obtain the SaCas9 nickase) is underlined and in bold. The residues K781, K967, and H1014 above (which can be mutated from E781, N967, and R1014 to obtain SaKKH Cas9) are underlined and in italics.

[0290] The polynucleotide-programmable nucleotide binding domain of the base editor may itself contain one or more domains. For example, the polynucleotide-programmable nucleotide binding domain may itself contain one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide-programmable nucleotide binding domain may contain an endonuclease or an exonuclease. In this disclosure, the term "exonuclease" refers to a protein or polypeptide capable of digesting nucleic acids (e.g., RNA or DNA) from their free ends, and the term "endonuclease" refers to a protein or polypeptide capable of catalyzing (e.g., cleaving) the inner regions of nucleic acids (e.g., DNA or RNA). In some embodiments, the endonuclease can cleave a single strand of a double-stranded nucleic acid. In some embodiments, the endonuclease can cleave both strands of a double-stranded nucleic acid molecule. In some embodiments, the polynucleotide-programmable nucleotide binding domain may be a deoxyribonuclease. In some embodiments, the polynucleotide-programmable nucleotide binding domain may be a ribonuclease.

[0291] In several embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain is capable of cleaving one, one, or two strands of the target nucleotide. In some cases, the polynucleotide programmable nucleotide binding domain may include a nickase domain. In this disclosure, the term "nickase" refers to a polynucleotide programmable nucleotide binding domain containing a nuclease domain that can only cleave one strand of a diploid nucleic acid molecule (e.g., DNA). In several embodiments, the nickase can be derived from the fully catalytically active (e.g., native) form of the polynucleotide programmable nucleotide binding domain by introducing one or more mutations into the active polynucleotide programmable nucleotide binding domain. For example, when the polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the Cas9-derived nickase domain may include a D10A mutation and a histidine residue at position 840. In this case, residue H840 retains catalytic activity and is thus capable of cleaving the single strand of a diploid nucleic acid. In another embodiment, the Cas9-derived nickase domain may include an H840A mutation, while the amino acid residue at position 10 remains D. In several embodiments, the nickase can be derived from the fully catalytically active (e.g., native) form of the polynucleotide programmable nucleotide binding domain by removing all or part of the nuclease domain that is not required for nickase activity. For example, when the polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the Cas9-derived nickase domain may include all or part of the deletion of the RuvC domain or the HNH domain.

[0292] A base editor comprising a polynucleotide programmable nucleotide-binding domain including a nicking enzyme domain can generate single-strand DNA breaks (nicks) at specific polynucleotide target sequences (e.g., determined by the complementary sequence of the bound guide nucleic acid). In some embodiments, the strand of the diploid target polynucleotide sequence cleaved by a base editor containing a nicking enzyme domain (e.g., a Cas9-derived nicking enzyme domain) is the strand not edited by the base editor (i.e., the strand cleaved by the base editor is the opposite of the strand containing the base to be edited). In other embodiments, a base editor containing a nicking enzyme domain (e.g., a Cas9-derived nicking enzyme domain) can cleave the strand of the DNA molecule targeted for editing. In this case, untargeted strands are not cleaved.

[0293] This disclosure also provides a base editor comprising a polynucleotide-programmable nucleotide-binding domain that is catalytically dead (i.e., unable to cleave a target polynucleotide sequence). In this disclosure, the terms "catalytically dead" and "nuclease dead" are used interchangeably to refer to a polynucleotide-programmable nucleotide-binding domain having one or more mutations and / or deletions that result in its inability to cleave a strand of nucleic acid. In some embodiments, the catalytically dead polynucleotide-programmable nucleotide-binding domain base editor may lack nuclease activity due to specific point mutations in one or more nuclease domains. For example, in a base editor comprising a Cas9 domain, Cas9 may contain both a D10A mutation and an H840A mutation. These mutations inactivate both nuclease domains, thus resulting in loss of nuclease activity. In other embodiments, the catalytically dead polynucleotide-programmable nucleotide-binding domain may contain one or more deletions of all or part of the catalytic domain (e.g., the RuvC1 and / or HNH domains). In a further embodiment, the catalytic death polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) and a complete or partial deletion of the nuclease domain.

[0294] This disclosure is also intended to cover mutations that can generate catalytic death polynucleotide programmable nucleotide binding domains from previously functional versions of the polynucleotide programmable nucleotide binding domain. For example, with catalytic death Cas9 (dCas9), variants are provided having mutations other than D10A and H840A that result in nuclease inactivation of Cas9. For example, such mutations include other amino acid substitutions in D10A and H840A, or other substitutions in the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). Based on the disclosure herein and knowledge of the art, additional suitable nuclease-inactivating dCas9 domains will be readily apparent to those skilled in the art and fall within the scope of this disclosure. Additional embodiments of such suitable nuclease-inactivating Cas9 domains include, but are not limited to, the D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains. (For example, see Prashant et al., "Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering." Nature Biotechnology. 2013; 31(9):833-838, the disclosure of which is incorporated herein by reference in its entirety.) In several embodiments, the dCas9 domain comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the same amino acid sequence as any of the dCas9 domains provided in this disclosure. In several embodiments, the dCas9 domain comprises an amino acid sequence having 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 mutations compared to any of the amino acid sequences listed in this disclosure.In several embodiments, the dCas9 domain comprises an amino acid sequence having 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, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical adjacent amino acid residues compared to any of the amino acid sequences listed in this disclosure.

[0295] Non-limiting embodiments of polynucleotide-programmable nucleotide-binding domains that can be incorporated into a base editor include CRISPR protein-derived domains, restriction-cutting nucleases, macronucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some cases, the base editor includes a polynucleotide-programmable nucleotide-binding domain containing a natural or modified protein or a portion thereof, which, through a bound guide nucleic acid, can bind to a nucleic acid sequence during CRISPR (i.e., clustered regularly spaced short palindromic repeats)-mediated nucleic acid modification. Such a protein is called a "CRISPR protein." Therefore, what is disclosed herein is a base editor containing a polynucleotide-programmable nucleotide-binding domain that contains all or part of a CRISPR protein (i.e., a base editor containing all or part of a CRISPR protein as a domain, also referred to as a "CRISPR protein-derived domain" of the base editor). The CRISPR protein-derived domain incorporated into the base editor can be modified compared to a wild-type or natural version of a CRISPR protein. For example, as described below, compared to the wild-type or natural version of a CRISPR protein, the CRISPR protein-derived domain may contain one or more mutations, insertions, deletions, rearrangements, and / or recombinations.

[0296] In several embodiments, the CRISPR protein-derived domain incorporated into the base editor is an endonuclease (e.g., deoxyribonuclease or ribonuclease) capable of binding the target polynucleotide when combined with a bound guide nucleic acid. In several embodiments, the CRISPR protein-derived domain incorporated into the base editor is a nicking enzyme capable of binding the target polynucleotide when combined with a bound guide nucleic acid. In several embodiments, the CRISPR protein-derived domain incorporated into the base editor is a catalytic death domain capable of binding the target polynucleotide when combined with a bound guide nucleic acid. In several embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is DNA. In several embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is RNA.

[0297] In several embodiments, the CRISPR protein-derived domain of the base editor may include all or part of a Cas9 derived from: *Corynebacterium ulcerans* (NCBI Refs: NC_015683.1, NC_017317.1); *Corynebacterium diphtheriae* (NCBI Refs: NC_016782.1, NC_016786.1); *Aphidida albicans* (NCBI Ref: NC_021284.1); *Prevotella intermedia* (NCBI Ref: NC_017861.1); *Taenia solium* (NCBI Ref: NC_021846.1); *Streptococcus dolphinus* (NCBI Ref: NC_021314.1); *Belia balolata* (NCBI Ref: NC_018010.1); *Campylobacter thermophilus* (NCBI Ref: NC_018721.1); *Streptococcus ... Ref: YP_820832.1); harmless Listeria (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1); Streptococcus pyogenes; or Staphylococcus aureus.

[0298] In several embodiments, the Cas9 domain is a Cas9 domain derived from Staphylococcus aureus (SaCas9). In several embodiments, SaCas9 is an active nuclease SaCas9, an inactive nuclease SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In several embodiments, SaCas9 contains an N579A mutation, or a corresponding mutation in any amino acid sequence provided in this disclosure.

[0299] In several embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain may bind to a nucleic acid sequence having a non-canonical PAM. In several embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain may bind to a nucleic acid sequence having an NNGRRT or NNNRRT PAM sequence. In several embodiments, the SaCas9 domain comprises one or more of the E781X, N967X, and R1014X mutations, or a corresponding mutation in any amino acid sequence provided in this disclosure, where X is any amino acid. In several embodiments, the SaCas9 domain comprises one or more of the E781K, N967K, and R1014H mutations, or one or more corresponding mutations in any amino acid sequence provided in this disclosure. In several embodiments, the SaCas9 domain comprises the E781K, N967K, and R1014H mutations, or a corresponding mutation in any amino acid sequence provided in this disclosure.

[0300] A base editor may include a domain derived from all or part of a Cas9 that is a high-fidelity Cas9. In several embodiments, the high-fidelity Cas9 of the base editor is an engineered Cas9 domain containing one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA compared to a corresponding wild-type Cas9 domain. A Cas9 domain with reduced electrostatic interaction with the sugar-phosphate backbone of DNA may have fewer off-target effects. In several embodiments, the Cas9 domain (e.g., a wild-type Cas9 domain) contains one or more mutations that reduce the association between the Cas9 domain and the sugar-phosphate backbone of DNA. In several embodiments, the Cas9 domain contains one or more mutations that reduce the association between the Cas9 domain and the sugar-phosphate backbone of DNA by 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 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or more.

[0301] As used herein, the term "guide polynucleotide" refers to a polynucleotide that is specific to a target sequence and can form a complex with a polynucleotide programmable nucleotide-binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is guide RNA. As used herein, the term "guide RNA (gRNA)" and its grammatical synonyms may refer to RNA that is specific to target DNA and can form a complex with a Cas protein. The RNA / Cas complex assists in "guided" the Cas protein to the target DNA. Cas9 / crRNA / tracrRNA endocleaves linear or circular dsDNA targets complementary to the spacer. Target strands not complementary to crRNA are first endocleaved and then exonerated 3'-5' trimmed. In nature, DNA binding and cleavage typically require both a protein and two RNAs. However, single-guide RNA ("sgRNA" or simply "gRNA") can be genetically modified to combine the states of both crRNA and tracrRNA into a single RNA. For example, see Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the full disclosure of which is incorporated herein by reference. Cas9 identifies short motifs (PAM or protospacer adjacent motifs) in CRISPR repeat sequences to help distinguish itself from non-self.

[0302] In some embodiments, the guiding polynucleotide is at least one single guiding RNA (“sgRNA” or “gRNA”). In some embodiments, the guiding polynucleotide is at least one tracrRNA. In some embodiments, the guiding polynucleotide does not require a PAM sequence to guide the polynucleotide programmable DNA binding domain (e.g., Cas9 or Cpf1) to the target nucleotide sequence.

[0303] The multinucleotide programmable nucleotide-binding domain (e.g., a CRISPR-derived domain) of the base editor disclosed herein can identify target nucleotide sequences by association with a guide polynucleotide. The guide polynucleotide (e.g., gRNA) is typically single-stranded and can be programmed to bind site-specifically (i.e., via complementary base pairing) to the target sequence of the polynucleotide, thereby guiding the base editor (which, along with the guide nucleic acid) to the target sequence. The guide polynucleotide can be DNA. The guide polynucleotide can be RNA. In some cases, the guide polynucleotide contains a natural nucleotide (e.g., adenosine). In some cases, the guide polynucleotide contains a non-natural (or unnatural) nucleotide (e.g., peptide nucleic acid or nucleotide analogue). In some cases, the target region length of the guide nucleic acid sequence can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The target region of a guiding nucleic acid can be 10 to 30 nucleotides long, or 15 to 25 nucleotides long, or 15 to 20 nucleotides long.

[0304] In several embodiments, the guide polynucleotide comprises two or more individual polynucleotides that interact with each other, for example, through complementary base pairing (e.g., dual guide polynucleotides). For example, the guide polynucleotide may comprise CRISPR RNA (crRNA) and trans-activated CRISPR RNA (tracrRNA). For example, the guide polynucleotide may comprise one or more trans-activated CRISPR RNAs (tracrRNA).

[0305] In type II CRISPR systems, nucleic acids are targeted by a CRISPR protein (e.g., Cas9). This typically requires complementary base pairing between a first RNA molecule (crRNA) (containing a sequence that recognizes the target sequence) and a second RNA molecule (trRNA), which contains repetitive sequences that form the backbone region stabilizing the guide RNA-CRISPR protein complex. This dual-guide RNA system can be used as a guide polynucleotide to guide the base editor disclosed herein to the target nucleotide sequence.

[0306] In several embodiments, the base editor provided in this disclosure utilizes a single-guided polynucleotide (e.g., gRNA). In several embodiments, the base editor provided in this disclosure utilizes a double-guided polynucleotide (e.g., double gRNA). In several embodiments, the base editor provided in this disclosure utilizes one or more guide polynucleotides (e.g., multiple gRNAs). In several embodiments, a single-guided polynucleotide is used in the different base editors described herein. For example, a single-guided polynucleotide may be used in a cytidine base editor and an adenosine base editor.

[0307] In other embodiments, the guiding polynucleotide may comprise both the polynucleotide target portion and the backbone portion of the nucleic acid in a single molecule (i.e., a single-molecule guiding nucleic acid). For example, a single-molecule guiding polynucleotide may be a single guiding RNA (sgRNA or gRNA). In this disclosure, the term guiding polynucleotide sequence is intended to encompass any single, double, or multiple-molecule nucleic acid that can interact with a base editor and guide the base editor to a target polynucleotide sequence.

[0308] Typically, a guide polynucleotide (e.g., a crRNA / trRNA complex or gRNA) comprises a "polynucleotide target segment" which includes a sequence capable of recognizing and binding to a target polynucleotide sequence, and a "protein-binding segment" whose polynucleotide programmable nucleotide-binding domain stabilizes the base editor within the guide polynucleotide. In some embodiments, the polynucleotide target segment of the guide polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating base editing in DNA. In other cases, the polynucleotide target segment of the guide polynucleotide recognizes and binds to an RNA polynucleotide, thereby facilitating base editing in RNA. In this disclosure, a "segment" refers to a segment or region of a molecule, such as an adjacent extension of a nucleotide in a guide polynucleotide. A segment also refers to a region / segment of a complex, such that a segment may contain regions of more than one molecule. For example, when the guide polynucleotide contains multiple nucleic acid molecules, a protein-binding segment may include all or part of multiple separate molecules, which, for example, hybridize along a complementary region. In several embodiments, a protein-binding segment comprising two separate DNA target RNA molecules may comprise (i) 40-75 base pairs of a first RNA molecule of length 100 base pairs; and (ii) 10-25 base pairs of a second RNA molecule of length 50 base pairs. Unless otherwise specifically defined herein, the definition of "segment" is not limited to a specific number of total base pairs, nor to any specific number of base pairs from a specified RNA molecule, nor to a specific number of separate molecules within a complex, and may include RNA molecule regions of any total length, and may include regions complementary to other molecules.

[0309] Guide RNA or guide polynucleotides may contain two or more RNAs, such as CRISPR RNA (crRNA) and trans-activated CRISPR RNA (tracrRNA). Guide RNA or guide polynucleotides may occasionally contain single-stranded RNA, or be a single guide RNA (sgRNA) formed by fusing crRNA with a portion (e.g., the functional portion) of tracrRNA. Guide RNA or guide polynucleotides may also be a dual RNA of crRNA and tracrRNA. Furthermore, crRNA can hybridize with target DNA.

[0310] As discussed earlier, guide RNA or guide polynucleotides can be expression products. For example, the DNA encoding guide RNA can be a vector containing a sequence that encodes the guide RNA. Guide RNA or guide polynucleotides can be transferred into cells by transfecting cells with isolated guide RNA or plasso DNA containing a sequence that encodes the guide RNA, and a promoter. Guide RNA or guide polynucleotides can also be transferred into cells in other ways, such as through viral-mediated gene delivery.

[0311] Guide RNA or guide polynucleotides can be isolated. For example, guide RNA can be transferred into cells or organisms in its isolated RNA form. Guide RNA can be produced by in vitro transcription using any in vitro transcription system known in the industry. Guide RNA can be transferred into cells in its isolated RNA form, rather than as a plastid containing the coding sequence.

[0312] Guide RNA or guide polynucleotide may contain three regions: a first region at the 5' end, which is complementary to a target site in the chromosomal sequence; a second inner region, which can form a stem-loop structure; and a third 3' region, which may be single-stranded. The first region of each guide RNA may also be different, allowing each guide RNA to guide the fusion protein to a specific target site. Furthermore, the second and third regions of each guide RNA may be identical throughout all guide RNAs.

[0313] The first region of the guide RNA or guide polynucleotide may be complementary to the sequence of the target site in the chromosomal sequence, such that the first region of the guide RNA can pair with the bases of the target site. In some cases, the first region of the guide RNA may contain 10 or more of about 10 nucleotides to 25 nucleotides (i.e., 10 nucleotides to 25 nucleotides; or about 10 nucleotides to about 25 nucleotides; or 10 nucleotides to about 25 nucleotides; or about 10 nucleotides to 25 nucleotides) or more. For example, the length of the base pairing region between the first region of the guide RNA and the target site in the chromosomal sequence may be or can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25 or more nucleotides. Occasionally, the length of the first region of the guide RNA may be or can be about 19, 20, or 21 nucleotides.

[0314] Guiding RNA or guiding polynucleotides may also contain a second region, which forms a secondary structure. For example, the secondary structure generated by the guiding RNA may include a stem (or hairpin) and a loop. The lengths of the loop and the stem can vary. For example, the length of the loop may range from about 3 to 10 nucleotides, and the length of the stem may range from about 6 to 20 nucleotides. The stem may contain one or more protrusions of 1 to 10 or about 10 nucleotides. The total length of the second region may range from about 16 to 60 nucleotides. For example, the length of the loop may be about 4 nucleotides, and the stem may be about 12 base pairs.

[0315] Guiding RNA or guiding polynucleotides may also be contained in a third region at the 3' end, which may be predominantly single-stranded. For example, the third region may occasionally be non-complementary to any chromosomal sequence in the cell of interest, and occasionally to the rest of the guiding RNA. Furthermore, the length of the third region can vary. The length of the third region may be more than or equal to about 4 nucleotides. For example, the length of the third region may range from or be between about 5 and 60 nucleotides.

[0316] Guide RNA or guide polynucleotides can target any exon or intron of a gene target. In some cases, the guide RNA targets exon 1 or 2 of the gene; in others, it targets exon 3 or 4. The composition may contain multiple guide RNAs, all of which target the same exon, or in some cases, multiple guide RNAs can target the same exon. Both exons and introns of the gene can be targeted.

[0317] Guide RNA or guide polynucleotides can target nucleic acid sequences of 20 or about 20 nucleotides. The target nucleic acid can be less than or about 20 nucleotides. The length of the target nucleic acid can be any length between at least and at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or 1-100 nucleotides. The length of the target nucleic acid can be any length between at most and at most about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or 1-100 nucleotides. The target nucleic acid sequence can be about 20 bases immediately preceding the 5' of the first nucleotide of the PAM. Guide RNA can target nucleic acid sequences. The length of the target nucleic acid can be at least or at least about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides.

[0318] Guided polynucleotides, such as guided RNA, can refer to nucleic acids capable of hybridizing to another nucleic acid, such as a target nucleic acid or a protospacer in the cellular genome. Guided polynucleotides can be RNA. Guided polynucleotides can be DNA. Guided polynucleotides can be programmed or configured to bind specifically to nucleic acid sequences. Guided polynucleotides may contain one polynucleotide chain and are called monoguided polynucleotides. Guided polynucleotides may contain two polynucleotide chains and are called diguided polynucleotides. Guided RNA can be introduced into cells or embryos as RNA molecules. For example, RNA molecules can be transcribed in vitro and / or can be chemically synthesized. RNA can be transcribed from synthetic DNA molecules, for example... Gene fragments. The guide RNA can then be introduced into cells or embryos as an RNA molecule. Guide RNA can also be introduced into cells or embryos in the form of non-RNA nucleic acid molecules (e.g., DNA molecules). For example, the DNA encoding the guide RNA can be operatively linked to a promoter control sequence for expression of the guide RNA in cells or embryos of interest. The RNA coding sequence can be operatively linked to a promoter sequence recognized by RNA polymerase III (Pol III). Plasmonic vectors that can be used to express guide RNA include, but are not limited to, the px330 and px333 vectors. In some cases, the plasmonic vector (e.g., the px333 vector) may contain at least two guide RNA encoding DNA sequences.

[0319] Methods for selecting, designing, and validating guide polynucleotides, such as guide RNAs and target sequences, are described in this disclosure and are known to those skilled in the art. For example, minimizing the effects of potential enzyme matrix contamination of deaminase domains (e.g., AID domains) in nucleoside base editor systems can minimize the number of residues that may be unintentionally targeted for deamination (e.g., off-target C residues that may reside on ssDNA within the target nucleic acid locus). Furthermore, software tools can be used to optimize gRNAs corresponding to target nucleic acid sequences, for example, minimizing overall off-target activity across the genome. For example, for the selection of various possible target domains using Streptococcus pyogenes Cas9, all off-target sequences (the aforementioned selected PAMs, such as NAG or NGG) can be recognized across the genome, containing up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mismatched base pairs. The first region of a gRNA complementary to the target site can be identified, and all first regions (e.g., crRNAs) can be ranked according to their predicted off-target scores; the highest-ranked target domain represents the one likely to have the greatest on-target activity and the least off-target activity. Candidate gRNAs can be evaluated using methods known in the industry and / or as described herein.

[0320] For non-limiting embodiments, the target DNA hybridization sequence in the crRNA of the guide RNA used for use with Cas9 can be identified using a DNA sequence search algorithm. gRNA design can be performed using a publicly available tool sequence alignment program (casoffinder) (as described in Bae S., Park J., & Kim J.-S. Cas-OFFinder: "A fast and versatile algorithm that searches for potential off-target sites of Cas9RNA-guidedendonucleases." Bioinformatics 30, 1473-1475 (2014)), using a customer-designed gRNA. After calculating its whole-genome off-target tendency, the guide is guided using a software score. Typically, a match range from perfect match to 7 mismatches is considered as a guide range of length 17 to 24. Once the off-target sites are determined, a cumulative score is calculated for each guide, and a summary list is output using a web interface. In addition to identifying potential target sites adjacent to PAM sequences, this software also identifies all PAM-adjacent sequences that differ from selected target sites by 1, 2, 3, or more than 3 nucleotides. For the target nucleic acid sequence, the genomic DNA sequence, such as the target gene, can be obtained, and repeaters can be screened using publicly available tools such as Repeatmasker. Repeatmasker searches for repeaters and low-complexity regions in the input DNA sequence. The output is a detailed annotation of the repeaters present in the specified query sequence.

[0321] Following identification, the first region of the guide RNA, such as crRNA, can be stratified based on its distance from the target site, its incoherence, and the presence of a 5' nucleotide that closely matches the associated PAM sequence (e.g., 5'G, based on recognition of close matches in the human genome containing associated PAMs such as NGG PAM for Streptococcus pyogenes, NNGRRT or NNGRRV PAM for Staphylococcus aureus). As used in this disclosure, incoherence refers to the minimum number of sequences in the human genome that mismatch the target sequence. "High incoherence" or "good incoherence" may, for example, refer to a 20-mer target domain that contains no identical sequences in the human genome other than the intended target, nor any sequences in the target sequence that contain one or two mismatches. Targeting domains with good incoherence can be selected to minimize off-target DNA fragmentation.

[0322] In several embodiments, the notification subsystem may be used to detect base editing activity and test candidate guide polynucleotides. In several embodiments, the notification subsystem may include an analytical assay based on a notification gene, wherein base editing activity results in the expression of the notification gene. For example, the notification subsystem may include a notification gene containing a deactivated start codon, such as a mutation on a template strand from 3'-TAC-5' to 3'-CAC-5'. When the target C is successfully deaminated, the corresponding mRNA will be transcribed to 5'-AUG-3' instead of 5'-GUG-3', enabling the notification gene to be translated. Suitable notification genes will be readily apparent to those skilled in the art. Non-limiting embodiments of notification genes include genes encoding green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase, secretory alkaline phosphatase (SEAP), or any other gene whose expression is detectable and readily apparent to those skilled in the art. The notification subsystem can be used to test many different gRNAs to determine which residue(s) an individual deaminase will target relative to the target DNA sequence. sgRNAs targeting non-template sequences can also be tested to evaluate the off-target effects of specific base-editing proteins, such as Cas9 deaminase fusion proteins. In some embodiments, these gRNAs can be engineered such that the mutated start codon will not pair with the gRNA bases. The guide polynucleotide may comprise a standard ribonucleotide, a modified ribonucleotide (e.g., pseudouridine), a ribonucleotide isomer, and / or a ribonucleotide analog. In some embodiments, the guide polynucleotide may comprise at least one detectable label. The detectable label may be a fluorescent group (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags, or suitable fluorescent dyes), a detection tag (e.g., biotin, digoxigenin, etc.), a quantum particle, or a gold particle.

[0323] Guide polynucleotides can be chemically synthesized, enzymatically synthesized, or a combination thereof. For example, guide RNA can be synthesized using a solid-phase synthesis method based on standard phosphorylation. Alternatively, guide RNA can be synthesized in vitro by operatively linking the DNA encoding the guide RNA to a promoter control sequence (which is recognized by a phage RNA polymerase). Examples of suitable phage promoter sequences include the T7, T3, SP6 promoter sequences, or variants thereof. In embodiments where the guide RNA comprises two separate molecules (e.g., crRNA and tracrRNA), the crRNA can be chemically synthesized, and the tracrRNA can be enzymatically synthesized.

[0324] In several embodiments, the base editor system may comprise a plurality of guide polynucleotides, such as gRNAs. For example, the gRNAs may target one or more target loci contained in the base editor system (e.g., at least 1 gRNA, at least 2 gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 gRNAs, at least 50 gRNAs). These plurality of gRNA sequences may be tandemly arranged and preferably separated by direct repeat sequences.

[0325] The DNA sequence encoding guide RNA or guide polynucleotides can also be part of a vector. Furthermore, the vector may contain additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selectable marker sequences (e.g., GFP or antibiotic resistance genes such as puromycin), origin of replication, etc. The DNA sequence encoding guide RNA can also be linear. The DNA sequence encoding guide RNA or guide polynucleotides can also be circular.

[0326] In several embodiments, one or more components of the base editor system may be encoded by DNA sequences. These DNA sequences may be introduced into an expression system, such as a cell, either together or separately. For example, DNA sequences encoding a polynucleotide programmable nucleotide binding domain and a guide RNA may be introduced into a cell, and the individual DNA sequences may be separate molecular components (e.g., one vector containing a polynucleotide programmable nucleotide binding domain coding sequence and a second vector containing a guide RNA coding sequence); or both may be the same molecular component (e.g., one vector containing coding (and regulatory) sequences for both the polynucleotide programmable nucleotide binding domain and the guide RNA).

[0327] Leading polynucleotides may contain one or more modifications to provide novel or enhanced features to nucleic acids. Leading polynucleotides may contain nucleic acid affinity tags. Leading polynucleotides may contain synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0328] In some cases, the gRNA or guide polynucleotide may contain modifications. Modifications can be made at any position on the gRNA or guide polynucleotide. More than one modification may be made to a single gRNA or guide polynucleotide. Post-modification quality control of the gRNA or guide polynucleotide may be performed. In some cases, quality control may include PAGE, HPLC, MS, or any combination thereof.

[0329] Modifications to gRNA or guide polynucleotides can include substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or any combination thereof.

[0330] gRNA or guide polynucleotides can also be modified through the following: 5' adenosine, 5' guanosine triphosphate cap, 5' N7-methylguanosine triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' thiophosphate, 5' phosphate, 5' thiophosphate, cis-trans thymidine binary, ternary, C12 spacer, C3 spacer, C6 spacer, d spacer, PC spacer, r spacer, spacer 18, spacer 9, 3'-3' modification, 5'-5' modification, abase desaturation site, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesterol TEG, dethiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, Dibiotin, PC-Biotin, 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, carboxyl linker, thiol linker, 2'-deoxyribonucleoside analog purine, 2'-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methylribonucleoside analog, sugar-modified analog, swing / universal base, fluorescent dye label, 2'-fluoroRNA, 2'-O-methylRNA, methyl phosphonate, phosphodiester DNA, phosphodiester RNA, thiophosphate DNA, thiophosphate RNA, UNA, pseudouridine-5'-triphosphate, 5'-methylcytidine-5'-triphosphate, or any combination thereof.

[0331] In some cases, the modification is permanent. In others, it is temporary. In some cases, multiple modifications can be made to gRNA or guide polynucleotides. Modifications to gRNA or guide polynucleotides can alter the physicochemical properties of nucleotides, such as their conformation, polarity, hydrophobicity, chemical reactivity, base pairing interactions, or any combination thereof.

[0332] Modifications can also involve phosphate-thiophosphate substitution. In some cases, the native phosphodiester bond may be susceptible to rapid degradation by cellular nucleases, while modifications using phosphate-thiophosphate (PS) bonds to replace nucleotide links may provide greater stability against hydrolysis by cellular degradation. Modifications can improve the stability of gRNAs or lead polynucleotides. Modifications can also enhance biological activity. In some cases, phosphate-thiophosphate-enhanced RNA gRNAs can inhibit RNase A, RNase T1, fetal bovine serum nuclease, or any combination thereof. These properties make PS-RNA gRNAs useful for applications where there is a high probability of exposure to nucleases in vivo or in vitro. For example, a phosphate-thiophosphate (PS) bond can be inserted between the last 3-5 nucleotides at the 5' or "-" end of the gRNA, which can inhibit exonuclease degradation. In some cases, phosphate-thiophosphate bonds can be added throughout the gRNA to reduce attack by endonucleases.

[0333] Adjacent units of the original spacer basis

[0334] The term "protospacer adjacent motif (PAM)" or PAM-like motif refers to the 2-6 base pairs of DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. In some embodiments, the PAM may be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM may be a 3' PAM (i.e., located downstream of the 5' end of the protospacer).

[0335] The protospacer adjacent motif (PAM) or PAM-like motif refers to the 2-6 base pairs of DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. In some embodiments, the PAM can be 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., located downstream of the 5' end of the protospacer). The PAM sequence is essential for target binding, but the exact sequence depends on the type of Cas protein. The base editor provided in this disclosure may contain a CRISPR protein-derived domain capable of binding nucleotide sequences containing canonical or non-canonical protospacer adjacent motif (PAM) sequences. The PAM site is a nucleotide sequence adjacent to the target polynucleotide sequence. Several versions of this disclosure provide a base editor that contains all or part of a CRISPR protein with different PAM specificities. For example, typical Cas9 proteins, such as Cas9 (spCas9) derived from Streptococcus pyogenes, require a canonical NGGPAM sequence to bind to a specific nucleic acid region, where N in NGG represents adenine (A), thymine (T), guanine (G), or cytosine (C), and G represents guanine. The PAM can be CRISPR protein specific and can differ between different base editors containing different CRISPR protein-derived domains. The PAM can be the 5' or 3' of the target sequence. The PAM can be upstream or downstream of the target sequence. The PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides in length. Often, the length of the PAM is between 2 and 6 nucleotides.

[0336] In several embodiments, the Cas9 domain is a Cas9 domain (SpCas9) derived from Streptococcus pyogenes. In several embodiments, the SpCas9 domain is an active nuclease SpCas9, an inactive nuclease SpCas9 (SpCas9d), or a SpCas9 nickase (SpCas9n). In several embodiments, SpCas9 contains a D9X mutation, or a corresponding mutation in any of the amino acid sequences provided in this disclosure, where X is any amino acid (except D). In several embodiments, SpCas9 contains a D9A mutation, or a corresponding mutation in any of the amino acid sequences provided in this disclosure. In several embodiments, the SpCas9 domain, SpCas9d domain, or SpCas9n domain can bind to nucleic acid sequences having a non-canonical PAM. In several embodiments, the SpCas9 domain, SpCas9d domain, or SpCas9n domain can bind to nucleic acid sequences having an NGG, NGA, or NGCG PAM sequence. In several embodiments, the SpCas9 domain comprises one or more of the mutations D1135X, R1335X, and T1337X, or a corresponding mutation in any of the amino acid sequences provided in this disclosure, wherein X is any amino acid. In several embodiments, the SpCas9 domain comprises one or more of the mutations D1135E, R1335Q, and T1337R, or a corresponding mutation in any of the amino acid sequences provided in this disclosure. In several embodiments, the SpCas9 domain comprises one or more of the mutations D1135E, R1335Q, and T1337R, or a corresponding mutation in any of the amino acid sequences provided in this disclosure, wherein X is any amino acid. In several embodiments, the SpCas9 domain comprises one or more of the D1135V, R1335Q, and T1337R mutations, or a corresponding mutation in any of the amino acid sequences provided in this disclosure. In several embodiments, the SpCas9 domain comprises one or more of the D1135V, R1335Q, and T1337R mutations, or a corresponding mutation in any of the amino acid sequences provided in this disclosure. In several embodiments, the SpCas9 domain comprises one or more of the D1135V, G1218X, R1335X, and T1337X mutations, or a corresponding mutation in any of the amino acid sequences provided in this disclosure, where X is any single amino acid. In several embodiments, the SpCas9 domain comprises one or more of the D1135V, G1218R, R1335Q, and T1337R mutations, or a corresponding mutation in any of the amino acid sequences provided in this disclosure.In several embodiments, the SpCas9 domain includes one or more of the mutations D1135V, G1218R, R1335Q, and T1337R, or the corresponding mutation in any of the amino acid sequences provided in this disclosure.

[0337] In several embodiments, the Cas9 domain of any of the fusion proteins provided in this disclosure comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cas9 polypeptide described herein. In several embodiments, the Cas9 domain of any of the fusion proteins provided in this disclosure comprises the amino acid sequence of any Cas9 polypeptide described herein. In several embodiments, the Cas9 domain of any of the fusion proteins provided in this disclosure is composed of the amino acid sequence of any Cas9 polypeptide described herein.

[0338] The amino acid sequence of the SpCas9 protein example that can bind to the PAM sequence is as follows.

[0339] The amino acid sequence of the SpCas9 example combining PAM is as follows:

[0340]

[0341]

[0342] The amino acid sequence of the SpCas9n example combining PAM is as follows:

[0343]

[0344] The amino acid sequence of the SpEQR Cas9 example combining PAM is as follows:

[0345]

[0346] In this sequence, residues E1135, Q1335, and R1337, which can be obtained by mutations in D1135, R1335, and T1337 to produce SpEQR Cas9, are underlined and in bold.

[0347] The amino acid sequence of the SpVQR Cas9 example combining PAM is as follows:

[0348]

[0349]

[0350] In this sequence, residues V1135, Q1335, and R1337, which can be obtained by mutations in D1135, R1335, and T1337 to obtain SpVQR Cas9, are underlined and in bold.

[0351] The amino acid sequence of the SpVRER Cas9 example combining PAM is as follows:

[0352]

[0353]

[0354] The amino acid sequence of the SpVRQR Cas9 example combining PAM is as follows:

[0355]

[0356] The residues V1135, R1218, Q1335, and R1337 above can be mutated from D1135, G1218, R1335, and T1337 to obtain SpVRQR Cas9, which is underlined and in bold below.

[0357] In several embodiments, the Cas9 domain is a recombinant Cas9 domain. In several embodiments, the recombinant Cas9 domain is a SpyMacCas9 domain. In several embodiments, the SpyMacCas9 domain is an active SpyMacCas9 nuclease, an inactive SpyMacCas9 nuclease (SpyMacCas9d), or a SpyMacCas9 nickase (SpyMacCas9n). In several embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to nucleic acid sequences having a non-canonical PAM. In several embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to nucleic acid sequences having an NAA PAM sequence.

[0358] SpyMacCas9 Implementation

[0359]

[0360] High-fidelity Cas9 domain

[0361] This document discloses several forms that provide high-fidelity Cas9 domains. In several embodiments, the high-fidelity Cas9 domain is a genetically modified Cas9 domain containing one or more mutations that, compared to the corresponding wild-type Cas9 domain, reduces the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA. Without being bound by any particular theory, a high-fidelity Cas9 domain with reduced electrostatic interaction with the sugar-phosphate backbone of DNA may have fewer off-target effects. In several embodiments, the Cas9 domain (e.g., the wild-type Cas9 domain) contains one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA. In several embodiments, the Cas9 domain comprises one or more mutations that reduce the association between the Cas9 domain and the sugar-phosphate backbone of DNA by 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 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%.

[0362] In several embodiments, any of the Cas9 fusion proteins provided in this disclosure includes one or more of the mutations N497X, R661X, Q695X, and / or Q926X, or a corresponding mutation in any of the amino acid sequences provided in this disclosure, wherein X is any single amino acid. In several embodiments, any of the Cas9 fusion proteins provided in this disclosure includes one or more of the mutations N497A, R661A, Q695A, and / or Q926A, or a corresponding mutation in any of the amino acid sequences provided in this disclosure. In several embodiments, any of the Cas9 fusion proteins provided in this disclosure includes the D10A mutation, or a corresponding mutation in any of the amino acid sequences provided in this disclosure. For example, high-fidelity Cas9 domains have been described in Kleinstiver, BP, et al. "High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects." Nature 529, 490-495 (2016); and Slaymaker, IM, et al. "Rationally engineered Cas9 nucleases with improved specificity." Science 351, 84-88 (2015), the full text of which is incorporated herein by reference.

[0363] Compared to Cas9, high-fidelity Cas9 domain mutations are displayed in bold and underlined.

[0364]

[0365]

[0366] In some cases, the mutant Cas9 protein carries mutations of H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, resulting in a reduced ability of the peptide to cleave target DNA or RNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind target DNA (e.g., single-stranded target DNA). As for another non-limiting embodiment, in some cases, the mutant Cas9 protein carries mutations of D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, resulting in a reduced ability of the peptide to cleave target DNA or RNA. This Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retains the ability to bind target DNA (e.g., single-stranded target DNA). In some cases, when the mutant Cas9 protein carries mutations such as W476A and W1126A, or mutations such as P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, the mutant Cas9 protein cannot effectively bind to the PAM sequence. Therefore, in some such cases, when this mutant Cas9 protein is used in a binding method, the method does not require the PAM sequence. In other words, in some cases, when this mutant Cas9 protein is used in a binding method, the method may include guide RNA, but the method may be performed without the presence of the PAM sequence (and therefore, the binding specificity is provided by the target segment of the guide RNA). Other residues can be mutated to achieve the above effect (i.e., inactivating one or another nuclease portion). As for non-limiting embodiments, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 may be modified (i.e., substituted). Furthermore, mutations other than alanine substitution are preferable.

[0367] In some embodiments, the CRISPR protein-derived domain of the base editor may comprise all or part of a Cas9 protein with a canonical PAM sequence (NGG). In other embodiments, the CRISPR protein-derived domain of the base editor may employ a non-canonical PAM sequence. Such sequences are described in the industry and will be readily apparent to those skilled in the art. For example, the Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, BP, et al., "Engineered CRISPR-Cas9 nucleases with altered PAM specificities" (Nature 523, 481-485 (2015); and Kleinstiver, BP, et al., "Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition" (Nature Biotechnology 33, 1293-1298 (2015), the full text of which is incorporated herein by reference.

[0368] In several embodiments, a PAM recognized by the CRISPR protein-derived domain of the base editor can be provided to the cell on a separate oligonucleotide to an insert (e.g., an AAV insert) encoding the base editor. In such cases, the PAM provided on the separate oligonucleotide allows the splitting of the target sequence, which would otherwise not be able to be split because no adjacent PAM exists on the same polynucleotide of the target sequence.

[0369] In one implementation, *Streptococcus pyogenes* Cas9 (SpCas9) can be used as a CRISPR endonuclease for gene editing. Other endonucleases may also be used. In some cases, different endonucleases can be used to target specific gene targets. In some cases, synthetic SpCas9-derived variants without the NGG PAM sequence can be used. Furthermore, other Cas9 variants from various species have been identified, and these "SpCas9" variants can bind various PAM sequences, which are also useful as disclosed herein. For example, relatively large SpCas9s (approximately 4 kb of coding sequence) have also been found to carry SpCas9 cDNA that cannot be efficiently expressed in cells. Conversely, the coding sequence of *Staphylococcus aureus* Cas9 (SaCas9) is approximately 1 kb shorter than that of SpCas9, potentially allowing for efficient intracellular expression. Similar to SpCas9, the SaCas9 endonuclease can modify target genes in both in vitro mammalian cells and in vivo mice. In some cases, Cas proteins can target different PAM sequences. In others, the target gene may be adjacent to a Cas9 PAM, such as 5'-NGG. In still others, other Cas9 proteins have different PAM requirements. For example, other PAMs such as Streptococcus thermophilus (5'-NNAGAA for CRISPR1 and 5'-NGGNG for CRISPR3) and Neisseria meningitidis (5'-NNNNGATT) have also found adjacent target genes.

[0370] In several embodiments, for the *Streptococcus pyogenes* system, the target gene sequence may precede (i.e., at 5') the 5'-NGG PAM, and a 20-nt guide RNA sequence may pair with the opposing base pair to mediate Cas9 cleavage adjacent to the PAM. In some cases, the adjacent cleavage may be or may be about 3 base pairs upstream of the PAM. In some cases, the adjacent cleavage may be or may be about 10 base pairs upstream of the PAM. In some cases, the adjacent cleavage may be or may be about 0-20 base pairs upstream of the PAM. For example, the adjacent cleavage may be immediately upstream of the PAM, at 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, or 30 base pairs. The adjacent cleavage may also be 1 to 30 base pairs downstream of the PAM.

[0371] Fusion proteins containing nuclear localization sequences (NLS)

[0372] Vectors encoding CRISPR enzymes containing one or more nuclear localization sequences (NLS) can be used. For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NLSs may be used. CRISPR enzymes may contain an NLS at or near the N-terminus, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NLSs at or near the C-terminus, or any combination thereof (e.g., one or more NLSs at the N-terminus and one or more NLSs at the C-terminus). When more than one NLS is present, each may be independently selected independently, such that a single NLS may exist in more than one copy, and / or one or more other NLSs may be combined to exist in more than one copy.

[0373] The CRISPR enzyme used in this method may contain approximately 6 NLS. An NLS is considered to be close to the N-terminus or C-terminus when the amino acid closest to the NLS is within approximately 50 amino acids along the polypeptide chain from the N-terminus or C-terminus, for example, within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 amino acids.

[0374] In several embodiments, the NLS comprises the amino acid sequence PKKKRKVEGADKRTADGSEFES PKKKRKV, KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRKPKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0375] In several embodiments, the NLS is present in the linker, or the NLS is adjacent to the linker, such as the linker described herein. In several embodiments, the N-terminal or C-terminal NLS is a bipartite NLS. A bipartite NLS comprises two clusters of basic amino acids separated by a relatively short spacer sequence (hence bipartite -2-part, unlike a single-part NLS). The nucleoprotein NLS, KR[PAATKKAGQA]KKKK, is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids separated by a spacer of about 10 amino acids. The sequence of an example of a bipartite NLS is as follows:

[0376] PKKKRKVEGADKRTADGSEFES PKKKRKV.

[0377] In some embodiments, the fusion protein of the present invention does not contain a spacer base sequence. In some embodiments, a spacer base sequence is present between one or more of the domains or proteins.

[0378] The PAM sequence can be any PAM sequence known in the industry. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNGATT, NNAGAAW, or NAAMC. Y is pyrimidine; N is any nucleotide base; W is A or T.

[0379] Nucleoside base editing domain

[0380] This article describes a base editor comprising a fusion protein including a polynucleotide programmable nucleotide-binding domain and a nucleoside base editing domain (e.g., a deaminase domain). The base editor can be programmed to edit one or more bases in a target polynucleotide sequence via guided polynucleotide interactions capable of recognizing the target sequence. Once the target sequence is identified, the base editor anchors to the polynucleotide to be edited, and then the deaminase domain component of the base editor edits the target base.

[0381] In some embodiments, the nucleoside base editing domain is a deaminase domain. In some cases, the deaminase domain may be cytosine deaminase or cytidine deaminase. In some embodiments, "cytosine deaminase" or "cytidine deaminase" may be used interchangeably. In some cases, the deaminase domain may be adenine deaminase or adenine deaminase. In some embodiments, "adenine deaminase" or "adenine deaminase" may be used interchangeably. Details of the nucleoside base editing protein are described in International PCT Applications Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), the full text of which is incorporated herein by reference. Please also refer to Komor, AC, et al., "Programmable editing of target bases in genomic DNA without double-strand DNA division" Nature 533, 420-424 (2016); Gaudelli, NM, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA division" Nature 551, 464-471 (2017); and Komor, AC, et al., "Improved base excision repair inhibition and C:G-to-T:A base editor from phage μGam protein with higher efficiency and product purity" Science Advances 3: eaao4774 (2017), the full contents of which are incorporated herein by reference.

[0382] C to T editor

[0383] In several embodiments, the base editor disclosed herein comprises a fusion protein containing a cytidine deaminase domain capable of deaminating a target cytidine (C) base of a polynucleotide to produce uridine (U), which has thymine base-pairing properties. In several embodiments, for example, where the polynucleotide is double-stranded (e.g., DNA), the uridine base can then be substituted with a thymine base (e.g., via a cellular repair mechanism) to obtain a C:G to T:A conversion. In other embodiments, the C to U deamination of the nucleic acid via the base editor is not accompanied by a U to T substitution.

[0384] The deamination of a target C in a polynucleotide to obtain U is a non-limiting embodiment of a type of base editing performed by the base editor described herein. In another embodiment, a base editor containing a cytidine deaminase domain can mediate the conversion of a cytosine (C) base to a purine (G) base. For example, the U of the polynucleotide produced by deaminating cytidine via the cytidine deaminase domain of the base editor can be excised from the polynucleotide via a base excision repair mechanism (e.g., via a uracil DNA glycosylase (UDG) domain), creating a debasement site. The nucleoside base opposite the debasement site can then be substituted with another base (such as C) by, for example, a translesion polymerase (e.g., via a cellular repair mechanism). While the nucleoside base opposite the debasement site is typically substituted with C, other substitutions (e.g., A, G, or T) are also possible.

[0385] Therefore, in several embodiments, the base editor described herein includes a deaminase domain (e.g., a cytidine deaminase domain) capable of deaminating a target C in a polynucleotide to U. Furthermore, as detailed later, the base editor may include additional domains that, in several embodiments, assist in the conversion of the U obtained from deamination to T or G. For example, a base editor including a cytidine deaminase domain may further include a uracil glycosylase inhibitor (UGI) domain to mediate the substitution of U for T, completing the C-to-T base editing event. In another embodiment, the base editor may incorporate a translesion polymerase to improve the efficiency of C-to-G base editing, because the translesion polymerase assists in binding C relative to the debasement site (i.e., resulting in G binding to the debasement site, completing the C-to-G base editing event).

[0386] A base editor containing a cytidine deaminase domain can deaminate a target C in any polynucleotide, including DNA, RNA, and DNA-RNA hybrids. Typically, cytidine deaminase catalyzes the C nucleoside base located in the single-stranded portion of a polynucleotide. In some embodiments, the entire polynucleotide containing the target C can be single-stranded. For example, a cytidine deaminase incorporated into a base editor can deaminate the target C in a single-stranded RNA polynucleotide. In other embodiments, a base editor containing a cytidine deaminase domain can act on a double-stranded polynucleotide, but the target C can be located in the polynucleotide portion that is in a single-stranded state during the deamination reaction. For example, in an embodiment where the NAGPB domain contains a Cas9 domain, during the formation of the Cas9-gRNA-target DNA complex, several nucleotides may remain unpaired, resulting in the formation of a Cas9 "R-loop complex". These unpaired nucleotides can form single-stranded DNA bubbles, which can be used as an enzyme substrate for single-stranded specific nucleotide deaminases (e.g., cytidine deaminase).

[0387] In several embodiments, the cytidine deaminase of the base editor may comprise all or part of an apolipoprotein B mRNA editing complex (APOBEC) family of deaminases. APOBEC is an evolutionarily conserved family of cytidine deaminases. Members of this family are C- to U-editing enzymes. The N-terminal domain of APOBEC-like proteins is the catalytic domain, while the C-terminal domain is the pseudocatalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain and is essential for cytidine deamination. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D ("APOBEC3E" hereafter refers to this), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-inducible (cytidine) deaminase. In several embodiments, the deaminase incorporated into the base editor comprises all or part of the APOBEC1 deaminase. In several embodiments, the deaminase incorporated into the base editor comprises all or part of APOBEC2 deaminase. In several embodiments, the deaminase incorporated into the base editor comprises all or part of APOBEC3 deaminase. In several embodiments, the deaminase incorporated into the base editor comprises all or part of APOBEC3A deaminase. In several embodiments, the deaminase incorporated into the base editor comprises all or part of APOBEC3B deaminase. In several embodiments, the deaminase incorporated into the base editor comprises all or part of APOBEC3C deaminase. In several embodiments, the deaminase incorporated into the base editor comprises all or part of APOBEC3D deaminase. In several embodiments, the deaminase incorporated into the base editor comprises all or part of APOBEC3E deaminase. In several embodiments, the deaminase incorporated into the base editor comprises all or part of APOBEC3F deaminase. In several embodiments, the deaminase incorporated into the base editor comprises all or part of APOBEC3G deaminase. In several embodiments, the deaminase incorporated into the base editor comprises all or part of APOBEC3H deaminase. In several embodiments, the deaminase incorporated into the base editor comprises all or part of APOBEC4 deaminase. In several embodiments, the deaminase incorporated into the base editor comprises all or part of activation-inducible deaminase (AID). In several embodiments, the deaminase incorporated into the base editor comprises all or part of cytidine deaminase 1 (CDA1). It should be understood that the base editor may comprise a deaminase derived from any organism (e.g., human or rat). In several embodiments, the deaminase domain of the base editor is derived from human, chimpanzee, orangutan, monkey, cow, dog, rat, or mouse. In several embodiments, the deaminase domain of the base editor is derived from rat (e.g., rat APOBEC1). In several embodiments, the deaminase domain of the base editor is human APOBEC1.In some embodiments, the deaminase domain of the base editor is pmCDA1.

[0388] The amino acid and nucleic acid sequences of pmCDA1 are shown below.

[0389] >tr|A5H718|A5H718_PETMA cytosine deaminase OS=hagmomon OX=7757PE=2SV=1 amino acid sequence:

[0390]

[0391] Nucleic acid sequence: >EF094822.1 Hagfish isolate PmCDA.21 cytosine deaminase mRNA, complete CDS:

[0392]

[0393] The amino acid and nucleic acid sequences of the coding sequence (CDS) of human activation-induced cytidine deaminase (AID) are shown below.

[0394] >tr|Q6QJ80|Q6QJ80_HUMAN activation-induced cytidine deaminase OS=Homo sapiens OX=9606GN=AICDA PE=2SV=1 amino acid sequence:

[0395]

[0396] Nucleic acid sequence: >NG_011588.1:5001-15681 Homo sapiens activation-induced cytidine deaminase (AICDA), RefSeqGene (LRG_17) on chromosome 12:

[0397]

[0398]

[0399]

[0400]

[0401]

[0402] According to the embodiments disclosed herein, other embodiments of deaminases that can fuse to Cas9 are provided below. It should be understood that in some embodiments, active domains of individual sequences may be used, for example, domains without localization signals (nuclear localization sequences, no nuclear output signals, cytoplasmic localization signals).

[0403] Human AID:

[0404]

[0405] (Underlined: Nuclear localization sequence; Double underlined: Nuclear output signal)

[0406] Mouse AID:

[0407]

[0408] (Underlined: Nuclear localization sequence; Double underlined: Nuclear output signal)

[0409] Canine AID:

[0410]

[0411] (Underlined: Nuclear localization sequence; Double underlined: Nuclear output signal)

[0412] Niu AID:

[0413]

[0414] (Underlined: Nuclear localization sequence; Double underlined: Nuclear output signal) Rat AID:

[0415]

[0416] (Underlined: Nuclear localization sequence; Double underlined: Nuclear output signal) Mouse APOBEC3:

[0417]

[0418] (Italicized: Nucleic acid editing domain)

[0419] Rat APOBEC-3:

[0420]

[0421] (Italicized: Nucleic acid editing domain)

[0422] Rhesus monkey APOBEC-3G:

[0423]

[0424] (Italics: Nucleic acid editing domain; underlined: Cytoplasmic localization signal) Chimpanzee APOBEC-3G:

[0425]

[0426] (Italics: Nucleic acid editing domain; underlined: cytoplasmic localization signal) Green Monkey APOBEC-3G:

[0427]

[0428] (Italics: Nucleic acid editing domain; underlined: Cytoplasmic localization signal) Human APOBEC-3G:

[0429]

[0430] (Italics: Nucleic acid editing domain; underlined: Cytoplasmic localization signal) Human APOBEC-3F:

[0431]

[0432] (Italicized: Nucleic acid editing domain)

[0433] Human APOBEC-3B:

[0434]

[0435] (Italicized: Nucleic acid editing domain)

[0436] Rat APOBEC-3B:

[0437]

[0438] Bovine APOBEC-3B:

[0439]

[0440] Chimpanzee APOBEC-3B:

[0441]

[0442] Human APOBEC-3C:

[0443]

[0444] (Italicized: Nucleic acid editing domain)

[0445] Apobec-3C (Gorilla):

[0446]

[0447] Human APOBEC-3A:

[0448]

[0449] (Italicized: Nucleic acid editing domain)

[0450] Rhesus monkey APOBEC-3A:

[0451]

[0452] (Italicized: Nucleic acid editing domain)

[0453] Bovine APOBEC-3A:

[0454]

[0455] (Italicized: Nucleic acid editing domain) Human APOBEC-3H:

[0456]

[0457] (Italicized: Nucleic acid editing domain)

[0458] Rhesus monkey APOBEC-3H:

[0459]

[0460] Human APOBEC-3D:

[0461]

[0462] (Italicized: Nucleic acid editing domain)

[0463] Human APOBEC-1:

[0464]

[0465] Mouse APOBEC-1:

[0466]

[0467] Rat APOBEC-1:

[0468]

[0469] Human APOBEC-2:

[0470]

[0471] Mouse APOBEC-2:

[0472]

[0473] Rat APOBEC-2:

[0474]

[0475] Bovine APOBEC-2:

[0476]

[0477] Hagfish CDA1 (pmCDA1):

[0478]

[0479] APOBEC3G D316R D317R:

[0480]

[0481] Human APOBEC3G Chain A:

[0482]

[0483] Human APOBEC3G Chain A D120R D121R:

[0484]

[0485] Several patterns disclosed herein are based on the understanding that, for example, by making point mutations in the deaminase domain, the catalytic activity of the deaminase domain in any of the fusion proteins described herein can be modulated to affect the sustained synthetic capacity of the fusion protein (e.g., a base editor). For example, mutations that reduce (but do not eliminate) the catalytic activity of the deaminase domain within a base-editing fusion protein make it less likely for the deaminase domain to catalyze the deamination of residues adjacent to the target residue, thus narrowing the deamination window. The ability to narrow the deamination window can prevent unintended deamination of residues adjacent to a specific target residue, reducing or preventing off-target effects.

[0486] For example, in several embodiments, the APOBEC deaminase incorporating a base editor may contain one or more mutations selected from the group consisting of H121X, H122X, R126X, R126X, R118X, W90X, W90X, and R132X of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase, wherein X is any amino acid. In several embodiments, the APOBEC deaminase incorporating a base editor may contain one or more mutations selected from the group consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase.

[0487] For example, in several embodiments, the APOBEC deaminase incorporating a base editor may contain one or more mutations selected from the group consisting of D316X, D317X, R320X, R320X, R313X, W285X, W285X, and R326X of hAPOBEC3G; or one or more corresponding mutations of another APOBEC deaminase, wherein X is any amino acid. In several embodiments, the APOBEC deaminase incorporating a base editor may contain one or more mutations selected from the group consisting of D316R, D317R, R320A, R320E, R313A, W285A, W285Y, and R326E of hAPOBEC3G; or one or more corresponding mutations of another APOBEC deaminase.

[0488] In several embodiments, the APOBEC deaminase incorporating a base editor may contain the H121R and H122R mutations of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain the R126A mutation of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain the R126E mutation of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain the R118A mutation of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain the W90A mutation of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporated with a base editor may contain a W90Y mutation in rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporated with a base editor may contain an R132E mutation in rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporated with a base editor may contain both W90Y and R126E mutations in rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporated with a base editor may contain both R126E and R132E mutations in rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporated with a base editor may contain both W90Y and R132E mutations in rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase that incorporates a base editor may contain the W90Y, R126E, and R132E mutations of rAPOBEC1; or one or more corresponding mutations in another APOBEC deaminase.

[0489] In several embodiments, the APOBEC deaminase incorporating a base editor may contain the D316R and D317R mutations of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain the R320A mutation of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain the R320E mutation of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain the R313A mutation of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain the W285A mutation of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain the W285Y mutation of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain the R326E mutation of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain both the W285Y and R320E mutations of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain both the R320E and R326E mutations of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain the W285Y and R326E mutations of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase. In several embodiments, the APOBEC deaminase incorporating a base editor may contain the W285Y, R320E, and R326E mutations of hAPOBEC3G; or one or more corresponding mutations in another APOBEC deaminase.

[0490] A variety of modified cytidine deaminases are commercially available, including, but not limited to, SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, VRER-BE3, YE1-BE3, EE-BE3, YE2-BE3, and YEE-BE3 (plastos 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, and 85177) derived from Addgene.

[0491] Details of the C-to-T nucleoside base editing protein are described in International PCT Application Nos. PCT / US2016 / 058344 (WO2017 / 070632) and Komor, AC, et al., "Programmable editing of target bases in genomic DNA without double-strand DNA division" Nature 533, 420-424 (2016), the full text of which is incorporated herein by reference.

[0492] A to G editors

[0493] In several embodiments, the base editor described herein may include a deaminase domain encompassing adenosine deaminase. This adenosine deaminase domain of the base editor assists in editing A nucleoside bases into G nucleoside bases by deaminating adenine (A) to inosine (I), which has the base-pairing properties of guanine (G). Adenosine deaminase deaminizes (i.e., removes the amino group) of deoxyadenosine residues in deoxyribonucleic acid (DNA).

[0494] In several embodiments, the nucleoside base editor provided herein can be manufactured by fusing one or more protein domains together, thereby generating a fusion protein. In some embodiments, the fusion protein provided herein includes one or more features that improve the base editing activity of the fusion protein (e.g., efficiency, selectivity, and specificity). For example, the fusion protein provided herein may include a Cas9 domain having reduced nuclease activity. In several embodiments, the fusion protein provided herein may have a non-nuclease-active Cas9 domain (dCas9), or a Cas9 domain that cleaves one strand of a diploid DNA molecule, referred to as Cas9 cleavage enzyme (nCas9). Without being bound by any particular theory, the presence of a catalytic residue (e.g., H840) maintains the activity of Cas9 in cleaving non-edited (e.g., non-deamination) strands containing T opposite to target A. Mutations in the catalytic residues of Cas9 (e.g., D10 to A10) prevent the cleavage of edited strands containing target A residues. These Cas9 variants can generate single-strand DNA breaks (nicks) at specific locations based on target sequences defined by gRNA, leading to the repair of the non-edited strand and ultimately resulting in T-to-C alterations on the non-edited strand. In several embodiments, the A-to-G base editor further comprises an inosine base excision repair inhibitor, such as a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine-specific nuclease. Without being bound by any particular theory, the UGI domain or the catalytically inactive inosine-specific nuclease can inhibit or prevent base excision repair of deamination-adenosine residues (e.g., inosine), which may improve the activity or efficiency of the base editor.

[0495] Base editors containing adenosine deaminase can act on any polynucleotide, including DNA, RNA, and DNA-RNA hybrids. In some embodiments, base editors containing adenosine deaminase can deaminate target A of a polynucleotide containing RNA. For example, a base editor may contain an adenosine deaminase domain capable of deaminating target A of RNA polynucleotides and / or DNA-RNA hybrid polynucleotides. In one embodiment, the adenosine deaminase incorporated into the base editor comprises all or part of an adenosine deaminase that acts on RNA (ADAR, e.g., ADAR1 or ADAR2). In another embodiment, the adenosine deaminase incorporated into the base editor comprises all or part of an adenosine deaminase that acts on tRNA (ADAT). Base editors containing an adenosine deaminase domain can also deaminate the A nucleoside base of a DNA polynucleotide. In one embodiment, the adenosine deaminase of the base editor comprises all or part of ADAT containing one or more mutations that allow the ADAT to deaminate target A in DNA. For example, a base editor may contain all or part of ADAT (EcTadA) derived from E. coli, which contains one or more of the following mutations: D108N, A106V, D147Y, E155V, L84F, H123Y, I157F, or a corresponding mutation in another adenosine deaminase.

[0496] Adenosine deaminases can be derived from any suitable organism (e.g., *Escherichia coli*). In several embodiments, the adenosine deaminase is a naturally occurring adenosine deaminase comprising one or more mutations corresponding to any mutations provided in this disclosure (e.g., mutations in ecTadA). Corresponding residues in any homologous protein can be identified, for example, by sequence alignment and deamination of homologous residues. Mutations in any naturally occurring adenosine deaminase corresponding to any mutations provided in this disclosure (e.g., any mutations identified in ecTadA) can be generated accordingly.

[0497] In a particular implementation, TadA is any of the TadA described in PCT / US2017 / 045381 (WO2018 / 027078) (the entire disclosure of this specification is incorporated herein by reference).

[0498] In some embodiments, adenosine deaminase comprises the following amino acid sequence:

[0499]

[0500] It is named "TadA reference sequence".

[0501] In several embodiments, the TadA deaminase is a whole-celled Escherichia coli TadA deaminase. For example, in some embodiments, the adenosine deaminase comprises the following amino acid sequence:

[0502]

[0503] However, it should be understood that the additional adenosine deaminases useful in this application will be readily apparent to those skilled in the art and fall within the scope of this disclosure. For example, the adenosine deaminase may be an adenosine deaminase homolog (ADAT) that acts on tRNA. Not limiting, the amino acid sequence of the ADAT homolog examples includes the following:

[0504] Staphylococcus aureus TadA:

[0505]

[0506] Bacillus subtilis TadA:

[0507]

[0508] Salmonella typhimurium (S. typhimurium) TadA:

[0509]

[0510] Shewanella putrefaciens (S. putrefaciens) TadA:

[0511]

[0512] Haemophilus influenzae F3031 (H. influenzae) TadA:

[0513]

[0514] C. crescentus TadA:

[0515]

[0516] Geobacter sulfurreducens (G. sulfurreducens) TadA:

[0517]

[0518] TadA 7.10:

[0519]

[0520] In several embodiments, the adenosine deaminase is derived from prokaryotes. In several embodiments, the adenosine deaminase is derived from bacteria. In several embodiments, the adenosine deaminase is derived from *Escherichia coli*, *Staphylococcus aureus*, *Salmonella typhimurium*, *Shewanella putrefactive*, *Haemophilus influenzae*, *Strychnos nucleus*, or *Bacillus subtilis*. In several embodiments, the adenosine deaminase is derived from *Escherichia coli*.

[0521] In one embodiment, the fusion protein of the present invention comprises wild-type TadA linked to TadA 7.10, which is linked to the Cas9 nickase. In a particular embodiment, the fusion protein comprises a single TadA 7.10 domain (e.g., provided as a monomer). In other embodiments, the ABE7.10 editor comprises TadA 7.10 and TadA (wt), which is capable of generating a heterodimer.

[0522] In several embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the amino acid sequences listed in any adenosine deaminase provided in this disclosure. It should be understood that the adenosine deaminases provided in this disclosure may include one or more mutations (e.g., any of the mutations provided in this disclosure). This document provides any deaminase domain having a certain percentage of identity plus any of the mutations or combinations described herein. In several embodiments, the adenosine deaminase comprises an amino acid sequence having a comparative reference sequence or any adenosine deaminase provided in this disclosure, having 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 mutations. In several embodiments, the adenosine deaminase comprises an amino acid sequence having at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical adjacent amino acid residues compared to any of the amino acid sequences known in the art or described herein.

[0523] In several embodiments, the adenosine deaminase comprises a D108X mutation, or a corresponding mutation in another adenosine deaminase, relative to the TadA reference sequence, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises a D108N, D108V, D108A, or D108Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase. However, it should be understood that additional deaminases can similarly be aligned to recognize homologous amino acid residues that are provided in this disclosure as mutagenic.

[0524] In several embodiments, the adenosine deaminase comprises the A106X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises the A106V mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0525] In several embodiments, the adenosine deaminase comprises an E155X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises an E155D, E155G, or E155V mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0526] In several embodiments, the adenosine deaminase comprises a D147X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises a D147Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0527] It should be understood that any mutations provided in this disclosure (e.g., based on the amino acid sequence of the TadA reference sequence) can be introduced into other adenosine deaminases, such as Staphylococcus aureus TadA (saTadA) or other adenosine deaminases (e.g., bacterial adenosine deaminases). Any mutations identified in the TadA reference sequence can be made in other adenosine deaminases having homologous amino acid residues. It should also be understood that any mutations provided in this disclosure can be made individually or in any combination in the TadA reference sequence or other adenosine deaminases.

[0528] For example, relative to the TadA reference sequence, adenosine deaminases contain mutations such as D108N, A106V, E155V, and / or D147Y, or corresponding mutations in another adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises the following groups of mutations (mutation groups separated by ";"), or corresponding mutations in another adenosine deaminase: D108N and A106V; D108N and E155V; D108N and D147Y; A106V and E155V; A106V and D147Y; E155V and D147Y; D108N, A106V and E55V; D108N, A106V and D147Y; D108N, E55V and D147Y; A106V, E55V and D147Y; and D108N, A106V, E55V and D147Y. However, it should be understood that any combination of the corresponding mutations provided in this disclosure (e.g., ecTadA) can be made in the adenosine deaminase.

[0529] In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises one or more of the mutations H8X, T17X, ​​L18X, W23X, L34X, W45X, R51X, A56X, E59X, E85X, M94X, I95X, V102X, F104X, A106X, R107X, D108X, K110X, M118X, N127X, A138X, F149X, M151X, R153X, Q154X, I156X, and / or K157X, or one or more corresponding mutations in another adenosine deaminase, wherein the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises one or more of the following mutations: H8Y, T17S, L18E, W23L, L34S, W45L, R51H, A56E or A56S, E59G, E85K or E85G, M94L, 1951, V102A, F104L, A106V, R107C or R107H or R107P, D108G or D108N or D108V or D108A or D108Y, K110I, M118K, N127S, A138V, F149Y, M151V, R153C, Q154L, I156D, and / or K157R, or one or more corresponding mutations in another adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises one or more of the mutations H8X, D108X, and / or N127X, or one or more corresponding mutations in another adenosine deaminase, wherein the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises one or more of the mutations H8Y, D108N, and / or N127S, or one or more corresponding mutations in another adenosine deaminase.

[0530] In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises one or more of the mutations H8X, R26X, M61X, L68X, M70X, A106X, D108X, A109X, N127X, D147X, R152X, Q154X, E155X, K161X, Q163X, and / or T166X, or one or more corresponding mutations in another adenosine deaminase, wherein the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises one or more of the following mutations: H8Y, R26W, M61I, L68Q, M70V, A106T, D108N, A109T, N127S, D147Y, R152C, Q154H or Q154R, E155G or E155V or E155D, K161Q, Q163H, and / or T166P, or one or more corresponding mutations in another adenosine deaminase.

[0531] In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises 1, 2, 3, 4, 5, or 6 mutations selected from the group consisting of H8X, D108X, N127X, D147X, R152X, and Q154X, or a corresponding mutation in another adenosine deaminase, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises 1, 2, 3, 4, 5, 6, 7, or 8 mutations selected from the group consisting of H8X, M61X, M70X, D108X, N127X, Q154X, E155X, and Q163X, or a corresponding mutation in another adenosine deaminase, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises 1, 2, 3, 4, or 5 mutations selected from the group consisting of H8X, D108X, N127X, E155X, and T166X, or a corresponding mutation in another adenosine deaminase, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.

[0532] In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises 1, 2, 3, 4, 5, or 6 mutations selected from the group consisting of H8X, A106X, and D108X, or a corresponding mutation in another adenosine deaminase, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises 1, 2, 3, 4, 5, 6, 7, or 8 mutations selected from the group consisting of H8X, R126X, L68X, D108X, N127X, D147X, and E155X, or a corresponding mutation in another adenosine deaminase, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises 1, 2, 3, 4, or 5 mutations selected from the group consisting of H8X, D108X, A109X, N127X, and E155X, or a corresponding mutation in another adenosine deaminase, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.

[0533] In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises 1, 2, 3, 4, 5, or 6 mutations selected from the group consisting of H8Y, D108N, N127S, D147Y, R152C, and Q154H, or a corresponding mutation in another adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises 1, 2, 3, 4, 5, 6, 7, or 8 mutations selected from the group consisting of H8Y, M61I, M70V, D108N, N127S, Q154R, E155G, and Q163H, or a corresponding mutation in another adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises 1, 2, 3, 4, or 5 mutations selected from the group consisting of H8Y, D108N, N127S, E155V, and T166P, or a corresponding mutation in another adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises 1, 2, 3, 4, 5, or 6 mutations selected from the group consisting of H8Y, A106T, D108N, N127S, E155D, and K161Q, or a corresponding mutation in another adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises 1, 2, 3, 4, 5, 6, 7, or 8 mutations selected from the group consisting of H8Y, R126W, L68Q, D108N, N127S, D147Y, and E155V, or a corresponding mutation in another adenosine deaminase. In several embodiments, relative to the TadA reference sequence, the adenosine deaminase comprises 1, 2, 3, 4, or 5 mutations selected from the group consisting of H8Y, D108N, A109T, N127S, and E155G, or a corresponding mutation in another adenosine deaminase.

[0534] It should be understood that any mutations provided in this disclosure, and any additional mutations (e.g., based on the TadA reference sequence amino acid sequence), can be introduced into other adenosine deaminases. Any mutations provided in this disclosure may be made individually or in any combination in the TadA reference sequence or other adenosine deaminases.

[0535] Details of the A-to-G nucleoside base editing protein are described in International PCT Application Nos. PCT / 2017 / 045381 (WO2018 / 027078) and Gaudelli, NM, et al., "Programmable base editing of A-T to G-C in genomic DNA without DNA division" Nature 551, 464-471 (2017), the disclosure of which is incorporated herein by reference in its entirety.

[0536] In several embodiments, the adenosine deaminase comprises one or more mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase. In several embodiments, the adenosine deaminase comprises the A106V and D108N mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In several embodiments, the adenosine deaminase comprises the R107C and D108N mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In several embodiments, the adenosine deaminase comprises the H8Y, D108N, N127S, D147Y, and Q154H mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In several embodiments, the adenosine deaminase comprises the H8Y, R24W, D108N, N127S, D147Y, and E155V mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In several embodiments, the adenosine deaminase comprises the D108N, D147Y, and E155V mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In several embodiments, the adenosine deaminase comprises the H8Y, D108N, and N127S mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In several embodiments, the adenosine deaminase comprises the A106V, D108N, D147Y, and E155V mutations in the TadA reference sequence, or corresponding mutations in another adenosine deaminase.

[0537] In several embodiments, the adenosine deaminase comprises one or more of the mutations a, S2X, H8X, I49X, L84X, H123X, N127X, I156X, and / or K160X in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises one or more of the mutations S2A, H8Y, I49F, L84F, H123Y, N127S, I156F, and / or K160S in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0538] In several embodiments, the adenosine deaminase comprises an L84X mutant adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises an L84F mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0539] In several embodiments, the adenosine deaminase comprises the H123X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises the H123Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase. In several embodiments, the adenosine deaminase comprises the I157X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises the I157F mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0540] In several embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations in the TadA reference sequence selected from the group consisting of L84X, A106X, D108X, H123X, D147X, E155X, and I156X, or a corresponding mutation in another adenosine deaminase, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations in the TadA reference sequence selected from the group consisting of S2X, I49X, A106X, D108X, D147X, and E155X, or a corresponding mutation in another adenosine deaminase, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations in the TadA reference sequence selected from the group consisting of H8X, A106X, D108X, N127X, and K160X, or a corresponding mutation in another adenosine deaminase, wherein X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.

[0541] In several embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations in the TadA reference sequence selected from the group consisting of L84F, A106V, D108N, H123Y, D147Y, E155V, and I156F, or a corresponding mutation in another adenosine deaminase. In several embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations in the TadA reference sequence selected from the group consisting of S2A, I49F, A106V, D108N, D147Y, and E155V, or a corresponding mutation in another adenosine deaminase.

[0542] In several embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations in the TadA reference sequence selected from the group consisting of H8Y, A106T, D108N, N127S, and K160S, or a corresponding mutation in another adenosine deaminase.

[0543] In several embodiments, the adenosine deaminase comprises one or more of the E25X, R26X, R107X, A142X, and / or A143X mutations in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, wherein the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises one or more of the following mutations in the TadA reference sequence: E25M, E25D, E25A, E25R, E25V, E25S, E25Y, R26G, R26N, R26Q, R26C, R26L, R26K, R107P, R07K, R107A, R107N, R107W, R107H, R107S, A142N, A142D, A142G, A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q and / or A143R, or one or more corresponding mutations in another adenosine deaminase. In several embodiments, the adenosine deaminase comprises one or more of the mutations described herein in the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase.

[0544] In several embodiments, the adenosine deaminase comprises an E25X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises an E25M, E25D, E25A, E25R, E25V, E25S, or E25Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0545] In several embodiments, the adenosine deaminase comprises an R26X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises an R26G, R26N, R26Q, R26C, R26L, or R26K mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0546] In several embodiments, the adenosine deaminase comprises an R107X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises an R107P, R07K, R107A, R107N, R107W, R107H, or R107S mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0547] In several embodiments, the adenosine deaminase comprises the A142X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises the A142N, A142D, or A142G mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0548] In several embodiments, the adenosine deaminase comprises the A143X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises the A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q, and / or A143R mutations in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0549] In several embodiments, the adenosine deaminase comprises one or more of the following mutations in the TadA reference sequence: H36X, N37X, P48X, I49X, R51X, M70X, N72X, D77X, E134X, S146X, Q154X, K157X, and / or K161X, or one or more corresponding mutations in another adenosine deaminase, wherein the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises one or more of the following mutations in the TadA reference sequence: H36L, N37T, N37S, P48T, P48L, I49V, R51H, R51L, M70L, N72S, D77G, E134G, S146R, S146C, Q154H, K157N, and / or K161T, or one or more corresponding mutations in another adenosine deaminase.

[0550] In several embodiments, the adenosine deaminase comprises an H36X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises an H36L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0551] In several embodiments, the adenosine deaminase comprises an N37X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises an N37T or N37S mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0552] In several embodiments, the adenosine deaminase comprises a P48X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises a P48T or P48L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0553] In several embodiments, the adenosine deaminase comprises an R51X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises an R51H or R51L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0554] In several embodiments, the adenosine deaminase comprises the S146X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises the S146R or S146C mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0555] In several embodiments, the adenosine deaminase comprises a K157X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises a K157N mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0556] In several embodiments, the adenosine deaminase comprises a P48X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises a P48S, P48T, or P48A mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0557] In several embodiments, the adenosine deaminase comprises the A142X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises the A142N mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0558] In several embodiments, the adenosine deaminase comprises a W23X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises a W23R or W23L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0559] In several embodiments, the adenosine deaminase comprises an R152X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, wherein X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In several embodiments, the adenosine deaminase comprises an R152P or R152H mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0560] In one embodiment, adenosine deaminase may comprise the mutations H36L, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N. In several embodiments, relative to the TadA reference sequence, adenosine deaminase comprises combinations of the following mutations, wherein the mutations in the combination are separated by "-", and the combinations of mutations are enclosed in parentheses: (A106V_D108N), (R107C_D108N).

[0561]

[0562]

[0563]

[0564]

[0565] In some embodiments, the fusion proteins provided in this disclosure include one or more features that improve the base editing activity of the fusion protein. For example, any of the fusion proteins provided in this disclosure may include a Cas9 domain with reduced nuclease activity. In several embodiments, any of the fusion proteins provided in this disclosure may have a Cas9 domain that does not have nuclease activity; or a Cas9 domain that cleaves one strand of a diploid DNA molecule, referred to as Cas9 cleavage enzyme (nCas9).

[0566] Cytidine deaminase

[0567] In one embodiment, the fusion protein of the present invention comprises a cytidine deaminase. In several embodiments, the cytidine deaminase provided in this disclosure is capable of deaminating cytosine or 5-methylcytosine to uracil or thymine. In one embodiment, the cytidine deaminase provided in this disclosure is capable of deaminating cytosine in DNA. The cytidine deaminase can be derived from any suitable organism. In several embodiments, the cytidine deaminase is a naturally occurring cytidine deaminase comprising one or more mutations corresponding to any mutation provided in this disclosure. Those skilled in the art will be able to identify corresponding residues in any homologous protein, for example, by sequence alignment and deamination of homologous residues. Accordingly, those skilled in the art will be able to generate mutations corresponding to any mutation provided in this disclosure in any naturally occurring cytidine deaminase. In several embodiments, the cytidine deaminase is derived from prokaryotes. In several embodiments, the cytidine deaminase is derived from bacteria. In several embodiments, the cytidine deaminase is derived from mammals (e.g., humans).

[0568] In several embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the cytidine deaminase amino acid sequences listed in this disclosure. It should be understood that the cytidine deaminases provided in this disclosure may include one or more mutations (e.g., any of the mutations provided in this disclosure). This document provides any deaminase domain having a certain percentage of identity plus any of the mutations described herein, or combinations thereof. In several embodiments, the cytidine deaminase comprises an amino acid sequence having a comparative reference sequence or any cytidine deaminase provided in this disclosure, having a mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more. In several embodiments, the cytidine deaminase comprises an amino acid sequence having at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical adjacent amino acid residues compared to any of the amino acid sequences known in the art or described herein.

[0569] The fusion protein of the present invention comprises a nucleic acid editing domain. In several embodiments, the nucleic acid editing domain is capable of catalyzing a C-to-U base change. In several embodiments, the nucleic acid editing domain is a deaminase domain. In several embodiments, the deaminase is a cytidine deaminase or adenosine deaminase. In several embodiments, the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In several embodiments, the deaminase is APOBEC1 deaminase. In several embodiments, the deaminase is APOBEC2 deaminase. In several embodiments, the deaminase is APOBEC3 deaminase. In several embodiments, the deaminase is APOBEC3A deaminase. In several embodiments, the deaminase is APOBEC3B deaminase. In several embodiments, the deaminase is APOBEC3C deaminase. In several embodiments, the deaminase is APOBEC3D deaminase. In several embodiments, the deaminase is APOBEC3E deaminase. In several embodiments, the deaminase is APOBEC3F deaminase. In several embodiments, the deaminase is APOBEC3G deaminase. In several embodiments, the deaminase is APOBEC3H deaminase. In several embodiments, the deaminase is APOBEC4 deaminase. In several embodiments, the deaminase is an activation-inducible deaminase (AID). In several embodiments, the deaminase is a vertebrate deaminase. In several embodiments, the deaminase is an invertebrate deaminase. In several embodiments, the deaminase is a human, chimpanzee, orangutan, monkey, bovine, dog, rat, or mouse deaminase. In several embodiments, the deaminase is a human deaminase. In several embodiments, the deaminase is a rat deaminase, for example, rAPOBEC1. In several embodiments, the deaminase is anguillarum deaminase 1 (pmCDA1). In several embodiments, the deaminase is human APOBEC3G. In several embodiments, the deaminase is a fragment of human APOBEC3G. In several embodiments, the deaminase is a variant of human APOBEC3G containing the D316R / D317R mutation. In several embodiments, the deaminase is a fragment of human APOBEC3G and a corresponding mutation containing the D316R / D317R mutation. In several embodiments, the nucleic acid editing domain is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the deaminase domain of any deaminase described herein.

[0570] Cas9 domain of nucleoside base editor

[0571] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) is a Cas9 domain. Non-limiting embodiments of the Cas9 domain are provided in this disclosure. The Cas9 domain can be a nuclease-active Cas9 domain, a nuclease-inactivating Cas9 domain, or a Cas9 cleavage enzyme. In several embodiments, the Cas9 domain is a nuclease-active Cas9 domain. For example, the Cas9 domain can be a Cas9 domain that cleaves two strands of a diploid nucleic acid (e.g., two strands of a diploid DNA molecule). In several embodiments, the Cas9 domain comprises any of the amino acid sequences listed in this disclosure. In several embodiments, the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the amino acid sequences listed in this disclosure. In several embodiments, the Cas9 domain comprises an amino acid sequence having, compared to any of the amino acid sequences listed in this disclosure, a mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more. In several embodiments, the Cas9 domain comprises an amino acid sequence having 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, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical adjacent amino acid residues compared to any of the amino acid sequences listed in this disclosure.

[0572] In several embodiments, the Cas9 domain is a nuclease-inactivating Cas9 domain (dCas9). For example, the dCas9 domain can bind to a diploid nucleic acid molecule (e.g., via a gRNA molecule) without cleaving either strand of the diploid nucleic acid molecule. In several embodiments, the nuclease-inactivating dCas9 domain comprises the D10X and H840X mutations of the amino acid sequences listed in this disclosure, or the corresponding mutations of any amino acid sequences provided in this disclosure, where X represents any amino acid change. In several embodiments, the nuclease-inactivating dCas9 domain comprises the D10A and H840A mutations of the amino acid sequences listed in this disclosure, or the corresponding mutations of any amino acid sequences provided in this disclosure.

[0573] In several embodiments, the Cas9 domain is a Cas9 cleavage enzyme. The Cas9 cleavage enzyme may be a Cas9 protein capable of cleaving a single strand of a diploid nucleic acid molecule (e.g., a diploid DNA molecule). In several embodiments, the Cas9 cleavage enzyme cleaves the target strand of the diploid nucleic acid molecule, meaning the Cas9 cleavage enzyme cleaves the strand that is base-paired (complementary) to a gRNA (e.g., sgRNA) bound to Cas9. In several embodiments, the Cas9 cleavage enzyme contains a D10A mutation and a histidine residue at position 840. In several embodiments, the Cas9 cleavage enzyme cleaves the non-target and non-base-editing strand of the diploid nucleic acid molecule, meaning the Cas9 cleavage enzyme cleaves the strand that is not base-paired to a gRNA (e.g., sgRNA) bound to Cas9. In several embodiments, the Cas9 cleavage enzyme contains an H840A mutation and an aspartic acid residue at position 10, or a corresponding mutation. In several embodiments, the Cas9 nicking enzyme comprises an amino acid sequence that is identical to any of the Cas9 nicking enzymes provided in this disclosure, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%. Any suitable Cas9 nicking enzyme will be readily apparent to those skilled in the art based on the disclosure herein and knowledge of the art, and falls within the scope of the disclosure herein.

[0574] Cas9 domain with reduced exclusivity

[0575] Typically, Cas9 proteins, such as Cas9 (spCas9) derived from Streptococcus pyogenes, require a canonical NGG PAM sequence to bind to a specific nucleic acid region, where "N" in "NGG" stands for adenine (A), thymine (T), or cytosine (C), and "G" stands for guanine. This can limit the ability to edit desired bases within a genome. In some embodiments, the base-editing fusion proteins provided in this disclosure may need to be placed at precise locations, such as a region containing the target base upstream of the PAM. See, for example, Komor, AC, et al., "Programmable base editing of target bases in genome DNA without double-stranded DNA division," Nature 533, 420-424 (2016), the entire disclosure of which is incorporated herein by reference. Accordingly, in some embodiments, any of the fusion proteins provided in this disclosure may contain a Cas9 domain capable of binding nucleotide sequences that do not contain a canonical (e.g., NGG) PAM sequence. The Cas9 domain, which is incorporated into non-canonical PAM sequences, has been described in the industry and is readily understood by those skilled in the art. For example, Cas9 domains binding to non-canonical PAM sequences have been described in Kleinstiver, BP, et al., "Modified CRISPR-Cas9 with altered PAM specificity" Nature 523, 481-485 (2015); and Kleinstiver, BP, et al., "Expanding the target range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition" Nature Biotechnology 33, 1293-1298 (2015); Nishimasu, H., et al., "Engineered CRISPR-Cas9 nuclease with expanded targeting space" Science. 2018 Sep 21; 361(6408):1259-1262; Chatterjee, P., et al., "Minimal PAM specificity of highly similar SpCas9 congeners" The specificity of PAM variants is highly similar to SpCas9 ortholog”)Sci Adv.2018Oct 24;4(10):eaau0766.doi:10.1126 / sciadv.aau0766, each of which is incorporated herein by reference in its entirety. Several PAM variants are described in Table 1 below.

[0576] Table 1. Cas9 protein and corresponding PAM sequence

[0577] Mutants PAM spCas9 NGG spCas9-VRQR NGA spCas9-VRER NGCG xCas9(sp) NGN saCas9 NNGRRT saCas9-KKH NNNRRT spCas9-MQKSER NGCG spCas9-MQKSER NGCN spCas9-LRKIQK NGTN spCas9-LRVSQK NGTN spCas9-LRVSQL NGTN SpyMacCas9 NAA Cpf1 5' (TTTV)

[0578] Cas9 complex with guide RNA

[0579] This disclosure provides several forms of complexes comprising any of the fusion proteins provided herein and a guide RNA (e.g., a guide RNA targeting a gene of interest). Any method for linking the fusion protein domains may be employed (e.g., self-form (GGGS)). n (GGGGS) n and (G) n Highly flexible linking base form (EAAAK) n (SGGS) n ,、SGSETPGTSESATPES (e.g., see Guilinger JP, Thompson DB, Liu DR. "Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification." Nat. Biotechnol. 2014; 32(6):577-82, the full disclosure of which is incorporated herein by reference) and (XP) n A more rigid linker is used to achieve the optimal length for nucleoside base editor activity. In several embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In several embodiments, the linker comprises (GGS). n The motif, where n is 1, 3, or 7. In several embodiments, the Cas9 domain of the fusion protein provided in this disclosure is fused via a linker comprising the amino acid sequence SGSETPGTSESATPES:.

[0580] In several embodiments, the guiding nucleic acid (e.g., guiding RNA) is 15-100 nucleotides in length and contains a sequence of at least 10 adjacent nucleotides that are complementary to the target sequence. In several embodiments, the guiding RNA is 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 nucleotides in length. In several embodiments, the guide RNA comprises a sequence of 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, or 40 adjacent nucleotides, which are complementary to the target sequence. In several embodiments, the target sequence is a DNA sequence. In several embodiments, the target sequence is a sequence in the genome of bacteria, yeast, fungi, insects, plants, or animals. In several embodiments, the target sequence is a sequence in the genome of humans. In several embodiments, the 3' end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In several embodiments, the target sequence is a sequence in the genome of humans. In several embodiments, the 3' end of the target sequence is immediately adjacent to a non-canonical PAM sequence (e.g., sequences listed in Table 1 or 5'-NAA-3'). In some embodiments, the guiding nucleic acid (e.g., guiding RNA) is complementary to a sequence in the gene of interest.

[0581] Several embodiments of this disclosure provide methods of using fusion proteins or complexes provided herein. For example, the methods provided in several embodiments of this disclosure include contacting a DNA molecule with any of the fusion proteins provided herein and with at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides in length and contains a sequence of at least 10 adjacent nucleotides that is complementary to a target sequence. In several embodiments, the 3' end of the target sequence is adjacent to an AGC, GAG, TTT, GTG, or CAA sequence. In several embodiments, the 3' end of the target sequence is adjacent to an NGA, NAA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, NGTN, NGTN, or 5' (TTTV) sequence.

[0582] It is important to understand that the numbering of specific positions or residues within an individual sequence depends on the specific protein and numbering scheme used. Numbering may differ, for example, between a precursor to a mature protein and the mature protein itself, and sequence differences between species may affect numbering. Through well-known methods, such as sequence alignment and homologous residue determination, skilled personnel will be able to identify individual residues in any homologous protein and in individual coding nucleic acids.

[0583] Those skilled in the art will readily recognize that, in order to target any of the fusion proteins described herein to a target site, such as a site containing the mutation to be edited, the fusion protein is typically expressed together with a guide RNA. As explained in further detail herein, the guide RNA typically comprises a tracrRNA framework that allows Cas9 binding; and a guide sequence that imparts sequence specificity to the Cas9:nuclease / domain fusion protein. Alternatively, the guide RNA and tracrRNA may be provided separately as two nucleic acid molecules. In several embodiments, the guide RNA comprises a structure in which the guide sequence includes a sequence complementary to the target sequence. The guide sequence is typically 20 nucleotides in length. Based on the disclosure herein, suitable guide RNA sequences for targeting Cas9:nuclease / domain fusion proteins to specific genomic target sites will be readily apparent to those skilled in the art. Such suitable guide RNA sequences typically include a guide sequence that is complementary to a nucleic acid sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. This disclosure provides several examples of suitable guide RNA sequences for targeting any provided fusion protein to a specific target sequence.

[0584] Methods of using fusion proteins containing a Cas9 domain and cytidine deaminase or adenosine deaminase

[0585] Several embodiments of this disclosure provide methods of using fusion proteins or complexes provided herein. For example, several embodiments of this disclosure provide methods comprising contacting a DNA molecule encoding a protein of interest with any of the fusion proteins provided herein, and contacting at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides in length and contains a sequence of at least 10 adjacent nucleotides that is complementary to the target sequence. In several embodiments, the 3' end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In several embodiments, the 3' end of the target sequence is not immediately adjacent to a canonical PAM sequence (NGG). In several embodiments, the 3' end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence. In several embodiments, the 3' end of the target sequence is immediately adjacent to an NGA, NAA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, NGTN, NGTN, or 5' (TTTV) sequence.

[0586] Additional domain

[0587] The base editor described herein may include any domain that facilitates nucleoside base editing, nucleoside base modification, or alteration of polynucleotides. In several embodiments, the base editor includes a polynucleotide programmable nucleotide-binding domain (e.g., Cas9), a nucleoside base editing domain (e.g., a deaminase domain), and one or more additional domains. In some cases, the additional domains may assist the enzymatic or catalytic function of the base editor, the binding function of the base editor, or act as an inhibitor of cellular structures (e.g., enzymes) that may interfere with the desired base editing outcome. In several embodiments, the base editor may include a nuclease, nickase, recombinase, deaminase, methyltransferase, methylase, acetylase, acetyltransferase, transcription activator, or transcription repressor domain.

[0588] In several embodiments, the base editor may include a uracil glycosylase inhibitor (UGI) domain. The UGI domain can, for example, improve the efficiency of a base editor containing a cytidine deaminase domain by inhibiting the conversion of U, formed by C deamination, back to a C nucleoside base. In some cases, the DNA repair response in the presence of U:G heterodiploid DNA may result in reduced nucleoside base editing efficiency in the cell. In such cases, uracil DNA glycosylase (UDG) catalyzes the removal of U from the cell's own DNA, which can trigger base excision repair (BER), mostly resulting in the reversal of U:G pairs to C:G pairs. In such cases, BER can be inhibited in a base editor containing one or more domains that bind to single strands, block the edited base, inhibit UGI, inhibit BER, protect the edited base, and / or promote the repair of non-edited strands. Thus, this document discloses a base editor fusion protein contemplated to include a UGI domain.

[0589] In several embodiments, the base editor comprises all or part of a double-strand break (DSB) binding protein as a domain. For example, the DSB binding protein may include the Gam protein of phage μ, which binds to the DSB terminus and protects the DSB from degradation. See Komor, AC, et al., "Improved base excision repair inhibition and C:G-to-T:A base editor obtained from phage μGam protein with higher efficiency and product purity", Science Advances 3:eaao4774 (2017); the disclosure of which is incorporated herein by reference in its entirety.

[0590] In several embodiments, the base editor comprises all or part of a nucleic acid polymerase (NAP) as a domain. For example, the base editor may comprise all or part of a prokaryotic NAP. In several embodiments, the NAP or a portion thereof bound to the base editor is a DNA polymerase. In several embodiments, the NAP or a portion thereof bound to the base editor has transpathogenic polymerase activity. In several embodiments, the NAP or a portion thereof bound to the base editor is a transpathogenic DNA polymerase. In several embodiments, the NAP or a portion thereof bound to the base editor is a Rev7, Rev1 complex, polymerase I, polymerase K, or polymerase N. In several embodiments, the NAP or a portion thereof bound to the base editor is a eukaryotic polymerase α, β, γ, δ, ε, γ, N, I, K, λ, μ, or V component. In several embodiments, the NAP or a portion thereof incorporated into the base editor contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a nucleic acid polymerase (e.g., a transpathic DNA polymerase).

[0591] Base Editor System

[0592] The base editor system provided in this disclosure includes the following steps: (a) contacting a target nucleotide sequence of an individual's polynucleotide (e.g., double-stranded DNA or RNA, single-stranded DNA or RNA) with a base editor system comprising a nucleoside base editor (e.g., an adenosine base editor or a cytidine base editor) and a guide polynucleotide (e.g., gRNA), wherein the target nucleotide sequence contains the targeted nucleoside base pair; (b) inducing strand separation of the target region; (c) converting the first nucleoside base of the target nucleoside base pair in the single strand of the target region to a second nucleoside base; and (d) cleaving no more than one strand of the target region, wherein a third nucleoside base complementary to the first nucleoside base is replaced by a fourth nucleoside base complementary to the second nucleoside base. It should be understood that in some embodiments, step (b) is omitted. In some embodiments, the targeted nucleoside base pair is a plurality of nucleoside base pairs in one or more genes. In several embodiments, the base editor system provided in this disclosure is capable of multiplexing multiple nucleoside base pairs in one or more genes. In several embodiments, the multiple nucleoside base pairs are in the same gene. In several embodiments, the multiple nucleoside base pairs are in one or more genes, wherein at least one gene is located at a different locus.

[0593] In several embodiments, the cleaved single strand (cut strand) hybridizes to a guide nucleic acid. In several embodiments, the cleaved single strand is the opposite of the strand containing the first nucleoside base. In several embodiments, the base editor comprises Cas9. In several embodiments, the first base is adenine, and the second base is not G, C, A, or T. In several embodiments, the second base is inosine.

[0594] The base editor system disclosed herein provides a novel method for genome editing that uses a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, cytidine deaminase, and a base excision repair inhibitor to induce programmable single nucleotide (C→T or A→G) changes in DNA without generating double-stranded DNA breaks, without requiring a donor DNA template, and without inducing excessive random insertions or deletions.

[0595] This disclosure provides systems, compositions, and methods for editing nucleoside bases using a base editor system. In several embodiments, the base editor system comprises (1) a base editor (BE) including a polynucleotide programmable nucleotide binding domain and a nucleoside base editing domain (e.g., a deaminase domain) for editing nucleoside bases; and (2) a guide polynucleotide (e.g., guide RNA) together with the polynucleotide programmable nucleotide binding domain. In several embodiments, the base editor system comprises a cytidine base editor (CBE). In several embodiments, the base editor system comprises an adenosine base editor (ABE). In several embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In several embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In several embodiments, the nucleoside base editing domain is a deaminase domain. In some cases, the deaminase domain may be cytosine deaminase or cytidine deaminase. In several embodiments, the terms "cytosine deaminase" and "cytidine deaminase" are used interchangeably. In some cases, the deaminase domain may be adenine deaminase or adenosine deaminase. In several embodiments, the terms "adenine deaminase" and "adenosine deaminase" are used interchangeably. Details of the nucleoside base editing protein are described in International PCT Applications Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), the full text of which is incorporated herein by reference. Please also refer to Komor, AC, et al., "Programmable editing of target bases in genomic DNA without double-strand DNA division," Nature 533, 420-424 (2016); Gaudelli, NM, et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA division," Nature 551, 464-471 (2017); and Komor, AC, et al., "Improved base excision repair inhibition and C:G-to-T:A base editor from phage μGam protein with higher efficiency and product purity," Science Advances 3:eaao4774 (2017); the full text of which is incorporated herein by reference.

[0596] In several embodiments, the base editor inhibits base excision repair of the edited strand. In several embodiments, the base editor protects or binds to the unedited strand. In several embodiments, the base editor contains UGI activity. In several embodiments, the base editor contains nicking enzyme activity. In several embodiments, the intended editing of the base pair is upstream of the PAM site. In several embodiments, the intended editing of the base pair is nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 upstream of the PAM site. In several embodiments, the intended editing of the base pair is downstream of the PAM site. In several embodiments, the intended editing of the base pair is nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 downstream of the PAM site.

[0597] In several embodiments, the method does not require a canonical (e.g., NGG) PAM site. In several embodiments, the nucleoside base editor includes a linker or spacer. In several embodiments, the linker or spacer is 1-25 amino acids long. In several embodiments, the linker or spacer is 5-20 amino acids long. In several embodiments, the linker or spacer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long.

[0598] In several embodiments, the target region includes a target window containing a target nucleoside base pair. In several embodiments, the target window contains 1-10 nucleotides. In several embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In several embodiments, the intended editing of the base pairs takes place within the target window. In several embodiments, the target window contains the intended editing of the base pairs. In several embodiments, the method is performed using any of the base editors provided in this disclosure. In several embodiments, the target window is a deamination window.

[0599] In several embodiments, the base editor is a cytidine base editor (CBE). In several embodiments, non-limiting examples of the CBE are BE1 (APOBEC1-XTEN-dCas9), BE2 (APOBEC1-XTEN-dCas9-UGI), BE3 (APOBEC1-XTEN-dCas9(A840H)-UGI), BE3-Gam, saBE3, saBE4-Gam, BE4, BE4-Gam, saBE4, or saB4E-Gam. BE4 extends the linker APOBEC1-Cas9n(D10A) to 32 amino acids, extends the Cas9n-UGI linker to 9 amino acids, and adds a second copy of UGI to the C-terminus of a construct having another 9-amino acid linker, thus becoming a single base editor construct. Base editors saBE3 and saBE4 have Streptococcus pyogenes Cas9n(D10A), which is replaced by Staphylococcus aureus Cas9n(D10A). BE3-Gam, saBE3-Gam, BE4-Gam, and saBE4-Gam have 174 residues of the Gam protein, which are fused to the N-terminus of BE3, saBE3, BE4, and saBE4 via a 16-amino acid XTEN linker.

[0600] In several embodiments, the base editor is an adenosine base editor (ABE). In several embodiments, the adenosine base editor is capable of deamination of adenine in DNA. In several embodiments, the ABE is generated via APOBEC1, a component in which BE3 is replaced by natural or modified E. coli TadA, human ADAR2, mouse ADA, or human ADAT2. In several embodiments, the ABE comprises an evolved TadA variant. In several embodiments, the ABE is ABE 1.2 (TadA*-XTEN-nCas9-NLS). In several embodiments, TadA* comprises the A106V and D108N mutations.

[0601] In several embodiments, ABE is a second-generation ABE. In several embodiments, ABE is ABE2.1, which contains the additional mutations D147Y and E155V in TadA* (TadA*2.1). In several embodiments, ABE is ABE2.2, where ABE2.1 is fused to a catalytically inactivated version of human alkyladenine DNA glycosylase (AAG with the E125Q mutation). In several embodiments, ABE is ABE2.3, where ABE2.1 is fused to a catalytically inactivated version of E. coli Endo V (inactivated with the D35A mutation). In several embodiments, ABE is ABE2.6, which has a linker twice as long as the linker in ABE2.1 (32 amino acids, (SGGS)2-XTEN-(SGGS)2). In several embodiments, ABE is ABE2.7, which is ABE2.1 plugged with an additional wild-type TadA monomer. In several embodiments, ABE is ABE2.8, which is ABE2.1 plugged with an additional TadA*2.1 monomer. In several embodiments, ABE is ABE2.9, which is an evolved TadA (TadA*2.1) directly fused to the N-terminus of ABE2.1. In several embodiments, ABE is ABE2.10, which is wild-type TadA directly fused to the N-terminus of ABE2.1. In several embodiments, ABE is ABE2.11, which is ABE2.9 having an inactivated E59A mutation at the N-terminus of the TadA* monomer. In several embodiments, ABE is ABE2.12, which is ABE2.9 having an inactivated E59A mutation in the internal TadA* monomer.

[0602] In some embodiments, ABE is a third-generation ABE. In some embodiments, ABE is ABE3.1, which is ABE2.3 with three additional TadA mutations (L84F, H123Y, and I157F).

[0603] In some embodiments, ABE is a fourth-generation ABE. In some embodiments, ABE is ABE4.3, which is ABE3.1 with an additional TadA mutation A142N (TadA*4.3).

[0604] In several embodiments, the ABE is a fifth-generation ABE. In several embodiments, the ABE is ABE5.1, which is generated by introducing a set of allelic mutations (H36L, R51L, S146C, and K157N) from surviving clones into ABE3.1. In several embodiments, the ABE is ABE5.3, which has a heterodimeric construct containing wild-type E. coli TadA fused into an internally evolved TadA*. In several embodiments, the ABE is ABE5.2, ABE5.4, ABE5.5, ABE5.6, ABE5.7, ABE5.8, ABE5.9, ABE5.10, ABE5.11, ABE5.12, ABE5.13, or ABE5.14, as shown in Table 2 below. In several embodiments, the ABE is a sixth-generation ABE. In some embodiments, ABE is ABE6.1, ABE6.2, ABE6.3, ABE6.4, ABE6.5, or ABE6.6, as shown in Table 2 below. In some embodiments, ABE is the seventh generation ABE. In some embodiments, ABE is ABE7.1, ABE7.2, ABE7.3, ABE7.4, ABE7.5, ABE7.6, ABE7.7, ABE7.8, ABE7.9, or ABE7.10, as shown in Table 2 below.

[0605] Table 2. Genotypes of ABE

[0606]

[0607]

[0608] In several embodiments, the base editor is a fusion protein comprising a multinucleotide programmable nucleotide-binding domain (e.g., a Cas9-derived domain) fused to a nucleoside base editing domain (e.g., all or part of a deaminase domain). In several embodiments, the base editor further comprises a domain comprising all or part of a uracil glycosylase inhibitor (UGI). In several embodiments, the base editor comprises a domain comprising all or part of a uracil-binding protein (UBP), such as uracil DNA glycosylase (UDG). In several embodiments, the base editor comprises a domain comprising all or part of a nucleic acid polymerase. In several embodiments, the nucleic acid polymerase or a portion thereof bound to the base editor is a transpathogenic DNA polymerase.

[0609] In several embodiments, a domain of the base editor may comprise multiple domains. For example, a base editor comprising a polynucleotide programmable nucleotide binding domain derived from Cas9 may comprise a REC leaflet, a NUC leaflet corresponding to the REC leaflet, and a NUC leaflet of wild-type or natural Cas9. In another embodiment, the base editor may comprise one or more of the RuvCI domain, BH domain, REC1 domain, REC2 domain, RuvCII domain, L1 domain, HNH domain, L2 domain, RuvCIII domain, WED domain, TOPO domain, or CTD domain. In several embodiments, one or more domains of ...

Claims

1. A method for editing a SERPINA1 polynucleotide comprising a single nucleotide polymorphism (SNP) associated with alpha-1 antitrypsin deficiency (A1AD) in vitro or in vitro, wherein the SNP is located in the SERPINA1 polynucleotide and results in the expression of a PiZ mutant of an alpha-1 antitrypsin (A1AT) protein having a lysine at amino acid position 342 or a PiS mutant of the A1AT protein having a valine at amino acid position 264, the method comprising contacting the SERPINA1 polynucleotide with a base editor complexed with one or more guide polynucleotides, wherein the base editor comprises, from N-terminus to C-terminus, apolipoprotein B mRNA editing complex 1 (APOBEC1) cytidine deaminase domain, a first linker, a Streptococcus pyogenes Cas9 nickase polynucleotide programmable DNA binding domain specific for a protospacer-adjacent motif having the sequence 5'-NGG-3', a second linker, a uracil glycosylase inhibitor (UGI) domain, a third linker, a second UGI domain, and a nuclear localization signal (NLS), and wherein the one or more guide polynucleotides target the base editor to deaminize the SERPINA1 polynucleotide cytidine at position 1455, thereby inducing a mutation of the A1AT protein encoded by the SERPINA1 polynucleotide at amino acid position 374 to isoleucine.

2. The method of claim 1, wherein the first linker consists of 32 amino acids.

3. The method of claim 2, wherein the first linking base consists of an amino acid sequence of SGGSSGGSSGSETPGTSESATPESSGGSSGGS.

4. The method of claim 1, wherein the second linker and the third linker consist of 9 amino acids.

5. The method of claim 1, wherein the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV.

6. The method of claim 1, wherein the APOBEC1 cytidine deaminase domain is a rat APOBEC-1 cytidine deaminase domain.

7. The method of claim 6, wherein the APOBEC1 cytidine deaminase domain consists of the following amino acid sequence: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIY IARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK.

8. The method of claim 1, wherein the contacting is in a cell.

9. The method of claim 8, wherein the cell is a eukaryotic cell.

10. The method of claim 8, wherein the cell is a mammalian cell.

11. The method of claim 8, wherein the cell is a human cell.

12. The method of claim 1, wherein the Streptococcus pyogenes Cas9 nickase comprises the amino acid substitution D10A.

13. The method of any one of claims 1 to 11, wherein the cytidine deaminase domain is capable of deaminating cytidine in deoxyribonucleic acid.

14. The method of claim 13, wherein the cytidine deaminase domain is a modified cytidine deaminase domain that does not occur in nature.

15. The method of any one of claims 1 to 11, wherein the one or more guide polynucleotides comprise CRISPR RNA (crRNA) and trans-micromolecule RNA, wherein the crRNA comprises a nucleic acid sequence complementary to a SERPINA1 nucleic acid sequence, and the SERPINA1 nucleic acid sequence comprises a SNP associated with A1AD. 16 . The method of any one of claims 1 to 11 , wherein the guide polynucleotide is a single guide RNA, and the sgRNA comprises a nucleic acid sequence complementary to the SERPINA1 nucleic acid sequence encoding methionine 374.

17. A cell produced by introducing into the interior of the cell: introducing a base editor, or a polynucleotide encoding the base editor, into the cell, wherein the base editor comprises, from N-terminus to C-terminus, an apolipoprotein B mRNA editing complex 1 (APOBEC1) cytidine deaminase domain, a first linker, a Streptococcus pyogenes Cas9 nickase polynucleotide programmable DNA binding domain specific for a protospacer-adjacent motif having the sequence 5'-NGG-3', a second linker, a uracil glycosylase inhibitor (UGI) domain, a third linker, a second UGI domain, and a nuclear localization signal (NLS); and One or more guide polynucleotides of the base editor are targeted to deaminize cytidine at nucleic acid position 1455 of a SERPINA1 polynucleotide, thereby inducing a methionine to isoleucine mutation at amino acid position 374 of an alpha-1 antitrypsin (A1AT) protein encoded by the SERPINA1 polynucleotide in a cell, wherein the cell comprises a single nucleotide polymorphism (SNP) associated with alpha-1 antitrypsin deficiency (A1AD), and wherein the SNP is located in the SERPINA1 polynucleotide and results in expression of a PiZ mutant of the A1AT protein having lysine at amino acid position 342 or a PiS mutant of the A1AT protein having valine at amino acid position 264.

18. The cell of claim 17, wherein the first linker consists of 32 amino acids.

19. The cell of claim 18, wherein the first linker consists of an amino acid sequence of SGGSSGGSSGSETPGTSESATPESSGGSSGGS.

20. The cell of claim 17, wherein the second linker and the third linker consist of 9 amino acids.

21. The cell of claim 17, wherein the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV.

22. The cell of claim 17, wherein the APOBEC1 cytidine deaminase domain is a rat APOBEC-1 cytidine deaminase domain.

23. The cell of claim 22, wherein the APOBEC1 cytidine deaminase domain consists of the following amino acid sequence: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIY IARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK.

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

25. The cell of any one of claims 17 to 24, wherein the cell or a progenitor thereof is an induced pluripotent stem cell.

26. The cell of claim 25, wherein the hepatocyte expresses A1AT polypeptide.

27. The cell of claim 17, wherein the cell is from an individual suffering from A1AD.

28. The cell of claim 17, wherein the cell is a mammalian cell.

29. The cell of claim 17, wherein the cell is a human cell.

30. The cell of claim 17, wherein the cell is selected for deamination of cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide.

31. The cell of claim 17, wherein the S. pyogenes Cas9 nickase comprises the amino acid substitution D10A.

32. The cell of claim 17, wherein the cytidine deaminase domain is capable of deaminating cytidine in DNA.

33. The cell of claim 32, wherein the cytidine deaminase domain is a modified cytidine deaminase domain that does not occur in nature.

34. The cell of claim 17, wherein the one or more guide polynucleotides comprise CRISPR RNA (crRNA) and trans-acting microRNA, wherein the crRNA comprises a nucleic acid sequence complementary to the cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide.

35. The cell of claim 17, wherein the guide polynucleotide is a single guide RNA, the sgRNA comprising a nucleic acid sequence complementary to the cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide.

36. An isolated cell or cell population proliferated or expanded from a cell as claimed in any one of claims 17 to 35.

37. A base editor or a polynucleotide encoding the base editor and one or more guide polynucleotides for the manufacture of a medicament for treating A1AD in an individual in need thereof, wherein the individual comprises a SERPINA1 polynucleotide comprising a single nucleotide polymorphism (SNP) associated with alpha-1 antitrypsin deficiency (A1AD), wherein the SNP is located in the SERPINA1 polynucleotide and results in the expression of a PiZ mutant of an alpha-1 antitrypsin (A1AT) protein having a lysine at amino acid position 342 or a PiS mutant of an A1AT protein having a valine at amino acid position 264, The base editor comprises, from the N-terminus to the C-terminus, an apolipoprotein B mRNA editing complex 1 (APOBEC1) cytidine deaminase domain, a first linker, a Streptococcus pyogenes Cas9 nickase polynucleotide programmable DNA binding domain specific for a protospacer adjacent motif having the sequence 5'-NGG-3', a second linker, a uracil glycosylase inhibitor (UGI) domain, a third linker, a second UGI domain, and a nuclear localization signal (NLS), and The one or more guide polynucleotides target the base editor to achieve a change in the cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide, thereby inducing a mutation of methionine to isoleucine at amino acid position 374 of the A1AT protein encoded by the SERPINA1 polynucleotide.

38. The use of claim 37, wherein the first linker consists of 32 amino acids.

39. The use as claimed in claim 38, wherein the first linking group consists of an amino acid sequence of SGGSSGGSSGSETPGTSESATPESSGGSSGGS.

40. The use as claimed in claim 37, wherein the second linker and the third linker consist of 9 amino acids.

41. The use of claim 37, wherein the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV.

42. The use of claim 37, wherein the APOBEC1 cytidine deaminase domain is a rat APOBEC-1 cytidine deaminase domain.

43. The use of claim 42, wherein the APOBEC1 cytidine deaminase domain consists of the following amino acid sequence: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIY IARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK.

44. The use of claim 37, wherein the subject is a mammal.

45. The use of claim 37, wherein the subject is a human.

46. ​​The use of claim 37, wherein the Streptococcus pyogenes Cas9 nickase comprises the amino acid substitution D10A.

47. The method of claim 37, wherein the cytidine deaminase domain is capable of deaminating cytidine in deoxyribonucleic acid.

48. The use of claim 47, wherein the cytidine deaminase domain is a modified cytidine deaminase domain that does not occur in nature.

49. The use of claim 47 or 48, wherein the cytidine deaminase domain is an APOBEC deaminase domain.

50. The use of claim 37, wherein the one or more guide polynucleotides comprise CRISPR RNA (crRNA) and trans-microRNA, wherein the crRNA comprises a nucleic acid sequence complementary to a SERPINA1 nucleic acid sequence.

51. The use of claim 37, wherein the guide polynucleotide is a single guide RNA, the sgRNA comprising a nucleic acid sequence complementary to the cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide.

52. The use of claim 50, wherein the cytidine deaminase domain is a modified cytidine deaminase domain that does not occur in nature.

53. The use of claim 50 or 51, wherein the cytidine deaminase domain is an APOBEC deaminase domain.

54. The use of claim 50, wherein the base editor is complexed with a single guide RNA, the sgRNA comprising a nucleic acid sequence complementary to the cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide.

55. A method for producing hepatocytes in vitro, comprising (a) introducing into a hepatocyte progenitor cell comprising a single nucleotide polymorphism (SNP) associated with alpha-1 antitrypsin deficiency (A1AD), wherein the SNP is located in a SERPINA1 polynucleotide and results in the expression of a PiZ mutant of the alpha-1 antitrypsin (A1AT) protein having a lysine at amino acid position 342 or a PiS mutant of the A1AT protein having a valine at amino acid position 264, A base editor, or a Cas9 polynucleotide encoding the base editor, wherein the base editor comprises, from N-terminus to C-terminus, an apolipoprotein B mRNA editing complex 1 (APOBEC1) cytidine deaminase domain, a first linker, a Streptococcus pyogenes Cas9 nickase polynucleotide programmable DNA binding domain specific for a protospacer-adjacent motif having the sequence 5'-NGG-3', a second linker, a uracil glycosylase inhibitor (UGI) domain, a third linker, a second UGI domain, and a nuclear localization signal (NLS); and one or more guide polynucleotides, wherein the one or more guide polynucleotides target the base editor to deaminize the cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide, thereby inducing a mutation of methionine to isoleucine at amino acid position 374 of the A1AT protein encoded by the SERPINA1 polynucleotide, and (b) Differentiating the hepatocyte progenitor cells into hepatocytes.

56. The method of claim 55, wherein the first linker consists of 32 amino acids.

57. The method of claim 56, wherein the first linking group consists of an amino acid sequence of SGGSSGGSSGSETPGTSESATPESSGGSSGGS.

58. The method of claim 55, wherein the second linker and the third linker consist of 9 amino acids.

59. The method of claim 55, wherein the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV.

60. The method of claim 55, wherein the APOBEC1 cytidine deaminase domain is a rat APOBEC-1 cytidine deaminase domain.

61. The method of claim 60, wherein the APOBEC1 cytidine deaminase domain consists of the following amino acid sequence: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIY IARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK.

62. The method of claim 55, wherein the hepatocyte progenitor cells express A1AT polypeptide.

63. The method of any one of claims 55-62, wherein the hepatocyte progenitor cells are obtained from an individual with A1AD.

64. The method of claim 55, wherein the hepatocyte progenitor cell is a mammalian cell.

65. The method of claim 55, wherein the hepatocyte progenitor cells are human cells.

66. The method of claim 55, wherein the polynucleotide programmable DNA binding domain is an inactivated nuclease or nickase.

67. The method of claim 55, wherein the S. pyogenes Cas9 nickase comprises the amino acid substitution D10A.

68. The method of claim 55, wherein the cytidine deaminase domain is capable of deaminating cytidine in deoxyribonucleic acid (DNA).

69. The method of claim 55, wherein the one or more guide polynucleotides comprise CRISPR RNA (crRNA) and trans-micromolecule RNA, wherein the crRNA comprises a nucleic acid sequence complementary to the cytidine at nucleic acid position 1455 of the SERPINA1 polynucleotide.

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