Method for editing single nucleotide polymorphism by using programmable base editor system

Abstract

The present invention provides a composition and a method of using a base editor comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain that binds a guide polynucleotide. The invention also provides a base editor system for editing nucleobases of a target nucleotide sequence.

Description

[0001] This application is a divisional application of the PCT patent application entered into China with Chinese patent application number 201980046479.3, invention name “Method for editing single nucleotide polymorphisms using a programmable base editor system”, and application date May 11, 2019.

[0002] Related applications

[0003] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 670,588, filed May 11, 2018, U.S. Provisional Application No. 62 / 780,838, filed December 17, 2018, and U.S. Provisional Application No. 62 / 817,986, filed March 13, 2019, the entire contents of which are incorporated herein by reference. Background Art

[0004] For most known genetic diseases, to study or solve the root cause of the disease, it is necessary to correct point mutations in the target locus, rather than random destruction of genes. Current genome editing technology uses clustered regularly interspaced short palindromic repeats (CRISPR) system to introduce double-stranded DNA breaks at the target locus as the first step of gene correction. In response to double-stranded DNA breaks, the cellular DNA repair process usually causes random insertions or deletions (Indels) of the DNA cut site by non-homologous end joining (NHEJ). Although most genetic diseases are caused by point mutations, current point mutation correction methods are inefficient and usually cause a large number of random insertions and deletions (Indels) on the target locus, which is due to the response of the cell to dsDNA breaks. Therefore, there is a need for an improved form of genome editing that is more efficient and has fewer by-products, such as random insertions or deletions (Indels) or translocations.

[0005] Alpha-1 antitrypsin deficiency (A1AD) is a genetic disease in which pathogenic mutations in the SERPINA1 gene, which encodes the alpha-1 antitrypsin (A1AT) protein, result in reduced protein production in individuals with the disease. A1AT is a particularly good inhibitor of neutrophil elastase, which protects tissues and organs such as the lungs from elastin degradation. As a result, the elastin in the lungs of patients with A1AD is more susceptible to degradation 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 a protease inhibitor.

[0006] The most common pathogenic A1AT variant is a guanine to adenine (G→A) mutation in the SERPINA1 gene, resulting in a glutamic acid to lysine substitution at amino acid position 342 of the A1AT protein. This substitution causes the protein to misfold and aggregate within hepatocytes. Ultimately, the toxic aggregates can lead to liver damage and cirrhosis. Although liver toxicity can be addressed through gene knockout (CRISPR / ZFN / TALEN) or gene knockout (siRNA), these approaches do not address the lung pathology. Although lung pathology can be addressed with protein replacement therapy, this therapy does not address liver toxicity. Gene therapy is also insufficient to address A1AT gene defects. Because the livers of A1AD patients are already burdened by a significant disease burden caused by endogenous A1AT aggregation, gene therapy to increase A1AT in the liver would be counterproductive. Therefore, there is a need for a method of treating patients with A1AD that simultaneously addresses both the lung pathology and liver toxicity associated with the disease.

[0007] References

[0008] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Unless otherwise indicated, the publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety. Summary of the Invention

[0009] As described herein, the present invention provides a composition and method for accurately correcting pathogenic amino acids in proteins associated with diseases or disorders using a programmable nucleobase editor. In a specific aspect, the composition and method can be used to treat alpha-1 antitrypsin deficiency (A1AD). In one embodiment, the composition and method for treating A1AD utilizes an adenosine (A) base editor (ABE-(NGC variant)) to accurately correct harmful single nucleotide polymorphisms (SNPs) in the endogenous SERPINA1 gene. In one embodiment, the composition and method correct the harmful mutation E342K, which affects the activity and function of the encoded alpha-1 antitrypsin (A1AT) protein. This correction simultaneously eliminates the pathogenic protein burden in the liver and restores lung function protein.

[0010] On the one hand, the present invention provides a method for editing a SERPINA1 polynucleotide comprising a single nucleotide polymorphism (SNP) associated with alpha 1 antitrypsin deficiency (A1AD), wherein the method includes contacting the SERPINA1 polynucleotide with a base editor, the base editor being compounded with one or more guide polynucleotides, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein the one or more guide polynucleotides target the base editor, thereby changing the A·T to G·C of the SNP in the SERPINA1 gene associated with A1AD. In one embodiment, the method includes contacting a cell, such as a eukaryotic cell, a mammalian cell, or a human cell. In another embodiment, the cell is in vivo or in vitro.

[0011] On the other hand, the present invention features a cell produced by introducing a base editor, a polynucleotide encoding the base editor, into a cell or its progenitor cell, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain. And one or more guiding polynucleotides targeting the base editor to cause an A·T to G·C change in a SNP in a gene associated with A1AD (eg, the SERPINA1 gene). In one embodiment, the cell produced is a hepatocyte. In another embodiment, the cell or its progenitor cell is an embryonic stem cell, an induced pluripotent stem cell, or a hepatocyte. In another embodiment, the hepatocyte expresses an A1AT polypeptide. In another embodiment, the cell is from a subject with AlAD. In another embodiment, the cell is a mammalian cell or a human cell.

[0012] In another aspect, the invention features a method of treating A1AD in a subject, the method comprising administering to the subject in need thereof a cell as described in the aspects and embodiments described above. In one embodiment, the cell is autologous, allogeneic, or xenogeneic to the subject.

[0013] In another aspect, the invention features an isolated cell or population of cells propagated or expanded from the cell of any of the above aspects.

[0014] In another aspect, the present invention features a method for treating A1AD in a subject, wherein the method comprises administering a base editor or a polynucleotide encoding the base editor to a subject in need thereof, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and one or more guide polynucleotides targeting the base editor to cause an A·T to G·C change in a SNP in a gene associated with A1AD (e.g., the SERPINA1 gene). In one embodiment, the subject is a mammal or a human. In another embodiment, the method involves delivering a base editor or a polynucleotide encoding a base editor and one or more guide polynucleotides to a cell of the subject. 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 an A1AT polypeptide containing a mutation.

[0015] In another aspect, the present invention features a method for producing hepatocytes or progenitor cells thereof, wherein the method involves (a) introducing an induced pluripotent stem cell or hepatocyte progenitor cell containing a SNP in a gene associated with A1AD (e.g., the SERPINA1 gene), a base editor, or a polynucleotide encoding a base editor, wherein the base editor comprises a polynucleotide programmable nucleotide binding domain and an adenosine deaminase domain; and one or more guide polynucleotides, wherein the one or more guide polynucleotides target the base editor to cause an A·T to G·C change in the SNP associated with A1AD; (b) differentiating the induced pluripotent stem cell or hepatocyte progenitor cell into a hepatocyte. In one embodiment, the method involves differentiating an induced pluripotent stem cell into a hepatocyte or its progenitor cell. In another embodiment, the induced pluripotent stem cell comprises an E342K mutation. In another embodiment, the hepatocyte progenitor cell is obtained from a subject suffering from A1AD. In another embodiment, the hepatocyte or hepatocyte progenitor cell is a mammalian cell or a human cell.

[0016] In another aspect, the compositions and methods described herein for use in base editors (BEs) include polypeptides comprising an amino acid sequence: (i)

[0017]

[0018] In another aspect, the invention features a guide RNA (gRNA) comprising the following nucleic acid sequence:

[0019] 5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUU-3';

[0020] 5'-ACCAUCGACAAGAAAGGGACUGAGUUUUAUAGAGAGUAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUU-3';

[0021] 5'-CCAUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGGCUUUU-3';

[0022] 5'-CAUCGACAAGAAAGGGACUGAGUUUUAGAGGAGAGUAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUU-3';

[0023] 5'-AUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUU-3';

[0024] 5′-UCGACAAGAAAGGGACUGAGUUUUAGAGGUGAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3′; and

[0025] 5'-CGACAAGAAAGGGACUGAGUUUUAGAGGUGAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUU-3'.

[0026] In another aspect, the invention features a protein-nucleic acid complex comprising the base editor of any of the preceding aspects or embodiments described herein and a guide RNA.

[0027] In various embodiments of the above aspects or any other aspects of the invention described herein, the A·T to G·C change at the SNP associated with A1AD changes the lysine in the A1AT polypeptide to glutamate. In various embodiments of the above-described aspects or any other aspects of the invention described herein, the SNP associated with A1AD results in the expression of an A1AT polypeptide with lysine at amino acid position 342. In another embodiment, the correction of the base editor is to replace the lysine at position 342 of the A1AT polypeptide associated with A1AD with glutamate. In various embodiments of the above aspects of the invention described above or any other aspects of the invention, the polynucleotide programmable DNA binding domain is a modified Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof.

[0028] In various embodiments of the above aspects of the invention described above or any other aspects of the invention, the polynucleotide programmable DNA binding domain comprises a modified SpCas9 with an altered protospacer adjacent motif (PAM) specificity. In various embodiments of the above aspects or any other aspects of the invention described herein, the modified SpCas9 is specific for the nucleic acid sequence 5'-AGC-3'. In various embodiments of the above aspects of the invention described herein or any other aspects, the modified SpCas9 comprises one or more of the amino acid substitutions D1332A and D1135M, S1136Q, G1218K, E1219F, D1332A, R1335E and T1337R, or the corresponding amino acids thereof. In various embodiments of the above aspects of the invention described above or any other aspects of the invention, the polynucleotide programmable DNA binding domain comprises a variant of SpCas9 with an altered protospacer adjacent motif (PAM) specificity. In various embodiments of the above aspects or any other aspects of the invention described herein, the variant of SpCas9 is specific for the nucleic acid sequence 5'-NGC-3'. In various embodiments of the above aspects or any other aspects of the invention described herein, the modified SpCas9 comprises amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E and T1337R, or their corresponding amino acid substitutions. In various embodiments of the above aspects or any other aspects of the invention described herein, the polynucleotide programmable DNA binding domain is a nuclease inactive or nickase variant. In various embodiments of the above aspects of the invention described above or any other aspects of the invention, the nickase variant comprises an amino acid substitution D10A or its corresponding amino acid substitution. In various embodiments of the above aspects of the invention described above or any other aspects of the invention, the adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA). In various embodiments of the above aspects or any other aspects of the invention described herein, the adenosine deaminase is a modified adenosine deaminase that does not exist in nature. In various embodiments of the above aspects or any other aspects of the invention described herein, the adenosine deaminase is a TadA deaminase (e.g., TadA*7.10). In various embodiments of the above aspects or any other aspects of the invention described herein, the one or more guide RNAs comprise CRISPR RNA (crRNA) and trans-encoded small RNA (tracrRNA), wherein the crRNA comprises a nucleic acid sequence complementary to a SERPINA1 nucleic acid sequence, wherein the SERPINA1 nucleic acid sequence comprises a SNP associated with A1AD.In various embodiments of the above aspects, the base editor is complexed with a single-stranded guide RNA (sgRNA), which comprises a nucleic acid sequence complementary to a SERPINA1 nucleic acid sequence, wherein the SERPINA1 nucleic acid sequence comprises a SNP associated with A1AD.

[0029] On the other hand, the present invention provides a base editor system for correcting pathogenic single nucleotide polymorphisms (SNPs) in genes, wherein the base editor system comprises (a) a base editor comprising: (i) a polynucleotide programmable DNA binding domain, and (ii) a deaminase domain capable of deaminating a pathogenic SNP or its complementary nucleobase; (b) a guide polynucleotide bound to the polynucleotide programmable DNA binding domain, wherein the guide polynucleotide targets the base editor to a target polynucleotide sequence, at least a portion of which is located in the gene or its reverse complementary sequence; wherein deamination of the pathogen SNP or its complementary nucleobase results in conversion of the pathogen SNP to its wild-type allele, thereby correcting the pathogenic mutation, such as listed in Tables 3A and 3B herein.

[0030] On the other hand, provided herein is a method for correcting a pathogenic single nucleotide polymorphism (SNP) in a gene, wherein the method comprises: contacting with a target nucleotide sequence, at least a portion of which is located in the gene or its reverse complementary sequence, wherein the base editor comprises: (i) a polynucleotide-programmable DNA binding domain that binds to a guide polynucleotide that targets the base editor to a target polynucleotide sequence, at least a portion of which is located in the gene or its reverse complementary sequence (ii) a deaminase domain capable of deaminating the pathogenic SNP or its complementary nucleobase; and editing the pathogenic SNP by deaminating the pathogenic SNP or its complementary nucleobase after targeting the base editor to the target nucleotide sequence, wherein deaminating the pathogenic SNP or its complementary nucleobase causes the pathogenic SNP to be converted into its wild-type allele, thereby correcting the pathogenic mutation, such as listed in Table 3A or Table 3B herein.

[0031] On the other hand, the present invention provides a method for treating a genetic disease in a subject by correcting a pathogenic single nucleotide polymorphism (SNP) in a gene, wherein the method comprises administering a base editor or a polynucleotide encoding the base editor to a subject in need thereof, wherein the base editor comprises: (i) a polynucleotide-programmable DNA binding domain, and (ii) a deaminase domain capable of deaminating the pathogenic SNP or its complementary nucleobase; administering a guide polynucleotide to the subject, wherein the guide polynucleotide targets the base editor to a target nucleotide sequence, at least a portion of the target nucleotide sequence being located in the gene or its reverse complement sequence; and editing the pathogenic SNP by deaminating the pathogenic SNP or its complementary nucleobase after targeting the base editor to the target nucleotide sequence, wherein deaminating the pathogenic SNP or its complementary nucleobase causes the pathogenic SNP to be converted to its wild-type allele, thereby correcting the pathogenic mutation listed in Table 3A or 3B, and treating the genetic disease.

[0032] Provided herein is a method for producing cells, tissues or organs for treating genetic diseases in subjects in need thereof by correcting pathogenic single nucleotide polymorphisms (SNPs) in genes of the cells, tissues or organs, wherein the method comprises: contacting the cells, tissues or organs with a base editor, wherein the base editor comprises: (i) a polynucleotide-programmable DNA binding domain, and (ii) a deaminase domain capable of deaminating the pathogenic SNP or its complementary nucleobase; and contacting the cells, tissues or organs with a guide polynucleotide, wherein the guide polynucleotide targets the base editor to a target nucleotide sequence, at least a portion of which is located in the gene or its reverse complement; and editing the pathogenic SNP by deaminating the pathogenic SNP or its complementary nucleobase after targeting the base editor to the target nucleotide sequence, wherein deaminating the pathogenic SNP or its complementary nucleobase causes the pathogenic SNP to be converted to its wild-type allele, thereby correcting a pathogenic mutation such as listed in Table 3A or 3B, and producing cells, tissues or organs for treating genetic diseases. In some embodiments, the method further comprises administering the cells, tissue, or organ to the subject. In some embodiments, the cells, tissue, or organ are autologous to the subject. In some embodiments, the cells, tissue, or organ are allogeneic to the subject. In some embodiments, the cells, tissue, or organ are xenogeneic to the subject.

[0033] In some embodiments, the pathogenic SNP is associated with Stargardt disease; optionally, the pathogenic SNP is in the ABCA4 gene; and, optionally, the pathogenic mutation comprises A1038V, L541P, G1961E, or a combination thereof. In some embodiments, the pathogenic SNP is associated with pseudoxanthoma elasticum. Optionally, the pathogenic SNP is in the ABCC6 gene; and, optionally, the pathogenic mutation comprises R1141*. In some embodiments, the pathogenic SNP is associated with medium-chain acyl-CoA dehydrogenase deficiency; optionally, the pathogenic SNP is in the ACADM gene; and, optionally, the pathogenic mutation comprises K329E. In some embodiments, the pathogenic SNP is associated with severe combined immunodeficiency; optionally, the pathogenic SNP is in the ADA gene; and, optionally, the pathogenic mutation comprises G216R, Q3*, or a combination thereof.

[0034] In some embodiments, the pathogenic SNP is associated with primary hypohematuria. Optionally, the pathogenic SNP is in the AGXT gene; and, optionally, the pathogenic mutation includes G170R. In some embodiments, the pathogenic SNP is associated with autosomal recessive hypercholesterolemia; optionally, the pathogenic SNP is in the ARH gene; and, optionally, the pathogenic mutation includes Q136*. In some embodiments, the pathogenic SNP is associated with heterochromatic leukodystrophy. Optionally, the pathogenic SNP is in the ARSA gene; and, optionally, the pathogenic mutation includes P426L, c.459+1G>A, or a combination thereof. In some embodiments, the pathogenic SNP is associated with Marteauz-Lamy Syndrome (MSPVI); optionally, the pathogenic SNP is in the ARSB gene; and, optionally, the pathogenic mutation includes Y210C. In some embodiments, the pathogenic SNP is associated with type I citrullinemia; for example, type I citrullinemia. Optionally, the pathogenic SNP is in the ASS gene; and, optionally, the pathogenic mutation comprises G390R. In some embodiments, the pathogenic SNP is associated with Darier's disease. Optionally, the pathogenic SNP is in the ATP2A2 gene; and, optionally, the pathogenic mutation comprises N767S.

[0035] In some embodiments, the pathogenic SNP is associated with classic homocystinuria; optionally, the pathogenic SNP is in the CBS gene; optionally, the pathogenic mutation includes G307S, T191M, or a combination thereof. In some embodiments, the pathogenic SNP is associated with cystic fibrosis; optionally, the pathogenic SNP is in the CFTR gene; optionally, the pathogenic mutation includes G551D, W1282*, R553*, R117H, or a combination thereof. In some embodiments, the pathogenic SNP is associated with choroidal hyperplasia; optionally, the pathogenic SNP is in the CHM gene; and, optionally, the pathogenic mutation includes R293*, R270*, A117A, or a combination thereof. In some embodiments, the pathogenic SNP is associated with neuronal ceroidlipofuscinosis (NCL). Optionally, the pathogenic SNP is in the CLN2 gene; and, optionally, the pathogenic mutation includes R208*. In some embodiments, the pathogenic SNP is associated with autosomal dominant deafness; optionally, the pathogenic SNP is in the COCH gene; and, optionally, the pathogenic mutation includes G88E. In some embodiments, the pathogenic SNP is associated with carnitine palmitoyltransferase II deficiency; optionally, the pathogenic SNP is in the CPT2 gene; and, optionally, the pathogenic mutation includes S113L.

[0036] In some embodiments, the pathogenic SNP is associated with cystinosis. Optionally, the pathogenic SNP is in the CTNS gene; and, optionally, the pathogenic mutation includes W138*. In some embodiments, the pathogenic SNP is associated with autosomal recessive deafness; optionally, the pathogenic SNP is in the CX30 gene; and, optionally, the pathogenic mutation includes T5M. In some embodiments, the pathogenic SNP is associated with autosomal recessive deafness; optionally, the pathogenic SNP is in the DFNB59 gene; and, optionally, the pathogenic mutation includes R183W. In some embodiments, the pathogenic SNP is associated with isolated agammaglobulinemia; optionally, the pathogenic SNP is in the E47 gene; and the pathogenic mutation includes E555K. In some embodiments, the pathogenic SNP is associated with congenital factor XI deficiency; optionally, the pathogenic SNP is in the F11 gene; and, optionally, the pathogenic mutation comprises E117*, F283L, or a combination thereof. In some embodiments, the pathogenic SNP is associated with congenital factor V deficiency; optionally, the pathogenic SNP is in the F5 gene; and, optionally, the pathogenic mutation comprises R506Q, R534Q, or a combination thereof. In some embodiments, the pathogenic SNP is associated with congenital factor VII deficiency; optionally, the pathogenic SNP is in the F7 gene; and, optionally, the pathogenic mutation comprises A294V, C310F, R304Q, Q100R, or a combination thereof.

[0037] In some embodiments, the pathogenic SNP is associated with hemophilia A; optionally, the pathogenic SNP is in the F8 gene; and, optionally, the pathogenic mutation comprises R2169H, R1985Q, R2178C, R550C, or a combination thereof. In some embodiments, the pathogenic SNP is associated with hemophilia B; optionally, the pathogenic SNP is in the F9 gene; and, optionally, the pathogenic mutation comprises T342M, R294Q, R43Q, R191H, G106S, A279T, R75*, R294*, R379Q, or a combination thereof. In some embodiments, the pathogenic SNP is associated with tyrosinemia type 1; optionally, the pathogenic SNP is in the FAH gene; and, optionally, the pathogenic mutation comprises P261L. In some embodiments, the pathogenic SNP is associated with an autosomal dominant hypophosphatemia disorder; optionally, the pathogenic SNP is in the FGF23 gene; and, optionally, the pathogenic mutation comprises R176Q.

[0038] In some embodiments, the pathogenic SNP is associated with von Gierke disease; optionally, the pathogenic SNP is in the G6PC gene; and, optionally, the pathogenic mutation comprises Q347*. In some embodiments, the pathogenic SNP is associated with Mediterranean G6PD deficiency; optionally, the pathogenic SNP is in the G6PD gene; and, optionally, the pathogenic mutation comprises S188D. In some embodiments, the pathogenic SNP is associated with Morquio syndrome (MPS IVA); optionally, the pathogenic SNP is in the GALNS gene; and, optionally, the pathogenic mutation comprises R386C. In some embodiments, the pathogenic SNP is associated with classic galactosemia; optionally, the pathogenic SNP is in the GALT gene; and, optionally, the pathogenic mutation comprises Q188R.

[0039] In some embodiments, the pathogenic SNP is associated with Gaucher disease; optionally, the pathogenic SNP is in the GBA gene; and, optionally, the pathogenic mutation comprises N370S, L444P, or a combination thereof. In some embodiments, the pathogenic SNP is associated with glutaryl-CoA dehydrogenase deficiency; optionally, the pathogenic SNP is in the GCDH gene; and, optionally, the pathogenic mutation comprises R138G, M263V, R402W, or a combination thereof. In some embodiments, the pathogenic SNP is associated with glycine encephalopathy; optionally, the pathogenic SNP is in the GLDC gene; and, optionally, the pathogenic mutation comprises A389V, G771R, T269M, or a combination thereof. In some embodiments, the pathogenic SNP is associated with cone-rod dystrophy; optionally, the pathogenic SNP is in the GUCY2D gene; and, optionally, the pathogenic mutation comprises R838C. In some embodiments, the causative SNP is associated with Sly syndrome (MPSVII); optionally, the causative SNP is in the GUSB gene; and, optionally, the causative mutation comprises L175F.

[0040] In some embodiments, the pathogenic SNP is associated with sickle cell disease; optionally, the pathogenic SNP is in the HBB gene; and, optionally, the pathogenic mutations include E26K; E7K; c.-138C>T; IVS2,654C>T; or a combination thereof. In some embodiments, the pathogenic SNP is associated with intermittent porphyria; optionally, the pathogenic SNP is in the HMBS gene; and, optionally, the pathogenic mutations include R173W. In some embodiments, the pathogenic SNP is associated with Lesch-Nyhan syndrome; optionally, the pathogenic SNP is in the HPRT1 gene; and, optionally, the pathogenic mutations include R51*, R170*, or a combination thereof. In some embodiments, the pathogenic SNP is associated with Hunter syndrome; optionally, the pathogenic SNP is in the IDS gene; and, optionally, the pathogenic mutations include R88C, G374G, or a combination thereof. In some embodiments, the pathogenic SNP is associated with Hurler syndrome (MPS1). Optionally, the pathogenic SNP is in the IDUA gene; and, optionally, the pathogenic mutation comprises Q70*.

[0041] In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in the IMPDH1 gene; and, optionally, the pathogenic mutation includes D226N. In certain embodiments, the pathogenic SNP is associated with Andersen-Tawil syndrome; optionally, the pathogenic SNP is in the KCNJ2 gene; and, optionally, the pathogenic mutation includes R218W. In some embodiments, the pathogenic SNP is associated with Meesmann epithelial corneal dystrophy; therefore, the SNP is associated with Meesmann epithelial corneal dystrophy. Optionally, the pathogenic SNP is in the KRT12 gene; and, optionally, the pathogenic mutation includes L132P. In some embodiments, the pathogenic SNP is associated with Parkinson's disease; optionally, the pathogenic SNP is in the LRRK2 gene; and, optionally, the pathogenic mutation includes G2109S. In some embodiments, the pathogenic SNP is associated with Rett syndrome; optionally, the pathogenic SNP is in the MECP2 gene; and, optionally, the pathogenic mutation comprises R106W, R133C, R306C, R168*, R255*, or a combination thereof. In some embodiments, the pathogenic SNP is associated with Sanfilippo Syndrome B (MPSIIIB); optionally, the pathogenic SNP is in the NAGLU gene; and, optionally, the pathogenic mutation comprises R297*, Y140C, or a combination thereof.

[0042] In some embodiments, the pathogenic SNP is associated with CADASIL syndrome; optionally, the pathogenic SNP is in the NOTCH3 gene; and, optionally, the pathogenic mutation comprises R90C, R141C, or a combination thereof. In some embodiments, the pathogenic SNP is associated with blue cone monochromacy; optionally, the pathogenic SNP is in the OPN1LW gene; and, optionally, the pathogenic mutation comprises C203R. In some embodiments, the pathogenic SNP is associated with phenylketonuria; optionally, the pathogenic SNP is in the PAH gene; and, optionally, the pathogenic mutation comprises R408W, I65T, R261Q, IVS10-11G>A, or a combination thereof. In some embodiments, the pathogenic SNP is associated with Usher syndrome type 1F; optionally, the pathogenic SNP is in the PCDH15 gene; and, optionally, the pathogenic mutation comprises R245*. In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in the PDE6A gene; and, optionally, the pathogenic mutation comprises V685M, D670G, or a combination thereof.

[0043] In some embodiments, the pathogenic SNP is associated with Pendred syndrome; optionally, the pathogenic SNP is in the PDS gene; and, optionally, the pathogenic mutation comprises L236P; c.1001+1G>A; IVS8,+1G>A, or a combination thereof. In some embodiments, the pathogenic SNP is associated with Variegate porphyria; optionally, the pathogenic SNP is in the PPOX gene; and, optionally, the pathogenic mutation comprises R59W. In some embodiments, the pathogenic SNP is associated with neuronal lipofuscinosis type 1; optionally, the pathogenic SNP is in the PPT1 gene; and, optionally, the pathogenic mutation comprises R151*. In some embodiments, the pathogenic SNP is associated with Creutzfeldt-Jakob Disease (CJD); optionally, the pathogenic SNP is in the PRNP gene; and, optionally, the pathogenic mutation comprises M129V, P102L, D178N, or a combination thereof. In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in the PRPF3 gene; and, optionally, the pathogenic mutation comprises T494M. In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in the PRPF8 gene; and, optionally, the pathogenic mutation comprises H2309R.

[0044] In some embodiments, the pathogenic SNP is associated with hereditary chronic pancreatitis; optionally, the pathogenic SNP is in the PRSS1 gene; and, optionally, the pathogenic mutation includes R122H. In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in the RHO gene; and, optionally, the pathogenic mutation includes P347L, D190N, or a combination thereof. In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in the RP1 gene; and, optionally, the pathogenic mutation includes R667*. In some embodiments, the pathogenic SNP is associated with Leber congenital amaurosis type 2; optionally, the pathogenic SNP is in the RPE65 gene; and, optionally, the pathogenic mutation includes R44*; IVS1, GA, +5; or a combination thereof. In some embodiments, the causative SNP is associated with Diamond-Blackfan anaemia; optionally, the causative SNP is in the RPS19 gene; and, optionally, the causative mutation comprises R62Q.

[0045] In some embodiments, the pathogenic SNP is associated with X-linked retinoblastoma; optionally, the pathogenic SNP is in the retinal toxin (RS1) gene; and, optionally, the pathogenic mutation includes R102W, R141C, or a combination thereof. In some embodiments, the pathogenic SNP is associated with A1AD; optionally, the pathogenic SNP is in the SERPINA1 gene; and, optionally, the pathogenic mutation includes E342K, R48C (R79C), or a combination thereof. In some embodiments, the pathogenic SNP is associated with Sanfilippo Syndrome A (MPSIIIA); optionally, the pathogenic SNP is in the SGSH gene; and, optionally, the pathogenic mutation includes R74C. In some embodiments, the causative SNP is associated with Niemann-Pick disease type A; optionally, the causative SNP is in the SMPD1 gene; and, optionally, the causative mutation comprises L302P.

[0046] In some embodiments, the pathogenic SNP is associated with autosomal dominant Parkinson's disease; optionally, the pathogenic SNP is in the SNCA gene; and, optionally, the pathogenic mutation comprises A53T. In some embodiments, the pathogenic SNP is associated with familial amyotrophic lateral sclerosis (ALS). Optionally, the pathogenic SNP is in the superoxide dismutase 1 (SOD1) gene; and, optionally, the pathogenic mutation comprises A4V, H46R, G37R, or a combination thereof. In some embodiments, the pathogenic SNP is associated with autosomal dominant deafness; optionally, the pathogenic SNP is in the TECTA gene; and, optionally, the pathogenic mutation comprises Y1870C. In some embodiments, the pathogenic SNP is associated with autosomal recessive deafness; optionally, the pathogenic SNP is in the TMC1 gene; and the pathogenic mutation comprises Y182C. In some embodiments, the pathogenic SNP is associated with ATTR amyloidosis; optionally, the pathogenic SNP is in the TTR gene; and, optionally, the pathogenic mutation comprises V50M / V30M. In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa / Usher syndrome type 1C; optionally, the pathogenic SNP is in the USH1C gene; and, optionally, the pathogenic mutation comprises V72V.

[0047] In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in the USH2a gene; and, optionally, the pathogenic mutation comprises C759F. In some embodiments, the pathogenic SNP is associated with myotubular myopathy; optionally, the pathogenic SNP is in the MTM1 gene; and, optionally, the pathogenic mutation comprises c.1261-10A>G.

[0048] In some embodiments, any base editor system or method provided herein may further include a second guide polynucleotide for editing additional nucleobases. In some embodiments, the additional nucleobase is not in a gene. In some embodiments, the additional nucleobase is located in a gene. In some embodiments, the additional nucleobase is located in a protein coding region. In some embodiments, the additional nucleobase is located in a protein non-coding region. In some embodiments, the protein non-coding region is a gene regulatory component. In some embodiments, the deaminase domain is a cytidine deaminase domain or an adenosine deaminase domain. In some embodiments, the deaminase domain is a cytidine deaminase domain. In some embodiments, the deaminase domain is an adenosine deaminase domain. In some embodiments, the adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA). In some embodiments, the guide polynucleotide comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the guide polynucleotide comprises a CRISPR RNA (crRNA) sequence, a transactivated CRISPR RNA (tracrRNA) sequence, or a combination thereof.

[0049] In some embodiments, any base editor system or method provided herein may further include a second guide polynucleotide. In some embodiments, the second guide polynucleotide includes ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the second guide polynucleotide includes a CRISPR RNA (crRNA) sequence, a transactivated CRISPR RNA (tracrRNA) sequence, or a combination thereof. In some embodiments, the second guide polynucleotide targets the base editor to a second target nucleotide sequence.

[0050] In some embodiments, in any base editor system or method provided herein, 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 some embodiments, the polynucleotide programmable DNA binding domain is nuclease dead. In some embodiments, the polynucleotide programmable DNA binding domain is a nickase. In some embodiments, the polynucleotide programmable DNA binding domain comprises a Cas9 domain. In some embodiments, the Cas9 domain comprises nuclease dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease active Cas9. In some embodiments, the Cas9 domain comprises a Cas9 nickase. In some embodiments, the polynucleotide programmable DNA binding domain is a designed or modified polynucleotide programmable DNA binding domain.

[0051] In some embodiments, any base editor system or method provided herein can further comprise a second base editor. In some embodiments, the second base editor comprises a different deaminase domain than the base editor.

[0052] In some embodiments, in any of the methods provided herein, editing results in less than 20% indel formation. In some embodiments, editing results in less than 15% indel formation. In some embodiments, editing results in less than 10% indel formation. In some embodiments, editing results in less than 5% indel formation. In some embodiments, editing results in less than 4% indel formation. In some embodiments, editing results in less than 3% indel formation. In some embodiments, editing results in less than 2% indel formation. In some embodiments, editing results in less than 1% indel formation. In some embodiments, editing results in less than 0.5% indel formation. In some embodiments, editing results in less than 0.1% indel formation. In some embodiments, editing does not result in a translocation.

[0053] On the one hand, the present invention provides a method for editing a G6PC polynucleotide comprising a single nucleotide polymorphism (SNP) associated with glycogen storage disease type 1a (GSD1a), the method comprising contacting the G6PC polynucleotide with a base editor, the base editor being complexed with one or more guide polynucleotides, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein the one or more guide polynucleotides target the base editor to cause an A·T to G·C change of the SNP associated with GSD1a. In one embodiment, the A·T to G·C change at the SNP associated with glycogen storage disease type 1a (GSD1a) changes glutamine (Q) to a non-glutamine (X) amino acid. In various embodiments of the above aspects described herein or any other aspect of the invention, the A·T to G·C change at the SNP associated with glycogen storage disease type 1a (GSD1a) changes arginine (R) to a non-arginine (X) in the G6PC polypeptide.

[0054] In various embodiments of the foregoing aspects or any other aspect of the invention described herein, the SNP associated with GSD1a results in the expression of a G6PC polypeptide having a non-glutamine (X) amino acid at position 347 or a non-arginine (X) amino acid at position 83. In one embodiment, the base editor correction is a substitution of a non-glutamine amino acid (X) for the glutamine at position 347. In another embodiment, the base editor correction is a substitution of a non-arginine amino acid (X) for the arginine at position 83.

[0055] In various embodiments of the above aspects of the invention described above or any other aspects of the invention, the polynucleotide programmable DNA binding domain is a modified Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof. In various embodiments of the above aspects of the invention described above or any other aspects of the invention, the polynucleotide programmable DNA binding domain comprises a modified SpCas9 with an altered protospacer adjacent motif (PAM) specificity. In one embodiment, the modified SpCas9 is specific for the nucleic acid sequence 5'-NGA-3' or 5'-NGG-3'. In various embodiments of the above aspects of the invention described herein or any other aspects, the adenosine deaminase is ABE7.10.

[0056] In one aspect, a cell is produced by introducing a base editor into the cell or a progenitor thereof, and a polynucleotide encoding the base editor into the cell, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and one or more guide polynucleotides targeting the base editor to cause an A·T to G·C change of a SNP associated with glycogen storage disease type 1a (GSD1a). In various embodiments of the above or any other aspect of the invention described herein, the cell is a hepatocyte, a hepatocyte precursor, or an iPS cell-derived hepatocyte.

[0057] In various embodiments of the above aspects of the invention described above or any other aspects of the invention, the cell is from a subject suffering from GSD1a. In various embodiments of the above aspects of the invention described above or any other aspects of the invention, the cell has a Q347X mutation. In various embodiments of the above aspects of the invention described herein or any other aspects of the invention, the A·T to G·C change at the SNP associated with GSD1a changes glutamine to a non-glutamine (X) amino acid. In various embodiments of the above aspects of the invention described herein or any other aspects of the invention, the A·T to G·C change at the SNP associated with GSD1a changes the arginine in the G6PC polypeptide to a non-arginine (X) amino acid. In various embodiments of the above aspects of the invention described above or any other aspects of the invention, the SNP associated with GSD1a results in the expression of a G6PC polypeptide having a non-glutamine (X) amino acid at position 347 or a non-arginine (X) amino acid at position 83.

[0058] In one aspect, the invention provides a method of treating glycogen storage disease type 1a (GSD1a) or von Gierke's disease in a subject in need thereof, the method comprising administering to the subject a cell of the various embodiments of the above or any other aspect of the invention described herein.

[0059] On the other hand, the present invention provides a method for producing hepatocytes or progenitor cells thereof, the method comprising: (a) introducing an induced pluripotent stem cell or hepatocyte progenitor cell comprising a SNP associated with GSD1a, a base editor, or a polynucleotide encoding the base editor, wherein the base editor comprises a polynucleotide programmable nucleotide binding domain and an adenosine deaminase domain; and one or more guide polynucleotides, wherein the one or more guide polynucleotides target the base editor to cause an A·T to G·C change of the SNP associated with GSD1a; (b) differentiating the induced pluripotent stem cell or hepatocyte progenitor cell into a hepatocyte. On the other hand, the method comprises differentiating the induced pluripotent stem cell into a hepatocyte or its progenitor cell. In various embodiments, the induced pluripotent stem cell of step (a) comprises a Q347X mutation.

[0060] In various embodiments of the above aspects or any other aspects of the invention described herein, the hepatocyte progenitor cells are obtained from a subject suffering from GSD1a. In various embodiments, the hepatocytes or hepatocyte progenitor cells are mammalian cells or human cells. In various embodiments of the above aspects of the invention described above or any other aspects of the invention, the A·T to G·C change at the SNP associated with GSD1a changes glutamine to a non-glutamine (X) amino acid or changes arginine to a non-arginine (X) amino acid in the G6PC polypeptide. In various embodiments, the SNP associated with GSD1a results in the expression of a G6PC polypeptide with a non-glutamine (X) amino acid at position 347. In various embodiments, the SNP associated with GSD1a results in the expression of a G6PC polypeptide with an arginine (X) amino acid at position 83. In various embodiments, the SNP associated with GSD1a replaces glutamine with a non-glutamine (X) amino acid. In various embodiments, the SNP associated with GSD1a replaces arginine with a non-arginine (X) amino acid. In various embodiments of the foregoing aspects or any other aspect of the invention described herein, the cell is selected for an A·T to G·C change of a SNP associated with GSD1a.

[0061] On the one hand, the present invention provides a method for editing an IDUA polynucleotide, wherein the IDUA polynucleotide comprises a single nucleotide polymorphism (SNP) associated with mucopolysaccharidosis type 1 (MPS1), the method comprising contacting the IDUA polynucleotide with a base editor, and the base editor one or more guide polynucleotides are complexed, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein the one or more guide polynucleotides target the base editor to cause an A·T to G·C change of the SNP associated with MPS1. In one embodiment, the polynucleotide programmable DNA binding domain is a modified Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof. In another embodiment, the polynucleotide programmable DNA binding domain comprises a modified SpCas9 with an altered protospacer adjacent motif (PAM) specificity. In various embodiments of the above aspects of the invention described above or any other aspects of the invention, the modified SpCas9 is specific to the nucleic acid sequence 5'-NGG-3'. In various embodiments of the above aspects of the invention described herein or any other aspects, the adenosine deaminase is ABE7.10. In various embodiments, the guide polynucleotide comprises the human nucleic acid sequence ACTCTaGGCAGAGGTCTCAA AGG. In various embodiments, the guide polynucleotide comprises the mouse nucleic acid sequence GCTCTaGGCCGAAGTGTCGC AGG.

[0062] On the one hand, a base editor is introduced into a cell or its progenitor cell, and a polynucleotide encoding the base editor is introduced into the cell to produce the cell, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and one or more guiding polynucleotides targeting the base editor to cause an A·T to G·C change of a SNP associated with mucopolysaccharidosis type 1 (MPS1). In various embodiments, the cell is a stem cell, a stem cell precursor, or an induced pluripotent stem cell (iPSC). In various embodiments of the above-described aspects of the invention or any other aspect of the invention, the cell is from a subject with MPS1.

[0063] In another aspect, the invention provides a method of treating MPS1 in a subject in need thereof, the method comprising administering to the subject a cell of the above aspect or any other aspect of the invention described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The features of the present application are particularly set forth in the appended claims. The features and advantages of the present invention may be better understood by reference to the following detailed description, which sets forth illustrative embodiments in which the principles of the present application are applied, and the accompanying drawings:

[0065] Figure 1 This diagram compares healthy subjects and patients with alpha-1 antitrypsin deficiency (A1AD). In healthy subjects, alpha-1 antitrypsin (A1AT) protects the lungs from proteases, and the liver releases alpha-1 antitrypsin into the blood. In patients with A1AD, the lack of a properly functioning A1AT protein leads to lung damage. Furthermore, the accumulation of abnormal A1AT in liver cells can lead to cirrhosis.

[0066] Figure 2 Typical ranges of serum alpha-1 antitrypsin (A1AT) levels are shown for different genotypes (normal (MM); heterozygous carriers of alpha-1 antitrypsin deficiency (MZ, SZ); and homozygous deficiency (SS, ZZ)). The serum alpha-1 antitrypsin (AAT) concentration is expressed in μM on the left "y" axis, which is common in the literature. The right "y" axis shows the approximate conversion of serum AAT concentrations to mg / dL units commonly reported by clinical laboratories and other measurement techniques (turbidimetry or radial immunodiffusion).

[0067] Figures 3A to 3C Shows base editing target sequences and their association with precise correction of disease-causing mutations in the SERPINA1 gene, which encodes the A1AT protein. Figure 3A A precise correction base editing strategy for mutations in the SERPINA1 gene encoding A1AT is shown. A7 ("Target A") can be edited to restore the wild-type (WT) phenotype. In some cases, the "A" nucleobases A5 / A7 can be edited to introduce amino acid D341G into the A1AT protein. In some cases, A7 / A8 can be edited to introduce amino acid E342G into the A1AT protein. Figure 3B Provided are a nucleic acid sequence showing the location and encoded amino acid of the target A nucleobase within the SERPINA1 gene, and a graph showing the levels (ng / ml) of A1AT secreted from HEK293T cells expressing wild-type (WT) or A1AT variants comprising E342K, D341G, or E342G. Figure 3C is a graph showing the elastase activity in wild-type (WT) A1AT protein and the elastase activity in A1AT variants containing E342K or D341G.

[0068] Figure 4Schematic diagram showing the strategy for developing DNA deoxyadenosine deaminases starting with the TadA tRNA deaminase. Shown are the Escherichia coli library, a plasmid library containing mutant ecTadA (TadA*) genes fused to dCas9, and a selection plasmid for targeted A·T to G·C mutations to repair antibiotic resistance genes. Mutations from surviving TadA* variants were introduced into the ABE architecture for base editing in human cells.

[0069] Figure 5 Provided is a nucleic acid sequence showing the position and encoded amino acid of the target "A" nucleobase within the SERPINA1 gene, and a graph showing the percentage of editing at position A5 or A7 in the SERPINA1 gene as a function of guide RNA length.

[0070] Figure 6A and 6B Describes a library of SpCas9 mutants generated to enrich for mutations within the PAM-interacting (PI) domain of Cas9. The library can be screened for SpCas9s with altered PAM specificity.

[0071] Figure 7 Schematic diagram showing the correction strategy for the Q347X mutation using base editors to convert A>G at the target site using NGG and NGAPAM recognition sequences (highlighted). Accurate correction produces a TAG>CAG (stop codon>glutamine) transition.

[0072] Figure 8A and 8B Provides a transfection schedule for the maturation cycle of GSD1a-based iPSC-derived hepatocytes. Figure 8A A timeline of the transfection schedule is provided showing representative time points for seeding, transfection, and cell harvest. Figure 8B Images showing mature GSD1aiPSc-derived hepatocytes at day 5 and day 7.

[0073] Figure 9A and 9B Data are presented and show precise correction of base editing of G6PC Q347X of GSD1a. Figure 9A Provided are nucleic acid sequences showing the locations of target and bystander "A" nucleobases within the G6PC gene and the corresponding NGG and NGA PAM sequences, and a graph showing the percentage base editing efficiency of G6PC Q347X in HEK293T cells for ABE-bystander, indels, and nuclease-indels on target using either an NGA PAM or an NGG PAM. Figure 9BProvided are graphs showing base editing efficiencies for ABE-on-target, ABE-bystander, indels, and nuclease indels in patient iPSC-derived hepatocytes using NGA PAM or NGG PAM in G6PCQ347X. The solid lines represent the mean of the experiments.

[0074] Figure 10 Provided are nucleic acid sequences showing the positions of target and bystander "A" nucleobases in the G6PC gene and the corresponding GGA PAM sequence, and graphs showing the percentage of A>G base editing for patient G6PC Q347X in iPSc-derived hepatocytes for ABE-On target, ABE-Bystander, and insertion of mRNA variants.

[0075] Figure 11 Provided are graphs showing the percentage of base editing efficiency in the mouse and human IDUA genes using the ABE7.10 base editor. DETAILED DESCRIPTION

[0076] The description and examples herein illustrate embodiments of the present application in detail. It should be understood that the application is not limited to the specific embodiments described herein and may vary. Those skilled in the art will recognize that there are many variations and modifications within the scope of the present application.

[0077] All terms are as would be understood by one skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in this application.

[0078] Unless otherwise indicated, the implementation of some embodiments disclosed herein employs conventional techniques within the technical scope of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (FM Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: APractical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988); Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (RI Freshney, ed. (2010)).

[0079] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0080] Although various features of the present application may be described in the context of a single embodiment, the features may also be provided independently or in any suitable combination. Conversely, for clarity, the present application may be described herein in the context of separate embodiments, but the application may also be implemented in a single embodiment.

[0081] definition

[0082] The following definitions are supplementary to those in the art and are specific to the present application and should not be attributed to any related or unrelated circumstances, such as any commonly owned patents or applications. Although any methods and materials similar or equivalent to those described herein can be used in practice to test the present application, preferred materials and methods are described herein. Therefore, the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0083] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. The following references provide general definitions of many of the terms used in the present 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).

[0084] In this application, the use of the singular includes the plural, unless expressly stated otherwise. It must be noted that the singular forms "a," "an," and "the" used in this specification include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the use of the term "include" as well as other forms (e.g., "includes," "comprising," and "included") is not restrictive.

[0085] As used in this specification and claims, the words "comprise" (and any form of including, such as "comprises" and "comprises"), "have" (and any form of having, such as "have" and "has"), "include" (and any form of containing, such as "includes" and "include"), or "contain" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present application, and vice versa. In addition, the compositions of the present application can be used to implement the methods of the present application.

[0086] The term "about" or "approximately" means that a particular value is determined by one skilled in the art to be within an acceptable error range, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, preferably within 5 times, and more preferably within 2 times. Where specific values ​​are described in the application and claims, the term "about" should be assumed to mean that the specific value is within an acceptable error range unless otherwise indicated.

[0087] References in the specification to "some embodiments," "one embodiment," "an embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some, but not necessarily all, embodiments of the disclosure.

[0088] "Adenosine deaminase" refers to a polypeptide or fragment thereof that can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolysis of adenosine to inosine or the hydrolysis of deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase (e.g., engineered adenosine deaminase, evolved adenosine deaminase) provided herein can be from any organism, such as bacteria.

[0089] "Agent" refers to any small molecule compound, antibody, nucleic acid molecule or polypeptide or fragment thereof.

[0090] "Ameliorate" means to reduce, inhibit, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0091] "Alteration" refers to a change (increase or decrease) in the expression level or activity of a gene or polypeptide, as detected by standard art-known methods (e.g., the methods described herein). As used herein, an alteration 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.

[0092] An "analog" is a molecule that is not identical but has similar functional or structural features. For example, a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide while possessing certain biochemical modifications that enhance the analog's function relative to the naturally occurring polypeptide. Such biochemical modifications may increase the analog's protease resistance, membrane permeability, or half-life without altering, for example, ligand binding. Analogs may include unnatural amino acids.

[0093] As used herein, "administering" refers to providing one or more compositions described herein to a patient or subject. By way of example and not limitation, the composition can be administered by intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, or intramuscular (im) injection, and one or more of these routes can be employed. For example, parenteral administration can be by bolus injection or gradual infusion over time. Alternatively or concurrently, administration can be by oral route.

[0094] "Alpha-1 antitrypsin (A1AT) protein" refers to a polypeptide or fragment thereof having at least about 95% amino acid sequence identity to the amino acid sequence of UniProt Accession No. P01009. In certain embodiments, the A1AT protein comprises one or more alterations relative to the following reference sequence. In a specific embodiment, the A1AT protein associated with A1AD comprises the E342K mutation. An exemplary A1AT amino acid sequence is provided below (>sp|P01009|A1AT_human alpha-1-antitrypsin OS=Homo sapiens OX=9606GN=SERPINA1 PE=1SV=3):

[0095] MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFS

[0096] LYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGF

[0097] QELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQ

[0098] INDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTV

[0099] KVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFL

[0100] ENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKA

[0101] VLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK.

[0102] "Base editor (BE)" or "nucleobase editor (NBE)" refers to a reagent that binds to a polynucleotide and has nucleobase modification activity. In various embodiments, the base editor comprises a nucleobase modification polypeptide (e.g., a deaminase) and a polynucleotide programmable nucleotide binding domain that is bound to a guide polynucleotide (e.g., a guide RNA). In various embodiments, the reagent is a biomolecule complex comprising a protein domain with base editing activity, i.e., capable of modifying bases (e.g., deoxyribonucleic acid) in a nucleic acid molecule (e.g., A, T, C, G, or U). In some embodiments, the polynucleotide programmable DNA binding domain is fused or connected to a deaminase domain. In one embodiment, the reagent is a fusion protein comprising a domain with base editing activity. In another embodiment, a protein domain with base editing activity is connected to a guide RNA (e.g., through an RNA binding motif on the guide RNA, and an RNA binding domain fused to a deaminase). In some embodiments, the domain with base editing activity is capable of deaminating bases within a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating bases within a DNA molecule. In some embodiments, the base editor is capable of deaminating cytosine (C) or adenosine (A) within DNA. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the adenosine deaminase evolved from TadA. In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-related (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. In some embodiments, the base editor is fused to a base excision repair (BER) inhibitor. In some embodiments, the base excision repair inhibitor is a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the base excision repair inhibitor is an inosine base excision repair inhibitor. Detailed information about base editors is described in International PCT Patent Application No. PCT / 2017 / 045381 (International WO Patent Application No. 2018 / 027078) and International PCT / US2016 / 058344 (WO Patent Application No. 2017 / 070632), the entire contents of which are incorporated herein by reference.See also 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); Komor, AC et al. "Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity" Science Advances3:eaao4774 (2017) and Rees, HA et al. "Base editing: precision chemistry on the genome and transcriptome of living cells." Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0103] For example, the cytidine base editor CBE used in the base editing compositions, systems, and methods described herein has the following nucleic acid sequence (8877 base pairs) (Addgene, Watertown, MA; Komor AC, et al., 2017, Sci Adv., 30; 3(8):eaao4774.doi:10.1126 / sciadv.aao4774), as shown below. Also included are polynucleotide sequences having at least 95% or greater identity to the BE4 nucleic acid sequence.

[0104] 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg

[0105] 61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg

[0106] 121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact

[0107] 181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa

[0108] 241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta

[0109] 301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct

[0110] 361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag

[0111] 421 actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag

[0112] 481 gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg

[0113] 541 gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc

[0114] 601 aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt

[0115] 661 acctggtttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg

[0116] 721 tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac

[0117] 781 ccccgcaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg

[0118] 841 actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa

[0119] 901 gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc

[0120] 961 atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca

[0121] 1021 ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccacacattctctgg

[0122] 1081 gccaccgggt tgaaatctgg tggttcttct ggtggttcta gcggcagcgagactcccggg

[0123] 1141 acctcagagt ccgccacacc cgaaagttct ggtggttctt ctggtggttctgataaaaag

[0124] 1201 tattctattg gtttagccat cggcactaat tccgttggat gggctgtcataaccgatgaa

[0125] 1261 tacaaagtac cttcaaagaa atttaaggtg ttgggggaaca cagaccgtcattcgattaaa

[0126] 1321 aagaatctta tcggtgccct cctattcgat agtggcgaaa cggcagaggcgactcgcctg

[0127] 1381 aaacgaaccg ctcggagaag gtatacacgt cgcaagaacc gaatatgttacttacaagaa

[0128] 1441 atttttagca atgagatggc caaagttgac gattctttct ttcaccgtttggaagagtcc

[0129] 1501 ttccttgtcg aagaggacaa gaaacatgaa cggcacccca tctttggaaacatagtagat

[0130] 1561 gaggtggcat atcatgaaaa gtacccaacg atttatcacc tcagaaaaaagctagttgac

[0131] 1621 tcaactgata aagcggacct gaggttaatc tacttggctc ttgcccatatgataaaagttc

[0132] 1681 cgtgggcact ttctcattga gggtgatcta aatccggaca actcggatgtcgacaaactg

[0133] 1741 ttcatccagt tagtacaaac ctataatcag ttgtttgaag agaaccctataaatgcaagt

[0134] 1801 ggcgtggatg cgaaggctat tcttagcgcc cgcctctcta aatcccgacggctagaaaac

[0135] 1861 ctgatcgcac aattacccgg agagagaaa aatggttgt tcggtaccttatagcgctc

[0136] 1921 tcactaggcc tgacaccaaa tttaagtcg aacttcgact tagctgagatgccaattg

[0137] 1981 cagcttagta aggacacgta cgatgacgat ctcgacaatc tactggcacaaattggagat

[0138] 2041 cagtagcgg acttatttt ggctgccaaaaccttagcg atgcaatcctcctatctgac

[0139] 2101 atactgagag ttatactga gattaccaag gcgccgtta ccgcttcaatgatcaaagg

[0140] 2161 tacgatgaac atcaccaga cttgacactt ctcaagccc tagtccgtcagcaactgcct

[0141] 2221 gagaaata aggaaatatt ctttgatcag tcgaaaaacg ggtacgcaggttattattgac

[0142] 2281 ggcggagcga gtcagagga attctacaag tttatcaac ccatattagagagagatggat

[0143] 2341 gggacggag agttgcttgt aaaaccaat cgcgagatc tactgcgaaagcagcggact

[0144] 2401 ttcgacaacg gtagcattcc acatcaaatc cacttaggcg aattgcatgctatacttaga

[0145] 2461 aggcaggagg atttttatcc gttcctcaaa gacaatcgtg aaaagattgagaaaatccta

[0146] 2521 acctttcgca taccttacta tgtggggaccc ctggcccgag ggaactctcggttcgcatgg

[0147] 2581 atgacaaga agtccgaaga aacgattact ccatggaatt ttgaggaagttgtcgataa

[0148] 2641 ggtgcgtcag ctcaatcgtt catcgagagg atgaccaact ttgacaagaatttaccgaac

[0149] 2701 gaaaaagtat tgcctaagca cagtttactt tcgagtatt tcacagtgtacaatgaactc

[0150] 2761 acgaaagtta agtatgtcac tgagggcatg cgtaaacccg cctttctaagcggagaacag

[0151] 2821 aagaaagcaa tagtagtct gttattcaag accaaccgca aagtgacagttaagcaattg

[0152] 2881 aaagaggact actttagaa aattgaatgc ttcgattctg tcgagatctccggggtagaa

[0153] 2941 gatcgattta atgcgtcact tggtacgtat catgacctcc taagataataaagataag

[0154] 3001 gacttcctgg father's father's father's fathergttgactcttacc

[0155] 3061 ctctttgaag atcgggaaat gattgaggaa agactaaaaa catacgctcacctgttcgac

[0156] 3121 gataaggtta tgaaacagtt aaagaggcgt cgctatacgg gctggggacgattgtcgcgg

[0157] 3181 aaacttatca acgggataag agacaagcaa agtggtaaaa ctattctcgattttctaaag

[0158] 3241 agcgacggct tcgccaatag gaactttatg cagctgatcc atgatgactctttaaccttc

[0159] 3301 aaagaggata tacaaaaggc acaggtttcc ggacaagggg actcattgcacgaacatatt

[0160] 3361 gcgaatcttg ctggttcgcc agccatcaaa aagggcatac tccagacagtcaaagtagtg

[0161] 3421 gatgagctag ttaaggtcat gggacgtcac aaaccggaaa acattgtaatcgagatggca

[0162] 3481 cgcgaaaatc aaacgactca gaaggggcaa aaaaacagtc gagagcggatgaagagaata

[0163] 3541 gaagagggta ttaaagaact gggcagccag atcttaaagg agcatcctgtggaaaatacc

[0164] 3601 caattgcaga acgagaaact ttacctctat tacctacaaa atggaagggacatgtatgtt

[0165] 3661 gatcaggaac tggacataaa ccgtttatct gattacgacg tcgatcacattgtaccccaa

[0166] 3721 tcctttttga aggacgattc aatcgacaat aaagtgctta cacgctcggataagaaccga

[0167] 3781 gggaaaagtg acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaactattggcgg

[0168] 3841 cagctcctaa atgcgaaact gataacgcaa agaaagttcg ataacttaactaaagctgag

[0169] 3901 aggggtggct tgtctgaact tgacaaggcc ggatttatta aacgtcagctcgtggaaacc

[0170] 3961 cgccaaatca caaagcatgt tgcacagata ctagattccc gaatgaatacgaaatacgac

[0171] 4021 gagaacgata agctgattcg ggaagtcaaa gtaatcactt taaagtcaaaattggtgtcg

[0172] 4081 gacttcagaa aggattttca attctataaa gttagggaga taaataactaccaccatgcg

[0173] 4141 cacgacgctt atcttaatgc cgtcgtaggg accgcactca ttaagaaatacccgaagcta

[0174] 4201 gaaagtgagt ttgtgtatgg tgattacaaa gtttatgacg tccgtaagatgatcgcgaaa

[0175] 4261 agcgacagg agataggcaa ggctacagcc aaatactct tttattctacattatgaat

[0176] 4321 ttctttaga cggaatcac tctgcaac ggagagagat gcaacgacctttaattgaa

[0177] 4381 accaatgggg agacaggtga aatcgtatgg gataagggcc gggacttcgcgacggtgaga

[0178] 4441 aaagttttgt ccatgcccca agtcaacata gtaaagaaa ctgaggtgcagaccggaggg

[0179] 4501 tttcaagg atcgattct tccaaaagg atagtgata agctcatcgctcgtaaaag

[0180] 4561 gactgggacc cgaaaagta cggtggctc gatagcccta cagttgcctattctctgtccta

[0181] 4621 gtagtggcaa aagttgagaa gggaaaatcc aagaactga agtcagtcaagaatttg

[0182] 4681 gggataacga tttaggagcg ctcgtctttt gaaaagaacc ccatcgacttccttgaggcg

[0183] 4741 aaggttaca aggaagtaa aaaggatctc ataatttaac taccaagtatagtctgttt

[0184] 4801 gagttagaaa atggccgaaa acggatgttg gctagcgccg gagagcttcaaagggaac

[0185] 4861 gaactcgcac taccgtctaa atacgtgaat ttcctgtatt tagcgtcccattacgagaag

[0186] 4921 ttgaaaggtt cacctgaaga taacgaacag aagcaacttt ttgttgagcagcacaaacat

[0187] 4981 tatctcgacg aaatcataga gcaaatttcg gaattcagta agagagtcatcctagctgat

[0188] 5041 gccaatctgg acaaagtatt aagcgcatac aacaagcaca gggataaacccatacgtgag

[0189] 5101 caggcggaaa atattatcca tttgtttact cttaccaacc tcggcgctccagccgcattc

[0190] 5161 aagtattttg acacaacgat agatcgcaaa cgatacactt ctaccaaggaggtgctagac

[0191] 5221 gcgacactga ttcaccaatc catcacggga ttatatgaaa ctcggatagatttgtcacag

[0192] 5281 cttgggggtg actctggtgg ttctggagga tctggtggtt ctactaatctgtcagatatt

[0193] 5341 attgaaaagg agaccggtaa gcaactggtt atccaggaat ccatcctcatgctcccagag

[0194] 5401 gaggtggaag aagtcattgg gaacaagccg gaaagcgata tactcgtgcacaccgcctac

[0195] 5461 gacgagagca ccgacgagaa tgtcatgctt ctgactagcg acgcccctgaatacaagcct

[0196] 5521 tgggctctgg tcatacagga tagcaacggt gagaacaaga ttaagatgctctctggtggt

[0197] 5581 tctggaggat ctggtggttc tactaatctg tcagatatta ttgaaaggagaccggtaag

[0198] 5641 caactggtta tccaggaatc catcctcatg ctcccagagg aggtggaagaagtcattggg

[0199] 5701 aacaagccgg aaagcgatat actcgtgcac accgcctacg acgagagcaccgacgagaat

[0200] 5761 gtcatgcttc tgactagcga cgcccctgaa tacaagcctt gggctctggtcatacaggat

[0201] 5821 agcaacggtg agaacaagat taagatgctc tctggtggtt ctcccaagaagaagagaa

[0202] 5881 gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatcagcctcgactg

[0203] 5941 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttccttgaccctgg

[0204] 6001 aaggtgccac tccactgtc ctttcctaat aaaatgagga aattgcatcgcattgtctga

[0205] 6061 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaaggggaggattggg

[0206] 6121 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgaggcggaaagaa

[0207] 6181 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatcatggtcatagc

[0208] 6241 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacgagccggaagca

[0209] 6301 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaattgcgttgcgct

[0210] 6361 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatgaatcggccaac

[0211] 6421 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctcactgactcgc

[0212] 6481 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcggtaatacggt

[0213] 6541 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggccagcaaaagg

[0214] 6601 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgcccccctgacg

[0215] 6661 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacaggactataaagat

[0216] 6721 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgaccctgccgctta

[0217] 6781 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcatagctcacgct

[0218] 6841 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtgcacgaacccc

[0219] 6901 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtccaacccggtaa

[0220] 6961 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcagagcgaggtatg

[0221] 7021 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacactagaagaacag

[0222] 7081 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagttggtagctctt

[0223] 7141 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaagcagcagatta

[0224] 7201 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacggggtctgacgctc

[0225] 7261 aatgggaacga aaactcacgt taagggattt tggtcatgag attatcaaaaaaggatcttca

[0226] 7321 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtatatgagtaaa

[0227] 7381 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcgatctgtctat

[0228] 7441 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgatacgggaggggct

[0229] 7501 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccggctccagatt

[0230] 7561 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcctgcaactttat

[0231] 7621 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagttcgccagtta

[0232] 7681 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgctcgtcgtttg

[0233] 7741 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatgatcccccatgt

[0234] 7801 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagtaagttggccg

[0235] 7861 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtcatgccatccg

[0236] 7921 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaatagtgtatgc

[0237] 7981 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgccacatagcagaa

[0238] 8041 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctcaaggatcttac

[0239] 8101 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatcttcagcatctt

[0240] 8161 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgccgcaaaaaaagg

[0241] 8221 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaatattattgaa

[0242] 8281 gcatttatca gggttatgt ctcatgagcg gatacatatt tgaatgtatttagaaaaata

[0243] 8341 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtcgacggatcgg

[0244] 8401 gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctctgatgccgcat

[0245] 8461 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtagtgcgcgagca

[0246] 8521 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaatctgcttaggg

[0247] 8581 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttgacattgattat

[0248] 8641 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagcccatatatggagt

[0249] 8701 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaacgaccccccgcc

[0250] 8761 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggactttccattgac

[0251] 8821 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatc

[0252] In some embodiments, the BE4 nucleic acid sequence is selected from one of the following:

[0253] Original BE4

[0254]

[0255] BE4 codon optimization 1

[0256]

[0257] BE4 codon optimization 2

[0258]

[0259] "Base editing activity" refers to the activity of chemically changing a base within a polynucleotide. 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 or adenine deaminase activity, for example, converting A·T to G·C.

[0260] The term "base editor system" refers to a system for editing the nucleobases of a target nucleotide sequence. In various embodiments, the base editor (BE) system comprises (1) a polynucleotide programmable nucleotide binding domain and a deaminase domain for deaminating the nucleobase; (2) a guide polynucleotide (e.g., a guide RNA) is bound to the polynucleotide programmable nucleotide binding domain. In some embodiments, the base editor system comprises (1) a base editor (BE) comprising a polynucleotide programmable DNA binding domain and a deaminase domain for deaminating the nucleobase; (2) the guide RNA is bound to the polynucleotide programmable DNA 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 adenine or adenosine base editor (ABE).

[0261] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of an amino acid with another amino acid having a common property. A functional method for defining the shared properties between individual amino acids is to analyze the normalized frequency 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 analysis, amino acid groups can be defined in which the amino acids within the group are preferentially exchanged with each other and are therefore most similar in their effect on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid substitutions of amino acids, such as lysine replacing arginine and vice versa, so that a positive charge can be maintained; glutamic acid replacing aspartic acid and vice versa, thereby maintaining a negative charge; threonine replacing serine, to maintain free -OH; and aminamide replacing asparagine, thereby maintaining free –NH2.

[0262] As used interchangeably herein, the terms "coding sequence" or "protein coding sequence" refer to a segment of a polynucleotide that encodes a protein. The region or sequence has a start codon near the 5' end and a stop codon near the 3' end. Coding sequences may also be referred to as open reading frames.

[0263] "Cytidine deaminase" refers to a polypeptide or fragment thereof that catalyzes a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, a cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. Examples of cytidine deaminases include PmCDA1, derived from the marine lamprey (Petromyzon marinus) ("PmCDA1"), AID (activation-induced cytidine deaminase; AICDA), derived from mammals (e.g., humans, pigs, cattle, horses, monkeys, etc.), and APOBEC.

[0264] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, which catalyzes the hydrolysis of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytosine deaminase, which catalyzes the hydrolysis of cytosine to uracil. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolysis of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolysis of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, which catalyzes the hydrolysis of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, adenosine deaminase catalyzes the hydrolysis of adenosine in deoxyribonucleic acid (DNA). Adenosine deaminases (e.g., engineered adenosine deaminases, evolved adenosine deaminases) provided herein can be from any organism, such as bacteria. In some embodiments, the adenosine deaminase is from bacteria, such as Escherichia coli, Staphylococcus aureus, Streptococcus typhi, Streptococcus putrefaciens, Haemophilus influenzae, or Clostridium crescentum. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is essentially absent. For example, in some 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% identical to a naturally occurring deaminase. For example, deaminase domains are described in International Patent Application No. PCT / 2017 / 045381 (International Patent Application No. WO 2018 / 027078) and International Patent Application No. PCT / US2016 / 058344 (International Patent Application No. WO 2017 / 070632), each of which is incorporated herein by reference in its entirety.See also Komor, AC et al., "Programmable base editing of a target base ingenomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, NM et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage" Nature 551,464-471(2017); Komor, AC et al., "Improved baseexcision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:Abase editors with higher efficiency and product purity" Science Advances 3:eaao4774(2017) and Rees, HA et al., "Base editing: precision chemistry on thegenome and transcriptome of living cells." Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0265] "Detectable label" refers to a composition that, when attached to a molecule of interest, enables detection by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin, or haptens.

[0266] "Disease" refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include retinitis pigmentosa, Usher syndrome, sickle cell disease, beta-thalassemia, alpha-1 antitrypsin deficiency (A1AD), hepatoporosis, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, von Gierke's disease, Pompe disease, Gaucher's disease, Hurler's disease, cystic fibrosis, or chronic pain. In a specific embodiment, the disease is A1AD.

[0267] "Effective amount" refers to the amount of an agent or active compound described herein, such as a base editor described herein, that is alleviated relative to an untreated patient or an individual without the disease. "Treatment" required to improve the symptoms of the disease relative to an untreated patient or an individual without the disease, that is, a healthy individual. The effective amount of the active compound used to implement the present invention to treat the disease varies according to the mode of administration, age, weight, and overall health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. The dosage is referred to as an "effective" amount. In one embodiment, the effective amount is the amount of the base editor of the present invention sufficient to introduce a change in the gene of interest into a cell (eg, a cell in vitro or in vivo). In one embodiment, the effective amount is an amount that achieves a therapeutic effect (e.g., reduction or control of retinitis pigmentosa, Usher syndrome, sickle cell disease, beta thalassemia, alpha-1 antitrypsin deficiency (A1AD), hepatoporosis, medium chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, von Gierke disease, Pompe disease, Gaucher disease, Hurler disease, cystic fibrosis, or chronic pain or symptoms or conditions thereof). Such a therapeutic effect is not sufficient to alter the disease-causing gene in all cells of a subject, tissue, or organ, but is only sufficient to alter the disease-causing gene in about 1%, 5%, 10%, 25%, 50%, 75%, or more of the cells present in the subject, tissue, or organ. In one embodiment, the effective amount is sufficient to alleviate the symptoms of one or more diseases (e.g., retinitis pigmentosa, Usher syndrome, sickle cell disease, beta thalassemia, alpha-1 antitrypsin deficiency (A1AD), hepatoporosis, medium chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, von Gierke disease, Pompe disease, Gaucher disease, Hurler disease, cystic fibrosis, or chronic pain).

[0268] "Fragment" refers to a portion of a polypeptide or nucleic acid molecule. The portion preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of a reference nucleic acid molecule or polypeptide. A fragment can comprise 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.

[0269] "Hybridization" refers to hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds.

[0270] The terms "base repair inhibitor," "base repair inhibitor," "IBR," or their grammatical equivalents refer to proteins that can inhibit the activity of nucleic acid repair enzymes, such as base excision repair enzymes. In some embodiments, IBR is an inhibitor of inosine base excision repair. Exemplary inhibitors of base repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7 Endol, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is catalytically inactive EndoV or catalytically inactive hAAG. In some embodiments, the base repair inhibitor is catalytically inactive EndoV or catalytically inactive hAAG. In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein that can inhibit the uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain comprises a fragment of wild-type UGI or wild-type UGI. In some embodiments, the UGI protein provided herein includes a fragment of UGI and a protein homologous to UGI or a UGI fragment. In some embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. In some embodiments, the base repair inhibitor is a "catalytically inactivated inosine-specific nuclease" or a "dead inosine-specific nuclease." Without wishing to be bound by any particular theory, catalytically inactivated inosine glycosylases (e.g., alkyladenine glycosylase (AAG)) can bind to inosine, but cannot produce abasic sites or remove inosine, thereby spatially preventing the newly formed inosine from being partially subjected to DNA damage / repair mechanisms. In some embodiments, catalytically inactivated inosine-specific nucleases can bind to inosine in nucleic acids, but do not cut nucleic acids. Non-limiting exemplary catalytically inactivated inosine-specific nucleases include, for example, catalytically inactivated alkyladenine glycosylases (AAG nucleases) from humans and, for example, catalytically inactivated endonucleases V (EndoV nucleases) from Escherichia coli. In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0271] The terms "isolated," "purified," or "biologically pure" refer to a material that is free to varying degrees from components that normally accompany the material in its native state. "Isolated" refers to a degree of separation from the original source or surrounding environment. "Purified" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other substances such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that a nucleic acid or protein forms essentially a single band in an electrophoretic gel. For proteins that can be modified, such as for phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be purified separately.

[0272] An "isolated polynucleotide" refers to a nucleic acid (e.g., DNA) that is free of the genes flanking the genes in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. Thus, the term includes, for example, recombinant DNA incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryotic or eukaryotic organism; or as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion). Additionally, the term includes RNA molecules transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0273] An "isolated polypeptide" refers to a polypeptide of the present invention that has been separated from components with which it is naturally associated. Generally, a polypeptide is isolated when it is at least 60% free, by weight, of the proteins and naturally occurring organic molecules with which it is naturally associated. 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. An 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 such a polypeptide, or by chemically synthesizing the protein, and purity can be measured by any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0274] As used herein, the term "linker" may refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a linker that connects two molecules or parts, for example, two components of a protein complex or a ribonucleic acid 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). The linker can connect different components or different parts of a base editor system. For example, in some embodiments, the linker can connect the guidance polynucleotide binding domain of the polynucleotide programmable nucleotide binding domain and the catalytic domain of the deaminase. In some embodiments, the linker can connect the CRISPR polypeptide and the deaminase. In some embodiments, the linker can connect Cas9 and the deaminase. In some embodiments, the linker can connect dCas9 and the deaminase. In some embodiments, the linker can connect nCas9 and the deaminase. In some embodiments, the linker can connect the guide polynucleotide and the deaminase. In some embodiments, the linker can connect the deamination component and the polynucleotide programmable nucleotide binding component of the base editor system. In some embodiments, a linker can connect the RNA binding portion of the deamination component and the polynucleotide programmable nucleotide binding component of the base editor system. In some embodiments, a linker can connect the RNA binding portion of the abasic component and the RNA binding portion of the polynucleotide programmable nucleotide binding component of the base editor system. The linker may be located between or on both sides of two groups, molecules or other parts and connected to each by a covalent bond or non-covalent interaction, thereby connecting the two. In some embodiments, the linker can be an organic molecule, group, polymer or chemical part. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, the linker can comprise an aptamer capable of binding to a ligand. In some embodiments, the ligand can be a carbohydrate, peptide, protein or nucleic acid. In some embodiments, the linker can comprise an aptamer that can be derived from a riboswitch. The riboswitch from which the aptamer is derived can be selected from the group consisting of theophylline riboswitch, thiamine pyrophosphate (TPP) riboswitch, adenosylcobalamin (AdoCbl) riboswitch, S-adenosylmethionine (SAM) riboswitch, SAH riboswitch, flavin mononucleotide (FMN) riboswitch, tetrahydrofolate riboswitch, lysine riboswitch, glycine riboswitch, purine riboswitch, GlmS riboswitch, or pre-queosine 1 (PreQ1) riboswitch. In some embodiments, the linker can comprise an aptamer that binds to a polypeptide or protein domain, such as a polypeptide ligand.In some embodiments, the polypeptide ligand can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. In some embodiments, the polypeptide ligand can be part of a base editor system component. For example, a nucleobase editing component can include a deaminase domain and an RNA recognition motif.

[0275] In some embodiments, the joint can be amino acid or multiple amino acids (for example, peptide or protein). In some embodiments, the length of the joint can be about 5 to 100 amino acids, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. The length is 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90 or 90 to 100 amino acids. In some embodiments, the length of the joint can be about 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450 or 450 to 500 amino acids. Longer or shorter joints can also be considered.

[0276] In some embodiments, the linker connects the gRNA binding domain of the RNA programmable nuclease, including the Cas9 nuclease domain, and the catalytic domain of the nucleic acid editing protein (e.g., cytidine or adenosine deaminase). In some embodiments, the linker connects dCas9 and nucleic acid editing protein. For example, the linker is located between two groups, molecules or other parts or on the side of two groups, molecules or other parts, and is covalently bonded to each, thereby connecting the two. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, a group, a polymer or a chemical moiety. In some embodiments, the linker is 5 to 200 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190, or 200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence SGGS. In some embodiments, the linker comprises (SGGS)n 、(GGGS) n 、(GGGGS) n 、(G) n 、(EAAAK) n 、(GGS) n , SGSETPGTSESATPES or (XP) n motif, or any of the following combinations, wherein n is independently an integer between 1 and 30, and X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a plurality of proline residues and a length of 5 to 21, 5 to 14, 5 to 9, or 5 to 7 amino acids, such as PAPAP, PAPAPA, PAPAPAP, PAPAPAPA, P(AP)4, P(AP)7, P(AP) 10 This proline-rich linker is also known as a "rigid" linker.

[0277] In some embodiments, the domain of the base editor is fused via a linker comprising the following amino acid sequence:

[0278] SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP. In some embodiments, the base editor domain is fused via a linker comprising the amino acid sequence SGSETPGTSESATPES, also referred to as an XTEN linker. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSSPGSETPGTSESATPESSGGS SGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence

[0279] PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0280] As used herein, the term “mutation” refers to the substitution of a residue in a sequence of, for example, a nucleic acid or amino acid sequence by another residue, or the deletion or insertion of one or more residues in the sequence. Mutations are generally described herein by identifying the original residue, and the original residue is subsequently described by the position of the residue in the sequence and the identity of the newly replaced residue. The various methods provided herein for performing amino acid substitutions (mutations) are well known in the art and are provided, for example, by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2012)). In some embodiments, the currently disclosed base editors can effectively produce “expected mutations”, such as point mutations, in nucleic acids (e.g., nucleic acids within the genome of a subject) without producing a large number of unexpected mutations, such as unexpected point mutations. In some embodiments, the expected mutation is a mutation generated by a specific base editor (e.g., a cytidine base editor or an adenosine base editor) that is bound to a guide polynucleotide (e.g., gRNA) specifically designed to produce the expected mutation.

[0281] Typically, mutations generated or identified in a sequence (e.g., an amino acid sequence described herein) are numbered relative to a reference (or wild-type) sequence, i.e., a sequence that does not contain the mutation. Those skilled in the art will readily understand how to determine the position of mutations in amino acid and nucleic acid sequences relative to a reference sequence.

[0282] The term "non-conservative mutation" refers to amino acid substitutions between different groups, such as lysine for tryptophan or phenylalanine for serine. In this case, the non-conservative amino acid substitution preferably does not replace, interfere with, or inhibit the function of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, thereby increasing the biological activity of the functional variant compared to the wild-type protein.

[0283] The term "nuclear localization sequence," "nuclear localization signal," or "NLS" refers to an amino acid sequence that facilitates the import of a protein into the nucleus. Nuclear localization sequences are known in the art and are described, for example, in International Patent Application No. PCT / EP2000 / 011690 filed by Plank et al. on November 23, 2000, and International Patent Application No. WO 2001 / 038547 published on May 31, 2001, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS such as described in Koblan et al., Nature Biotech, 2018, doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence

[0284] KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0285] The terms "nucleobase," "nitrogen base," or "base," used interchangeably herein, refer to nitrogenous biological compounds that form nucleosides, which in turn are components of nucleotides. The ability of nucleobases to form base pairs and stack on each other directly leads to long helical structures, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleobases—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 modified (non-primary) bases. Non-limiting exemplary modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydromethylcytosine. Hypoxanthine and xanthine can be produced by the presence of mutagens, and both can be produced by deamination (replacing an amine group with a carbonyl group). Hypoxanthine can be modified by adenine. Xanthine can be modified by guanine. Uracil can be produced by deamination of cytosine. "Nucleoside" is composed of a nucleobase and a five-carbon 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 nucleobases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C) and pseudouridine (Ψ). "Nucleotide" is composed of a nucleobase, a five-carbon sugar (ribose or deoxyribose) and at least one phosphate group.

[0286] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to compounds comprising a core base and an acidic moiety (e.g., a polymer of nucleosides, nucleotides, or nucleotides). Typically, polymeric nucleic acids, such as nucleic acid molecules comprising three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to one another by phosphodiester bonds. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide," "polynucleotide," and "polynucleic acid" are used interchangeably to refer to a polymer (e.g., a string of at least three nucleotides) of nucleotides. In some embodiments, "nucleic acid" encompasses RNA and single-stranded and / or double-stranded DNA. Nucleic acid can be naturally occurring, such as in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be non-naturally occurring molecules, for example recombinant DNA or RNA, artificial chromosome, engineered genome or its fragment, or synthetic DNA, RNA, DNA / RNA hybrid, or comprise non-naturally occurring nucleotides or nucleosides.In addition, term " nucleic acid ", " DNA ", " RNA " and / or similar terms comprise nucleic acid analogs, for example, have the analog except the phosphodiester backbone. Nucleic acid can be purified from natural origin, uses recombinant expression system to produce, and optionally purification, chemical synthesis etc. In appropriate cases, for example, in the case of chemically synthesized molecules, nucleic acid can comprise nucleoside analogs, for example, have chemically modified base or sugar and backbone modification. Unless otherwise indicated, nucleotide sequence is shown with 5 ' to 3 ' direction. In some embodiments, the nucleic acid is or comprises a natural nucleoside (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); or a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5 -methylcytosine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O6-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalating groups; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0287] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" can be used interchangeably with "polynucleotide programmable nucleotide binding domain" to refer to a protein (e.g., DNA or RNA) that associates with a nucleic acid, such as a guide nucleic acid, guiding the napDNAbp to a specific nucleic acid sequence. For example, the Cas9 protein can be bound to a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as a nuclease-active 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), Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. Other nucleic acid programmable DNA binding proteins are also within the scope of the present application, although they may not be specifically listed in the present application. See, e.g., 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 entire contents of each of which are incorporated herein by reference.

[0288] As used herein, the term "nucleobase editing domain" or "nucleobase editing protein" refers to a protein or enzyme that catalyzes the modification of nucleobases in RNA or DNA, such as the deamination of cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine), and the deamination of hypoxanthine (or inosine) by adenine (or adenosine), as well as the addition and insertion of non-templated nucleotides. 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 can be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain can be an engineered or evolved nucleobase editing domain from a naturally occurring nucleobase editing domain. The nucleobase editing domain can be from any organism, such as bacteria, humans, chimpanzees, gorillas, monkeys, cows, dogs, rats, or mice. For example, nucleobase editing proteins are described in International Patent Application No. PCT / 2017 / 045381 (International Patent Application No. WO 2018 / 027078) and International Patent Application No. PCT / US2016 / 058344 (International Patent Application No. WO 2017 / 070632), each of which is incorporated herein by reference in its entirety. See also 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 and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the contents of which are incorporated herein by reference.

[0289] As used herein, "obtaining" and "obtaining a reagent" include synthesizing, purchasing or otherwise obtaining the reagent.

[0290] As used herein, "patient" or "subject" refers to a mammalian subject or individual diagnosed with, having, developing, or suspected of having or developing a disease or condition. In some embodiments, the term "patient" refers to a mammalian subject having a higher than average likelihood of having a disease or condition. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cows, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other animals that can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female.

[0291] A "patient in need thereof" or "subject in need thereof" herein refers to a patient diagnosed with or suspected of having a disease or disorder, such as, but not limited to, alpha-1 antitrypsin deficiency (A1AD).

[0292] The terms "pathogenic mutation," "pathogenic variant," "disease capsid mutation," "disease-causing variant," "deleterious mutation," or "susceptibility mutation" refer to genetic variations or mutations that increase an individual's susceptibility or predisposition to a disease or condition. In some embodiments, the pathogenic mutation comprises at least one wild-type amino acid substitution for at least one pathogenic amino acid in a protein encoded by a gene.

[0293] The terms "protein," "peptide," "polypeptide," and their grammatical equivalents are used interchangeably herein to refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids in length. A protein, peptide, or polypeptide may refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications, and the like. A protein, peptide, or polypeptide may also be a single molecule or may be a multimolecular complex. A protein, peptide, or polypeptide may be simply a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein can be located at the amino terminal (N-terminal) portion or the carboxyl terminal (C-terminal) protein of the fusion protein, thereby forming an amino terminal fusion protein or a carboxyl terminal fusion protein, respectively. Proteins can include different domains, such as nucleic acid binding domains (e.g., the gRNA binding domain of Cas9 that guides protein to bind to the target site) and nucleic acid cleavage domains or nucleic acid catalytic domains acid editing proteins. In some embodiments, the protein includes a protein portion, such as an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, such as a compound that can serve as a nucleic acid cleavage agent. In some embodiments, the protein is compounded or associated with nucleic acids such as RNA or DNA. Any protein provided herein can be produced by any method known in the art. For example, the protein provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising peptide linkers. Recombinant protein expression and purification methods are well known, including Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2012)) described in method, all of which are incorporated herein by reference.

[0294] The polypeptides and proteins disclosed herein (including functional parts and functional variants thereof) may comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, dihydroindole- 2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine. Polypeptides and proteins can be associated with post-translational modifications of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications include phosphorylation, acylation (including acetylation and formylation), glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation (including methylation and ethylation), ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bonds, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation, and iodination.

[0295] The term "polynucleotide programmable nucleotide binding domain" refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guiding polynucleotide (e.g., a guide RNA) that directs a polynucleotide programmable DNA binding domain, which guides the 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 bound to a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactivated Cas9 (dCas9). Non-limiting examples of nucleic acid-programmable DNA-binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpf1, Cas12b / C2c1 and Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i.Non-limiting examples of Cas enzymes 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, Csc Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, type II Cas effector protein, type V Cas effector protein, type VI Cas effector protein, CARF, DinG, homologs thereof, or modified or engineered forms thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of this application, even if they are not specifically listed in this application.

[0296] The term "recombinant" as used herein in the context of a protein or nucleic acid refers to a protein or nucleic acid that does not occur in nature and is the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence.

[0297] "Decrease" refers to a negative change of at least 10%, 25%, 50%, 75% or 100%.

[0298] "Reference" refers to standard or control conditions. In one embodiment, the reference is a wild-type or healthy cell.

[0299] "Reference sequence" is a defined sequence used as the basis for sequence comparison. A reference sequence can be a subset or all of a given sequence. For example, a fragment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence is typically at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence is typically at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides or about 300 nucleotides or any integer near or between them.

[0300] The terms "RNA programmable nuclease" and "RNA-guided nuclease" are used in conjunction with (e.g., bound to or associated with) one or more RNAs that are not targets for cleavage. In some embodiments, when an RNA programmable nuclease is complexed with RNA, it can be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. A gRNA that exists as a single-stranded RNA molecule can be referred to as a single-stranded guide RNA (sgRNA), although "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or a complex of two or more molecules. Typically, a gRNA that exists as a single-stranded RNA species contains two domains: (1) a domain that has homology to the target nucleic acid (e.g., and guides the Cas9 complex to bind to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence called tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is the same as or homologous to the tracrRNA provided by Jinek et al. in Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. Provisional Patent Application No. 61 / 874,682, filed on September 6, 2013, entitled "Switchable Cas9 Nucleases and Uses," and U.S. Patent Application No. 61 / 874,746, filed on September 6, 2013, entitled "Delivery System For Functional Nucleases," which are incorporated herein by reference in their entirety. In some embodiments, the gRNA comprises two or more of domains (1) and (2) and can be referred to as an "extended gRNA." For example, as described herein, an extended gRNA will, for example, bind to two or more Cas9 proteins and bind to a target nucleic acid at two or more different regions. The gRNA comprises a nucleotide sequence complementary to the target site, which mediates the binding of the nuclease / RNA complex to the target site and provides sequence specificity to the nuclease:RNA complex.In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 from Streptococcus pyogenes (Csn1) (see, e.g., “Complete genome sequence of anM1 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 H.S., 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 by trans-encoded small RNA and host factorRNase III.", Deltcheva E., Chylinski K., Sharma CM, Gonzales K., Chao Y., PirzadaZ.A., Eckert MR, Vogel J., Charpentier E., Nature 471: 602-607 (2011).

[0301] The term "single nucleotide polymorphism (SNP)" refers to a variation in a single-stranded nucleotide at a specific position in the genome, wherein each variation is present to a certain degree (e.g., >1%) in a population. For example, at a specific base position in the human genome, a C nucleotide may appear in most individuals, but in a minority of individuals, the position is occupied by an A. This means that there is a SNP at the specific position, and the two possible nucleotide variations, C or A, are considered alleles at the position. SNPs underlie differences in susceptibility to disease. The severity of a disease and how the human body responds to treatment are also manifestations of genetic variation. SNPs can fall within the coding region of a gene, the non-coding region of a gene, or the intergenic region (the region between genes). In some embodiments, due to the degeneracy of the genetic code, SNPs within a coding sequence do not necessarily change the amino acid sequence of the protein produced. There are two types of SNPs in the coding region: synonymous and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs do change the amino acid sequence of the protein. There are two types of non-synonymous SNPs: missense and nonsense. SNPs that are not located in protein-coding regions can still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNAs. Gene expression affected by this type of SNP is called an eSNP (expression SNP) and can occur upstream or downstream of the gene. Single nucleotide variants (SNVs) are variations in a single strand of nucleotides that occur with no frequency restriction and can occur in somatic cells. Somatic SNVs (e.g., caused by cancer) are also referred to as SNVs.

[0302] "SERPINA1 polynucleotide" refers to a nucleic acid molecule encoding an A1AT protein or a fragment thereof. The sequence of an exemplary SERPINA1 polynucleotide, which can be found in NCBI Accession No. NM_000295, is provided below:

[0303] 1 acaatgactc ctttcggtaa gtgcagtgga agctgtacac tgcccaggca aagcgtccgg

[0304] 61 gcagcgtagg cgggcgactc agatcccagc cagtggactt agcccctgtt tgctcctccg

[0305] 121 ataactgggg tgaccttggt taatattcac cagcagcctc ccccgttgcc cctctggatc

[0306] 181 cactgcttaa atacggacga ggacagggcc ctgtctcctc agcttcaggc accaccactg

[0307] 241 acctgggaca gtgaatcgac aatgccgtct tctgtctcgt ggggcatcct cctgctggca

[0308] 301 ggcctgtgct gcctggtccc tgtctccctg gctgaggatc cccagggaga tgctgcccag

[0309] 361 aagacagata catcccacca tgatcaggat cacccaacct tcaacaagat cacccccaac

[0310] 421 ctggctgagt tcgccttcag cctataccgc cagctggcac accagtccaa cagcaccaat

[0311] 481 atcttcttct ccccagtgag catcgctaca gcctttgcaa tgctctccct ggggaccaag

[0312] 541 gctgacactc acgatgaaat cctggagggc ctgaatttca acctcacgga gattccggag

[0313] 601 gctcagatcc atgaaggctt ccaggaactc ctccgtaccc tcaaccagcc agacagccag

[0314] 661 ctccagctga ccaccggcaa tggcctgttc ctcagcgagg gcctgaagct agtggataag

[0315] 721 tttttggagg atgttaaaaa gttgtaccac tcagaagcct tcactgtcaa cttcggggac

[0316] 781 accgaaggg ccaagaaaca gatcaacgat tacgtggaga agggtactca agggaaaatt

[0317] 841 gtggattgg tcaggagct tgacagagac acagttttg ctctggtga tcatcttc

[0318] 901 tttaaggca aatgggagag acccttgaa gtcaggaca ccgaggaga ggacttccac

[0319] 961 gtggaccagg tgaccaccgt gaggtgcct atgatgaagc gtttaggcat gtttaacatc

[0320] 1021 cagcactgta agaagctgtc cagctgggtg ctgctgatga atacctgggcaatgccacc

[0321] 1081 gccatctct tctgcctga tgagggaa ctacagcacc tggaaatgaactcaccac

[0322] 1141 gatatcatca ccaagttcct ggaaatgaa gagaaggt ctgccagcttacatttaccc

[0323] 1201 aactgtcca ttactggaac ctatgatctg aagagcgtcc tggtcactgggcatcact

[0324] 1261 aaggtctca gcaatggggc tgacctcc gggtcacag aggaggcacccctgaagctc

[0325] 1321 sneaky sneaky sneaky sneaky g no no. tgctggg

[0326] 1381 gccatgtttt tagaggccat acccatgtct atcccccccg aggtcaagttcaacaaaccc

[0327] 1441 tttgtcttct taatgattga acaaaatacc aagtctcccc tcttcatgggaaaagtggtg

[0328] 1501 aatcccaccc aaaaataact gcctctcgct cctcaacccc tcccctccatccctggcccc

[0329] 1561 ctccctggat gacattaaag aagggttgag ctggtccctg cctgcatgtgactgtaaatc

[0330] 1621 cctcccatgt tttctctgag tctccctttg cctgctgagg ctgtatgtgggctccaggta

[0331] 1681 acagtgctgt cttcgggccc cctgaactgt gttcatggag catctggctgggtaggcaca

[0332] 1741 tgctgggctt gaatccaggg gggactgaat cctcagctta cggacctgggcccatctgtt

[0333] 1801 tctggagggc tccagtcttc cttgtcctgt cttggagtcc ccaagaaggaatcacagggg

[0334] 1861 aggaaccaga taccagccat gaccccaggc tccaccaagc atcttcatgtccccctgctc

[0335] 1921 atcccccact cccccccacc cagagttgct catcctgcca gggctggctgtgcccacccc

[0336] 1981 aaggctgccc tcctgggggc cccagaactg cctgatcgtg ccgtggcccagttttgtggc

[0337] 2041 atctgcagca acacaagaga gaggacaatg tcctcctctt gacccgctgtcacctaacca

[0338] 2101 gactcgggcc ctgcacctct caggcacttc tggaaaatga ctgaggcagattcttcctga

[0339] 2161 agcccattct ccatggggca acaaggacac ctattctgtc cttgtccttccatcgctgcc

[0340] 2221 ccagaaagcc tcacatatct ccgtttagaa tcaggtccct tctccccagatgaagaggag

[0341] 2281 ggtctctgct ttgttttctc tatctcctcc tcagacttga ccaggcccagcaggccccag

[0342] 2341 aagaccatta ccctatatcc cttctcctcc ctagtcacat ggccataggcctgctgatgg

[0343] 2401 ctcaggaagg ccattgcaag gactcctcag ctatgggaga ggaagcacatcacccattga

[0344] 2461 cccccgcaac ccctcccttt cctcctctga gtcccgactg gggccacatgcagcctgact

[0345] 2521 tctttgtgcc tgttgctgtc cctgcagtct tcagagggcc accgcagctccagtgccacg

[0346] 2581 gcaggaggct gttcctgaat agcccctgtg gtaagggcca ggagagtccttccatcctcc

[0347] 2641 aaggccctgc taaaggacac agcagccagg aagtcccctg ggcccctagctgaaggacag

[0348] 2701 cctgctccct ccgtctctac caggaatggc cttgtcctat ggaaggcactgccccatccc

[0349] 2761 aaactaatct aggaatcact gtctaaccac tcactgtcat gaatgtgtacttaaaggatg

[0350] 2821 aggttgagtc ataccaaata gtgatttcga tagttcaaaa tggtgaaattagcaattcta

[0351] 2881 catgattcag tctaatcaat ggataccgac tgtttcccac acaagtctcctgttctctta

[0352] 2941 agcttactca ctgacagcct ttcactctcc acaaatacat taaagatatggccatcacca

[0353] 3001 agccccctag gatgacacca gacctgagag tctgaagacc tggatccaagttctgacttt

[0354] 3061 tccccctgac agctgtgtga ccttcgtgaa gtcgccaaac ctctctgagccccagtcatt

[0355] 3121 gctagtaaga cctgcctttg agttggtatg atgttcaagt tagataacaaaatgtttata

[0356] 3181 cccattagaa cagagaataa atagaactac atttcttgca

[0357] The PAM sequence is shown in italics and double underlined and shows the correct sequence after adenosine base editing.

[0358] "Specific binding" refers to a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid programmable DNA binding domain and a guide nucleic acid), compound, or molecule that recognizes and binds to a polypeptide and / or nucleic acid. Acidic molecules of the present invention, but do not substantially recognize and bind to other molecules in a sample, such as a biological sample.

[0359] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" refers to the formation of a double-stranded molecule pair between complementary polynucleotide sequences (e.g., the genes described herein) or portions thereof under various stringent conditions. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

[0360] For example, stringent salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of an organic solvent such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions will typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Varying other parameters, such as hybridization time, the concentration of detergents, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of vector DNA, is well known to those skilled in the art. Various degrees of stringency can be achieved by combining these various conditions as desired. In a preferred embodiment, hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will be conducted in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In a most preferred embodiment, hybridization will occur in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations on these conditions will be apparent to those skilled in the art.

[0361] For most applications, the stringency of the post-hybridization wash step will also vary. Wash stringency conditions can be defined by salt concentration and temperature. As described above, wash stringency can be increased by reducing the salt concentration or by increasing the temperature. For example, the stringent salt concentration of the wash step will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash step typically include 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 wash step will occur in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS at 25°C. In a more preferred embodiment, the wash step will be performed in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 42°C. In a more preferred embodiment, the wash step will be performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Other variations on these conditions will be 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.

[0362] "Substantially identical" refers to 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 more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid level to the sequence used for comparison.

[0363] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs of the Genetics Computer Group at the Biotechnology Center at the University of Wisconsin (1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine, lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, where e -3 and e -100 Probability scores between and indicate closely related sequences.

[0364] For example, COBALT is used with the following parameters:

[0365] a) Alignment parameters: gap penalty -11, -1 and gap penalty 5, -1,

[0366] b) CDD parameters: use RPS BLAST; Blast E value 0.003; search for conserved columns and recalculate, then

[0367] c) Query clustering parameters: use query clustering; character length 4; maximum cluster distance 0.8; regular letters.

[0368] EMBOSS Needle, for example, uses the following parameters:

[0369] a) Matrix: BLOSUM62;

[0370] b) Open spaces: 10;

[0371] c) Expanded space: 0.5;

[0372] d) Output format: in pairs;

[0373] e) Final Gap Penalty: False;

[0374] f) Final slot opened: 10; and

[0375] g) Final vacancy expansion: 0.5.

[0376] "Subject" refers to a mammal, including but not limited to a human or a non-human mammal, such as a cow, horse, dog, sheep, or cat.

[0377] The term "target site" refers to a sequence within a nucleic acid molecule that is modified by a base editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein comprising a deaminase (e.g., cytidine or adenine deaminase).

[0378] Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybrids to target DNA cleavage sites, these proteins can, in principle, target any sequence specified by a guide RNA. Methods for performing site-specific cleavage (e.g., modifying genomes) using RNA-programmable nucleases (e.g., Cas9) are known in the art (e.g., see Cong, L. et al., Multiplex genome engineering using CRISPR / Cas systems., Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9., Science 339, 823-826 (2013); Hwang, WY et al., Efficient genome editing in zebrafish using a CRISPR-Cas system., Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems., Nucleic acids research (2013); Jiang, W. et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems., Nature biotechnology 31, 233-239 (2013); the entire contents of each are incorporated herein by reference).

[0379] As used herein, the term "treat" refers to alleviating or improving the disease and / or symptoms associated therewith or obtaining the desired pharmacological and / or physiological effects. It should be understood that, although not excluded, treating a disease or condition does not require the complete elimination of the disease, condition or symptoms associated therewith. In some embodiments, the effect is therapeutic, that is, but not limited to, the effect partially or completely alleviates (reduces), reduces (diminishes), eliminates (abrogates), alleviates (abates), alleviates (alleviates), alleviates (decreases) the intensity of the disease or cures (cures) the disease and / or is attributed to the adverse symptoms of the disease. In some embodiments, the effect is preventive, that is, the effect protects or prevents the occurrence or recurrence of a disease or condition. For this reason, the method disclosed in the present invention includes administering a therapeutically effective amount of a composition as described herein.

[0380] "Uracil glycosylase inhibitor" refers to an agent that inhibits the uracil excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds to the host uracil-DNA glycosylase and prevents the removal of uracil residues from DNA.

[0381] 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or a subrange of 50.

[0382] The recitation of chemical groups in any definition of a variable herein includes defining the variable as any single group or combination of the listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0383] Any composition or method provided herein can be combined with one or more of any other compositions or methods provided herein.

[0384] DNA editing has become a viable approach to altering disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms worked by inducing DNA double-strand breaks (DSBs) at specified genomic sites and relying on endogenous DNA repair pathways to determine the outcome of the product in a semi-random manner, resulting in a complex population of genetic products. Although precise user-defined repair outcomes can be achieved through the homology-directed repair (HDR) pathway, many challenges have prevented the use of HDR for efficient repair in therapeutically relevant cell types. In practice, this is inefficient relative to the competing, error-prone non-homologous end joining pathway. In addition, HDR is strictly restricted to the G1 and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells. As a result, it has proven difficult or impossible to efficiently change genomic sequences in these populations in a user-defined, programmable manner.

[0385] Nucleobase editors

[0386] Disclosed herein are base editors or core base editors for editing, modifying or changing the target nucleotide sequence of a polynucleotide. Described herein are core base editors or base editors comprising a polynucleotide programmable nucleotide binding domain and a core base editing domain. When combined with a bound guidance polynucleotide (e.g., gRNA), the polynucleotide programmable nucleotide binding domain can specifically bind to a target polynucleotide sequence (i.e., by complementary base pairing between the bases of the bound guide nucleic acid and the bases of the target polynucleotide sequence), thereby positioning the base editor to the target nucleic acid sequence desired to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.

[0387] Polynucleotide programmable nucleotide binding domain

[0388] The term "polynucleotide programmable nucleotide binding domain" refers to a protein associated with a nucleic acid (e.g., DNA or RNA), such as a guide polynucleotide (e.g., a guide RNA), which guides the polynucleotide programmable nucleotide 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. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cpf1 protein.

[0389] It should be understood that the polynucleotide programmable nucleotide binding domain can also include nucleic acid programmable proteins that bind RNA. For example, the polynucleotide programmable nucleotide binding domain can bind to a nucleic acid that guides the polynucleotide programmable nucleotide binding domain to RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of this application, although they are not specifically listed in this application.

[0390] The polynucleotide programmable nucleotide binding domain of the base editor itself may include one or more domains. For example, the polynucleotide programmable nucleotide binding domain may include one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain may include an endonuclease or an exonuclease. In this article, the term "exonuclease" refers to a protein or polypeptide capable of digesting nucleic acids (e.g., RNA or DNA) from the free end, and the term "endonuclease" refers to a protein or polypeptide capable of catalyzing (e.g., cutting) an internal region. In nucleic acids (e.g., DNA or RNA). In some embodiments, an endonuclease can cut a single strand of a double-stranded nucleic acid. In some embodiments, an endonuclease can cut two chains 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.

[0391] In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain can cut zero, one or two chains of the target polynucleotide.In some cases, the polynucleotide programmable nucleotide binding domain may include a nickase domain.Herein, the term "nickase" refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain, which is only capable of cutting one of the two chains in a double-stranded nucleic acid molecule (such as DNA). In some embodiments, a nickase can be derived from the complete catalytic activity (such as natural) form of a polynucleotide programmable nucleotide binding domain by introducing one or more mutations into an active polynucleotide programmable nucleotide binding domain. For example, in the case where a polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the nickase domain derived from Cas9 can include a D10A mutation and histidine at position 840. In this case, residue H840 retains catalytic activity and can therefore cut a single strand of a nucleic acid duplex. In another example, the nickase domain derived from Cas9 can include an H840A mutation, and the amino acid residue at position 10 is still D. In some embodiments, nickase can be derived from the complete catalytic activity (e.g., natural) form of polynucleotide.A programmable nucleotide binding domain is achieved by removing all or part of the nuclease domain that is not needed for nickase activity.For example, in the case where the polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the nickase domain derived from Cas9 can include all or part of the deletion of RuvC domain or HNH domain.

[0392] The amino acid sequence of an exemplary catalytically active Cas9 is as follows:

[0393]

[0394] Therefore, the base editor comprising a polynucleotide programmable nucleotide binding domain includes a nucleotidase domain, which is capable of producing single-stranded DNA breaks (nicks) on a specific polynucleotide target sequence (e.g., determined by the complementary sequence of the bound guide nucleic acid). In some embodiments, the chain of the nucleic acid duplex target polynucleotide sequence cut by a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) is a chain not edited by a base editor (i.e., the fragment cut by the base editor is opposite to the chain comprising the base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) can cut a chain targeted to the edited DNA molecule. In this case, the non-targeted chain will not be cut.

[0395] Also provided herein are base editors comprising polynucleotide programmable nucleotide binding domains, which are catalytically dead (i.e., unable to cut the target polynucleotide sequence). In this article, the terms "catalytically dead" and "nuclease dead" are used interchangeably and refer to polynucleotide programmable nucleotide binding domains having one or more mutations and / or deletions that cause them to be unable to cut nucleic acid chains. In some embodiments, due to specific point mutations in one or more nuclease domains, the catalytically dead polynucleotide programmable nucleotide binding domain base editor may lack nuclease activity. For example, in the case where the base editor comprises a Cas9 domain, Cas9 may simultaneously comprise a D10A mutation and an H840A mutation. Such mutations inactivate the two nuclease domains, resulting in the loss of nuclease activity. In other embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain may comprise one or more deletions of all or part of a catalytic domain (e.g., RuvC1 and / or HNH domain). In a further embodiment, the catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) and all or part of a nuclease domain.

[0396] Also contemplated herein are mutations of polynucleotide programmable nucleotide binding domains that are capable of producing catalytically dead polynucleotide programmable nucleotide binding domains from a previous functional form of the polynucleotide programmable nucleotide binding domain. For example, in the case of catalytically dead Cas9 ("dCas9"), variants with mutations other than D10A and H840A are provided, which result in nuclease-inactivated Cas9. For example, such mutations include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). Based on the present application and the knowledge in the art, other suitable nuclease-inactivated dCas9 domains will be apparent to those skilled in the art and are within the scope of the present application. Such additional exemplary suitable nuclease-inactivating Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., 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 entire contents of which are incorporated herein by reference).

[0397] Non-limiting examples of polynucleotide programmable nucleotide binding domains that can be incorporated into base editors include CRISPR protein-derived domains, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some cases, the base editor comprises a polynucleotide programmable nucleotide binding domain comprising a natural or modified protein or portion thereof that is capable of binding to a nucleic acid sequence (i.e., clustered regularly spaced short palindromic repeats) mediated nucleic acid modification during the CRISPR process by a bound guide nucleic acid. Such proteins are referred to herein as “CRISPR proteins.” Therefore, disclosed herein are base editors comprising polynucleotide programmable nucleotide binding domains, wherein the polynucleotide programmable nucleotide binding domains comprise all or part of a CRISPR protein (i.e., a base editor comprising all or part of a CRISPR protein as a domain, also referred to as a “CRISPR protein-derived domain” of a base editor). The domain of the CRISPR protein incorporated into the base editor can be modified compared to a wild-type or natural version of the CRISPR protein. For example, as described below, a domain derived from a CRISPR protein can comprise one or more mutations, insertions, deletions, rearrangements, and / or recombination relative to a wild-type or native form of the CRISPR protein.

[0398] CRISPR is an adaptive immune system that provides protection against mobile genetic components (viruses, transposable elements, and conjugative plasmids). The CRISPR cluster contains a spacer, a sequence complementary to the previously mobile component, and targets the invading nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, proper processing of pre-crRNA requires an anti-coding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. tracrRNA serves as a guide for RNase 3 to assist in pre-crRNA processing. Cas9 / crRNA / tracrRNA then performs endonucleolytic cleavage of a linear or circular dsDNA target complementary to the spacer. Target strands that are not complementary to the crRNA are first cleaved endonucleolytically, followed by 3'-5' exonucleolysis. In practice, DNA binding and cleavage typically require a protein and two RNAs. However, single-stranded guide RNAs ("sgRNAs" or simply "gRNAs") can be engineered to integrate aspects of both crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), incorporated herein by reference in its entirety. Cas9 recognizes a short motif (PAM or protospacer adjacent motif) in the CRISPR repeat sequence to help distinguish self from non-self.

[0399] In some embodiments, the methods described herein can utilize engineered Cas proteins. A guide RNA (gRNA) is a short synthetic RNA that is synthesized by Cas to bind a desired scaffold sequence and a user-defined The genomic target of the Cas protein is determined in part by the specificity of the gRNA targeting sequence for the genomic target compared to the rest of the genome.

[0400] In some embodiments, the gRNA scaffold sequence is as follows: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAUAAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0401] In some embodiments, the domain derived from CRISPR protein incorporated into the base editor is an endonuclease (e.g., deoxyribonuclease or ribonuclease) capable of binding to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the domain derived from CRISPR protein incorporated into the base editor is a nickase capable of binding to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the domain derived from CRISPR protein incorporated into the base editor is a catalytic death domain capable of binding to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the target polynucleotide bound to a domain derived from a CRISPR protein of a base editor is DNA. In some embodiments, the target polynucleotide bound to a domain derived from a CRISPR protein of a base editor is RNA.

[0402] Cas proteins that can be used herein include class 1 and class 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, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, C mr4, 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 / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h and Cas12i CARF, DinG, homologs or modifications thereof. Unmodified CRISPR enzymes can have DNA cleavage activity, such as Cas9, which has two functional endonuclease domains: RuvC and HNH. The CRISPR enzyme can direct cleavage of one or both strands at a target sequence, for example, within the target sequence and / or within the complement of the target sequence. For example, the CRISPR enzyme can direct cleavage of one or both strands at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or a plurality of base pairs from the first or last nucleotide of the target sequence.

[0403] Vectors encoding CRISPR enzymes that are mutated relative to the corresponding wild-type enzyme can be used so that the mutated CRISPR enzyme lacks the ability to cut one or both strands of a target polynucleotide containing a target sequence. Cas9 can 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 to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from Streptococcus pyogenes). Cas9 can refer to a polypeptide having at most or about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., from Streptococcus pyogenes). Cas9 may refer to a wild-type or modified form of the Cas9 protein, which may include amino acid changes, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0404] In some embodiments, the CRISPR protein-derived domain of the base editor may include a domain from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Ref: 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_021284.1); Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1), all or part of Cas9 of Streptococcus pyogenes or Staphylococcus aureus.

[0405] Cas9 domains of nucleobase editors

[0406] The term "Cas9" or "Cas9 domain" refers to a protein comprising a Cas9 protein or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and / or a gRNA binding domain of Cas9). Cas9 nucleases are sometimes also referred to as casn1 nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)-related nucleases. An exemplary Cas9 is Streptococcus pyogenes Cas9 (spCas9), whose amino acid sequence is as follows.

[0407] (Bottom line: HNH domain; double bottom line: RuvC domain)

[0408] The Cas9 nuclease sequence and structure are well known to those skilled in the art (e.g., see “Complete genome sequence of an M1 strain of Streptococcus pyogenes.”, Ferretti et al., 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 by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski J., et al., “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease inadaptive bacterial immunity.", Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821 (2012), each of which is incorporated herein by reference). Cas9 orthologs have been described in various species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus.Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on the disclosure, and such Cas9 nucleases and sequences include those from Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0409] In some aspects, nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-restrictive exemplary Cas9 domains are provided herein. The Cas9 domain can be a nuclease active Cas9 domain, a nuclease inactive Cas9 domain or a Cas9 nickase. In some embodiments, the Cas9 domain is a nuclease active domain. For example, the Cas9 domain can be a Cas9 domain that cuts two chains of a double-stranded nucleic acid (for example, two chains of a double-stranded DNA molecule). In some embodiments, the Cas9 domain includes any amino acid sequence as described herein. In some embodiments, the amino acid sequence included in the Cas9 domain 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 amino acid sequence listed herein. In some embodiments, the Cas9 domain comprises 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 herein. In some embodiments, the Cas9 domain comprises a polypeptide 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 contiguous amino acid residues compared to any of the amino acid sequences listed herein.

[0410] In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, the protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or a fragment thereof are referred to as "Cas9 variants". Cas9 variants have homology with Cas9 or a fragment thereof. For example, Cas9 variants are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical under the following conditions. It has at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity with wild-type Cas9. In some embodiments, the Cas9 variant can 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 changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some 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 96%, at least 97%, at least 98%, at least 99%, at least 99.5% of the amino acid length of the corresponding wild-type Cas9. In some embodiments, the fragment is at least 100 amino acids long. In some 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 1300 amino acids in length.

[0411] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of the Cas9 protein, such as one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but only comprise one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and other suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0412] The Cas9 protein can be associated with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactivated 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.

[0413]

[0414]

[0415] (Bottom line: HNH domain; double bottom line: RuvC domain)

[0416] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0417]

[0418]

[0419] (Single bottom line: HNH domain; double bottom line: RuvC domain).

[0420] In some embodiments, the wild-type Cas9 corresponds to Cas9 of Streptococcus pyogenes (NCBI reference sequence: NC_002737.2 (nucleotide sequence below); and Uniprot reference sequence: Q99ZW2 (amino acid sequence below):

[0421]

[0422]

[0423] (Bottom line: HNH domain; double bottom line: RuvC domain)

[0424] In some embodiments, Cas9 is derived from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheriae (NCBI Ref: NC_016782.1, NC_016786.1); Spiroplasma aphididae (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spirochete spirochetes (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Bellella balticola (NCBI Ref: NC_018010.1); Campylobacter contortus (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria monocytogenes (NCBI Ref: YP_820832.1), and Streptococcus pyogenes (NCBI Ref: YP_820832.1). Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1), or Neisseria meningitidis (NCBI Ref: YP_002342100.1), or Cas9 from any other organism.

[0425] It will be appreciated that other Cas9 proteins, including variants and homologs thereof (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is a nuclease-dead Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease-active Cas9.

[0426] In some embodiments, the Cas9 domain is a nuclease-free Cas9 domain (dCas9). For example, the dCas9 domain can bind to a double-stranded nucleic acid molecule (for example, by a gRNA molecule) without cutting any chain of the double-stranded nucleic acid molecule. In some embodiments, the nuclease-free dCas9 domain comprises a D10X mutation and an H840X mutation of the amino acid sequence listed herein, or a corresponding mutation in any amino acid sequence provided herein, wherein X is any amino acid change. In some embodiments, the nuclease-free dCas9 domain comprises a D10A mutation and an H840A mutation of the amino acid sequence listed herein, or a corresponding mutation in any amino acid sequence provided herein. As an example, the nuclease-free Cas9 domain comprises the amino acid sequence listed in the cloning vector pPlatTET-gRNA2 (accession number BAV54124).

[0427] The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows:

[0428]

[0429] (See, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell., 2013; 152(5): 1173-83, which is incorporated herein by reference in its entirety).

[0430] In some embodiments, the Cas9 nuclease has an inactivated (e.g., inactivated) DNA cleavage domain, i.e., the Cas9 is a nickase, referred to as a "nCas9" protein (as a "nickase" Cas9). Nuclease-inactivated Cas9 proteins can be interchangeably referred to as "dCas9" proteins (for nuclease "dead" Cas9) or catalytically inactive Cas9. Methods for generating Cas9 proteins (or fragments thereof) with inactivated DNA cleavage domains 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 of which is incorporated herein by reference in its entirety. For example, it is known that the DNA cleavage domain of Cas9 includes two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cuts the strand complementary to the gRNA, while the RuvC1 subdomain cuts the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivated 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)).

[0431] In some embodiments, the dCas9 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 dCas9 domain provided herein. In some embodiments, the Cas9 domain comprises 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 herein. In some embodiments, the Cas9 domain comprises a polypeptide 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 contiguous amino acid residues compared to any of the amino acid sequences listed herein.

[0432] In some embodiments, the dCas9 corresponds to or comprises, in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9.

[0433] In some embodiments, the dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A):

[0434] (Single bottom line: HNH domain; double bottom line: RuvC domain).

[0435] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequence provided above, or at the corresponding position in any of the amino acid sequences provided herein.

[0436] In other embodiments, dCas9 variants are provided having mutations other than D10A and H840A, e.g., resulting in nuclease-inactivated Cas9 (dCas9). For example, such mutations include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs 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 some embodiments, dCas9 variants are provided having an amino acid sequence that is shorter or longer by 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, about 100 amino acids, or more.

[0437] In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase can be a Cas9 protein that can only cut a chain of a double-stranded nucleic acid molecule (for example, a double-stranded DNA molecule). In some embodiments, the Cas9 nickase cuts the target chain of the double-stranded nucleic acid molecule, which means that the Cas9 nickase cuts the chain that is base-paired (complementary) with the gRNA (for example, sgRNA) bound to Cas9. In some embodiments, the Cas9 nickase includes a D10A mutation and has histidine at position 840. In some embodiments, the Cas9 nickase cuts the chain of the non-target, non-base editor of the double-stranded nucleic acid molecule, which means that the Cas9 nickase cuts the chain that is not base-paired with the gRNA (for example, sgRNA) bound to Cas9. In some embodiments, the Cas9 nickase includes an H840A mutation and has an aspartic acid residue or a corresponding mutation at position 10. In some embodiments, the amino acid sequence included in the Cas9 nickase 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 Cas9 nickase provided herein. Based on the application and the knowledge in the art, other suitable Cas9 nickases are apparent to those skilled in the art and are within the scope of the application.

[0438] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows:

[0439]

[0440] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., nanoarchaea), which constitute the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, the programmable nucleotide binding protein can be a CasX or CasY protein, which has been described in, for example, Burstein et al., "New CRISPR-Cas systems from uncultivated microbes.", Cell Res, February 21, 2017, doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, many CRISPR-Cas systems have been identified, including the Cas9 first reported in the archaeal domain of life. This differentiated Cas9 protein was found in the seldom-studied nanoarchaea and is part of an active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, have been found, which are the most compact systems discovered so far. In some embodiments, in the base editor system described herein, Cas9 is replaced by CasX or a variant of CasX. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasY or a variant of CasY. It should be understood that other RNA-guided DNA binding proteins can be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of this application.

[0441] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be CasX or CasY protein. In some embodiments, napDNAbp is CasX protein. In some embodiments, napDNAbp is CasY protein. In some embodiments, the amino acid sequence included in napDNAbp 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 naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide binding protein is a naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide binding protein comprises an amino acid sequence having 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% identity to any CasX or CasY protein described herein. It will be appreciated that CasX and CasY from other bacterial species may also be used in accordance with the present application.

[0442] An exemplary CasX ((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53)tr|F0NN87|F0NN87_SULIHCRISPR-associated Casx protein OS=Sulfolobus icelandica (strain HVE10 / 4) GN=SiH_0402PE=4SV=1) amino acid sequence is as follows:

[0443] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0444] An exemplary CasX (>tr|F0NH53|F0NH53_SULIR CRISPR-associated protein, Casx OS=Sulfolobus icelandica (strain REY15A) GN=SiRe_0771 PE=4SV=1) amino acid sequence is as follows:

[0445] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKV SEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0446] Delta-Proteobacterial CasX

[0447] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQ KWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0448] An exemplary CasY ((ncbi.nlm.nih.gov / protein / APG80656.1)>APG80656.1 CRISPR-associated protein CasY [uncultured bacteria from the phylum Thresholdiae]) amino acid sequence is as follows:

[0449]

[0450] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of the microbial CRISPR-Cas system. Single effectors of the microbial CRISPR-Cas system include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Generally, 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 single protein effectors. For example, Cas9 and Cpf1 are Class 2 effectors. In addition to Cas9 and Cpf1, Shmakov et al., "Discovery and Functional Characterization of DiverseClass 2CRISPR Cas Systems", Mol.Cell, November 5, 2015; 60(3): 385-397 also describe three different Class 2 CRISPR-Cas systems, the entire contents of which are incorporated herein by reference. The effectors Cas12b / C2c1 and Cas12c / C2c3 of two systems contain a RuvC-like endonuclease domain associated with Cpf1. The third system contains an effector with two predicate HEPN RNase domains. Unlike the CRISPR RNA produced by Cas12b / C2c1, the production of mature CRISPR RNA is independent of tracrRNA. Cas12b / C2c1 relies on both CRISPR RNA and tracrRNA for DNA cleavage.

[0451] The crystal structure of A. acidocaldarius Cas12b / C2c1 (AacC2c1) in complex with a chimeric single-molecule guide RNA (sgRNA) has been reported. For example, see Liu et al., "C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism", Mol. Cell, January 19, 2017; 65(2): 310-322, the entire contents of which are incorporated herein by reference. The crystal structure of A. acidocaldarius C2c1 bound to target DNA in a ternary complex has also been reported. For example, see Yang et al., "PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease", Cell, December 15, 2016; 167(7): 1814-1828, the entire contents of which are incorporated herein by reference. A catalytically competent conformation of AacC2c1 with target and non-target DNA strands has been independently trapped and placed in a single RuvC catalytic pocket, where Cas12b / C2c1-mediated cleavage results in a seven-nucleotide staggering of the target DNA. Structural comparisons between the Cas12b / C2c1 ternary complex and previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms employed by the CRISPR-Cas9 system.

[0452] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any fusion protein provided herein can be Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, napDNAbp is Cas12b / C2c1 protein. In some embodiments, napDNAbp is Cas12c / C2c3 protein. In some 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 naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, napDNAbp is a naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the 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 any of the napDNAbp sequences provided herein. It should be understood that Cas12b / C2c1 or Cas12c / C2c3 from other bacterial species can also be used according to the present application.

[0453] A Cas12b / C2c1 ((uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2|C2C1_ALIAGCRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acidophilus (strain ATCC 49025 / DSM 3922 / CIP106132 / NCIMB 13) GN=c2c1 PE=1SV=1) amino acid sequence is as follows:

[0454]

[0455] BhCas12b (Bacillus histidinidum) NCBI reference sequence: WP_095142515

[0456]

[0457] In some embodiments, Cas12b is BvCas12B, which is a variant of BhCas12b and comprises the following changes relative to BhCas12B: S893R, K846R, and E837G. BvCas12b (Bacillus V3-13) NCBI reference sequence: WP_101661451.1

[0458] RLETKKTPDERKKAIHKLMEIVQSYDSWTASQKEVWEKELNLLTNMAAFNDEIWKESLVELHHRIEPYVGQIVSKWRKGLSEGRKNLAGISMWNIDELEDTRRLLISWSKRSRTPG EANRIETDEPFGSSLLQHIQNVKDDRLKQMANLIIMTALGFKYDKEEKDRYKRWKETYPACQIILFENLNRYLFNLDRSRRENSRLMKWAHRSIPRTVSMQGEMFGLQVGDVRSEY SSRFHAKTGAPGIRCHALTEEDLKAGSNTLKRLIEDGFINESELAYLKKGDIIPSQGGELFVTLSKRYKKDSDNNELTVIHADINAAQNLQKRFWQQNSEVYRVPCQLARMGEDKL YIPKSQTETIKKYFGKGSFVKNNTEQEVYKWEKSEKMKIKTDTTFDLQDLDGFEDISKTIELAQEQQKKYLTMFRDPSGYFFNNETWRPQKEYWSIVNNIIKSCLKKKILSNKVEL

[0459] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes a second conformational change upon target binding that positions the nuclease domains to cleave the opposite strand of the target DNA. The end result of Cas9-mediated DNA cleavage 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 general repair pathways: (1) the efficient but error-prone non-homologous end joining (NHEJ) pathway; or (2) the less efficient but high-fidelity homology-directed repair (HDR) pathway.

[0460] The "efficiency" of non-homologous end joining (NHEJ) and / or homology-directed repair (HDR) can be calculated by any convenient method. For example, in some cases, the efficiency can be expressed as a percentage of successful HDR. For example, a surveyor nuclease assay can be used to generate cleavage products, and the ratio of product to substrate can be used to calculate the percentage. For example, as a result of successful HDR, a surveyor nuclease can be used that directly cuts DNA containing newly integrated restriction sequences. The more substrate that is cut, the higher the HDR percentage (the higher the HDR efficiency). As an illustrative example, the score (percentage) of HDR can be calculated using the following equation [(cleavage product) / (substrate plus cleavage product)] (e.g., (b+c) / (a+b+c), where "a" is the band intensity of the DNA substrate and "b" and "c" are the cleavage products.

[0461] In some cases, efficiency can be expressed as the success rate of NHEJ. For example, the T7 endonuclease I assay can be used to generate cleavage products, and the ratio of product to substrate can be used to calculate the percentage of NHEJ. T7 endonuclease cleaves mismatched heteroduplex DNA caused by hybridization of wild-type and mutant DNA strands (NHEJ generates small random insertions or deletions (Indels) at the original break site). A higher degree of cleavage indicates a higher percentage of NHEJ (higher NHEJ efficiency). As an illustrative example, the fraction (percentage) of NHEJ can be calculated using the following equation: (1-(1-(b(c+c) / (a+b+c))1 / 2)×100, where “a” is the band intensity of the DNA substrate and “b” and “c” are the cleavage products (Ran et al., Cell, 2013 Sep 12;154(6):1380-9; Ran et al., Nat Protoc., 2013 Nov;8(11):2281–2308).

[0462] The NHEJ repair pathway is the most active repair mechanism, frequently causing small nucleotide insertions or deletions (Indels) at DSB sites. The stochastic nature of NHEJ-mediated DSB repair has important practical implications, as cell populations expressing Cas9 and gRNA or guide polynucleotides can result in a wide variety of mutations. In most cases, NHEJ generates small insertions in the target DNA, leading to amino acid deletions, insertions, or frameshift mutations, resulting in premature 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.

[0463] While NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to generate specific nucleotide changes, ranging from single nucleotide changes to large insertions, such as the addition of fluorophores or tags.

[0464] To utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered to the cell type of interest along with gRNA and Cas9 or Cas9 nickase. The repair template can contain the desired edit as well as additional homologous sequences immediately upstream and downstream of the target (called left and right homology arms). The length of each homology arm can depend on the size of the introduced change, with larger insertions requiring longer homology arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. Even in cells expressing Cas9, gRNA, and an exogenous repair template, the efficiency of HDR is generally low (<10% of modified alleles). Because HDR occurs during the S and G2 phases of the cell cycle, the efficiency of HDR can be improved by synchronizing cells. Chemical or genetic inhibitors of genes involved in NHEJ can also increase the frequency of HDR.

[0465] In some embodiments, Cas9 is a modified Cas9. A given gRNA targeting sequence may have other sites with partial homology throughout the genome. These sites are called off-target sites and need to be considered when designing the gRNA. In addition to optimizing gRNA design, CRISPR specificity can also be improved by modifying Cas9. Cas9 produces double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. The Cas9 nickase is a D10A mutant of SpCas9 that retains one nuclease domain and produces DNA nicks instead of DSBs. The nickase system can also be combined with HDR-mediated gene editing for specific gene editing.

[0466] In some cases, Cas9 is a variant of Cas9 protein. When compared with the amino acid sequence of wild-type Cas9 protein, the amino acid sequence of the variant Cas9 polypeptide differs by one amino acid (e.g., with deletion, insertion, substitution, fusion). In some cases, the variant Cas9 polypeptide has an amino acid change (e.g., deletion, insertion or substitution) that reduces the nuclease activity of the Cas9 polypeptide. 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% nuclease. The activity of the corresponding wild-type Cas9 protein. In some cases, the variant Cas9 protein does not have substantial nuclease activity. When the subject Cas9 protein is a variant Cas9 protein without substantial nuclease activity, it can be referred to as "dCas9".

[0467] In some cases, the variant Cas9 protein has reduced nuclease activity. For example, the variant Cas9 protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the endonuclease activity of a wild-type Cas9 protein, such as a wild-type Cas9 protein.

[0468] In some cases, the variant Cas9 protein can cut the complementary chain of the guide target sequence, but has a reduced ability to cut the non-complementary chain of the double-stranded guide target sequence. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant Cas9 protein has D10A (aspartic acid with glutamic acid at amino acid position 10), so it can cut the complementary chain of the double-stranded guide target sequence, but the ability to cut the non-complementary chain of the double-stranded guide target sequence is reduced (therefore, when the variant Cas9 protein cuts the double-stranded target nucleic acid, it causes a single-strand break (SSB) instead of a double-strand break (DSB)) (for example, see Jinek et al., Science. 2012 August 17; 337 (6096): 816-21).

[0469] In some cases, variant Cas9 protein can cut the non-complementary chain of double-stranded guidance target sequence, but has the ability of reducing the complementary chain of cutting guidance target sequence.For example, variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, variant Cas9 protein has H840A (histidine to alanine at amino acid position 840) mutation, and therefore can cut the non-complementary chain of guidance target sequence, but has the ability of reducing the cutting target sequence. Guidance target sequence complementary chain (therefore, when variant Cas9 protein cuts double-stranded guidance target sequence, SSB will be produced instead of DSB). Such Cas9 protein cutting guides the ability of target sequence (such as single-stranded guidance target sequence) to reduce, but retains the ability to bind to guide target sequence (such as single-stranded guidance target sequence).

[0470] In some cases, the ability of the variant Cas9 protein to cut the complementary and non-complementary chains of the double-stranded target DNA is reduced. As a non-limiting example, in some cases, the variant Cas9 protein has both a D10A mutation and an H840A mutation, so that the polypeptide has a reduced ability to cut the complementary and non-complementary chains of the double-stranded target DNA. Such a Cas9 protein has a reduced ability to cut the target DNA (e.g., single-stranded target DNA), but retains the ability to bind to the target DNA (e.g., single-stranded target DNA).

[0471] As another non-limiting example, in some cases, the variant Cas9 protein has W476A and W1126A mutations, such that the polypeptide has a reduced ability to cut target DNA. Such a Cas9 protein has a reduced ability to cut target DNA (e.g., single-stranded target DNA), but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0472] As another non-limiting example, in some cases, the variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, such that the polypeptide has a reduced ability to cut target DNA. Such Cas9 proteins have a reduced ability to cut target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0473] As another non-limiting example, in some cases, the variant Cas9 protein has H840A, W476A and W1126A mutations, so that the polypeptide has the ability to cut the target DNA reduced. The ability of such Cas9 protein to cut target DNA (such as single-stranded target DNA) is reduced, but the ability to bind target DNA (such as single-stranded target DNA) is retained. As another non-limiting example, in some cases, the variant Cas9 protein carries H840A, D10A, W476A and W1126A mutations, so that the ability of the polypeptide to cut target DNA is reduced. The ability of such Cas9 protein to cut target DNA (such as single-stranded target DNA) is reduced, but the ability to bind target DNA (such as single-stranded target DNA) is retained. In some embodiments, variant Cas9 has restored catalytic His residues at position 840 in Cas9 HNH domain (A840H).

[0474] As another non-limiting example, in some cases, variant Cas9 protein with H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the ability of the polypeptide to cut target DNA is reduced. The ability of such Cas9 protein to cut target DNA (such as single-stranded target DNA) is reduced, but the ability to bind target DNA (such as single-stranded target DNA) is retained. As another non-limiting example, in some cases, variant Cas9 protein with D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the polypeptide has the ability to cut target DNA reduced. The ability of such Cas9 protein to cut target DNA (such as single-stranded target DNA) is reduced, but the ability to bind target DNA (such as single-stranded target DNA) is retained. In some cases, when the variant Cas9 protein carries W476A and W1126A mutations, or the variant Cas9 protein carries P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, the variant Cas9 protein cannot effectively bind to the PAM sequence. Therefore, in some such cases, when using this variant Cas9 protein in a binding method, the method does not require a PAM sequence. In other words, in some cases, when this Cas9 variant protein is used in a binding method, the method may include a guide RNA, but the method can be performed in the absence of a PAM sequence (thus, the specificity of the binding is provided by the targeting fragment of the guide RNA). Other residues can be mutated to obtain the above-mentioned effects (i.e., to inactivate one or another nuclease portion). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986 and / or A987 may be altered (i.e., substituted). Likewise, mutations other than alanine substitutions are also suitable.

[0475] In some embodiments, a variant Cas9 protein with reduced catalytic activity (e.g., when the Cas9 protein has D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986 and / or if it is A987 mutation, such as D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A and / or D986A), the Cas9 variant protein can still bind to the target DNA in a site-specific manner (because as long as it retains the ability to interact with the guide RNA, it can still be guided to the target DNA sequence by the guide RNA).

[0476] In some embodiments, the variant Cas protein can be spCas9, spCas9-VRQR, spCas9-VRER, xCas9(sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.

[0477] Alternatives to S. pyogenes Cas9 could include RNA-guided endonucleases of the Cpf1 family, which have shown cleavage activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the class II CRISPR / Cas system. This acquired immunity mechanism is found in Prevotella and Francisella. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, Cpf1-mediated DNA cleavage results in a double-strand break with a short 3' overhang. The staggered cleavage pattern of Cpf1 opens the possibility of directional gene transfer, similar to traditional restriction enzyme cloning, and can improve gene editing efficiency. Like the aforementioned Cas9 variants and orthologs, Cpf1 can also expand the number of CRISPR-targeted sites in AT-rich regions or AT genomes lacking the NGG PAM sites supported by SpCas9. The Cpf1 locus comprises a mixed α / β domain, followed by a helical region, a RuvC-II, and a zinc finger-like domain, RuvC-I. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, Cpf1 lacks the HNH endonuclease domain, and its N-terminus lacks the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain structure reveals that Cpf1 is functionally unique and is classified as a Class 2, Type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to type II systems than to type I and type III systems. Functional Cpf1 does not require transactivating CRISPR RNA (tracrRNA), so only CRISPR (crRNA) is required. This is beneficial for genome editing because Cpf1 is not only smaller than Cas9, but also has a smaller sgRNA molecule (about half that of Cas9). Compared to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex can cut the target DNA or RNA by recognizing the original spacer adjacent to 5'-YTN-3'. After identifying the PAM, Cpf1 introduces a sticky end-like DNA double-strand break with a 4 or 5 nucleotide overhang.

[0478] Some aspects of the present application provide a fusion protein comprising a domain acting as a nucleic acid programmable DNA binding protein, which can be used to guide a protein (e.g., a base editor) to a specific nucleic acid (e.g., DNA or RNA) sequence. In a specific embodiment, the fusion protein comprises 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 / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, and Cas12i. An example of a programmable polynucleotide binding protein with a PAM specificity different from that of Cas9 is a cluster of regularly spaced short palindromic repeats from Prevotella and Francisella 1 (Cpf1). Similar to Cas9, Cpf1 is also a Class 2 CRISPR effector. It has been shown that Cpf1 mediates a powerful DNA interference with features different from Cas9. Cpf1 is a single-stranded RNA-guided endonuclease lacking tracrRNA that utilizes a T-rich adjacent protospacer motif (TTN, TTTN or YTN). In addition, Cpf1 cuts DNA by staggered DNA double-strand breaks. Among the 16 Cpf1 family proteins, two enzymes from Acidaminococcus and Lachnospira were shown to have effective genome editing activity in human cells. Cpf1 proteins are known in the art and have been previously described, for example, by Yamano et al., "Crystal structure of Cpf1 in complex with guide RNA and target DNA." Cell. (165) 2016, p. 949-962; the entire contents of which are incorporated herein by reference.

[0479] Also useful in the compositions and methods of the present invention are nuclease-free Cpf1 (dCpf1) variants, which can be used as a polynucleotide binding protein domain that guides nucleotide sequence programming. The Cpf1 protein has a RuvC-like nuclease domain similar to the RuvC domain of Cas9, but does not have an HNH nuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. It is shown in Zetsche et al., Cell., 163, 759-771, 2015 (incorporated herein by reference) that the RuvC-like domain of Cpf1 is responsible for cutting the DNA chain and the inactivation of the RuvC-like domain. Cpf1 nuclease activity is inactivated. For example, mutations corresponding to D917A, E1006A or D1255A in Novicida novicida Cpf1 inactivate Cpf1 nuclease activity. In some embodiments, the dCpf1 of the present application comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A or D917A / E1006A / D1255A. It should be understood that according to the present invention, any mutation can be used, such as a substitution mutation, deletion or insertion that inactivates the RuvC domain of Cpf1. In some embodiments, the nucleic acid programmable nucleotide binding protein of any fusion protein provided herein can be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is a nuclease-inactivated Cpf1 (dCpf1). In some 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 a Cpf1 sequence disclosed herein. In some 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 a Cpf1 sequence disclosed herein, and comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It will be understood that Cpf1 from other bacterial species may also be used in accordance with the present application.

[0480] The amino acid sequence of wild-type Francisella novicida Cpf1 is shown below. D917, E1006, and D1255 are in bold and underlined.

[0481]

[0482] The following is the amino acid sequence of Cpf1 D917A from Francisella novogenes. (A917, E1006, and D1255 are bold and underlined).

[0483]

[0484]

[0485] The following is the amino acid sequence of Cpf1 E1006A from Francisella novo (D917, A1006, and D1255 are bold and underlined).

[0486]

[0487] Below is the amino acid sequence of Cpf1 D1255A from Francisella novae. (The D917, E1006, and A1255 mutation positions are shown in bold and underlined).

[0488]

[0489] The amino acid sequence of Francisella novogenes Cpf1 D917A / E1006A is shown below (A917, A1006, and D1255 are bold and underlined).

[0490]

[0491]

[0492] The following is the amino acid sequence of Francisella novogenes Cpf1 D917A / D1255A (A917, E1006, and A1255 are bold and underlined).

[0493]

[0494]

[0495] The amino acid sequence of Francisella novogenes Cpf1 E1006A / D1255A is shown below (D917, A1006, and A1255 are bold and underlined).

[0496]

[0497] The following is the amino acid sequence of Cpf1 D917A / E1006A / D1255A from Francisella novogenes. (A917, A1006, and A1255 are bold and underlined).

[0498]

[0499]

[0500] In some embodiments, one of the Cas9 domains present in the fusion protein can be replaced by a guide nucleotide sequence programmable DNA binding protein domain that does not require a PAM sequence.

[0501] In some embodiments, the Cas domain is a Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, a nuclease-inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 domain comprises an N579A mutation or a corresponding mutation in any of the amino acid sequences provided herein.

[0502] In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence with an atypical PAM. In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence with a NNGRRT or NNGRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of E781X, N967X, and R1014X mutations or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more E781K, N967K, and R1014H mutations in any amino acid sequence provided herein, or one or more corresponding mutations. In some embodiments, the SaCas9 domain comprises E781K, N967K, or R1014H mutations or corresponding mutations in any amino acid sequence provided herein.

[0503] The amino acid sequence of an exemplary SaCas9 is as follows:

[0504]

[0505]

[0506] In the sequence, the underlined and bold residue N579 can be mutated (e.g., to A579) to generate the SaCas9 nickase.

[0507] The amino acid sequence of an exemplary SaCas9n is as follows:

[0508]

[0509] In the sequence, residue A579, which can be mutated from N579 to generate the SaCas9 nickase, is underlined and bolded.

[0510] The amino acid sequence of an exemplary SaKKH Cas9 is as follows:

[0511]

[0512] Residue A579 is underlined and bolded above and can be mutated from N579 to generate the SaCas9 nickase. Residues K781, K967, and H1014 are underlined and italicized above and can be mutated from E781, N967, and R1014 to generate the SaKKH Cas9.

[0513] High-fidelity Cas9 domain

[0514] Some aspects of the present application provide high-fidelity Cas9 domains. In some embodiments, the high-fidelity Cas9 domain is an engineered Cas9 domain comprising one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA relative to the corresponding wild-type Cas9 domain. The high-fidelity Cas9 domain with reduced electrostatic interaction with the sugar-phosphate backbone of DNA may have fewer off-target effects. In some embodiments, the Cas9 domain (e.g., a wild-type Cas9 domain) comprises one or more mutations that reduce the association between the Cas9 domain and the sugar-phosphate backbone of DNA. In some embodiments, the Cas9 domain comprises one or more mutations that reduce the association of the Cas9 domain with 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%.

[0515] In some embodiments, any Cas9 fusion proteins provided herein include one or more of N497X, R661X, Q695X and / or Q926X mutations or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, any Cas9 fusion proteins provided herein include one or more of N497A, R661A, Q695A and / or Q926A mutations or corresponding mutations in any amino acid sequence provided herein. In some embodiments, the Cas9 domain includes a D10A mutation or a corresponding mutation in any amino acid sequence provided herein. Cas9 domains with high fidelity are known in the art and will be apparent to those skilled in the art. For example, 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 entire contents of each are incorporated herein by reference.

[0516] In some embodiments, the modified Cas9 is a high-fidelity Cas9 enzyme. In some embodiments, the high-fidelity Cas9 enzyme is SpCas9 (K855A), eSpCas9 (1.1), SpCas9-HF1, or a super-precise Cas9 variant (HypaCas9). The modified Cas9 eSpCas9 (1.1) contains alanine substitutions that weaken the interaction between the HNH / RuvC groove and the non-target DNA strand, thereby preventing strand separation and cutting at off-target sites. Similarly, SpCas9-HF1 reduces off-target editing through alanine substitutions that disrupt the interaction between Cas9 and the DNA phosphate backbone. HypaCas9 contains mutations in the REC3 domain (SpCas9N692A / M694A / Q695A / H698A) that increase Cas9 proofreading and target recognition. All three high-fidelity enzymes produce fewer off-target edits compared to wild-type Cas9.

[0517] Exemplary high-fidelity Cas9 is provided below.

[0518] High-fidelity Cas9 domain mutations relative to Cas9 are shown in bold and underlined.

[0519]

[0520] Guide polynucleotide

[0521] As used herein, the term "(one or more) guide polynucleotides" refers to a polynucleotide that can be 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 a guide RNA. As used herein, the term "guide RNA (gRNA)" and its grammatical equivalents can refer to an RNA that can be specific to a target DNA and can form a complex with a Cas protein. The RNA / Cas complex can help "guide" the Cas protein to the target DNA. Cas9 / crRNA / tracrRNA endonuclease cuts a linear or circular dsDNA target that is complementary to a spacer. The target chain that is not complementary to the crRNA is first cut into the nucleic acid, and then cuts 3'-5' in the nucleic acid exonuclease. In fact, DNA binding and cutting generally require protein and two RNAs. However, single-stranded guide RNA ("sgRNA" or simply "gNRA") can be modified so that each aspect of crRNA and tracrRNA is integrated into a single RNA species. See, e.g., Jinek M. et al., Science 337:816-821 (2012), which is incorporated herein by reference in its entirety.Cas9 recognizes a short motif (PAM or protospacer adjacent motif) in the CRISPR repeat sequence to help distinguish self from non-self. The Cas9 nuclease sequence and structure are well known to those skilled in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti, JJ et al., Natl. Acad. Sci. USA 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and hostfactor RNase III.” Deltcheva E. et al., Nature 471:602-607 (2011); and Jinek M. et al., “Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, Streptococcus pyogenes and Streptococcus thermophilus.Based on the disclosure, other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art, and such Cas9 nucleases and sequences include those from Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5,726-737; the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease has an inactivated (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase.

[0522] In some embodiments, the guide polynucleotide is at least one single guide RNA ("sgRNA" or "gRNA"). In some embodiments, the guide polynucleotide is at least one tracrRNA. In some embodiments, the guide 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.

[0523] The polynucleotide programmable nucleotide binding domains (e.g., CRISPR-derived domains) of base editors disclosed herein can be identified by binding to a guidance polynucleotide. Guidance polynucleotides (e.g., gRNA) are typically single-stranded and can be programmed to specifically bind to the target sequence site of the polynucleotide (i.e., by complementary base pairing), thereby guiding the nucleic acid of the base editor target sequence bound to the guidance. The guidance polynucleotide can be DNA. The guidance polynucleotide can be RNA. In some cases, the guidance polynucleotide includes natural nucleotides (e.g., adenosine). In some cases, the guidance polynucleotide includes non-natural (or non-natural) nucleotides (e.g., peptide nucleic acid or nucleotide analogs). In some cases, the targeting region of the guidance 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 in length. The length of the targeting region of the guidance nucleic acid can be between 10 and 30 nucleotides, or between 15 and 25 nucleotides, or between 15 and 20 nucleotides.

[0524] In some embodiments, the guide polynucleotide comprises two or more separate polynucleotides that can interact with each other through, for example, complementary base pairing (e.g., double-stranded guide polynucleotides). For example, the guide polynucleotide can comprise a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA). For example, the guide polynucleotide can comprise one or more transactivating CRISPR RNAs (tracrRNAs).

[0525] In a type II CRISPR system, targeting nucleic acids by a CRISPR protein (e.g., Cas9) 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) containing a repeat sequence that forms a scaffold region that stabilizes the guide RNA-CRISPR protein complex. Such a double-stranded guide RNA system can be used as a guide polynucleotide to guide the base editor disclosed herein to the target polynucleotide sequence.

[0526] In some embodiments, the base editors provided herein utilize single-stranded guidance polynucleotides (e.g., gRNA). In some embodiments, the base editors provided herein utilize double-stranded guidance polynucleotides (e.g., double-stranded gRNA). In some embodiments, the base editors provided herein utilize one or more guidance polynucleotides (e.g., multiple gRNAs). In some embodiments, single-stranded guidance polynucleotides are used for different base editors described herein. For example, single-stranded guidance polynucleotides can be used for cytidine base editors and adenosine base editors.

[0527] In other embodiments, the guide polynucleotide can comprise both the polynucleotide targeting portion of the nucleic acid and the scaffold portion of the nucleic acid in a single molecule (i.e., a single-molecule guide nucleic acid). For example, the single-molecule guide polynucleotide can be a single-stranded guide RNA (sgRNA or gRNA). As used herein, the term "guide polynucleotide sequence" encompasses any single-molecule, double-molecule, or multi-molecule nucleic acid that is capable of interacting with and guiding a target polynucleotide sequence to the target polynucleotide sequence.

[0528] Typically, a guiding polynucleotide (e.g., a crRNA / trRNA complex or gRNA) comprises a "polynucleotide targeting fragment" and a "protein binding fragment," wherein the fragment comprises a sequence capable of recognizing and binding to a target polynucleotide sequence. The guiding polynucleotide is stabilized within the polynucleotide programmable nucleotide binding domain component of the base editor. In some embodiments, the polynucleotide targeting segment of the guiding polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating the editing of bases in the DNA. In other cases, the polynucleotide targeting segment of the guiding polynucleotide recognizes and binds to an RNA polynucleotide, thereby facilitating the editing of bases in the RNA. As used herein, a "fragment" refers to a portion or region of a molecule, such as a continuous stretch of nucleotides in a guiding polynucleotide; a fragment may also refer to a region / portion of a complex, such that a fragment may comprise a region greater than or equal to 1. For example, where the guiding polynucleotide comprises a plurality of nucleic acid molecules, its protein binding segment may include, for example, all or a portion of a plurality of separate molecules that hybridize along a region of complementarity. A binding segment of a DNA-targeting RNA comprising two separate molecules can comprise (i) 40 to 75 base pairs of a first RNA molecule that is 100 base pairs in length; and (ii) 10 to 25 base pairs of a second RNA molecule that is 50 base pairs in length. The definition of "segment," unless otherwise expressly defined in a particular context, is not limited to a particular number of total base pairs, nor is it limited to any particular number of base pairs from which to proceed. For a given RNA molecule, it is not limited to a particular number of separate molecules in the complex, and can include regions of RNA molecules of any total length, and can include regions that are complementary to other molecules.

[0529] The guide RNA or guide polynucleotide may include two or more RNAs, for example, CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The guide RNA or guide polynucleotide may sometimes include single-stranded RNA, or a single guide RNA (sgRNA) formed by fusing a portion (e.g., a functional portion) of crRNA and tracrRNA. The guide RNA or guide polynucleotide may also be a double-stranded RNA comprising crRNA and tracrRNA. In addition, crRNA can hybridize with target DNA.

[0530] As described above, the guide RNA or guide polynucleotide can be an expression product. For example, the DNA encoding the guide RNA can be a vector comprising a sequence encoding the guide RNA. The guide RNA or guide polynucleotide can be transferred into the cell by transfecting the cell with an isolated guide RNA or a plasmid DNA comprising a sequence encoding the guide RNA and a promoter. The guide RNA or guide polynucleotide can also be transferred into the cell in other ways, such as using viral-mediated gene delivery.

[0531] The guide RNA or guide polynucleotide can be isolated. For example, the guide RNA can be transfected into a cell or organism in the form of isolated RNA. The guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the art. The guide RNA can be transferred to the cell in the form of isolated RNA rather than a plasmid containing the coding sequence of the guide RNA.

[0532] The guide RNA or guide polynucleotide can comprise three regions: a first region at the 5' end that can be complementary to the target site in the chromosomal sequence; a second internal region that can form a stem-loop structure; and a third 3' region that can be single-stranded. The first region of each guide RNA can also be different, so that each guide RNA guides the fusion protein to a specific target site. In addition, the second and third regions of each guide RNA can be the same in all guide RNAs.

[0533] The first region of the guide RNA or the guide polynucleotide can be complementary to the sequence of the target site in the chromosome sequence so that the first region of the guide RNA can be base paired with the target site. In some cases, the first region of the guide RNA may include about 10 nucleotides to 25 nucleotides or about 10 nucleotides to 25 nucleotides (i.e., 10 nucleotides to nucleotides; or about 10 nucleotides to about 25 nucleotides; or 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 base pairing region between the first region of the guide RNA and the target site in the chromosome sequence can be or can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25 or more nucleotide lengths. Sometimes, the length of the first region of the guide RNA can be or can be about 19, 20 or 21 nucleotides.

[0534] The guide RNA or guide polynucleotide can also include a second region that forms a secondary structure. For example, the secondary structure formed by the guide RNA can include a stem (or hairpin) and a loop. The length of the loop and the stem can vary. For example, the length of the loop can be in the range of about 3 to 10 nucleotides or about 3 to 10 nucleotides, and the length of the stem can be in the range of about 6 to 20 base pairs or about 6 to 20 base pairs. The stem can include one or more 1 to 10 or about 10 nucleotides. The length of the total length of the second region can be in the range of about 16 to 60 nucleotides or 16 to 60 nucleotides. For example, the length of the loop can be or can be about 4 nucleotides, and the length of the stem can be or can be about 12 base pairs.

[0535] The guide RNA or guide polynucleotide may also include a third region at the 3' end that may be substantially single-stranded. For example, the third region may not be complementary to any chromosomal sequence in the target cell and may not be complementary to the rest of the guide RNA. In addition, the length of the third region may vary. The length of the third region may be greater than or greater than about 4 nucleotides. For example, the length of the third region may be in the range of about 5 to 60 nucleotides.

[0536] The guide RNA or guide polynucleotide can target any exon or intron of a gene target. In some cases, the guide can target exon 1 or exon 2 of a gene; the guide can target exon 3 or 4 of a gene. A composition can contain multiple guide RNAs that all target the same exon, or in some cases, can contain multiple guide RNAs that can target different exons. Exons and introns of a gene can be targeted.

[0537] The guide RNA or guide polynucleotide can target a nucleic acid sequence of 20 nucleotides or about 20 nucleotides. The target nucleic acid can be less than or about 20 nucleotides. The target nucleic acid can be at least or at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 in length, or between 1 and 100 nucleotides in length. The target nucleic acid can be at most or about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or any value between 1 and 100 in length. The target nucleic acid sequence can be or can be 20 bases 5' of the first nucleotide of the PAM. The guide RNA can target a nucleic acid sequence. The target nucleic acid can be at least or at least about 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, or 1 to 100 nucleotides.

[0538] A guide polynucleotide, such as a guide RNA, can refer to a nucleic acid that can hybridize with another nucleic acid, such as a target nucleic acid or a protospacer in the genome of a cell. A guide polynucleotide can be RNA. A guide polynucleotide can be DNA. A guide polynucleotide can be programmed or designed to bind to a nucleic acid sequence specifically at a site. A guide polynucleotide can comprise a polynucleotide chain and can be referred to as a single-stranded guide polynucleotide. A guide polynucleotide can comprise two polynucleotide chains and can be referred to as a double-stranded guide polynucleotide. A guide RNA can be introduced into a cell or embryo as an RNA molecule. For example, an RNA molecule can be transcribed in vitro and / or can be chemically synthesized. A guide RNA can be produced from a synthetic DNA molecule (e.g., Gene fragment) transcribes RNA. The guide RNA can then be introduced into the cell or embryo as an RNA molecule. The guide RNA can also be introduced into the cell or embryo in the form of a non-RNA nucleic acid molecule, such as a DNA molecule. For example, the DNA encoding the guide RNA can be operably linked to a promoter control sequence to express the guide RNA in the target cell or embryo. The RNA coding sequence can be operably linked to a promoter sequence recognized by RNA polymerase III (Pol III). Plasmid vectors that can be used to express guide RNA include but are not limited to px330 vectors and px333 vectors. In some cases, the plasmid vector (e.g., px333 vector) may contain at least two DNA sequences encoding the guide RNA.

[0539] The method of selecting, designing and verifying guiding polynucleotides, such as guide RNA and targeting sequences are described in this article and are known to those skilled in the art. For example, in order to minimize the impact of the potential substrate of the deaminase domain in the abasic enzyme domain (e.g., AID domain), the number of residues of deamination can be unintentionally targeted (e.g., the C residues of off-target may minimize the residues on the ssDNA in the target nucleic acid locus). In addition, software tools can be used to optimize the gRNA corresponding to the target nucleic acid sequence, such as to minimize the off-target activity of the entire genome. For example, for each possible targeting domain selection performed using Streptococcus pyogenes Cas9, all off-target sequences (before the PAM selected, such as NAG or NGG) can be identified in the entire genome, including up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) of mismatched base pairs. The first region of the gRNA complementary to the target site can be identified, and all first regions (e.g., crRNA) can be ranked according to their total predicted off-target scores; the highest-ranked positioning domain represents the activity with the most target activity and the least target activity. Candidate targeting gRNAs can be functionally evaluated using methods known in the art and / or described herein.

[0540] As a non-limiting example, a DNA sequence search algorithm can be used to identify the target DNA hybridization sequence in the crRNA of the guide RNA used with Cas9. Custom gRNA design software based on the public tool cas-offer can be used for gRNA design, as described in Bae S., Park J., and Kim J.-S. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases, Bioinformatics 30, 1473-1475 (2014). The software scores the guide after calculating the tendency of the guide genome to deviate from the target. Typically, for guides ranging from 17 to 24 in length, matches from perfect matches to 7 mismatches are considered. Once the non-target site is determined by calculation, a total score is calculated for each guide and summarized as a table output interface using the web version. In addition to identifying potential target sites adjacent to the PAM sequence, the software also identifies all PAM adjacent sequences that differ from the selected target site by 1, 2, 3 or more nucleotides. The genomic DNA sequence of the target nucleic acid sequence, such as the target gene, can be obtained and can be screened for repetitive elements using publicly available tools such as the RepeatMasker program. RepeatMasker searches for repetitive elements and low-complexity regions in the input DNA sequence. The output is a detailed annotation of the repeats present in the given query sequence.

[0541] After identification, the first region of the guide RNA, e.g., crRNA, can be classified based on their distance from the target site, their orthogonality, and the presence of a 5' nucleotide that closely matches a relevant PAM sequence (e.g., based on identifying a closely matching 5'G in the human genome that contains a relevant PAM, such as the NGG PAM of Streptococcus pyogenes, the NNGRRT or NNGRRV PAM of Staphylococcus aureus). As used herein, orthogonality refers to the number of sequences in the human genome that contain the minimum number of mismatches with the target sequence. "High level of orthogonality" or "good orthogonality" can, for example, refer to a 20-mer targeting domain that has no identical sequence in the human genome other than the intended target, nor any sequence that contains one or two mismatches in the target. Targeting domains with good orthogonality can be selected to minimize the cleavage of off-target DNA.

[0542] In some embodiments, a reporter system can be used to detect base editing activity and test candidate guide polynucleotides. In some embodiments, the reporter system can include a reporter gene-based assay, wherein the base editing activity results in the expression of the reporter gene. For example, the reporter system can include a reporter gene that includes an inactivated start codon, such as a mutation from 3'-TAC-5' to 3'-CAC-5' on the template chain. After the target C is successfully deaminated, the corresponding mRNA will be transcribed as 5'-AUG-3' instead of 5'-GUG-3', thereby enabling the translation of the reporter gene. Suitable reporter genes are obvious to those skilled in the art. Non-limiting examples of reporter genes include genes encoding green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase, secretory alkaline phosphatase (SEAP), or any other gene expressed that is obvious to those skilled in the art. The reporter system can be used to test many different gRNAs, for example, to determine which residue of the target DNA sequence the corresponding deaminase will target. sgRNAs targeting the non-template strand can also be tested to evaluate specific base editing proteins (e.g., Cas9 deaminase fusion proteins. In some embodiments, such gRNAs can be designed so that the mutated start codon will not base pair with the gRNA. The guide polynucleotide can comprise standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine), ribonucleotide isomers and / or ribonucleotide analogs. In some embodiments, the guide polynucleotide can comprise at least one detectable label. The detectable label can be a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags or suitable fluorescent dyes), a detection label (e.g., biotin, digoxigenin, etc.), quantum dots, or gold particles.

[0543] The guide polynucleotide can be chemically synthesized, enzymatically synthesized, or a combination thereof. For example, a solid phase synthesis method based on standard phosphoramidites can be used to synthesize guide RNA. Alternatively, the guide RNA can be synthesized in vitro by operably connecting the DNA encoding the guide RNA to a promoter control sequence recognized by a phage RNA polymerase. Examples of suitable phage promoter sequences include T7, T3, SP6 promoter sequences, or variants thereof. In embodiments where the guide RNA comprises two separate molecules (e.g., crRNA and tracrRNA), crRNA can be chemically synthesized and tracrRNA can be enzymatically synthesized.

[0544] In some embodiments, the base editor system can include multiple guide polynucleotides, for example, gRNAs. For example, gRNAs can target one or more target loci (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 g gRNAs) included in the base editor system. The multiple gRNA sequences can be arranged in series and are preferably separated by direct repeats.

[0545] The DNA sequence encoding the guide RNA or guide polynucleotide can also be part of the vector. In addition, the vector can contain other expression control sequences (such as enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selective marker sequences (such as GFP or antibiotic resistance genes, such as puromycin), replication origins, etc. The DNA molecule encoding the guide RNA can also be linear. The DNA molecule encoding the guide RNA or guide polynucleotide can also be circular.

[0546] In some embodiments, one or more components of the base editor system can be encoded by a DNA sequence. Such DNA sequences can be introduced into an expression system, such as a cDNA. One unit, together or separately. For example, a DNA sequence encoding a polynucleotide programmable nucleotide binding domain and a guide RNA can be introduced into a cell, and each DNA sequence can be part of a separate molecule (e.g., a vector comprising a polynucleotide programmable nucleotide binding domain encoding sequence and another vector comprising a guide RNA encoding sequence or both can be part of the same molecule (e.g., a vector comprising a polynucleotide programmable nucleotide binding domain and a coding (and regulatory) sequence of a guide RNA).

[0547] The guidance polynucleotide may comprise one or more modifications to provide a nucleic acid with new or enhanced characteristics. The guidance polynucleotide may comprise a nucleic acid affinity tag. The guidance polynucleotide may comprise synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0548] In some cases, the gRNA or guide polynucleotide may include modifications. Modifications can be made at any position of the gRNA or guide polynucleotide. More than one modification can be made to a single-stranded gRNA or guide polynucleotide. After modification, the gRNA or guide polynucleotide can be quality controlled. In some cases, quality control can include PAGE, HPLC, MS, or any combination thereof.

[0549] Modifications of the gRNA or guide polynucleotide can be substitutions, insertions, deletions, chemical modifications, physical modifications, stabilization, purification, or any combination thereof.

[0550] It can also be modified by 5' adenylate, 5' guanosine triphosphate cap, 5' N7-methylguanosine triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' phosphorothioate, 5' phosphate, 5' phosphorothioate, cis-thymine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, d spacer, PC spacer, r spacer, 18 spacer 9, 3'-3' modification, 5'-5' modification, no base, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesterol TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye The gRNA or guide polynucleotide is modified with QC-1, QSY-21, QSY-35, QSY-7, QSY-9, a carboxyl linker, a thiol linker, a 2'-deoxyribonucleoside analog purine, a 2'-deoxyribonucleoside analog pyrimidine, a ribonucleoside analog, a 2'-O-methyl ribonucleoside analog, a sugar-modified analog, a wobble / universal base, a fluorescent dye label, 2'-fluoro RNA, 2'-O-methyl RNA, a methyl phosphonate, a phosphodiester deoxyribonucleic acid, a phosphodiester RNA, a phosphorothioate DNA, a phosphorothioate RNA, UNA, pseudouridine 5'-triphosphate, 5'-methylcytidine 5'-triphosphate, or any combination thereof.

[0551] In some cases, the modification is permanent. In other cases, the modification is temporary. In some cases, multiple modifications are made to the gRNA or guide polynucleotide. The gRNA or guide polynucleotide modifications can change the physicochemical properties of the nucleotide, such as its conformation, polarity, hydrophobicity, chemical reactivity, base pairing interactions, or any combination thereof.

[0552] The modification can also be a phosphorothioate substitute. In some cases, natural phosphodiester bonds may be susceptible to rapid degradation by cellular nucleases, and modifying the internucleotide bond with a phosphorothioate (PS) bond substitute is more stable to hydrolysis by cellular degradation. The modification can increase the stability of the gRNA or guide polynucleotide. The modification can also enhance biological activity. In some cases, phosphorothioate-enhanced RNA gRNA can inhibit RNase A, RNase T1, calf serum nuclease, or any combination thereof. These properties allow the use of PS-RNA gRNA in applications where exposure to nucleases is highly likely in vivo or in vitro. For example, a phosphorothioate (PS) bond can be introduced between the last 3-5 nucleotides of the 5' or " end of the gRNA, which can inhibit exonuclease degradation. In some cases, phosphorothioate bonds can be added throughout the gRNA to reduce attack by endonucleases.

[0553] Protospacer-adjacent motif

[0554] The term "spacer adjacent motif (PAM)" or PAM-like motif refers to a 2-6 base pair DNA sequence that immediately follows 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).

[0555] A protospacer adjacent motif (PAM) or PAM-like motif refers to a DNA sequence of 2-6 base pairs that follows 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., upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (i.e., downstream of the 5' end of the protospacer). The PAM sequence is crucial for target binding, but the exact sequence depends on the type of Cas protein.

[0556] The base editors provided herein may include a CRISPR protein-derived domain that is capable of binding to a nucleotide sequence comprising a typical or atypical protospacer adjacent motif (PAM) sequence. A PAM site is a nucleotide sequence close to a target polynucleotide sequence. Some aspects of the present application provide base editors comprising all or part of a CRISPR protein with different PAM specificities. For example, a typical Cas9 protein, such as Cas9 (spCas9) of Streptococcus pyogenes, requires a typical NGG PAM sequence to bind to a specific nucleic acid region, where the "N" in "NGG" is adenine (A), thymine (T), guanine (G) or cytosine (C), and G is guanine. PAM can be CRISPR protein-specific and can be different between different base editors comprising different CRISPR protein-derived domains. PAM can be 5' or 3' of the target sequence. PAM can be upstream or downstream of the target sequence. The length of PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. Typically, the length of PAM is between 2-6 nucleotides.

[0557] In some embodiments, the Cas9 domain is a Cas9 domain of Streptococcus pyogenes (SpCas9). In some embodiments, the SpCas9 domain is a nuclease-active SpCas9, a nuclease-inactive SpCas9 (SpCas9d) or a SpCas9 nickase (SpCas9n). In some embodiments, SpCas9 comprises a D9X mutation or a corresponding mutation in any amino acid sequence provided herein, wherein X is any amino acid except D. In some embodiments, SpCas9 comprises a D9A mutation or a corresponding mutation. In any amino acid sequence provided herein. In some embodiments, the SpCas9 domain, SpCas9d domain or SpCas9n domain can bind to a nucleic acid sequence with an atypical PAM. In some embodiments, the SpCas9 domain, SpCas9d domain or SpCas9n domain can bind to a nucleic acid sequence with an NGG, NGA or NGCG PAM sequence. In some embodiments, the SpCas9 domain comprises one or more of D1135X, R1335X, and T1336X mutations or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1135E, R1335Q, and T1336R mutations or corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SpCas9 domain comprises D1135E, R1335Q, and T1336R mutations, or corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SpCas9 domain comprises one or more of D1135X, R1335X, and T1336X mutations or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1135V, R1335Q, and T1336R mutations or corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SpCas9 domain comprises D1135V, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of D1135X, G1217X, R1335X, and T1336X mutations, or corresponding mutations in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1135V, G1217R, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises D1135V, G1217R, R1335Q, and T1336R mutations, or corresponding mutations in any of the amino acid sequences provided herein.

[0558] In some embodiments, the Cas9 domain of any fusion protein provided herein comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80% amino acid sequence, and has 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% identity with the Cas9 polypeptide described herein. In some embodiments, the Cas9 domain of any fusion protein provided herein comprises the amino acid sequence of any Cas9 polypeptide described herein. In some embodiments, the Cas9 domain of any fusion protein provided herein consists of the amino acid sequence of any Cas9 polypeptide described herein.

[0559] The amino acid sequence of an exemplary PAM-bound SpCas9 is as follows:

[0560]

[0561] The amino acid sequence of an exemplary PAM-bound SpCas9n is as follows:

[0562]

[0563] An exemplary PAM-binding amino acid sequence of SpEQR Cas9 is as follows:

[0564]

[0565] In this sequence, residues E1135, Q1335, and R1337, which can be mutated from D1135, R1335, and T1337 to generate SpEQRCas9, are underlined and bolded.

[0566] The amino acid sequence of an exemplary PAM-bound SpVQR Cas9 is as follows:

[0567]

[0568]

[0569] In this sequence, residues V1135, Q1335, and R1336, which can be mutated from D1135, R1335, and T1336 to generate SpVQRCas9, are underlined and bolded.

[0570] The amino acid sequence of an exemplary PAM-bound SpVRER Cas9 is as follows:

[0571]

[0572]

[0573] In some embodiments, the Cas9 domain is a recombinant Cas9 domain. In some embodiments, the recombinant Cas9 domain is a SpyMacCas9 domain. In some embodiments, the SpyMacCas9 domain is a nuclease-active SpyMacCas9, a nuclease-inactive SpyMacCas9 (SpyMacCas9d), or a SpyMacCas9 nickase (SpyMacCas9n). In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence with an atypical PAM. In some embodiments, the SpyMacCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence with an NAA PAM sequence.

[0574] Exemplary SpyMacCas9

[0575]

[0576] In some cases, variant Cas9 protein carries H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1218A mutations, so that the ability of the polypeptide to cut target DNA or RNA is reduced. The ability of such Cas9 protein to cut target DNA (such as single-stranded target DNA) is reduced, but the ability to bind target DNA (such as single-stranded target DNA) is retained. As another non-limiting example, in some cases, variant Cas9 protein carries D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1218A mutations, so that the ability of the polypeptide to cut target DNA is reduced. The ability of such Cas9 protein to cut target DNA (such as single-stranded target DNA) is reduced, but the ability to bind target DNA (such as single-stranded target DNA) is retained. In some cases, when the variant Cas9 protein carries W476A and W1126A mutations, or the variant Cas9 protein carries P475A, W476A, N477A, ​​D1125A, W1126A and D1218A mutations, the variant Cas9 protein cannot effectively bind to the PAM sequence. Therefore, in some such cases, when such variant Cas9 protein is used in a binding method, the method does not require a PAM sequence. In other words, in some cases, when such a Cas9 variant protein is used in a binding method, the method may include a guide RNA, but the method may be performed in the absence of a PAM sequence (the specificity of the binding is therefore provided by the targeting fragment of the guide RNA). Other residues can be mutated to obtain the above-mentioned effects (i.e., to inactivate one or another nuclease portion). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986 and / or A987 may be altered (i.e., substituted). Likewise, mutations other than alanine substitutions are also suitable.

[0577] In some embodiments, the domain of the CRISPR protein source of the base editor may include all or part of a Cas9 protein with a standard PAM sequence (NGG). In other embodiments, the Cas9-derived domain of the base editor may adopt an atypical PAM sequence. Such sequences have been described in the art and are obvious to those skilled in the art. For example, in Kleinstiver, BP et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities”, Nature 523, 481-485 (2015), Cas9 domains that bind to atypical PAM sequences have been described. Kleinstiver, BP et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are incorporated herein by reference.

[0578] In some examples, a PAM recognized by a CRISPR protein-derived domain of a base editor disclosed herein can be provided to the cell on a separate oligonucleotide encoding an insert (e.g., an AAV insert) of the base editor. In this case, providing the PAM on a separate oligonucleotide can allow cleavage of a target sequence that would otherwise be inaccessible because there is no adjacent PAM on the same polynucleotide as the target sequence.

[0579] In one embodiment, Streptococcus pyogenes Cas9 (SpCas9) can be used as a CRISPR nuclease for genome engineering. However, other methods can be used. In some cases, different nucleases can be used to target certain genomic targets. In some cases, synthetic SpCas9 derivatives with non-NGG PAM sequences can be used. In addition, other Cas9 orthologs from various species have been identified, and these "non-SpCas9s" can bind to a variety of PAM sequences, which can also be used in this application. For example, the relatively large size of SpCas9 (approximately 4kb coding sequence) will result in plasmids carrying SpCas9 cDNA being unable to be effectively expressed in cells. In contrast, the coding sequence of Staphylococcus aureus Cas9 (SaCas9) is approximately 1 kilobase shorter than that of SpCas9, which may enable it to be effectively expressed in cells. Similar to SpCas9, SaCas9 endonucleases are able to modify target genes in mammalian cells in vitro and in vivo. In some cases, Cas proteins can target different PAM sequences. In some cases, the target gene may be adjacent to the Cas9 PAM (e.g., 5'-NGG). In other cases, other Cas9 orthologs may have different PAM requirements, for example, other PAMs such as those found in Streptococcus thermophilus (5'-NNAGAA for CRISPR1 and 5'-NGGNG for CRISPR3) and Neisseria meningitidis (5'-NNNNGATT).

[0580] In some embodiments, for the Streptococcus pyogenes system, the target gene sequence can be before (i.e., 5' to) 5'-NGG PAM, and the 20-nt guide RNA sequence can be mediated by the opposite chain base pairing to guide the Cas9 cutting adjacent to the PAM. In some cases, the adjacent fragment can be or can be about 3 base pairs upstream of the PAM. In some cases, the adjacent fragment can be or can be about 10 base pairs upstream of the PAM. In some cases, the adjacent fragment can be upstream of the PAM or can be about 0-20 base pairs upstream of the PAM. For example, adjacent cutting can be next to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20PAM upstream 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs. The adjacent fragment can also be 1 to 30 base pairs downstream of the PAM.

[0581] Fusion proteins containing nuclear localization sequences (NLS)

[0582] In some embodiments, the fusion protein provided herein further comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, such as a nuclear localization sequence (NLS). In one embodiment, a bipartite NLS is used. In some embodiments, the NLS comprises an amino acid sequence that helps to import a protein comprising the NLS into the nucleus (e.g., by nuclear transport). In some embodiments, any fusion protein provided herein further comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the Cas9 domain. In some embodiments, the NLS is fused to the C-terminus of the nCas9 domain or the dCas9 domain. In some embodiments, the NLS is fused to the N-terminus of a deaminase. In some embodiments, the NLS is fused to the C-terminus of a deaminase. In some embodiments, the NLS is fused to the C-terminus of a deaminase. In some embodiments, the NLS is fused to the fusion protein through one or more joints. In some embodiments, the NLS is fused to a fusion protein without a joint. In some embodiments, the NLS comprises the amino acid sequence of any NLS sequence provided or cited herein. Additional nuclear localization sequences are known in the art and will be apparent to those skilled in the art. For example, NLS sequences are described in Plank et al., PCT / EP2000 / 011690, the contents of which are incorporated herein by reference as exemplary nuclear localization sequences. In some embodiments, the NLS comprises the amino acid sequence PKKKRKVEGADKRTADGSEFESPKKKRKV, KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRKPKKRKKVK, or KMLKNRLCFLYQLY. In some embodiments, the NLS is present in a linker or is flanked by a linker, such as a linker described herein. In some embodiments, the N-terminal or C-terminal NLS is a bipartite NLS. Bipartite NLSs consist of two basic amino acid clusters separated by a relatively short spacer sequence (hence, bipartite - 2 parts, while monopartite NLSs do not). The NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK, is a prototype of a ubiquitous bipartite signal: two basic amino acid clusters separated by a spacer of approximately 10 amino acids. The sequence of an exemplary bipartite NLS is as follows: PKKKRKVEGADKRTADGSEFES PKKKRKV.

[0583] In some embodiments, the fusion proteins of the invention do not comprise a linker sequence.In some embodiments, there is a linker sequence between one or more domains or proteins.

[0584] Should be understood that fusion rotein of the present invention can comprise one or more additional features.For example, in some embodiments, fusion rotein can comprise inhibitor, cytoplasm localization sequence, export sequence (for example nuclear export sequence or other localization sequences), and can be used for solubilizing, purifying or detecting the sequence tag of fusion rotein.Suitable protein tag provided herein includes but is not limited to biotin carboxylase carrier protein (BCCP) label, myc label, calmodulin label, FLAG label, hemagglutinin (HA) label, polyhistidine tag (also referred to as histidine tag or His- label), maltose binding protein (MBP) label, nus label, glutathione S-transferase (GST) label, green fluorescent protein (GFP) label, thioredoxin label, S label, Softags (for example, Softag 1, Softag 3), streptococcus label, biotin ligase label, F1AsH label, V5 label and SBP label.For those skilled in the art, other suitable sequences are apparent.In some embodiments, fusion rotein comprises one or more His tags.

[0585] Vectors encoding CRISPR enzymes comprising one or more nuclear localization sequences (NLSs) can be used. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NLSs can be used or approximately used. The CRISPR enzyme can comprise an NLS at or near the end of the molecule and about or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NLSs at or near the carboxy terminus and combinations of these (e.g., one or more NLSs at the amino terminus and one or more NLSs at the carboxy terminus). When more than one NLS is present, they can be selected independently of one another such that a single NLS can be present in more than one copy and / or in one or more copies with one or more other NLSs.

[0586] The CRISPR enzyme used in the method can comprise about 6 NLSs. An NLS is considered to be near the N-terminus or C-terminus when the amino acid closest to the NLS is within about 50 amino acids of the polypeptide chain from the N-terminus or C-terminus, such as within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 amino acids.

[0587] In some embodiments, the NLS comprises the amino acid sequence

[0588] PKKKRKVEGADKRTADGSEFESPKKKRKV, KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRKPKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.

[0589] In some embodiments, the NLS is present in a linker or is flanked by a linker, such as those described herein. In some embodiments, the N-terminal or C-terminal NLS is a bipartite NLS. A bipartite NLS comprises two basic amino acid clusters separated by a relatively short spacer sequence (thus, a bipartite - 2 parts, whereas a monopartite NLS does not). The NLS KR[PAATKKAGQA]KKKK of nucleoplasmin is a prototype of a ubiquitous bipartite signal: two basic amino acid clusters separated by a spacer of approximately 10 amino acids. The sequence of an exemplary bipartite NLS is as follows: PKKKRKVEGADKRTADGSEFES PKKKRKV.

[0590] In some embodiments, the fusion proteins of the invention do not comprise a linker sequence.In some embodiments, there is a linker sequence between one or more domains or proteins.

[0591] The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NGRRR(N), TTTV, TYCV, TYCV, TATV, NNNGTAT, NNAGAAW, or NAAAC. Y is a pyrimidine; N is any nucleotide base; and W is A or T.

[0592] Nucleobase editing domain

[0593] Described herein are base editors comprising fusion proteins comprising a polynucleotide programmable nucleotide binding domain and a nucleoside base editing domain (e.g., a deaminase domain). By interacting with a guidance polynucleotide capable of recognizing a target sequence, the base editor can be programmed to edit one or more bases in a target polynucleotide sequence. Once the target sequence is identified, the base editor is anchored to the polynucleotide to be edited, and the deaminase domain component of the base editor can then edit the target base.

[0594] In some embodiments, the nucleobase editing domain is a deaminase domain. In some cases, the deaminase domain can be a cytosine deaminase or a cytidine deaminase. In some embodiments, the terms "cytosine deaminase" and "cytidine deaminase" can be used interchangeably. In some cases, the deaminase domain can be an adenine deaminase or an adenosine deaminase. In some embodiments, the terms "adenine deaminase" and "adenosine deaminase" can be used interchangeably. The details of the nucleobase editing protein are described in International PCT Patent Application No. PCT / 2017 / 045381 (WO International Patent Application No. 2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), the entire contents of which are incorporated herein by reference. See also Komor, AC et al. “Programmable editing of a target base in genomic DNAwithout 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 excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the contents of which are incorporated herein by reference in their entirety.

[0595] C to T Edit

[0596] In some embodiments, the base editors disclosed herein include a fusion protein comprising a cytidine deaminase that is capable of deaminating a target cytidine (C) base of a polynucleotide to produce uridine (U), which has the base pairing properties of thymine. In some embodiments, for example, where the polynucleotide is double-stranded (e.g., DNA), the uridine base can then be replaced by a thymidine base (e.g., by a cellular repair mechanism) to produce a C:G to T:A conversion. In other embodiments, when the base editor deaminates C in a nucleic acid to U, it cannot replace U with T.

[0597] Deamination of the target C in a polynucleotide to produce U is a non-limiting example of a base editing type that can be performed by a base editor as described herein. In another example, a base editor comprising a cytidine deaminase domain can mediate the conversion of a cytosine (C) base to a guanine (G) base. For example, the U of the polynucleotide produced by the deamination of the cytosine of the cytosine deaminase domain of the base editor can be excised from the polynucleotide by a base excision repair mechanism (e.g., by a uracil DNA glycosylase (UDG) domain), resulting in a baseless site. The nucleobase opposite to the abasic site can then be replaced with another base, such as C, by, for example, a transfer lesion polymerase (e.g., by a base repair mechanism). Although the nucleobase opposite to the abasic site is typically substituted with C, other substitutions (e.g., A, G, or T) can also occur.

[0598] Thus, in some embodiments, the base editors described herein comprise a deamination domain (e.g., a cytidine deaminase domain) capable of deaminating a target C in a polynucleotide to a U. In addition, as described below, the base editor may comprise other domains that promote the conversion of U produced by deamination to T or G. For example, a base editor comprising a cytidine deaminase domain may further comprise a uracil glycosylase inhibitor (UGI) domain to mediate the replacement of U with T, thereby completing a C to T base editing event. In another example, a base editor can be incorporated into a translesion polymerase to improve the efficiency of C-to-G base editing, because the translesion polymerase can promote the incorporation of C relative to the abasic site (i.e., resulting in the incorporation of G at the abasic site, completing the C-to-G base editing activity).

[0599] A base editor comprising a cytidine deaminase as a domain can deaminate a target C in any polynucleotide, including DNA, RNA, and DNA-RNA hybrids. Typically, a cytidine deaminase catalyzes a C core base located in the context of a single-stranded portion of a polynucleotide. In some embodiments, the entire polynucleotide comprising the target C can be single-stranded. For example, a cytidine deaminase incorporated into a base editor can deaminate a target C in a single-stranded RNA polynucleotide. In other embodiments, a base editor comprising a cytidine deaminase domain can act on a double-stranded polynucleotide, but the target C can be located in a portion of a polynucleotide that is in a single-stranded state during the deamination reaction. For example, in an embodiment in which the NAGPB domain comprises a Cas9 domain, several nucleotides may be unpaired during the formation of the Cas9-gRNA-target DNA complex, resulting in the formation of a Cas9 "R-loop complex". These unpaired nucleotides can form bubbles of single-stranded DNA, which can be used as a substrate for single-stranded specific nucleotide deaminases (e.g., cytidine deaminase).

[0600] In some embodiments, the cytidine deaminase of the base editor may include all or part of an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. APOBEC is an evolutionarily conserved family of cytidine deaminases. Members of the family are C to U (C-to-U) editing enzymes. Like proteins, the N-terminal domain of APOBEC is a catalytic domain, while the C-terminal domain is a pseudo-catalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain and is important for cytidine deamination. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (now called "APOBEC3E"), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase. In some embodiments, the deaminase incorporated into the base editor includes all or part of an APOBEC1 deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC2 deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3 deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3A deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3B deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3C deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3D deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3E deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3F deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3G deaminase. In some embodiments, the deaminase incorporated into the base editor includes all or part of an APOBEC3H deaminase. In some embodiments, the deaminase incorporated into the base editor includes all or part of an APOBEC4 deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or part of an activation-induced deaminase (AID). In some embodiments, the deaminase incorporated into the base editor comprises all or part of a cytidine deaminase 1 (CDA1). It should be understood that the base editor may include a deaminase from any suitable organism (eg, human or rat). In some embodiments, the deaminase domain of the base editor is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse.In some embodiments, the deaminase domain of the base editor is derived from rat (e.g., rat APOBEC1). In some embodiments, the deaminase domain of the base editor is human APOBEC1. In some embodiments, the deaminase domain of the base editor is pmCDA1.

[0601] The amino acid and nucleic acid sequences of PmCDA1 are shown below.

[0602] >tr|A5H718|A5H718_PETMA cytosine deaminase OS=sea lamprey OX=7757PE=2SV=1 amino acid sequence:

[0603] MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSG

[0604] TERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLK

[0605] IWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKT

[0606] LKRAEKRRSELSIMIQVKILHTTKSPAV

[0607] Nucleic acid sequence: >EF094822.1 Dipyrimidis maritima isolate PmCDA.21 cytosine deaminase mRNA, complete CDS:

[0608] TGACACGACACAGCCGTGTATATGAGGAAGGGTAGCTGGATGGGGGGGGGGGGAATACGTTCAGAGAGGACATTAGCGAGCGTCTTGTTGGTGGCCTTGAGTCTAGACACCTGCAGACATGACCGACGCTGAGTACGTGAGAATCCATGAGAAGTTGGACATCTACACGTTTAAGAAACAGTTTTTCAACA ACAAAAAATCCGTGTCGCATAGATGCTACGTCTCTTTGAATTAAAACGACGGGGTGAACGTAGAGCGTGTTTTGGGGGCTATGCTGTGAATAAACCACAGAGCGGGACAGAACGTGGAATTCACGCCGAAATCTTTAGCATTAGAAAAGTCGAAGAATACCTGCGCGACAACCCCGGACAATTCACGATAA ATTGGTACTCATCCTGGAGTCCTTGTGCAGATTGCGCTGAAAAGATCTTAGAATGGTATAACCAGGAGCTGCGGGGGAACGGCCACACTTTGAAAATCTGGGCTTGCAAACTCTATTACGAGAAAAATGCGAGGAATCAAATTGGGCTGTGGAACCTCAGAGATAACGGGGTTGGGTTGAATGTAATGGTA AGTGAACACTACCAAATGTTGCAGGAAAATATTCATCCAATCGTCGCACAATCAATTGAATGAGAATAGATGGCTTGAGAAGACTTTGAAGCGAGCTGAAAAACGACGGAGCGAGTTGTCCATTATGATTCAGGTAAAAATACTCCACACCACTAAGAGTCCTGCTGTTTAAGAGGCTATGCGGATGGTTTTC

[0609] The amino acid and nucleic acid sequences of the coding sequence (CDS) of human activation-induced cytidine deaminase (AID) are shown below.

[0610] >tr|Q6QJ80|Q6QJ80_HUMAN activation-induced cytidine deaminase OS=Homo sapiens OX=9606GN=AICDAPE=2SV=1 amino acid sequence:

[0611] MDSLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKAPV

[0612] The amino acid and nucleic acid sequences of the coding sequence (CDS) of human activation-induced cytidine deaminase (AID) are shown below.

[0613] >tr|Q6QJ80|Q6QJ80_HUMAN activation-induced cytidine deaminase OS=Homo sapiens OX=9606GN=AICDAPE=2SV=1 amino acid sequence:

[0614] MDSLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELL

[0615] FLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRK

[0616] AEPEGLRRLHRAGVQIAIMTFKAPV

[0617] Nucleic acid sequence: >NG_011588.1:5001-15681 Homo sapiens activation-induced cytidine deaminase (AICDA), RefSeqGene (LRG_17) on chromosome 12:

[0618]

[0619] Other exemplary deaminases that can be fused to Cas9 according to aspects of the present application are provided below. It should be understood that in some embodiments, active domains of corresponding sequences, such as domains without localization signals (nuclear localization sequences, no nuclear export signals, cytoplasmic localization signals), can be used.

[0620] Human AIDs:

[0621]

[0622] (Bottom line: nuclear localization sequence; double bottom line: nuclear export signal)

[0623] Mouse AID:

[0624]

[0625] (Bottom line: nuclear localization sequence; double bottom line: nuclear export signal)

[0626] Canine AIDs:

[0627]

[0628] (Bottom line: nuclear localization sequence; double bottom line: nuclear export signal)

[0629] Cattle AID:

[0630]

[0631] (Bottom line: nuclear localization sequence; double bottom line: nuclear export signal)

[0632] Rat AID:

[0633]

[0634] (Bottom line: nuclear localization sequence; double bottom line: nuclear export signal)

[0635] Mouse APOBEC-3

[0636] MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQIVRFLATHHNLSLDIFSSRLYNVQDPETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVEGRRMDPLSEEEFYSQFYNQRVKHLCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVTITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFGNLQLGPPMS

[0637] (Italic: nucleic acid editing domain)

[0638] Rat APOBEC-3:

[0639] MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNRLRYAIDRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQVLRFLATHHNLSLDIFSSRLYNIRDPENQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKKLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVERRRVHLLSEEEFYSQFYNQRVKHLCYYHGVKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVIITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLHRIKESWGLQDLVNDFGNLQLGPPMS

[0640] (Italic: nucleic acid editing domain)

[0641] Macaque APOBEC-3G:

[0642] MVEPMDPRTFVSNFNNRPILSGLNTVWLCCEVKTKDPSGPPLDAKIFQGKVYSKAKYHPEM RFLRWFHKWRQLHHDQEYKVTWYVSWSPCTRCANSVATFLAKDPKVTLTIFVARLYYFWKPDYQQALRILCQKRGGPHATMKIMNYNEFQDCWNKFVDGRGKPFKPRNNLPKHYTLLQATLGELLRHLMDPGTFTSNFNNKPWVSGQHETYLCY KVERLHNDTWVPLNQHRGFLRNQAPNIHGFPKGRHAELCFLDLIPFWKLDGQQYRVTCFTSWSPCFSCAQEMAKFISNNEHVSLCIFAARIYDDQGRYQEGLRALHRDGAKIAMMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI

[0643] (Italics: nucleic acid editing domain; bottom line: cytoplasmic localization signal)

[0644] Chimpanzee APOBEC-3G:

[0645] MKPHFRNPVERMYQDTFSDNFYNRPILSHRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYS KLKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDVATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTSNFNNELWVRGRHETYLCY EVERLHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLHQDYRVTCFTSWSPCFSCAQEMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLAKAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLEEHSQALSGRLRAILQNQNQGN

[0646] (Italics: nucleic acid editing domain; bottom line: cytoplasmic localization signal)

[0647] Green Monkey APOBEC-3G:

[0648] MNPQIRNMVEQMEPDIFVYYFNNRPILSGRNTVWLCYEVKTKDPSGPPLDANIFQGKLYPEAKDHPEMKFLHWFRKWRQLHRDQEYEVTWYVSWSPCTRCANSVATFLAEDPKVTLTIFVARLYYFWKPDYQQALRILCQERGGPHATMKIMNYNEFQHCWNEFVDGQGKPFKPRKNLPKHYTLLHATLGELLRHVMDPGTTFTSNFNNKPWVSGQRET YLCYKVERSHNDTWVLLNQHRGFLRNQAPDRHGFPKGRHAELCFDLIPFWKLDDQQYRVTCFTSWSPCFSCAQKMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLHRDGAKIAVMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI

[0649] (Italics: nucleic acid editing domain; bottom line: cytoplasmic localization signal)

[0650] Human APOBEC-3G:

[0651] MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYS ELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCY EVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN

[0652] (Italics: nucleic acid editing domain; bottom line: cytoplasmic localization signal)

[0653] Human APOBEC-3F:

[0654] MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQVYSQPEHHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYSEGQPFMPWYKFDDNYAFLHRTLKEILRNPMEAMYPHIFYFHFKNLRKAYGRNESWLCFTMEVVKHHSPVSWKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDFKYCWENFVYNDDEPFKPWKGLKYNFLFLDSKLQEILE

[0655] (Italic: nucleic acid editing domain)

[0656] Human APOBEC-3B:

[0657] MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGQVYFKPQYHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLSEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVTIMDYEEFAYCWENFVYNEGQQFMPWYKFDENYAFLHRTLKEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN

[0658] (Italic: nucleic acid editing domain)

[0659] Rat APOBEC-3B:

[0660] MQPQGLGPNAGMGPVCLGCSHRRPYSPIRNPLKKLYQQTFYFHFKNVRYAWGRKNNFLCYEVNGMDCALPVPLRQGVFRKQGHIHAELCFIYWFHDKVLRVLSPMEEFKVTWYMSWSPCSKCAEQVARFLAAHRNLSLAIFSSRLYYYLRNPNYQQKLCRLIQEGVHVAAMDLPEFKKCWNKFVDNDGQPFRPWMRLRINFSFYDCKLQEIFSRMNLLREDVFYLQFNNSHRVKPVQNRYYRRKSYLCYQLERANGQEPLKGYLLYKKGEQHVEILFLEKMRSMELSQVRITCYLTWSPCPNCARQLAAFKKDHPDLILRIYTSRLYFWRKKFQKGLCTLWRSGIHVDVMDLPQFADCWTNFVNPQRPFRPWNELEKNSWRIQRRLRRIKESWGL

[0661] Bovine APOBEC-3B:<C

[0662] DGWEVAFRSGTVLKAGVLGVSMTEGWAGSGHPGQGACVWTPGTRNTMNLLREVLFKQQFGNQPRVPAPYYRRKTYLCYQLKQRNDLTLDRGCFRNKKQRHAERFIDKINSLDLNPSQSYKIICYITWSPCPNCANELVNFITRNNHLKLEIFASRLYFHWIKSFKMGLQDLQNAGISVAVMTHTEFEDCWEQFVDNQSRPFQPWDKLEQYSASIRRRLQRILTAPI

[0663] Chimpanzee APOBEC-3B:

[0664] MNPQIRNPMEWMYQRTFYYNFENEPILYGRSYTWLCYEVKIRRGHSNLLWDTGVFRGQMYSQPEHHAEMCFLSWFCGNQLSAYKCFQITWFVSWTPCPDCVAKLAKFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYNEGQPFMPWYKFDDNYAFLHRTLKEIIRHLMDPDTFTFNFNNDPLVLRRHQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGQVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQVRASSLCMVPHRPPPPPQSPGPCLPLCSEPPLGSLLPTGRPAPSLPFLLTASFSFPPPASLPPLPSLSLSPGHLPVPSFHSLTSCSIQPPCSSRIRETEGWASVSKEGRDLG

[0665] Human APOBEC-3C:

[0666] MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRNQVDSETHCHAERCFLSWFCDDILSPNTKYQVTWYTSWSPCPDCAGEVAEFLARHSNVNLTIFTARLYYFQYPCYQEGLRSLSQEGVAVEIMDYEDFKYCWENFVYNDNEPFKPWKGLKTNFRLLKRRLRESLQ

[0667] (Italic: nucleic acid editing domain)

[0668] Gorilla APOBEC-3C

[0669] MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRNQVDSETHCHAERCFLSWECDDILSPNTNYQVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLYYFQDTDYQEGLRSLSQEGVAVKIMDYKDFKYCWENFVYNDDEPFKPWKGLKYNFRFLKRRLQEILE

[0670] Human APOBEC-3A:

[0671] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN

[0672] (Italic: nucleic acid editing domain)

[0673] Rhesus macaque APOBEC-3A:

[0674] MDGSPASRPRHLMDPNTFTFNFNNDLSVRGRHQTYLCYEVERLDNGTWVPMDERRGFLCNKAKNVPCGDYGCHVELRFLCEVPSWQLDPAQTYRVTWFISWSPCFRRGCAGQVRVFLQENKHVRLRIFAARIYDYDPLYQEALRTLRDAGAQVSIMTYEEFKHCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAILQNQGN

[0675] (Italic: nucleic acid editing domain)

[0676] Bovine APOBEC-3A:

[0677] MDEYTFTENFNNQGWPSKTYLCYEMERLDGDATIPLDEYKGFVRNKGLDQPEKPCHAELYFLGKIHSWNLDRNQHYRLTCFISWSPCYDCAQKLTTFLKENHHISLHILASRIYTHNRFGCHQSGLCELQAAGARITIMTFEDFKHCWETFVDHKGKPFQPWEGLNVKSQALCTELQAILKTQQN

[0678] (Italic: nucleic acid editing domain

[0679] Human APOBEC-3H:

[0680] MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENKKKCHAEICFINEIKSMGLDETQCYQVTCYLTWSPCSSCAWELVDFIKAHDHLNLGIFASRLYYHWCKPQQKGLRLLCGSQVPVEVMGFPKFADCWENFVDHEKPLSFNPYKMLEELDKNSRAIKRRLERIKIPGVRAQGRYMDILCDAEV

[0681] (Italic: nucleic acid editing domain)

[0682] Rhesus APOBEC-3H:

[0683] MALLTAKTFSLQFNNKRRVNKPYYPRKALLCYQLTPQNGSTPTRGHLKNKKKDHAEIRFINKIKSMGLDETQCYQVTCYLTWSPCPSCAGELVDFIKAHRHLNLRIFASRLYYHWRPNYQEGLLLLCGSQVPVEVMGLPEFTDCWENFVDHKEPPSFNPSEKLEELDKNSQAIKRRLERIKSRSVDVLENGLRSLQLGPVTPSSSIRNSR

[0684] Human APOBEC-3D:

[0685] MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGPVLPKRQSNHRQEVYFRFENHAEMCFLSWFCGNRLPANRRFQITWFVSWNPCLPCVVKVTKFLAEHPNVTLTISAARLYYYRDRDWRWVLRLHKAGARVKIMDYEDFAYCWENFVCNEGQPFMPWYKFDDNYASL HRTLKEILRNPMEAMYPHIFYFHFKNLLKACGRNESWLCFTMEVTKHHSAVFRKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLSQEGASVKIMGYKDFVSCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ

[0686] (slanted:nucleic acid editing field)

[0687] Human APOBEC-1:

[0688] MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPHILLATGLIHPSVAWR

[0689] APOBEC-1 mouse:

[0690] MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK

[0691] Rat APOBEC-1:

[0692] MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK

[0693] Human APOBEC-2:

[0694] MAQKEEAAVATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRNVEYSSGRNKTFLCYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALRYNVTWYVSSSPCAACADRIIKTLSKTKNLRLLILVGRLFMWEEPEIQAALKKLKEAGCKLRIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQENFLYYEEKLADILK

[0695] Mouse APOBEC-2:

[0696] MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYSSGRNKTFLCYVVEVQSKGGQAQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK Rat APOBEC-2:

[0697] MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYSSGRNKTFLCYVVEAQSKGGQVQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYLWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK Bovine APOBEC-2:

[0698] MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRNVEYSSGRNKTFLCYVVEAQSKGGQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALRYMVTWYVSSSPCAACADRIVKTLNKTKNLRLLILVGRLFMWEEPEIQAALRKLKEAGCRLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK Lamprey CDA1 (pmCDAl):

[0699] MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRG

[0700] ERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKI<00第14页,共22页01494>

[0701] LEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQC

[0702] CRKIFIQSSHNQ

[0703] LNENRWLEKTLKRAEKRRSELSFMIQVKILHTTKSPAV

[0704] Human APOBEC3G D316R D317R:

[0705] MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQ

[0706] VYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDP

[0707] KVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKFNYDEFQHCWSKFVYSQ

[0708] RELFEPWNNLPKYYILLHFMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVER

[0709] MHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTC

[0710] FTSWSPCFSCAQEMAKFISKKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISF T

[0711] YSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN Human APOBEC3G Chain A:

[0712] MDPPTFTFNFNNEPWWGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHG

[0713] FLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI

[0714] FTARIYDDQGRCQEGLRTLAEAGAKISFTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ

[0715] Human APOBEC3G chain A D120R D121R:

[0716] MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHG

[0717] FLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI

[0718] FTARIYRRQGRCQEGLRTLAEAGAKISFMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ

[0719] Some aspects of the present application are based on the following recognition: regulating the catalytic activity of the deaminase domain of any fusion protein described herein, for example, by affecting the synthetic ability of the fusion protein (e.g., base editor) by making point mutations in the deaminase domain. For example, a mutation that reduces but does not eliminate the catalytic activity of the deaminase domain in a base editing fusion protein can reduce the probability that the deaminase domain catalyzes the deamination of residues adjacent to the target residue, thereby narrowing the deamination window. The ability to narrow the deamination window can prevent undesirable deamination of residues adjacent to a specific target residue, which can reduce or prevent off-target effects.

[0720] For example, in some embodiments, the APOBEC deaminase incorporated into the base editor may comprise one or more mutations selected from H121X, H122X, R126X, R126X, R118X, W90X, W90X, and R132X of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase, where X is any amino acid. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise one or more mutations selected from H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase.

[0721] In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise one or more mutations selected from D316X, D317X, R320X, R320X, R313X, W285X, W285X, R326X of hAPOBEC3G, or one or more corresponding mutations. A mutation in another APOBEC deaminase, wherein X is any amino acid. In some embodiments, any fusion protein provided herein comprises an APOBEC deaminase comprising one or more mutations selected from D316R, D317R, R320A, R320E, R313A, W285A, W285Y, R326E of hAPOBEC3G, or one or more corresponding mutations. In another APOBEC deaminase.

[0722] In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise the H121R and H122R mutations of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the R126A mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the R126E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the R118A mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising a W90A mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising a W90Y mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising an R132E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising a W90Y and R126E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the R126E and R132E mutations of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the W90Y and R132E mutations of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the W90Y, R126E, and R132E mutations of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase.

[0723] In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the D316R and D317R mutations of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any fusion protein provided herein comprises an APOBEC deaminase comprising the R320A mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the R320E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the R313A mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the W285A mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the W285Y mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the R326E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the W285Y and R320E mutations of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the R320E and R326E mutations of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the W285Y and R326E mutations of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor may comprise an APOBEC deaminase comprising the W285Y, R320E, and R326E mutations of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase.

[0724] Many 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, available from Addgene (plasmids 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, 85177). In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC1 deaminase.

[0725] Detailed information on C to T nucleobase editing proteins is described in International PCT Patent Application No. PCT / US2016 / 058344 (International WO Patent Application No. 2017 / 070632) and Komor, AC et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature 533, 420-424 (2016), the entire contents of which are incorporated herein by reference.

[0726] A to GEdit

[0727] In some embodiments, the base editor described herein may include a deaminase domain, which includes an adenosine deaminase. This adenosine deaminase domain of the base editor can promote the editing of adenine (A) nucleoside base to guanine (G) nucleoside base by deaminating A to form inosine (I), exhibiting the base pairing properties of G. Adenosine deaminase is a method for deaminating (i.e., removing an amine group) adenine from deoxyadenosine residues in deoxyribonucleic acid (DNA).

[0728] In some embodiments, the base editor provided herein can be prepared by fusing one or more protein domains together to produce a fusion protein. In certain embodiments, the fusion protein provided herein includes one or more features that improve the base editing activity (e.g., efficiency, selectivity, and specificity) of the fusion protein. For example, the fusion protein provided herein may include a Cas9 domain with reduced nuclease activity. In some embodiments, the fusion protein provided herein may have a Cas9 domain (dCas9) that does not have nuclease activity or a Cas9 domain that cuts a chain of a double-stranded DNA molecule, referred to as Cas9 nickase (nCas9). Without wishing to be bound by any particular theory, the presence of catalytic residues (e.g., H840) maintains the activity of Cas9 cutting an unedited (e.g., non-deaminized) chain containing T relative to target A. The "residues" of the catalytic residues of Cas9 (e.g., D10 to A10) prevent the cutting of the editing chain containing the target A residue. Such Cas9 variants are able to produce single-stranded DNA breaks (gaps) at specific positions based on the target sequence defined by gRNA, resulting in the repair of the unedited chain, ultimately resulting in a non-edited sub-line. In some embodiments, the A to G base editor further comprises an inhibitor of inosine base excision repair, such as a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine-specific nuclease. Without wishing to be bound by any particular theory, the UGI domain or the catalytically inactive inosine-specific nuclease can inhibit or prevent base excision repair of deaminated adenosine residues (e.g., inosine), which can improve the activity or efficiency of the base editor.

[0729] A base editor comprising an adenosine deaminase can act on any polynucleotide, including DNA, RNA, and DNA-RNA hybrids. In certain embodiments, a base editor comprising an adenosine deaminase can deaminate a target A of a polynucleotide comprising RNA. For example, a base editor may include an adenosine deaminase domain capable of deaminating a target A of an RNA polynucleotide and / or a DNA-RNA hybrid polynucleotide. In one embodiment, the adenosine deaminase incorporated into the base editor includes all or part of an adenosine deaminase acting on RNA (ADAR, e.g., ADAR1 or ADAR2). In another embodiment, the adenosine deaminase incorporated into the base editor includes all or part of an adenosine deaminase acting on tRNA (ADAT). A base editor comprising an adenosine deaminase domain can also deaminate the A core base of a DNA polynucleotide. In one embodiment, the adenosine deaminase domain of the base editor includes all or part of an ADAT, which includes one or more mutations that allow ADAT to deaminate the target A in DNA. For example, a base editor can comprise all or a portion of ADAT from Escherichia coli (EcTadA) comprising one or more of the following mutations: D108N, A106V, D147Y, E155V, L84F, H123Y, I157F, or another mutant adenosine deaminase.

[0730] Adenosine deaminase can be derived from any suitable organism (e.g., E. coli). In some embodiments, the adenine deaminase is a naturally occurring adenosine deaminase comprising one or more mutations corresponding to any mutation provided herein (e.g., mutations in ecTadA). Corresponding residues in any homologous protein can be identified, for example, by sequence alignment and determination of homologous residues. Mutations in any naturally occurring adenosine deaminase corresponding to any mutation described herein (e.g., any mutation identified in ecTadA) can be generated accordingly (e.g., having homology to ecTadA).

[0731] TadA

[0732] In specific embodiments, the TadA is any of the TadAs described in PCT / US2017 / 045381 (WO2018 / 027078), which are incorporated herein by reference in their entirety.

[0733] In one embodiment, the fusion protein of the present invention comprises wild-type TadA linked to TadA7.10, which is linked to the Cas9 nickase. In a specific embodiment, the fusion protein comprises a single TadA7.10 domain (e.g., provided in monomeric form). In other embodiments, the ABE7.10 editor comprises TadA7.10 and TadA (wt) that can form heterodimers. The relevant sequences are as follows:

[0734] MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (referred to as "Ta").

[0735] TadA7.10:

[0736] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0737] In some 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 of the adenosine deaminases provided herein. It should be understood that the adenosine deaminases provided herein can include one or more mutations (e.g., any mutation provided herein). The present disclosure provides any deaminase domain having a certain percentage of identity, plus any mutation described herein, or a combination thereof. In some embodiments, the adenosine deaminase 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 a reference sequence or any adenosine deaminase provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 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 consecutive amino acid residues compared to any amino acid sequence known in the art or described herein.

[0738] In some embodiments, the TadA deaminase is a full-length E. coli TadA deaminase. For example, in certain embodiments, the adenosine deaminase comprises the amino acid sequence:

[0739] MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD.

[0740] However, it will be appreciated that additional adenosine deaminases that may be used in the present application will be readily apparent to those skilled in the art and are within the scope of the present application. For example, the adenosine deaminase may be a homolog of adenosine deaminase that acts on tRNA (ADAT). Without limitation, exemplary amino acid sequences of ADAT homologs include the following:

[0741] Staphylococcus aureus TadA:

[0742] MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN

[0743] Bacillus subtilis TadA:

[0744] MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEML

[0745] VIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGC S

[0746] GTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE

[0747] Salmonella typhimurium (S. typhimurium) TadA:

[0748] MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV

[0749] Shewanella putrefaciens (S. putrefaciens) TadA:

[0750] MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPT

[0751] AHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE

[0752] Haemophilus influenzae F3031 (Haemophilus influenzae, H. influenzae) TadA:

[0753] MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTΑΗAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFG

[0754] ASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK

[0755] Caulobacter crescentus (Caulobacter crescentus, C. crescentus) TadA:

[0756] MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADD

[0757] PKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI

[0758] Geobacter sulfurreducens (Geobacter sulfurreducens, G. sulphurredensens) TadA:

[0759] MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP

[0760] Examples of E. coli TadA (ecTadA) include the following:

[0761] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0762] In some embodiments, the adenosine deaminase is from a prokaryotic organism. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Bacillus crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from Escherichia coli.

[0763] In one embodiment, the fusion protein of the present invention comprises wild-type TadA connected to TadA7.10, which is connected to the Cas9 nickase. In a specific embodiment, the fusion protein comprises a single TadA7.10 domain (e.g., provided in monomeric form). In other embodiments, the ABE7.10 editor includes TadA7.10 and TadA (wt) that can form a heterodimer.

[0764] In some 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 herein. It should be understood that the adenosine deaminases provided herein can include one or more mutations (e.g., any mutation provided herein). The present disclosure provides any deaminase domain having a certain percentage of identity, plus any mutation described herein, or a combination thereof. In some embodiments, the adenosine deaminase 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 a reference sequence or any adenosine deaminase provided herein. In some 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 contiguous amino acid residues compared to any amino acid sequence known in the art or described herein.

[0765] It will be understood that any mutation provided herein (e.g., based on the TadA reference sequence) can be introduced into other adenosine deaminases (e.g., E. coli TadA (ecTadA)), Staphylococcus aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). It will be apparent to one skilled in the art that additional deaminases can be similarly aligned to identify homologous amino acid residues that can be mutated as provided herein. Thus, any mutation identified in the TadA reference sequence can be made in other adenosine deaminases (e.g., ecTadA) having homologous amino acid residues. It will also be understood that any mutation provided herein can be made in the TadA reference sequence or another adenosine deaminase, alone or in any combination.

[0766] In some embodiments, the adenosine deaminase comprises a D108X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108G, D108N, D108V, D108A, or D108Y mutation, or a corresponding mutation in another adenosine deaminase.

[0767] In some embodiments, the adenosine deaminase comprises an A106X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), wherein X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A106V mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., wild-type TadA or ecTadA).

[0768] In some embodiments, the adenosine deaminase comprises an E155X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), wherein the presence of X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E155D, E155G, or E155V mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).

[0769] In some embodiments, the a...

Claims

1. Use of a base editor complexed with one or more guided polynucleotides for the preparation of a drug for editing a SERPINA1 polynucleotide comprising single nucleotide polymorphisms associated with alpha-1 antitrypsin deficiency, in, The drug is in contact with the SERPINA1 polynucleotide, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein the one or more directing polynucleotides to target the base editor to cause the A·T to G·C alteration of the single nucleotide polymorphism associated with alpha-1 antitrypsin deficiency.

2. A cell produced by introducing the following into the cell or its progenitor cells: base editor, polynucleotide encoding the base editor, in, The base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and One or more guide polynucleotides targeting the base editor to cause the A·T to G·C alteration of the single nucleotide polymorphism associated with alpha-1 antitrypsin deficiency.

3. Use of a cell according to claim 2 for the preparation of a drug for the treatment of alpha-1 antitrypsin deficiency in a subject in need thereof, said drug being administered to said subject.

4. Use of a base editor or polynucleotide encoding the base editor and one or more guide polynucleotides in the preparation of a drug for the treatment of alpha-1 antitrypsin deficiency in a subject, in, The base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and Among them, the guide polynucleotide targets the base editor to cause A·T to G·C changes in single nucleotide polymorphisms associated with α-1 antitrypsin deficiency.

5. A method for producing hepatocytes or their progenitor cells, the method include: (a) Introduce the following (i) and (ii) to progenitor cells containing induced pluripotent stem cells or hepatocytes associated with α-1 antitrypsin deficiency, (i) a base editor or a polynucleotide encoding the base editor, wherein the base editor comprises a polynucleotide programmable nucleotide binding domain and an adenosine deaminase domain; and (ii) one or more guide polynucleotides, wherein the one or more guide polynucleotides target a base editor to cause the A·T to G·C alteration of the single nucleotide polymorphism associated with alpha-1 antitrypsin deficiency; and (b) The progenitor cells that differentiate the induced pluripotent stem cells or hepatocytes into hepatocytes.

6. A base editor, include: (i) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDE REVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADICAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDD LDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNF DKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFmqPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAkfLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPrAFKYFDTTIaRKeYrSTKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRK; and (ii) Adenosine deaminase domain.

7. A guide RNA comprising a nucleic acid sequence selected from the group consisting of: 5’-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAAAGGCUA GUCCGUUAUCAAC UUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3’; 5’-ACCAUCGACAAGAAAAGGGACUGAGUUUAGAGCUAGAAAAAAAAAGGCUAGUCCGUUAUCAACUUGAAAAAAAAAGGCUAGUCCGUUAUCAACUUGAAAAAAAAAGUGCACCGAGUCGGUGCUUUU-3’; 5’-CCAUCGACAAGAAAAGGGACUGAGUUUAGAGCUAGAAAAUAAAAGGCUAGUCCGUUAUCAACUUGAAAAAAA GUGGCACCGAGUCGGUGCUUUU-3’; 5’-CAUCGACAAGAAAAGGGACUGAGUUUAGAGCUAGAAAU AGCAAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGAAAAAAGUGCACCGAGUCGGUGCUUUU-3’; 5’-AUCGACAAGAAAAGGGACUGAGUUUAGAGCUAGAAAU AGCAAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGAAAAAAGUGCACCGAGUCGGUGCUUUU-3’; 5’-UCGACAAGAAAAGGGACUGAGUUUAGAGCUAGAAAUA GCAAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGAAAAAAGU GGCACCGAGUCGGUGCUUUU-3’; and 5’-CGACAAGAAAAGGGACUGAGUUUAGAGCUAGAAAUAGC AAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGAAAAAAGUGG CACCGAGUCGGUGCUUUU-3’.

8. A base editor system for correcting pathogenic single nucleotide polymorphisms in genes, in, The base editor system includes: (a) Base editor, including: (i) polynucleotide programmable DNA binding domain, and (ii) a deaminase domain that enables the deamination of the pathogenic single nucleotide polymorphism or its complementary nucleobase; and (b) a guide polynucleotide that binds to the polynucleotide programmable DNA binding domain, wherein the guide polynucleotide targets a base editor to a target polynucleotide sequence, at least a portion of the target polynucleotide sequence is located in the gene or its reverse complementary sequence; Wherein, deaminating the pathogenic single nucleotide polymorphism or its complementary nucleobase causes the pathogenic single nucleotide polymorphism to its wild-type allele, thereby correcting the pathogenic mutations listed in Table 3A or Table 3B.

9. Use of a base editor complexed with one or more guide polynucleotides for the preparation of a drug for editing a G6PC polynucleotide comprising a single nucleotide polymorphism associated with type 1a glycogen storage, the drug being in contact with the G6PC polynucleotide, in, The base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein the one or more guide polynucleotides to target the base editor to cause A·T to G·C changes in single nucleotide polymorphisms associated with type 1a glycogen storage.

10. Use of a base editor complexed with one or more guide polynucleotides for the preparation of a drug for editing an IDUA polynucleotide comprising a single nucleotide polymorphism associated with type 1 mucopolysaccharide storage disease, the drug being in contact with the IDUA polynucleotide, in, The base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein the one or more guide polynucleotides to target the base editor to cause A·T to G·C changes in single nucleotide polymorphisms associated with type 1 mucopolysaccharide storage disease.

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