Programmable adenine base editor and application thereof

Abstract

The invention provides a programmable adenine base editor, a system comprising the programmable adenine base editor and a use method of the programmable adenine base editor. Also provided are MPG and mutants thereof, which can be used in the programmable adenine base editor.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit and priority of the following patent applications: PCT patent application No. PCT / CN2022 / 092759, entitled “ENGINGEERED N-METHYLPURINE DNA GLYCOSYLASE(MPG)AND USES THEREOF,” filed on May 13, 2022, and PCT patent application No. PCT / CN2022 / 139699, entitled “PROGRAMMABLE ADENINE TRANSVERSION BASE EDITOR AND USES THEREOF,” filed on December 16, 2022, the entire contents of these patent applications, including any sequence listings and drawings, are incorporated herein by reference in their entirety.

[0003] Reference to electronic sequence listing

[0004] The present disclosure contains an electronic sequence listing ("HGP025PCT.xml", created on May 11, 2023 by "WIPOSequence" software in accordance with WIPO Standard ST.26), which is incorporated herein by reference in its entirety. According to WIPO Standard ST.26, the symbol "t" is used to represent both T in DNA and U in RNA (see "Table 1: List of Nucleotide Symbols", the definition of the symbol "t" is "thymine in DNA / uracil in RNA (t / u)"). Therefore, in the sequence listing prepared according to ST.26, in any case where the sequence is RNA, T in the sequence should be regarded as U. Background Art

[0005] Base editors are promising tools for precise base editing in basic research and therapeutic applications 1,2 Adenine base editors (ABE) and cytosine base editors (CBE) can achieve A:T to G:C and C:G to T:A conversions, respectively. 3,4 Recently, C to G base editors (CGBEs) were developed by replacing uracil DNA N-glycosylase inhibitor (UGI) with uracil DNA N-glycosylase (UNG) in cytosine base editors. 5-9 However, no editor can support base conversion including transitions and transversions. Base editors capable of A to T and A to C transversions are still needed to repair a large number of point mutations. 2 , which accounts for up to 27% of genetic diseases ( Figure 3). There is a need in the art for adenine base editors with expanded editing outcomes to facilitate, for example, A to C and A to T transversion editing (AYBE, Y=C or T), especially in mammalian cells.

[0006] Citation or identification of any document in this disclosure is not an admission that the document is available as prior art to the present disclosure. Each reference mentioned or cited in this disclosure is incorporated by reference in its entirety. Summary of the invention

[0007] In view of the above background technology, the present disclosure provides certain advantages and advances over the prior art. Although the present disclosure is not limited to specific advantages or functions, in one aspect, the present disclosure provides a base editor comprising:

[0008] (1) a nucleic acid programmable DNA binding domain (napDNAbd) capable of binding to a target dsDNA comprising a target A:T base pair, wherein the target A:T base pair comprises a target deoxyadenosine (dA) (first deoxyribonucleotide) in a protospacer sequence on a non-target strand (editing strand) of the target dsDNA and a deoxythymidine (dT) (second deoxyribonucleotide) in a target sequence on a target strand (non-editing strand) of the target dsDNA, wherein the protospacer sequence is completely reverse complementary to the target sequence;

[0009] (2) an adenine deaminase domain capable of deaminating adenine of the target deoxyadenosine to hypoxanthine; and

[0010] (3) a hypoxanthine excision domain capable of excising the hypoxanthine.

[0011] In another aspect, the present disclosure provides a system comprising:

[0012] (1) A base editor or a polynucleotide encoding the base editor, wherein the base editor comprises:

[0013] (a) a nucleic acid programmable DNA binding domain (napDNAbd) capable of binding to a target dsDNA comprising a target A:T base pair, wherein the target A:T base pair comprises a target deoxyadenosine (dA) (first deoxyribonucleotide) in a protospacer sequence on a non-target strand (editing strand) of the target dsDNA and a deoxythymidine (dT) (second deoxyribonucleotide) in a target sequence on a target strand (non-editing strand) of the target dsDNA, wherein the protospacer sequence is completely reverse complementary to the target sequence;

[0014] (b) an adenine deaminase domain capable of deaminating adenine of the target deoxyadenosine to hypoxanthine; and

[0015] (c) a hypoxanthine excision domain capable of excising said hypoxanthine; and

[0016] (2) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, wherein the guide nucleic acid comprises:

[0017] (a) a scaffold sequence capable of forming a complex with the napDNAbd; and

[0018] (b) is capable of hybridizing to a target sequence on a target strand of the target dsDNA, thereby directing the complex to a guide sequence of the target dsDNA.

[0019] In yet another aspect, the present disclosure provides a method for modifying a target dsDNA, the method comprising contacting the target dsDNA with a system,

[0020] The target dsDNA comprises a target A:T base pair, wherein the target A:T base pair comprises a target deoxyadenosine (dA) (first deoxyribonucleotide) in a protospacer sequence on a non-target strand (editing strand) of the target dsDNA and a deoxythymidine (dT) (second deoxyribonucleotide) in a target sequence on a target strand (non-editing strand) of the target dsDNA, wherein the protospacer sequence is completely reverse complementary to the target sequence;

[0021] The system comprises:

[0022] (1) A base editor or a polynucleotide encoding the base editor, wherein the base editor comprises:

[0023] (a) a nucleic acid programmable DNA binding domain (napDNAbd) capable of binding to the target dsDNA;

[0024] (b) an adenine deaminase domain capable of deaminating adenine of the target deoxyadenosine to hypoxanthine; and

[0025] (c) a hypoxanthine excision domain capable of excising said hypoxanthine; and

[0026] (2) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, wherein the guide nucleic acid comprises:

[0027] (1) a scaffold sequence capable of forming a complex with the napDNAbd; and

[0028] (2) capable of hybridizing to a target sequence on the target strand of the target dsDNA, thereby directing the complex to a guide sequence of the target dsDNA.

[0029] In yet another aspect, the present disclosure provides a polynucleotide encoding a base editor of the present disclosure and, optionally, a guide nucleic acid as defined in the present disclosure.

[0030] In yet another aspect, the present disclosure provides a vector comprising the polynucleotide of the present disclosure.

[0031] In yet another aspect, the present disclosure provides a complex comprising a base editor of the present disclosure and a guide nucleic acid defined in the present disclosure.

[0032] In yet another aspect, the present disclosure provides a cell comprising the base editor or system of the present disclosure, the polynucleotide of the present disclosure, the vector of the present disclosure, or the complex of the present disclosure.

[0033] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising:

[0034] (i) a base editor or system of the present disclosure, a polynucleotide of the present disclosure, a vector of the present disclosure, a complex of the present disclosure, or a cell of the present disclosure; and

[0035] (ii) a pharmaceutically acceptable excipient.

[0036] In yet another aspect, the present disclosure provides a method for treating a subject having or at risk of having a disease associated with a target deoxyadenosine of a target dsDNA, the method comprising administering (e.g., an effective amount) a system of the present disclosure to the subject, wherein the target deoxyadenosine is modified by the system, and the modification treats or prevents the disease.

[0037] In yet another aspect, the present disclosure provides the MPG defined in the present disclosure.

[0038] The details of one or more embodiments of the present disclosure are set forth in the following description. Other features or advantages of the present disclosure should be clear from the following drawings and detailed description of several embodiments and from the claims. It should be understood that, unless otherwise indicated, any aspect or embodiment of the present disclosure may be combined with any other aspect or embodiment of the present disclosure to constitute another embodiment explicitly or implicitly disclosed herein.

[0039] definition

[0040] The present disclosure will describe specific embodiments, but the present disclosure is not limited thereto, but only by the claims. Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those commonly understood by ordinary technicians in the field to which the present disclosure belongs. Unless otherwise indicated, the terms set forth below are generally understood in their ordinary meanings.

[0041] Overview

[0042] Nucleic acid programmable DNA binding protein (napDNAbp), such as Cas9, Cas12, IscB, is substantially capable of binding to target DNA (e.g., dsDNA) under the guidance of a guide nucleic acid (e.g., guide RNA) comprising a guide sequence targeting the target DNA. In some embodiments, the target DNA is eukaryotic.

[0043] Without wishing to be bound by theory, in some embodiments, the guide nucleic acid comprises a scaffold sequence responsible for forming a complex with the napDNAbp, and a guide sequence intentionally designed to be responsible for hybridizing with a target sequence of the target DNA, thereby directing the complex comprising the napDNAbp and the guide nucleic acid to the target DNA.

[0044] See also Fig.21 Depicted, an exemplary target dsDNA comprises a 5' to 3' single DNA strand comprising a first deoxyribonucleotide dA and a 3' to 5' single DNA strand comprising a second deoxyribonucleotide dT that base pairs with dA.

[0045] Exemplary guide nucleic acids are depicted as comprising a guide sequence and a scaffold sequence. The guide sequence is designed to hybridize with a portion of a 3' to 5' single DNA strand, and the guide sequence is thus "targeted" to this portion. Therefore, the 3' to 5' single DNA strand is referred to as the "target strand (TS)" of the target dsDNA, and the relative 5' to 3' single DNA strand is referred to as the "non-target strand (NTS)" of the target dsDNA. This portion of the target strand based on which the guide sequence is designed and to which the guide sequence can hybridize is referred to as the "target sequence", and the relative portion corresponding to this portion on the non-target strand is referred to as the "protospacer sequence", which is 100% (completely) reverse complementary to the target sequence.

[0046] Typically, nucleic acid sequences (eg, DNA sequences, RNA sequences) are written in a 5' to 3' direction / orientation.

[0047] For example, for the DNA sequence of ATGC, unless otherwise indicated, it is generally understood as 5'-ATGC-3'. Its reverse sequence is 5'-CGTA-3', its complete complement sequence is 5'-TACG-3', and its complete reverse complement sequence is 5'-GCAT-3'.

[0048] In general, unless otherwise indicated, the double-stranded sequence of a dsDNA may be represented by the sequence of its 5' to 3' single DNA strand, conventionally written in a 5' to 3' direction / orientation.

[0049] For example, for a dsDNA having a 5' to 3' single DNA strand of 5'-ATGC-3' and a 3' to 5' single DNA strand of 3'-TACG-5', the dsDNA can be simply represented as 5'-ATGC-3'.

[0050] 5'-----ATGC-----3'

[0051] 3'-----TACG-----5'

[0052] It should be noted that the 5' to 3' single DNA strand or the 3' to 5' single DNA strand of the dsDNA can be a non-target strand from which the protospacer sequence is selected.

[0053] In the sense of base editing, the strand where the target nucleotide to be edited is located is called the editing strand, and the opposite strand is called the non-editing strand. As used herein, the non-target strand is the editing strand, and the target strand is the non-editing strand.

[0054] Typically, for dsDNA, such as a gene, the 5' to 3' single DNA strand is the sense strand of the gene, and the 3' to 5' single DNA strand is the antisense strand of the gene. However, it should be noted that the sense strand or antisense strand of a gene can be a non-target strand from which the protospacer sequence is selected.

[0055] To hybridize to the target dsDNA, in one embodiment, the guide sequence of the guide nucleic acid is designed to have a sequence of 5'-AUGC-3' that is completely reverse complementary to the 3' to 5' strand of the target dsDNA, which sequence will be listed in ATGC in the electronic sequence listing, but is marked as an RNA sequence; and in another embodiment, the guide sequence of the guide nucleic acid is designed to have a sequence of 5'-GCAU-3' that is completely reverse complementary to the 5' to 3' strand of the target dsDNA, which sequence will be listed in GCAT in the electronic sequence listing, but is marked as an RNA sequence.

[0056] In the case where the guide sequence of the guide nucleic acid is completely reverse complementary to the target sequence and the target sequence is completely reverse complementary to the protospacer sequence, the guide sequence is identical to the protospacer sequence except for U in the guide sequence (due to its RNA nature) and T in the corresponding protospacer sequence (due to its DNA nature). According to WIPO Standard ST.26, the symbol "t" is used to represent both T in DNA and U in RNA (see "Table 1: List of Nucleotide Symbols", the definition of the symbol "t" is "thymine in DNA / uracil in RNA (t / u)"). Therefore, in the sequence table of the present disclosure prepared according to ST.26, such a guide sequence can be listed in the same order as the corresponding protospacer sequence. For convenience, a single SEQ ID NO in the sequence table can be used to represent both such a guide sequence and the protospacer sequence, although such a single SEQ ID NO can be marked as DNA or RNA in the sequence table. When referring to such a SEQ ID NO showing a protospacer sequence / guide sequence, it refers to the protospacer sequence as a DNA sequence or the guide sequence as an RNA sequence according to the context, regardless of whether it is marked as DNA or RNA in the sequence table.

[0057] the term

[0058] Unless otherwise specified, all technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs. Throughout the specification, several terms defined in the following paragraphs are used. Other definitions are also found in the body of the specification.

[0059] As used herein, the term "nucleic acid programmable DNA binding domain (napDNAbd)" can be used interchangeably with "nucleic acid programmable DNA binding protein (napDNAbp)" and refers to a protein that can be associated (e.g., bound) with a programmable nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid (e.g., gRNA), which is programmed to direct the protein to a specific sequence of a target DNA through an interaction (e.g., hybridization) between the programmable nucleic acid and the target DNA. napDNAbd can be indirectly associated (e.g., bound) with a target DNA through an interaction between a programmable nucleic acid and the target DNA.

[0060] As used herein, the terms "nucleic acid", "nucleic acid molecule" or "polynucleotide" are used interchangeably. They refer to polymers of deoxyribonucleotides or ribonucleotides or mixtures thereof in single-stranded or double-stranded form, and unless otherwise specified, also encompass known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides. These terms encompass nucleic acid-like structures with synthetic backbones as well as amplification products. Both DNA and RNA are polynucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deaza adenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioate and 5′-N-phosphoramidite bonds).

[0061] As used herein, the terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0062] As used herein, "fusion protein" refers to a protein created by joining two or more originally separate proteins or parts thereof. In some embodiments, a linker may be present between each protein.

[0063] As used herein, the term "heterologous" in reference to polypeptide domains refers to the fact that the polypeptide domains do not naturally occur together (e.g., in the same polypeptide). For example, in an artificially generated fusion protein, a polypeptide domain from one polypeptide can be fused with a polypeptide domain from a different polypeptide. Two polypeptide domains will be considered "heterologous" to each other because they do not naturally occur together.

[0064] As used herein, the term "nuclease" refers to a polypeptide capable of cleaving phosphodiester bonds between nucleotide subunits of a nucleic acid; the term "endonuclease" refers to a polypeptide capable of cleaving phosphodiester bonds within a polynucleotide chain.

[0065] As used herein, the term "guide nucleic acid" refers to a nucleic acid-based molecule capable of forming a complex with napDNAbp (e.g., Cas9, Cas12, IscB) (e.g., via a scaffold sequence of a guide nucleic acid), and comprises a sequence (e.g., a guide sequence) that is fully complementary to the target DNA to hybridize with the target DNA and guide the complex to the target DNA, the nucleic acid including but not limited to RNA-based molecules, such as guide RNA. As used herein, the terms "crRNA", "guide RNA (gRNA)", "single guide RNA (sgRNA)" and "RNA guidance" are used interchangeably. As used in the present disclosure, the term "guide sequence" is used interchangeably with the term "spacer sequence", and the term "scaffold sequence" is used interchangeably with the term "direct repeat (DR) sequence".

[0066] As used herein, the term "complex" refers to a grouping of two or more molecules. In some embodiments, the complex comprises a polypeptide and a nucleic acid that interact (e.g., bind, contact, adhere) with each other. As used herein, the term "complex" may refer to a grouping of a guide nucleic acid and a polypeptide (e.g., napDNAbp). As used herein, the term "complex" may refer to a grouping of a guide nucleic acid, a polypeptide (e.g., napDNAbp) and a target DNA.

[0067] As used herein, the term "activity" refers to biological activity. In some embodiments, the activity includes enzymatic activity, such as the catalytic ability of the effector. For example, the activity can include nuclease activity, such as DNA nuclease activity, dsDNA endonuclease activity, guide sequence-specific (on-target) dsDNA endonuclease activity, guide sequence-independent (off-target) dsDNA endonuclease activity.

[0068] As used herein, the term "cutting" refers to the breakage of the covalent backbone of a DNA molecule. Cutting can be performed by a variety of methods, including but not limited to enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-stranded and double-stranded cutting are possible, and double-stranded cutting can occur as a result of two different single-stranded cutting events. DNA cutting can result in the production of blunt ends or sticky ends.

[0069] As used herein, the meanings of "cleaved nucleic acid" or "modified nucleic acid" may overlap. Modified nucleic acid includes not only modification of single nucleotides, but also insertion or deletion of nucleic acid fragments.

[0070] As used herein, the term "on-target" refers to a predetermined or intended region of DNA that is bound, cut and / or edited, for example, by a base editor of the present disclosure.

[0071] As used herein, the term "off-target" refers to an unintended or unexpected region of DNA that is bound, cut, and / or edited, for example, by a base editor of the present disclosure. In some embodiments, a DNA region is an off-target region when it is different from a DNA region that is predetermined or expected to be bound, cut, and / or edited by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

[0072] As used herein, if a DNA sequence, e.g., 5'-ATGC-3', is transcribed into an RNA sequence, and each dT (deoxythymidine, or "T" for short) in the primary sequence of the DNA sequence is replaced with U (uridine), and each dA (deoxyadenosine, or "A" for short), dG (deoxyguanosine, or "G" for short), and dC (deoxycytidine, or "C" for short) is replaced with A (adenosine), G (guanosine), and C (cytidine), respectively, e.g., 5'-AUGC-3', then the DNA sequence is said in the present disclosure to "encode" the RNA sequence.

[0073] As used herein, the term "protospacer adjacent motif" or "PAM" refers to a short sequence (or motif) adjacent to the protospacer sequence on the non-target strand of dsDNA that is recognized by a complex comprising napDNAbp.

[0074] As used herein, the term "adjacent" includes the situation where there are no nucleotides between the original spacer sequence and the PAM, and the situation where there are a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides between the original spacer sequence and the PAM. As used herein, A is "adjacent" to B (adjacent to B), A is "adjacent" to the 5' of B, and A is "adjacent" to the 3' of B means that there are no nucleotides between A and B.

[0075] As described herein, the guide sequence is so designed to be able to hybridize with the target sequence substantially.As used herein, the term "hybridization (hybridize, hybridizing or hybridization)" refers to a reaction in which one or more polynucleotide sequences react to form a complex, which is stable via hydrogen bonds between the bases of one or more polynucleotide sequences.Hydrogen bonds can occur by Watson Crick base pairing, Hoogstein binding or in any other sequence-specific manner.The polynucleotide sequence that can hybridize with a given polynucleotide sequence is referred to as the "complementary sequence" of a given polynucleotide sequence.As used herein, the hybridization of a guide sequence and a target sequence is so stable that the napDNAbp of the guide nucleic acid complex with a functional domain (e.g., deaminase domain) comprising a guide sequence or associated (e.g., fusion) with napDNAbp acts (e.g., cutting, deamination) at or near a target sequence or its complementary sequence (e.g., target DNA sequence or its complementary sequence).

[0076] For the purpose of hybridization, in some embodiments, the guide sequence is reverse complementary to the target sequence. As used herein, the term "complementary" refers to the ability of the nucleobase of the first polynucleotide sequence (such as a guide sequence) to base pair with the nucleobase of the second polynucleotide sequence (such as a target sequence) by traditional Watson Crick base pairing. Two complementary polynucleotide sequences can be non-covalently bound under appropriate temperature and solution ionic strength conditions. In some embodiments, the first polynucleotide sequence (e.g., a guide sequence) has 100% (complete) complementarity with the second nucleic acid (e.g., a target sequence). In some embodiments, if the first polynucleotide sequence (e.g., a guide sequence) has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementarity with the second nucleic acid, the first polynucleotide sequence is complementary to the second nucleic acid. As used herein, the term "substantially complementary" refers to a degree of complementarity between a polynucleotide sequence (e.g., a guide sequence) and a second polynucleotide sequence (e.g., a target sequence) such that the first polynucleotide sequence (e.g., a guide sequence) can hybridize with sufficient affinity to the second polynucleotide sequence (e.g., a target sequence) to allow the napDNAbd complexed with the first polynucleotide sequence or a nucleic acid comprising the first polynucleotide sequence or a functional domain associated with (e.g., fused to) the napDNAbd to act (e.g., cleave, deaminize) on the target sequence or its complement (e.g., a target DNA sequence or its complement). In some embodiments, a guide sequence that is substantially complementary to a target sequence has 100% or less complementarity with the target sequence. In some embodiments, a guide sequence that is substantially complementary to a target sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the target sequence.

[0077] As used herein, the term "identity" refers to the overall correlation between polymer molecules, for example, between nucleic acid molecules (for example, DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, if the sequence of a polymer molecule has at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity, then they are considered to be "substantially identical" to each other. The calculation of the identity percentage of two nucleic acids or polypeptide sequences can be performed by comparing two sequences for the best comparison purpose (for example, a room can be introduced in one or both of the first sequence and the second sequence to achieve the best comparison, and different sequences can be ignored for the purpose of comparison). In some embodiments, the sequence length compared for the purpose of comparison is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or substantially 100% of the length of the reference sequence. The nucleotides at corresponding positions are then compared. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, GAP BLAST, and PSI-BLAST for amino acid sequences. In some embodiments, sequence identity is determined by global alignment, for example using the Needleman-Wunsch algorithm and

[0078] In some embodiments, sequence identity is calculated by local alignment, for example using the Smith-Waterman algorithm and the online tool at ebi.ac.uk / Tools / psa / emboss_water / .

[0079] As used herein, the term "variant" refers to an entity that shows significant structural identity with a reference entity (e.g., a wild-type sequence), but is structurally different from the reference entity in terms of the presence or level of one or more chemical moieties compared to the reference entity. In many embodiments, the variant is also functionally different from its reference entity. In general, whether a particular entity is appropriately regarded as a "variant" of a reference entity is based on its degree of structural identity with the reference entity. As understood by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. By definition, a variant is a unique chemical entity that shares one or more such characteristic structural elements. To name just a few examples, a polypeptide can have a characteristic sequence element that includes a plurality of amino acids that have a specified position relative to each other in a linear or three-dimensional space and / or contribute to a specific biological function; a nucleic acid can have a characteristic sequence element that includes a plurality of nucleotide residues that have a specified position relative to each other in a linear or three-dimensional space. For example, a variant polypeptide may differ from a reference polypeptide due to one or more differences in an amino acid sequence and / or one or more differences in a chemical moiety (e.g., carbohydrates, lipids, etc.) covalently attached to a polypeptide backbone. In some embodiments, the variant polypeptide shows an overall sequence identity with a reference polypeptide (e.g., a nuclease described herein), which is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Alternatively or in addition, in some embodiments, the variant polypeptide does not share at least one characteristic sequence element with the reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, the variant polypeptide shares one or more biological activities of the reference polypeptide, such as nuclease activity. In some embodiments, the variant polypeptide lacks one or more biological activities of the reference polypeptide. In some embodiments, compared with the reference polypeptide, the variant polypeptide shows a reduced level of one or more biological activities (e.g., nuclease activity, such as off-target nuclease activity). In some embodiments, if the polypeptide of interest has the same amino acid sequence as the parent, but has a small amount of sequence changes at a specific position, the polypeptide of interest is considered to be a "variant" of the parent or reference polypeptide. Typically, less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the residues in the variant are substituted compared to the parent or reference polypeptide. In some embodiments, the variant has 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 substituted residues compared to the parent or reference polypeptide. Variants often have a very small number (e.g., less than 5, 4, 3, 2 or 1) of substituted functional residues (i.e., residues involved in a specific biological activity).In some embodiments, compared with the parent or reference polypeptide, the variant has no more than 5, 4, 3, 2 or 1 additions or deletions, and often no additions or deletions. In addition, any addition or deletion is typically less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6 residues, and usually less than about 5, about 4, about 3 or about 2 residues. In some embodiments, the parent or reference polypeptide is wild type. The variant of polynucleotides or polypeptides can be naturally occurring, such as allelic variants, or it can be a variant known to be not naturally occurring. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis and by other recombinant methods known to those skilled in the art.

[0080] As used herein, the terms "non-naturally occurring" and "engineered" are used interchangeably and refer to human involvement. When these terms are used to describe a nucleic acid or polypeptide, it means that the nucleic acid or polypeptide is at least substantially free of at least one other component with which it is found in nature or in association with nature.

[0081] Conservative substitutions of non-critical amino acids of a protein can be made without affecting the normal function of the protein. Conservative substitutions refer to replacing an amino acid with a chemically or functionally similar amino acid. In some embodiments, conservative amino acid substitutions refer to amino acid substitutions that do not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. In some embodiments, "conservative substitutions" refer to amino acid substitutions made to amino acids in the following groups: i) methionine, isoleucine, leucine, valine, ii) phenylalanine, tyrosine, tryptophan, iii) lysine, arginine, histidine, iv) alanine, glycine, v) serine, threonine, vi) glutamine, asparagine and vii) glutamic acid, aspartic acid.

[0082] As used herein, the term "wild type" has the meaning generally understood by those skilled in the art, i.e., refers to the typical form of an organism, strain, gene or characteristic, which, when it occurs in nature, distinguishes it from a mutant or variant. It can be isolated from a source in nature without being intentionally modified.

[0083] As used herein, the description of "a variant or mutant (e.g., of MPG) comprising an amino acid mutation (e.g., substitution) at a given position (e.g., N169) of a given polypeptide (e.g., SEQ ID NO: 2)" or similar descriptions means that the polypeptide as shown in the amino acid sequence of the given polypeptide serves as a parent or reference polypeptide, and the variant is a variant of the parent or reference polypeptide and comprises an amino acid mutation at a position of the amino acid sequence of the variant corresponding to the given position of the amino acid sequence of the given polypeptide. The position of the amino acid mutation in the amino acid sequence of the variant may be the same as the given position of the given polypeptide, for example, when the variant comprises only an amino acid substitution compared to the given polypeptide and has the same length as the given polypeptide. The position of the amino acid mutation in the amino acid sequence of the variant may also be different from the given position of the given polypeptide, for example, when the variant comprises an N-terminal truncation compared to the given polypeptide and the first N-terminal amino acid of the variant does not correspond to the first N-terminal amino acid of the given polypeptide, but corresponds to an amino acid within the given polypeptide, but the position of the amino acid mutation can be determined by comparing the variant with the given polypeptide to identify the corresponding amino acid in its sequence, as understood by those skilled in the art. For example, if a variant has an N-terminal truncation of 20 amino acids compared to a given polypeptide, then the variant comprises an amino acid mutation at N169 of the given polypeptide means that the variant comprises an amino acid mutation at N149 of the variant, because N149 in the variant corresponds to N169 in the given polypeptide, as determined by alignment of the variant and the given polypeptide, where positions are numbered according to the amino acid sequence of SEQ ID NO:1.

[0084] As used herein, the description of a "variant or mutant (e.g., of MPG) comprising a given amino acid substitution (e.g., N169S) relative to a given polypeptide (e.g., SEQ ID NO: 2)" means that the polypeptide as shown in the amino acid sequence of the given polypeptide acts as a parent or reference polypeptide that does not comprise the given amino acid substitution, and the variant is a variant of the parent or reference polypeptide and comprises an amino acid substitution of the same substitution type as the given amino acid substitution at a position in the amino acid sequence of the variant corresponding to the position of the given amino acid substitution. For example, an MPG variant comprising the amino acid substitution N169S relative to SEQ ID NO: 2 refers to the fact that the amino acid sequence of SEQ ID NO: 2 comprises an amino acid N at position 169, while the MPG variant comprises an amino acid S at a position corresponding to N169 of the amino acid sequence of SEQ ID NO: 2, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO: 1. The correspondence of positions in two amino acid sequences as determined by alignment is explained in the previous paragraph.

[0085] Positions are numbered with respect to a given polypeptide as a parent or reference polypeptide. However, if specifically indicated, for example, "wherein positions are numbered with respect to the amino acid sequence of SEQ ID NO: 1," positions may also be numbered with respect to another polypeptide as a parent or reference polypeptide. For example, SEQ ID NO: 1 is a full-length wild-type human MPG with an N-terminal initial methionine (M), and SEQ ID NO: 2 is an N-terminal truncation of SEQ ID NO: 1, wherein the N-terminal initial methionine (M) is removed. The amino acid at position 169 of SEQ ID NO: 1 is N, and the corresponding amino acid N in SEQ ID NO: 2 is at position 168 of SEQ ID NO: 2. When an amino acid substitution is made at the amino acid N at position 168 of SEQ ID NO:2 based on SEQ ID NO:2 as a parent or reference polypeptide, the amino acid N can still be numbered according to SEQ ID NO:1 and referred to as N169 of SEQ ID NO:2, although the corresponding amino acid N is actually located at position 168 of SEQ ID NO:2, and such an amino acid substitution is referred to as N169X relative to SEQ ID NO:2 (wherein X represents the amino acid replacing N).

[0086] As used herein, the terms "upstream" and "downstream" refer to relative positions within a single nucleic acid (e.g., DNA) sequence in a nucleic acid. "Upstream" and "downstream" are respectively related to the 5' to 3' direction in which transcription occurs. For a first sequence and a second sequence present on the same strand of a single nucleic acid written in a 5' to 3' direction, when the 3' end of the first sequence is to the left of the 5' end of the second sequence, the first sequence is located upstream of the second sequence, and when the 5' end of the first sequence is to the right of the 3' end of the second sequence, the first sequence is located downstream of the second sequence. For example, a promoter is typically located upstream of a sequence under the regulation of the promoter; and on the other hand, a sequence under the regulation of the promoter is typically located downstream of the promoter.

[0087] As used herein, the term "regulatory element" refers to a DNA sequence that controls or affects one or more aspects of transcription and / or expression, and is intended to include promoters, enhancers, silencers, termination signals, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Regulatory elements include those that direct constitutive expression of nucleotide sequences in many types of host cells, and those that direct expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). Regulatory elements can also direct expression in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell type specific.

[0088] As used herein, the term "operably linked" refers to a juxtaposition in which the components described are in a relationship that allows them to function in the intended manner. A regulatory element that is "operably linked" to a functional element is associated in such a way that transcription, expression, and / or activity of the functional element is achieved under conditions that are compatible with the regulatory element. In some embodiments, an "operably linked" regulatory element is contiguous (e.g., covalently linked) with a functional element of interest; in some embodiments, the regulatory element trans-acts on a functional element of interest or is otherwise kept at a distance from the functional element of interest.

[0089] As used herein, the term "cell" is understood to refer not only to a particular individual cell, but also to the progeny or potential progeny of the cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not in fact be different from the parent cell, but are still included within the scope of the term.

[0090] As used herein, the term "in vivo" means inside an organism, and the term "ex vivo" or "in vitro" means outside an organism.

[0091] As used herein, the term "treatment" is a method for obtaining beneficial or desired results (including clinical results). For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, weakening the degree of the disease, stabilizing the disease (e.g., preventing or delaying the deterioration of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, reducing the recurrence rate of the disease, delaying or slowing down the progress of the disease, improving the disease state, providing relief (partial or complete) of the disease, reducing the dosage of one or more other drugs required for the treatment of the disease, delaying disease progression, increasing the quality of life and prolonging survival. The term also encompasses the pathological results of reducing diseases (such as cancer). The method of this disclosure contemplates any one or more of these therapeutic aspects of treatment.

[0092] As used herein, the term "disease" includes the terms "disorder" and "condition", and is not limited to those specific diseases that have been medically or clinically defined.

[0093] As used herein, reference to a value or parameter that is "not" generally means and describes a value or parameter that is "different from." For example, a method is not for treating cancer type X, meaning that the method can be used to treat cancers other than type X.

[0094] As used herein, the singular forms "a, an," and "the" include plural referents unless the context clearly dictates otherwise. That is, the articles "a, an," and "the" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, "an" means one element or more than one element, for example, two elements.

[0095] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to mean one or both of the alternatives, including both A and B, A or B, A (alone), and B (alone). Likewise, the term "and / or" in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0096] As used herein, when the term "about" precedes a series of numbers (e.g., about 1, 2, 3), it is understood that each of the series of numbers is modified by the term "about" (i.e., about 1, about 2, about 3). The term "about XY" as used herein has the same meaning as "about X to about Y".

[0097] As used herein, unless otherwise stated or otherwise apparent from the context, when referring to numbers, the terms "about" and "approximately" are used herein to include numbers that fall within a range of 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% in either direction (greater or less than) of the number (except where such number would exceed 100% of the possible value).

[0098] As used herein, a numerical range includes the end value of the range and each specific value within the range, for example, "16 to 100 nucleotides" includes 16 nucleotides and 100 nucleotides and each specific value between 16 and 100, for example, 17, 23, 34, 52, 78.

[0099] As used herein, unless the context requires otherwise, the terms "comprise", "include", "contain", and "have" should be understood to imply the inclusion of the stated element or group of elements but not the exclusion of any other element or group of elements. In certain embodiments, the terms "comprise", "include", "contain", and "have" are used synonymously.

[0100] As used herein, the phrase "consisting essentially of is intended to include any elements listed after the phrase "consisting essentially of," and is limited to other elements that do not interfere with or contribute to the activity or action specified in the present disclosure of the listed elements. Thus, the phrase "consisting essentially of is intended to indicate that the listed elements are required, but no other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed elements.

[0101] As used herein, the phrase “consisting of is meant to include, but is not limited to, any elements following the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required, and other elements may not be present.

[0102] As used herein, the term “consisting of also encompasses the terms “consisting essentially of” and “consisting of.” It should be understood that “comprising” the embodiments of the present disclosure described herein also includes “consisting essentially of” and “consisting of.”

[0103] Throughout the specification, references to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "another embodiment," or "a further embodiment," or combinations thereof, mean that a particular element, feature, structure, or characteristic described in conjunction with that embodiment is included in at least one embodiment of the present disclosure. Therefore, the aforementioned phrases that appear in different places throughout the specification do not necessarily all refer to the same embodiment. In addition, particular elements, features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0104] It is also noted that the claims may be drafted to exclude any optional element. As such, this description is intended to serve as an antecedent basis for use of exclusive terminology such as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] An understanding of certain features and advantages of the present disclosure will be gained by referring to the following detailed description, which sets forth illustrative embodiments in which the principles of the present disclosure may be utilized, and to the accompanying drawings, in which:

[0106] Figure 1 The engineering and optimization of adenine transversion base editors in some embodiments are shown. Figure 1a, Schematic diagram of potential pathways and editing outcomes for adenine transversion. After adenine deamination on the editing (non-target) strand by ABE8e and nicking on the non-editing (target) strand by Cas9 nickase (nCas9-D10A), MPG induces hypoxanthine excision followed by DNA repair / trans-lesion synthesis (TLS) and / or replication, resulting in different editing outcomes. I, deoxyinosine (the corresponding base is hypoxanthine (Hx)). MPG, N-methylpurine DNA glycosylase. AP, apurinic / apyrimidinic site. DSB, double-strand break. TLS: trans-lesion synthesis. Figure 1 b, Schematic design of reporter system and transversion base editor constructs for A to T editing detection. Y = C or T. P2A, 2A peptide from porcine teschovirus-1. Figure 1 c, Representative flow cytometry scatter plots showing the gating strategy for each base editor and EGFP + Percentage of cells. NT, non-target. Figure 1 d, EGFP + Percentage of cells. e, MFI (mean fluorescence intensity) of EGFP. Dashed line, mean value of wild-type MPG group. Fold change was calculated relative to the wild-type MPG group. au, arbitrary unit. Figure 1 d and Figure 1 In e, n=3. Figure 1 f, Schematic diagram of the mutagenesis and screening strategy. MPG-N169S was a constant mutation during two rounds of mutagenesis and screening. Figure 1 g and Figure 1 h, Performance of engineered AYBE variants measured by EGFP expression in rounds 1 and 2 of mutagenesis and screening. Each point represents the mean of three biological replicates for each AYBE variant. Dashed line, mean of the MPG-N169S group. Fold changes were calculated relative to the MPG-N169S group. Figure 1 i, Gradual improvement of AYBE-mediated EGFP activation. n=3. Dashed line, mean of wild-type MPG group. Fold changes were calculated relative to the wild-type MPG group. All values ​​are presented as mean±sem.

[0107] Figure 2 Shown is the characterization of the editing profile of AYBE by high-throughput target sequencing in some embodiments. Figure 2 a, Bar graph showing the on-target DNA base editing frequencies of adenine with the most A to C and / or A to T editing for ABE8e and AYBEv3 at the top 12 efficiently edited genomic sites in HEK293T cells. The editing frequencies of three independent replicates (n=3) for each base are shown side by side. Transfected mCherry-positive cells were sorted for further characterization. Figure 2 b–2d, in Figure 2 The editing purity of a's editing site from A to C (b), A to T (c), or A to G (d) by ABE8e and AYBEv3. Figure 2 e, from Figure 2 Frequency of A to C and A to T editing by AYBEv3 in protospacer positions 1-20 of the editing site of a (where PAM is at positions 21-23). ​​Individual dots represent individual replicates (n = 3 independent replicates / site). Boxes span the interquartile range (IQR) (25th to 75th percentiles), the horizontal line represents the median (50th percentile); and whiskers extend to the minimum and maximum values. Figure 2 f, gRNA-dependent off-target (OT) analysis comparing ABE8e and AYBEv3 at site 5 (HBG) and site 6 (EMX1) (n = 3). Note that the high-fidelity version 17 8e V106W For ABE8e and AYBEv3. Figure 2 g, gRNA-independent off-target editing detected by orthogonal R-loop assay at each R-loop site (n=3). Figure 2 h, Potential correction of nonsense mutations in DMD (Duchenne muscular dystrophy) by AYBEv3. Allele frequencies by on-target editing of AYBEv3 in a stable HEK293T cell line generated by lentiviral transduction. The red arrow indicates the targeted adenine for correction. The green arrow shows allele correction with potential therapeutic benefit. The values ​​on the right represent the frequency and read count of the mutant allele. Figure 2 i, Schematic diagram of potential pathways to increase A to T editing outcomes. Figure 2 j–2k, A to T editing results of Polη introduction (n=3). Figure 2 l, Diagram showing the types of point mutations obtainable with available base editors. All values ​​are presented as mean ± sem.

[0108] Figure 3 Shown are the distribution of human pathogenic SNP variants and demonstration of potential codon transversions using adenine transversion base editors in some embodiments. Figure 3 a, Base pair changes required to correct pathogenic human SNPs in the ClinVar database (accessed on July 23, 2022). Figure 3 b, Table of all potential codon transversions achieved by A to C or A to T editing. Potential outcomes for AYBE (adenine transversion base editor) are highlighted in blue.

[0109] Figure 4 The structures of the prototype AYBE and MPG in some embodiments are shown. Figure 4a, Prototype AYBE candidates designed in three orientations / configurations: MTC, TMC, and TCM. M=MPG, T=TadA8e, C=nCas9. Figure 4 b, EGFP of each prototype + All values ​​are presented as mean ± sem, n = 3. Figure 4 c, by AlphaFold( alphafold.com / entry / P29372 ) View of the predicted MPG structure in ribbon representation. The non-conserved N-terminal region (1-79aa) is intrinsically disordered, and the rest of the MPG region contributes to base excision and DNA binding activities.

[0110] Figure 5 Characterization of the A to C and A to T reporter systems in some embodiments is shown. Figure 5 a, Schematic construct design for detecting A to C editing. Figure 5 b, Representative flow cytometry scatter plots showing the gating strategy for each base editor at the splice acceptor site and BFP, respectively. + and EGFP + Percentage of cells. At least 2000 BFPs for each sample + Cells were analyzed. Figure 5 c, Representative flow cytometry scatter plots showing the gating strategy for each base editor and EGFP + The percentage of cells.

[0111] Figure 6 Shown is a gradual improvement in AYBE-mediated transversion editing at efficient residues shown on the endogenous genome and structures in some embodiments. Figure 6 a, A bar graph showing the frequency of on-target DNA base editing for ABE8e and different AYBE variants at site 3. The editing frequencies of three independent repeats (n=3) for each base are shown side by side. The original spacer position of the target adenine is highlighted in red. The percentage value below a specific adenine base indicates the average A to T (light green) or A to C (light blue) editing observed. Figure 6 b, in Figure 6 A to T and A to C results of ABE8e and different AYBE variants at the editing site A7 of a. Figure 6 c, using Figure 6 a The percentage of alleles containing insertions and / or deletions throughout the protospacer for various base editors. All values ​​are presented as mean ± sem. Transfected mCherry-positive cells were sorted for further characterization. Figure 6d, Positions of active residues of the AYBEv3 variant shown in magenta on the 3D structure. DNA from the deposited structure (PDB 1BNK, shown in grey) and the region 78–298 aa of the MPG protein predicted by AlphaFold (from Figure 4 c) overlapping structure.

[0112] Figure 7 Characterization of the AYBEv3 editing profile at the top 12 efficiently edited genomic sites in HEK293T cells in some embodiments is shown. The bar graph shows the on-target DNA base editing frequency of ABE8e and AYBEv3. The editing frequencies of three independent repeats (n=3) of each base are shown side by side. The original spacer position of the target adenine is highlighted in red. The percentage value below the specific adenine base indicates the average A to T (light green) or A to C (light blue) editing observed. The arrow indicates the adenine with the most transversion editing. Transfected mCherry-positive cells were sorted for further characterization of AYBEv3.

[0113] Figure 8 Characterization of AYBEv3 in some embodiments is shown. Figure 7 Frequency of A to G and A to Y editing in protospacer positions 1-20 of 1,2-diaminobenzidine (with PAM at positions 21-23) by ABE8e or AYBEv3 editors. Data are presented as median values.

[0114] Fig. 9 Characterization of the editing profile of AYBEv3 at the A7 or A8 position at seven additional genomic sites in some embodiments is shown. The bar graph shows the frequency of on-target DNA base editing for ABE8e and AYBEv3. The editing frequencies of three independent repeats (n=3) of each base are shown side by side. The original spacer position of the target adenine is highlighted in red. The percentage value below the specific adenine base indicates the average A to T (light green) or A to C (light blue) editing observed. The arrow indicates the adenine with the most transversion editing. Transfected mCherry-positive cells were sorted for further characterization of AYBEv3.

[0115] Fig.10Characterization of the editing profile of AYBEv3 at seven additional genomic sites is shown, with high editing activity at A5, A6, or A9 in some embodiments. The bar graph shows the frequency of on-target DNA base editing for ABE8e and AYBEv3. The editing frequencies of three independent repeats (n=3) of each base are shown side by side. The original spacer position of the target adenine is highlighted in red. Percent values ​​below a specific adenine base indicate the average A to T (light green) or A to C (light blue) editing observed. Arrows indicate the adenine with the most transversion editing. Transfected mCherry-positive cells were sorted for further characterization of AYBEv3.

[0116] Fig.11 Additional characterization of target editing activity in AYBEv3 in HEK293T cells is shown in some embodiments. Fig.11 a, Dot plots and box plots showing the combined distribution of A to C, A to T, A to G and indel frequencies for each nucleotide throughout the protospacer from experiments with ABE8e and AYBEv3 using 26 guide sequences. For indel frequencies, the percentage of alleles containing insertions and / or deletions for different sites throughout the protospacer is also presented at the bottom. Individual dots represent individual replicates (n = 3 independent replicates / site). Fig.11 b. Utilization Fig.11 Frequency of A to C and A to T editing at the NAN DNA motif using AYBEv3, which occurs at protospacer positions 6-9 at genomic loci. Each point indicates the mean of the motifs from the corresponding locus. The box spans the IQR (25th to 75th percentile), the horizontal line indicates the median (50th percentile), and the whiskers extend to the minimum and maximum values. The data points in the figure represent the full range of values ​​plotted. The subplots are from Figure 7 , 9 and the data shown in 10.

[0117] Fig.12 Shown is the allelic composition following treatment with AYBEv3 in some embodiments. Fig.12 a-12b, in HEK293T cells, expressed by AYBEv3 at site 33 ( Fig.12 a) and site 35 ( Fig.12 b) Allele frequency of target edits in the processed DNA. The values ​​on the right represent the frequency and read count of mutant alleles. Site 33 has multiple As within the target window, some of which are edited to Cs, while others are edited to Ts. AYBEv3 induces fewer bystander edits than ABE8e. A decrease in the percentage of alleles containing multiple edits simultaneously was observed after treatment with AYBEv3 compared to ABE8e.

[0118] Fig.13Characterization of the editing profile of AYBEv3 in HeLa, U2OS, and K562 cells in some embodiments is shown. The bar graph shows the frequency of on-target DNA base editing of ABE8e and AYBEv3 using 5 gRNAs targeting genomic sites in different cells. The editing frequencies of three independent repeats (n=3) of each base are shown side by side. The original spacer position of the target adenine is highlighted in red. The percentage value below the specific adenine base indicates the average A to T (light green) or A to C (light blue) editing observed. The arrow indicates the adenine with the most transversion editing. Transfected mCherry-positive cells were sorted for further characterization of AYBEv3.

[0119] Fig.14 Additional characterization of target editing activity in AYBEv3 in HeLa, U2OS, and K562 cells is shown in some embodiments. Fig.14 a, dot plots and box plots showing the combined distribution of A to C, A to T and indel frequencies for each nucleotide in the entire protospacer from experiments with AYBEv3 using 5 guide sequences. The box spans the IQR (25th to 75th percentile), the horizontal line indicates the median (50th percentile), and the whiskers extend to the minimum and maximum values. The data points in the figure represent the full range of values ​​plotted. A single point represents a single repeat (n = 3 independent repeats / site). Fig.14 b, A to C and A to T editing purity of AYBEv3 in different cells. n = 3. All values ​​are presented as mean ± sem. Fig.13 Data shown.

[0120] Fig.15 Off-target analysis of AYBEv3 in some embodiments is shown. Fig.15 a, Bar graph showing the frequency of on-target DNA base editing by ABE8e(TadA8e-V106W) and AYBEv3(TadA8e-V106W) at site 5 (HBG) and site 6 (EMX1) in HEK293T cells. Fig.15 b, Overview of the orthogonal R-loop assay. Fig.15 c, Bar graph showing the frequency of on-target DNA base editing by ABE8e and AYBEv3 at site 3. Fig.15 d, On-target base editing efficiency of ABE8e and AYBEv3 at A5 and A7 of site 3 in HEK293T cells. Points represent individual biological replicates, and bars represent mean ± sem, n = 3. Fig.15 a and Fig.15In c, the editing frequencies of three independent repeats (n=3) for each base are shown side by side. The original spacer position of the target adenine is highlighted in red. The percentage value below the specific adenine base indicates the average A to T (light green) or A to C (light blue) editing observed. The arrow indicates the adenine with the most transversion editing. Transfected mCherry-positive cells were sorted for further characterization of AYBEv3.

[0121] Fig.16 Potential correction of disease-associated transversion mutations using AYBEv3 in some embodiments is shown. Fig.16 a-16b, edited via A to C of AYBEv3 ( Fig.16 a) or A to T editing ( Fig.16 b) DNA sequencing chromatograms of corrected different disease-associated mutations. SAS, splice acceptor site. Arrows indicate the targeted adenine for correction. The corresponding results of the correction are shown below. Mutations in the DMD gene are associated with Duchenne muscular dystrophy. Mutations in the SLC26A4 gene are associated with autosomal recessive non-syndromic hearing loss 4. Mutations in the ATM gene are associated with ataxia-telangiectasia syndrome. Mutations in the TTN gene are associated with dilated cardiomyopathy 1G. Fig.16 c, Stacked bar graph showing the frequency of on-target DNA base editing of four targeted disease-associated mutations using AYBEv3 in a stable HEK293T cell line generated via lentiviral transduction. The editing frequency of the corrected bases is shown (n=3). All values ​​are presented as mean±sem. Fig.16 d–16f, Allele frequencies of on-target editing for different disease-associated mutations corrected by AYBEv3. Red arrows indicate targeted adenines for correction. Green arrows show allele corrections with potential therapeutic benefit. Values ​​on the right represent the frequency and read count of the mutant allele. Data shown are representative of three biological replicates.

[0122] Fig.17 Characterization of the editing profile of AYBEv3 together with Polη in some embodiments is shown. The bar graph shows the frequency of on-target DNA base editing for AYBEv3 and AYBEv3+Polη. The editing frequencies of three independent repeats (n=3) for each base are shown side by side. The original spacer position of the target adenine is highlighted in red. The percentage value below the specific adenine base indicates the average A to T (light green) or A to C (light blue) editing observed. The arrow indicates the adenine with the most transversion editing. Transfected mCherry-positive cells were sorted for further characterization of AYBEv3.

[0123] Fig.18Characterization of the editing profiles of AYBEs developed from ABE8e or ABEmax in some embodiments is shown. The bar graph shows the on-target DNA base editing frequencies of ABE8e and AYBEv3 using 10 genomic sites in HEK293T cells. The editing frequencies of three independent repeats (n=3) for each base are shown side by side. The original spacer position of the target adenine is highlighted in red. The percentage value below the specific adenine base indicates the average A to T (light green) or A to C (light blue) editing observed. The arrow indicates the adenine with the most transversion editing. Transfected mCherry-positive cells were sorted for further characterization of AYBEv3.

[0124] Fig.19 Shown is the transversion activity of AYBE containing an inactivated Cas9 compared to ABE8e in some embodiments.

[0125] Fig. 20 Shown is the transversion activity of AYBE containing Cas12i nickase compared to ABE8e in some embodiments.

[0126] Fig.21 Shown are an exemplary target dsDNA (comprising an exemplary target deoxyribonucleotide dA) prior to base editing in some embodiments, an exemplary guide nucleic acid, and an exemplary napDNAbp that is a nickase (but may not be a nickase in some other embodiments).

[0127] Fig. 22 An exemplary target dsDNA after base editing in some embodiments is shown (comprising an exemplary deoxyribonucleotide dC as a base editing result).

[0128] Fig.23 An exemplary target dsDNA after base editing (comprising an exemplary deoxyribonucleotide dT as a base editing result) in some embodiments is shown.

[0129] The drawings herein are for illustration purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION

[0130] Overview

[0131] The present disclosure provides a novel adenine base editor capable of expanding editing results, a system comprising the adenine base editor, and a method for using the same.

[0132] BE

[0133] In one aspect, the present disclosure provides a base editor comprising:

[0134] (1) a nucleic acid programmable DNA binding domain (napDNAbd) capable of binding to a target dsDNA comprising a target A:T base pair, wherein the target A:T base pair comprises a target deoxyadenosine (dA) (first deoxyribonucleotide) in a protospacer sequence on a non-target strand (editing strand) of the target dsDNA and a deoxythymidine (dT) (second deoxyribonucleotide) in a target sequence on a target strand (non-editing strand) of the target dsDNA, wherein the protospacer sequence is completely reverse complementary to the target sequence;

[0135] (2) an adenine deaminase domain capable of deaminating adenine of the target deoxyadenosine to hypoxanthine; and

[0136] (3) a hypoxanthine excision domain capable of excising the hypoxanthine.

[0137] The base editor includes a nucleic acid programmable DNA binding domain (napDNAbd). napDNAbd can be associated with a guide nucleic acid (e.g., a guide RNA), which positions / targets napDNAbd to a target DNA, which comprises a DNA chain (i.e., a target chain) complementary to the guide nucleic acid or a portion thereof (e.g., a guide sequence of a guide RNA). In other words, the guide nucleic acid "programs" napDNAbd to position and bind to the target DNA. The binding of napDNAbd of the base editor to the target DNA enables the functional domain of the base editor to approach the target DNA and act thereon as needed.

[0138] The components of the base editor are described in more detail below.

[0139] BE System

[0140] In another aspect, the present disclosure provides a system comprising:

[0141] (1) A base editor or a polynucleotide encoding the base editor, wherein the base editor comprises:

[0142] (a) a nucleic acid programmable DNA binding domain (napDNAbd) capable of binding to a target dsDNA comprising a target A:T base pair, wherein the target A:T base pair comprises a target deoxyadenosine (dA) (first deoxyribonucleotide) in a protospacer sequence on a non-target strand (editing strand) of the target dsDNA and a deoxythymidine (dT) (second deoxyribonucleotide) in a target sequence on a target strand (non-editing strand) of the target dsDNA, wherein the protospacer sequence is completely reverse complementary to the target sequence;

[0143] (b) an adenine deaminase domain capable of deaminating adenine of the target deoxyadenosine to hypoxanthine; and

[0144] (c) a hypoxanthine excision domain capable of excising said hypoxanthine; and

[0145] (2) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, wherein the guide nucleic acid comprises:

[0146] (a) a scaffold sequence capable of forming a complex with the napDNAbd; and

[0147] (b) is capable of hybridizing to a target sequence on a target strand of the target dsDNA, thereby directing the complex to a guide sequence of the target dsDNA.

[0148] In some embodiments, the system is a complex comprising a base editor complexed with a guide nucleic acid. In some embodiments, the complex further comprises a target dsDNA hybridized with a guide sequence. In some embodiments, the system is a composition comprising component (1) and component (2). The components of the system are described more particularly below. By relying on the hybridization between the guide sequence and the target dsDNA, the guide nucleic acid designed in this way can target the base editor comprising napDNAbp to the target dsDNA.

[0149] BE Method

[0150] In yet another aspect, the present disclosure provides a method for modifying a target dsDNA, the method comprising contacting the target dsDNA with a system,

[0151] The target dsDNA comprises a target A:T base pair, wherein the target A:T base pair comprises a target deoxyadenosine (dA) (first deoxyribonucleotide) in a protospacer sequence on a non-target strand (editing strand) of the target dsDNA and a deoxythymidine (dT) (second deoxyribonucleotide) in a target sequence on a target strand (non-editing strand) of the target dsDNA, wherein the protospacer sequence is completely reverse complementary to the target sequence;

[0152] The system comprises:

[0153] (1) A base editor or a polynucleotide encoding the base editor, wherein the base editor comprises:

[0154] (a) a nucleic acid programmable DNA binding domain (napDNAbd) capable of binding to the target dsDNA;

[0155] (b) an adenine deaminase domain capable of deaminating adenine of the target deoxyadenosine to hypoxanthine; and

[0156] (c) a hypoxanthine excision domain capable of excising said hypoxanthine; and

[0157] (2) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, wherein the guide nucleic acid comprises:

[0158] (1) a scaffold sequence capable of forming a complex with the napDNAbd; and

[0159] (2) capable of hybridizing to a target sequence on the target strand of the target dsDNA, thereby directing the complex to a guide sequence of the target dsDNA.

[0160] The components of the method are described in more detail below.

[0161] Identity of target deoxyadenosine

[0162] In some embodiments, the target dsDNA is wild-type.

[0163] In some embodiments, the target deoxyadenosine is native to the target dsDNA.

[0164] In some embodiments, the target deoxyadenosine is a mutation in the target dsDNA.

[0165] In some embodiments, the target deoxyadenosine is a pathogenic mutation in the target dsDNA.

[0166] In some embodiments, the target dsDNA is a target gene.

[0167] mechanism

[0168] Without wishing to be bound by any particular theory, it is believed that the adenine base editing ability of the base editors of the present disclosure relies on the ability of the hypoxanthine excision domain to excise hypoxanthine induced by deamination by the adenine deaminase domain.

[0169] In some embodiments, the target deoxyadenosine (first deoxyribonucleotide) is replaced by the base editor with a fourth deoxyribonucleotide different from the target deoxyadenosine (first deoxyribonucleotide).

[0170] In some embodiments, adenine of the target deoxyadenosine is deaminated by the adenine deaminase domain to form hypoxanthine in situ.

[0171] In some embodiments, the hypoxanthine excision domain is substantially capable of excision of hypoxanthine formed in situ by the adenine deaminase domain.

[0172] In some embodiments, the hypoxanthine excision domain is substantially capable of cleaving or hydrolyzing the glycosidic bond linking the in situ formed hypoxanthine and the deoxyribose of the target deoxyadenosine.

[0173] In some embodiments, the excision of the in situ formed hypoxanthine converts the target deoxyadenosine in the protospacer sequence into an abasic site having the sugar-phosphate backbone of the target deoxyadenosine.

[0174] In some embodiments, the target strand is nicked by the napDNAbd.

[0175] In some embodiments, the nick on the target strand induces a deletion in the target strand.

[0176] In some embodiments, the dsDNA is in a target cell.

[0177] In some embodiments, the deletion on the target strand is repaired, for example, by translesion synthesis (TLS) in the target cell using the protospacer sequence containing the abasic site as a repair template.

[0178] In some embodiments, during repair of the target strand, a third deoxyribonucleotide (e.g., dG, dA) different from deoxythymidine (dT) (the second deoxyribonucleotide) is formed in the target sequence at a site opposite to the abasic site in the protospacer sequence that serves as a repair template.

[0179] In some embodiments, the sugar-phosphate backbone of the target deoxyadenosine at the abasic site is removed, for example, by an enzyme in the target cell.

[0180] In some embodiments, after removal of the sugar-phosphate backbone of the target deoxyadenosine at the abasic site, a fourth deoxyribonucleotide (e.g., dC, dT) is formed at the abasic site in the protospacer sequence to base pair with the third deoxyribonucleotide (e.g., dG, dA) in the target sequence, such that the target deoxyadenosine in the protospacer sequence is replaced with the fourth deoxyribonucleotide (e.g., dA to dC, dA to dT).

[0181] In some embodiments, the third deoxyribonucleotide is dA, dC, or dG.

[0182] In some embodiments, the fourth deoxyribonucleotide is dT, dC, or dG.

[0183] In some embodiments, the substitution of the target deoxyadenosine to the fourth deoxyribonucleotide is a dA to dC, dA to dT, or dA to dG substitution.

[0184] In some embodiments, the replacement converts a stop codon to a non-stop codon or converts a non-stop codon to a stop codon.

[0185] In some embodiments, the stop codon is on the sense strand of the dsDNA.

[0186] In some embodiments, the replacement occurs on the sense strand or the non-sense strand of the dsDNA.

[0187] In some embodiments, the replacement occurs on the sense strand of the dsDNA, converting a stop codon on the sense strand to a non-stop codon or converting a non-stop codon on the sense strand to a stop codon.

[0188] In some embodiments, the replacement occurs on the nonsense strand of the dsDNA, converting a stop codon on the sense strand to a non-stop codon or converting a non-stop codon on the sense strand to a stop codon.

[0189] In some embodiments, the replacement occurs in a splice site (eg, splice donor, splice acceptor) of the target dsDNA.

[0190] In some embodiments, the substitution occurring in the splice site (eg, splice donor, splice acceptor) results in increased or decreased translation of a transcript transcribed from the target dsDNA.

[0191] BE orientation

[0192] In some embodiments, the base editor is a fusion protein.

[0193] In some embodiments, the base editor comprises, from N-terminus to C-terminus:

[0194] (1) the adenine deaminase domain, the napDNAbd and the hypoxanthine excision domain;

[0195] (2) the hypoxanthine excision domain, the adenine deaminase domain and the napDNAbd; or

[0196] (3). The adenine deaminase domain, the hypoxanthine excision domain and the napDNAbd.

[0197] In some embodiments, any two adjacent domains of (1), (2) or (3) are fused with or without a linker. Suitable linkers include, for example, those listed in WO 2020 / 181195, which is incorporated herein by reference in its entirety.

[0198] base excision domain

[0199] In some embodiments, the base editor comprises one, two, three or more hypoxanthine excision domains.

[0200] In some embodiments, the hypoxanthine excision domain is substantially capable of or has been engineered to be substantially capable of excising the hypoxanthine.

[0201] In some embodiments, the hypoxanthine excision domain comprises a glycosylase or a variant thereof.

[0202] In some embodiments, the glycosylase or variant thereof is substantially capable of or has been engineered to be substantially capable of cleaving the hypoxanthine.

[0203] Various glycosylases are known in the art, including those listed in WO 2020 / 181195, which is incorporated herein by reference in its entirety. Representative glycosylases include, for example, N-methylpurine DNA glycosylase (MPG), 8-oxoguanine DNA glycosylase (OGG1), methyl-CpG binding domain 4, DNA glycosylase (MBD4), thymine DNA glycosylase (TDG), uracil DNA glycosylase (UNG), single-stranded selective monofunctional uracil-DNA glycosylase 1 (SMUG1), mutY DNA glycosylase (MUTYH), nth-like DNA glycosylase 1 (NTHL1), nei-like DNA glycosylase 1 (NEIL1), nei-like DNA glycosylase 2 (NEIL2), nei-like DNA glycosylase 3 (NEIL3) and mutants or variants that can remove the hypoxanthine.

[0204] MPG

[0205] In some embodiments, the hypoxanthine excision domain comprises N-methylpurine DNA glycosylase protein (MPG).

[0206] In some embodiments, the MPG is substantially capable of or has been engineered to be substantially capable of cleaving the hypoxanthine.

[0207] In some embodiments, the MPG has or has been engineered to have substantially N-methylpurine DNA glycosylase activity.

[0208] In some embodiments, the MPG comprises the motif GxxYxxxxYGxxxxxN.

[0209] Non-limiting examples of MPGs include any MPG from any species selected from Table A. In some embodiments, the MPG is obtained from a species selected from Table A. In some embodiments, the MPG is a variant of an MPG obtained from a species selected from Table A.

[0210] Non-limiting examples of MPG include any MPG shown in Table B. In some embodiments, the MPG is a variant of human MPG (SEQ ID NO: 1 or 2) or any MPG shown in Table B.

[0211] In some embodiments, the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to any of the MPGs shown in SEQ ID NO: 1 or 2, or Table B.

[0212] In some embodiments, the MPG is contained in SEQ ID 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 ,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147, 148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, and / or 297, or amino acid substitutions at corresponding positions in MPG of another species other than human (e.g., a species other than human selected from Table A), wherein the positions are numbered according to the amino acid sequence of SEQ ID NO: 1.

[0213] In some embodiments, the MPG comprises an amino acid substitution at position N169 of SEQ ID NO:2, or at a corresponding position in MPG of another species other than human (e.g., a species other than human selected from Table A), wherein the position is numbered according to the amino acid sequence of SEQ ID NO:1.

[0214] In some embodiments, the amino acid substitution is with alanine (Ala / A) or serine (Ser / S).

[0215] In some embodiments, the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO: 28. In some embodiments, the MPG comprises the amino acid sequence of SEQ ID NO: 28.

[0216] In some embodiments, the MPG further comprises an amino acid substitution at position S198, K202, G203, S206 and / or K210 of SEQ ID NO:28, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:1.

[0217] In some embodiments, the amino acid substitution is a substitution with alanine (Ala / A).

[0218] In some embodiments, the MPG further comprises an amino acid substitution selected from S198A, K202A, G203A, S206A and K210A relative to SEQ ID NO:28, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:1.

[0219] In some embodiments, the MPG comprises amino acid substitutions N169S, S198A, K202A, G203A, S206A and K210A relative to SEQ ID NO:2, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:1.

[0220] In some embodiments, the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO: 30. In some embodiments, the MPG comprises the amino acid sequence of SEQ ID NO: 30.

[0221] In some embodiments, the MPG further comprises positions S78, P79, K80, G81, R110, T115, E116, R120, R138, G163, Q173, G174, D175, A177, E185, L187, E188, L190, E191, T192, Q195, S198, T199, R201, K202, V208, K210, R212, S213, 6. amino acid substitutions at K220, A226, N228, K229, S230, Q238, E240, A241, R246, L249, P251, E253, P254, A255, R272, P274, V279, R280, G281, V291, Q294, D295, T296, Q297 and / or A298, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:1.

[0222] In some embodiments, the amino acid substitution is with arginine (Arg / R) or lysine (Lys / K).

[0223] In some embodiments, the MPG further comprises a residue selected from the group consisting of S78R, P79R, K80R, G81R, R110K, T115R, E116R, R120K, R138K, G163R, Q173R, G174R, D175R, A177R, E185R, L187R, E188R, L190R, E191R, T192R, Q195R, S198R, T199R, R201K, K202R, V208R, K210R, R212K, Amino acid substitutions of S216R, K220R, A226R, N228R, K229R, S230R, Q238R, E240R, A241R, R246K, L249R, P251R, E253R, P254R, A255R, R272K, P274R, V279R, R280K, G281R, V291R, Q294R, D295R, T296R, Q297R and A298R, wherein the positions are numbered according to SEQ ID NO: 1.

[0224] In some embodiments, the MPG comprises amino acid substitutions N169S and G163R relative to SEQ ID NO:2, wherein the positions are numbered according to SEQ ID NO:1.

[0225] In some embodiments, the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO: 32. In some embodiments, the MPG comprises the amino acid sequence of SEQ ID NO: 32.

[0226] In some embodiments, the MPG comprises amino acid substitutions N169S, G163R, S198A, K202A, G203A, S206A and K210A relative to SEQ ID NO:2, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:1.

[0227] In some embodiments, the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO: 34. In some embodiments, the MPG comprises the amino acid sequence of SEQ ID NO: 34.

[0228] Adenine deaminase domain

[0229] Various adenine deaminases are known in the art, including, for example, those listed in WO 2020 / 181195, which is incorporated herein by reference in its entirety. Representative adenine deaminases include, for example, TadA and homologs and variants thereof, and APOBEC and homologs and variants thereof.

[0230] In some embodiments, the adenine deaminase domain comprises tRNA adenosine deaminase (TadA) or a functional variant or fragment thereof, such as TadA8e (SEQ ID NO: 3), TadA8.17, TadA8.20, TadA9, TadA8E V106W 、TadA8E V106W+D108Q TadA-CDa, TadA-CDb, TadA-CDc, TadA-CDd, TadA-CDe, TadA-dual, T AD AC-1.2, T AD AC-1.14, T AD AC-1.17, T AD AC-1.19, T AD AC-2.5, T AD AC-2.6, T AD AC-2.9, T AD AC-2.19, T AD AC-2.23, TadA8e-N46L, TadA8e-N46P.

[0231] In some embodiments, the adenine deaminase domain comprises an apolipoprotein B mRNA editing complex (APOBEC) family deaminase, activation-induced deaminase (AID), cytidine deaminase 1 from sea lamprey (Petromyzon marinus) (pmCDA1) or a functional variant or fragment thereof, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H.

[0232] In some embodiments, the adenine deaminase domain comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO:3.

[0233] In some embodiments, the adenine deaminase domain comprises the amino acid sequence of SEQ ID NO:3.

[0234] napDNAbd

[0235] Various napDNAbds are known in the art, including, for example, those listed in WO 2020 / 181195, which is incorporated herein by reference in its entirety. Representative napDNAbds include, for example, CRISPR-associated (Cas) proteins, IscB, IsrB, and TnpB.

[0236] In some embodiments, the napDNAbd substantially lacks dsDNA cleavage activity.

[0237] In some embodiments, the napDNAbd substantially lacks dsDNA cleavage activity and nickase activity.

[0238] In some embodiments, the napDNAbd has nickase activity.

[0239] In some embodiments, the napDNAbd has nickase activity to nick the target strand.

[0240] In some embodiments, the napDNAbd comprises a Cas nickase or an inactivated Cas of a Cas protein.

[0241] In some embodiments, the Cas protein is selected from Cas9 protein (e.g., SpCas9, SaCas9, GeoCas9, CjCas9, Cas9-KKH, circularly permuted Cas9, Argonaute (Ago), SmacCas9, Spy-macCas9, xCas9, SpCas9-NG); Cas12 protein (e.g., Cas12a, AsCas12a, LbCas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f (Cas14), Cas12g, Cas12h, Cas12i, xCas12i, Cas12Max, hfCas12Max, Cas12j, Cas12k, Cas12l, Cas12m, Cas12n, Cas12o, Cas12p, Cas12q, Cas12r, Cas12s, Cas12t, Cas12u, Cas12v, Cas12w, Cas12x, Cas12y, Cas12z); Cas13 protein (e.g., Cas13a, Cas13b, Cas13c, Cas13d, Cas13e, Cas13f, Cas13x, Cas13y); Csn2; and mutants thereof.

[0242] In some embodiments, the Cas nickase is a Cas9 nickase (nCas9), such as a SpCas9 nickase (SpCas9-D10A). In some embodiments, the napDNAbd comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO: 4. In some embodiments, the napDNAbd comprises an amino acid sequence of SEQ ID NO: 4.

[0243] In some embodiments, the inactivated Cas is an inactivated Cas9 (dCas9), such as an inactivated SpCas9 (SpCas9-D10A+H840A). In some embodiments, the napDNAbd comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO: 37. In some embodiments, the napDNAbd comprises the amino acid sequence of SEQ ID NO: 37.

[0244] In some embodiments, the Cas nickase is a Cas12i nickase (nCas12i) or an inactivated Cas12i (dCas12i), such as an inactivated Cas12i of an xCas12i polypeptide. In some embodiments, the napDNAbd comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO: 38. In some embodiments, the napDNAbd comprises an amino acid sequence of SEQ ID NO: 38.

[0245] In some embodiments, the napDNAbd comprises an IscB nickase (nIscB) or an inactivated IscB (dIscB) of an IscB protein (eg, OgeuIscB).

[0246] In some embodiments, the napDNAbd comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO:4, 37, or 38. In some embodiments, the napDNAbd comprises an amino acid sequence of SEQ ID NO:4, 37, or 38.

[0247] In some embodiments, the napDNAbd comprises a TnpB nickase or an inactivated TnpB of a TnpB protein.

[0248] Full-length base editor

[0249] In some embodiments, the base editor comprises an NLS at the N-terminus and / or C-terminus of the napDNAbp.

[0250] In some embodiments, the base editor comprises an NLS at the N-terminus and / or C-terminus of the hypoxanthine excision domain.

[0251] In some embodiments, the base editor comprises an NLS at the N-terminus and / or C-terminus of the adenine deaminase domain.

[0252] In some embodiments, the NLS is SV40NLS, bpSV40 NLS (e.g., SEQ ID NO: 11 or 12), or NP NLS (Xenopus laevis nucleoplasmin NLS, nucleoplasmin NLS). Additional NLSs suitable for the present disclosure or for connecting NLSs to any component of the base editors of the present disclosure include, for example, those listed in WO2020 / 181195, which is incorporated herein by reference in its entirety.

[0253] In some embodiments, the base editor comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to any one of SEQ ID NOs: 5, 6, 7, 29, 31, 33, and 35. In some embodiments, the base editor comprises an amino acid sequence of any one of SEQ ID NOs: 5, 6, 7, 29, 31, 33, and 35.

[0254] Target dsDNA

[0255] In some embodiments, the target deoxyadenosine is at a position of the protospacer sequence selected from the group consisting of position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, and combinations thereof.

[0256] In some embodiments, the target deoxyadenosine is at a position of the protospacer sequence selected from the group consisting of position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and combinations thereof.

[0257] In some embodiments, the target deoxyadenosine is at a position of the protospacer sequence selected from the group consisting of position 5, position 6, position 7, position 8, position 9, and combinations thereof.

[0258] In some embodiments, the target deoxyadenosine is at position 7 or 8 of the protospacer sequence.

[0259] In some embodiments, the target deoxyadenosine is the N2 nucleotide in a N1N2N3 motif, wherein N1, N2, or N3 is A, T, G, or C. In some embodiments, the target deoxyadenosine is deoxyadenosine (dA) in a CAA or CAG motif.

[0260] Protospacer sequence

[0261] In some embodiments, the protospacer sequence comprises about or at least about 16 consecutive nucleotides of the target dsDNA, such as about or at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more consecutive nucleotides of the target dsDNA, or in a numerical range between any two of the foregoing values, such as about 16 to about 50 or about 17 to about 22 consecutive nucleotides of the target dsDNA. In some embodiments, the protospacer sequence comprises about 20 consecutive nucleotides of the target dsDNA.

[0262] In some embodiments, the protospacer sequence is immediately 5' or 3' to a protospacer adjacent motif (PAM), which comprises the sequence 5'-NN-3', 5'-NNN-3', 5'-NNNN-3', 5'-NNNNN-3' or 5'-NNNNNN-3', wherein N is A, T, G or C.

[0263] In some embodiments, the protospacer sequence is immediately 5' to a protospacer adjacent motif (PAM), which comprises the sequence 5'-NGG-3', wherein N is A, T, G, or C.

[0264] In some embodiments, the protospacer sequence is immediately 3' to a protospacer adjacent motif (PAM), which comprises the sequence 5'-TTN-3', wherein N is A, T, G or C.

[0265] Guide sequence

[0266] In some embodiments, the guide sequence is about or at least about 16 nucleotides in length, such as about or at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more nucleotides in length, or a range of values ​​between any two of the aforementioned values, such as about 16 to about 50 nucleotides in length or about 17 to about 22 nucleotides in length. In some embodiments, the spacer sequence is about 20 nucleotides in length.

[0267] In some embodiments, (1) the guide sequence is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (perfectly) reverse complementary to the target sequence, optionally about 100% (perfectly) reverse complementary; (2) the guide sequence contains no more than 5, 4, 3, 2, or 1 mismatches with the target sequence or no mismatches; or (3) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (4) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (5) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (6) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (7) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (8) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (9) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (10) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (11) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (12) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (13) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (14) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (15) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (16) the guide sequence is at least about 10% (perfectly) reverse complementary to the target sequence; (17) the guide sequence is at least about 10 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides thereof, the PAM is immediately 5' to the protospacer sequence when at the 5' end of the guide sequence and immediately 3' to the protospacer sequence when at the 3' end of the guide sequence.

[0268] In some embodiments, the guide sequence comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence of any one of SEQ ID NOs: 40-89; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to a sequence of any one of SEQ ID NOs: 40-89. In some embodiments, the guide sequence comprises a polynucleotide sequence of any one of SEQ ID NOs: 40-89.

[0269] Scaffold sequence

[0270] For the purpose of the present disclosure, the support sequence is compatible with napDNAbd of the present disclosure, and can be compounded with napDNAbd. The support sequence can be a naturally occurring support sequence identified together with napDNAbd or a variant of the ability to be compounded with napDNAbd maintained by it. Generally, as long as the secondary structure of the variant is substantially the same as the secondary structure of the naturally occurring support sequence, the ability to be compounded with napDNAbd can be maintained. Nucleotide deletions, insertions or substitutions in the primary sequence of the support sequence may not necessarily change the secondary structure of the support sequence (for example, the relative position and / or size of the stem, protrusion and ring of the support sequence will not significantly deviate from the relative position and / or size of the original stem, protrusion and ring). For example, nucleotide deletions, insertions or substitutions can be in the protrusion or loop region of the support sequence, so that the overall symmetry of the protrusion and therefore the secondary structure remains roughly the same. Nucleotide deletions, insertions or substitutions can also be in the stem of the support sequence, so that the length of the stem does not significantly deviate from the length of the original stem (for example, adding or missing a base pair in each of the two stems corresponds to a total of 4 base changes).

[0271] In some embodiments, the scaffold sequence is 5' or 3' to the guide sequence.

[0272] In some embodiments, the scaffold sequence is compatible with the napDNAbp.

[0273] In some embodiments, the scaffold sequence has a secondary structure substantially identical to the secondary structure of SEQ ID NO: 13 or 39.

[0274] In some embodiments, the scaffold sequence comprises a polynucleotide sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 13 or 39. In some embodiments, the scaffold sequence comprises a polynucleotide sequence of SEQ ID NO: 13 or 39.

[0275] Translesion synthesis (TLS) polymerase

[0276] In some aspects, the base editors disclosed herein can be used in combination with trans-lesion synthesis (TLS) polymerases to improve the purity of the results. "Purity" means the percentage / ratio of a certain result among all possible results. For example, the purity of dT means the percentage / ratio of dT as a result among all possible results including, for example, dA, dT, dG, and dC. As listed in Table 4, TLS polymerases may have a tendency to incorporate various deoxyribonucleotides relative to the AP site during polymerization. By utilizing this tendency, the base editing results can be intentionally controlled to improve the purity of the results. For example, human Polη (SEQ ID NO: 36) is a TLS polymerase that preferentially incorporates dA relative to the AP site. In combination with human Polη, the base editing results can be adjusted to dT, thereby increasing the purity of dT.

[0277] Table 4. DNA polymerases used to incorporate perfect bases relative to the AP site.

[0278] DNA polymerase Perfect base relative to AP site Pol α(α) dA Polδ(δ) / PCNA dA Polγ(γ) dA Polη(η) dT and dA Polι(ι) dT, dG and dA Polκ(κ) dC and dA Polθ(θ) dA REV1 dC

[0279] In some embodiments, the base editor or system further comprises a translesion synthesis (TLS) polymerase or a recruitment domain capable of recruiting a TLS polymerase optionally fused to the base editor, or a coding sequence thereof.

[0280] Non-limiting examples of the TLS polymerase include Pol α (α), Pol β (β), Pol δ (δ) (PCNA), Pol γ (γ), Pol η (η), Pol ι (ι), Pol κ (κ), Pol λ (λ), Pol μ (μ), Pol ν (ν), Pol θ (θ), and REV1.

[0281] In some embodiments, the TLS polymerase comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO: 36. In some embodiments, the TLS polymerase comprises an amino acid sequence of SEQ ID NO: 36.

[0282] In some embodiments, a base editor or system further comprising a translesion synthesis (TLS) polymerase or a recruitment domain capable of recruiting a TLS polymerase causes the target deoxyadenosine (the first deoxyribonucleotide) to be replaced by dC, dT, or dG.

[0283] Additional cytidine deaminase domain

[0284] In some aspects, the base editor of the present disclosure can be used in combination with a cytidine deaminase domain to improve the purity of the result, especially the purity of dT. "Purity" means the percentage / ratio of a certain result among all possible results. For example, the purity of dT means the percentage / ratio of dC as a result among all possible results including, for example, dA, dT, dG, and dC. It is believed that the introduction of the cytidine deaminase domain can help to further convert the result dC into dT by C to T base editing. Therefore, in summary, there is a two-stage conversion, first, the target dA is converted to dC by A to C base editing as described herein, and secondly, dC is converted to dT by C to T base editing.

[0285] In some embodiments, the base editor or system further comprises a cytidine deaminase domain.

[0286] In some embodiments, the base editor or system further comprising the cytidine deaminase domain causes the target deoxyadenosine (the first deoxyribonucleotide) to be replaced by dT.

[0287] In some embodiments, the cytidine deaminase domain promotes the conversion of the fourth deoxyribonucleotide of dC to dT.

[0288] extend

[0289] In yet another aspect, the present disclosure provides a polynucleotide encoding a base editor of the present disclosure and, optionally, a guide nucleic acid as defined in the present disclosure.

[0290] In yet another aspect, the present disclosure provides a vector comprising the polynucleotide of the present disclosure.

[0291] In yet another aspect, the present disclosure provides a complex comprising a base editor of the present disclosure and a guide nucleic acid defined in the present disclosure.

[0292] In yet another aspect, the present disclosure provides a cell comprising the base editor or system of the present disclosure, the polynucleotide of the present disclosure, the vector of the present disclosure, or the complex of the present disclosure.

[0293] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising:

[0294] (i) a base editor or system of the present disclosure, a polynucleotide of the present disclosure, a vector of the present disclosure, a complex of the present disclosure, or a cell of the present disclosure; and

[0295] (ii) a pharmaceutically acceptable excipient.

[0296] In yet another aspect, the present disclosure provides a method for treating a subject having or at risk of having a disease associated with a target deoxyadenosine of a target dsDNA, the method comprising administering (e.g., an effective amount) a system of the present disclosure to the subject, wherein the target deoxyadenosine is modified by the system, and the modification treats or prevents the disease.

[0297] MPG

[0298] In yet another aspect, the present disclosure provides a MPG that is substantially capable of or has been engineered to be substantially capable of cleaving said hypoxanthine.

[0299] In some embodiments, the MPG is not wild-type human MPG (hMPG; SEQ ID NO: 1), hMPG-N169A, hMPG-N169S, hMPG-N169D, hMPG-N169H, or a variant thereof without the N-terminal initial methionine (M) (eg, SEQ ID NO: 2).

[0300] In some embodiments, the MPG has or has been engineered to have substantially N-methylpurine DNA glycosylase activity.

[0301] In some embodiments, the MPG comprises the motif GxxYxxxxYGxxxxxN.

[0302] In some embodiments, the MPG is obtained from a species selected from Table A.

[0303] In some embodiments, the MPG is a variant of MPG obtained from a species selected from Table A.

[0304] In some embodiments, the MPG is a variant of human MPG (SEQ ID NO: 1 or 2) or any MPG shown in Table B.

[0305] In some embodiments, the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to any of the MPGs shown in SEQ ID NO: 1 or 2, or Table B.

[0306] In some embodiments, the MPG is contained in SEQ ID 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 ,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147, 148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, and / or 297, or amino acid substitutions at corresponding positions in MPG of another species other than human (e.g., a species other than human selected from Table A), wherein the positions are numbered according to the amino acid sequence of SEQ ID NO: 1.

[0307] In some embodiments, the MPG comprises an amino acid substitution at position N169 of SEQ ID NO:2, or at a corresponding position in MPG of another species other than human (e.g., a species other than human selected from Table A), wherein the position is numbered according to the amino acid sequence of SEQ ID NO:1.

[0308] In some embodiments, the amino acid substitution is with alanine (Ala / A) or serine (Ser / S).

[0309] In some embodiments, the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO: 28. In some embodiments, the MPG comprises the amino acid sequence of SEQ ID NO: 28.

[0310] In some embodiments, the MPG further comprises an amino acid substitution at position S198, K202, G203, S206 and / or K210 of SEQ ID NO:28, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:1.

[0311] In some embodiments, the amino acid substitution is a substitution with alanine (Ala / A).

[0312] In some embodiments, the MPG further comprises an amino acid substitution selected from S198A, K202A, G203A, S206A and K210A relative to SEQ ID NO:28, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:1.

[0313] In some embodiments, the MPG comprises amino acid substitutions N169S, S198A, K202A, G203A, S206A and K210A relative to SEQ ID NO:2, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:1.

[0314] In some embodiments, the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO: 30. In some embodiments, the MPG comprises the amino acid sequence of SEQ ID NO: 30.

[0315] In some embodiments, the MPG further comprises positions S78, P79, K80, G81, R110, T115, E116, R120, R138, G163, Q173, G174, D175, A177, E185, L187, E188, L190, E191, T192, Q195, S198, T199, R201, K202, V208, K210, R212, S213, 6. amino acid substitutions at K220, A226, N228, K229, S230, Q238, E240, A241, R246, L249, P251, E253, P254, A255, R272, P274, V279, R280, G281, V291, Q294, D295, T296, Q297 and / or A298, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:1.

[0316] In some embodiments, the amino acid substitution is with arginine (Arg / R) or lysine (Lys / K).

[0317] In some embodiments, the MPG further comprises a residue selected from the group consisting of S78R, P79R, K80R, G81R, R110K, T115R, E116R, R120K, R138K, G163R, Q173R, G174R, D175R, A177R, E185R, L187R, E188R, L190R, E191R, T192R, Q195R, S198R, T199R, R201K, K202R, V208R, K210R, R212K, Amino acid substitutions of S216R, K220R, A226R, N228R, K229R, S230R, Q238R, E240R, A241R, R246K, L249R, P251R, E253R, P254R, A255R, R272K, P274R, V279R, R280K, G281R, V291R, Q294R, D295R, T296R, Q297R and A298R, wherein the positions are numbered according to SEQ ID NO: 1.

[0318] In some embodiments, the MPG comprises amino acid substitutions N169S and G163R relative to SEQ ID NO:2, wherein the positions are numbered according to SEQ ID NO:1.

[0319] In some embodiments, the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO: 32. In some embodiments, the MPG comprises the amino acid sequence of SEQ ID NO: 32.

[0320] In some embodiments, the MPG comprises amino acid substitutions N169S, G163R, S198A, K202A, G203A, S206A and K210A relative to SEQ ID NO:2, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:1.

[0321] In some embodiments, the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO: 34. In some embodiments, the MPG comprises the amino acid sequence of SEQ ID NO: 34.

[0322] Directing the regulation of nucleic acids

[0323] In some embodiments, the polynucleotide encoding the guide nucleic acid is DNA, RNA, or a DNA / RNA mixture.

[0324] "DNA / RNA mixture" refers to a nucleic acid comprising one or more modified or unmodified ribonucleotides and one or more modified or unmodified deoxyribonucleotides, whether or not consecutive. However, "DNA" or "RNA" may also refer to a DNA containing one or more modified or unmodified ribonucleotides, whether or not consecutive, or an RNA containing one or more modified or unmodified deoxyribonucleotides, whether or not consecutive.

[0325] In some embodiments, the guide nucleic acid is operably linked to or under the regulation of a promoter.

[0326] In some embodiments, the promoter is a broad spectrum promoter, a tissue-specific promoter, a cell type-specific promoter, a constitutive promoter, or an inducible promoter.

[0327] Suitable promoters are known in the art and include, for example, Cbh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, retroviral Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, elongation factor 1 alpha short (EFS) promoter, β-glucuronidase (GUSB) promoter, cytomegalovirus (CMV) immediate early (le) enhancer and / or promoter, chicken β-actin (CBA) promoter or derivatives thereof such as CAG promoter, CB promoter, (human) elongation factor 1 alpha-subunit (EF1α) promoter, ubiquitin C (UBC) promoter, prion promoter, neuron-specific enolase (NSE), neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, Platelet-derived growth factor (PDGF) promoter, platelet-derived growth factor B chain (PDGF-β) promoter, synapsin (Syn) promoter, synapsin 1 (Syn1) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, β-globin minigene nβ2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter, and myelin basic protein (MBP) promoter.

[0328] Regulation of base editors

[0329] In some embodiments, the polynucleotide encoding the base editor is DNA, RNA, or a DNA / RNA mixture. "DNA / RNA mixture" refers to a nucleic acid comprising one or more modified or unmodified ribonucleotides and one or more modified or unmodified deoxyribonucleotides, whether or not continuous. However, "DNA" or "RNA" may also refer to a DNA containing one or more modified or unmodified ribonucleotides, whether or not continuous, or an RNA containing one or more modified or unmodified deoxyribonucleotides, whether or not continuous.

[0330] In some embodiments, the polynucleotide encoding the base editor is operably linked to or under the regulation of a promoter.

[0331] In some embodiments, the promoter is a broad spectrum promoter, a tissue-specific promoter, a cell type-specific promoter, a constitutive promoter, or an inducible promoter.

[0332] Suitable promoters are known in the art and include, for example, the Cbh promoter, the Cba promoter, the pol I promoter, the pol II promoter, the pol III promoter, T7 promoter, U6 promoter, H1 promoter, retroviral Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, elongation factor 1 alpha short (EFS) promoter, β-glucuronidase (GUSB) promoter, cytomegalovirus (CMV) immediate early (Ie) enhancer and / or promoter, chicken β-actin (CBA) promoter or its derivatives such as CAG promoter, CB promoter, (human) elongation factor 1 alpha-subunit (EF1α) promoter, ubiquitin C (UBC) promoter, prion promoter, neuron-specific enolase (NSE), neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, platelet-derived growth factor (PDGF) promoter, platelet-derived growth factor B chain (PDGF-β) promoter, synaptophysin (Syn) promoter, human synapsin (hSyn) promoter, synapsin 1 (Syn1) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, β-globin minigene nβ2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter, myelin basic protein (MBP) promoter, OTOF promoter, GRK1 promoter, CRX promoter, NRL promoter, MECP2 promoter, mMECP2 promoter, hMECP2 promoter, APP promoter and RCVRN promoter.

[0333] deliver

[0334] Depending on practical needs, various delivery methods can be applied to the base editor of the present disclosure or the system of the present disclosure.

[0335] In yet another aspect, the present disclosure provides a delivery system comprising (1) a base editor of the present disclosure, a polynucleotide of the present disclosure, or a system of the present disclosure; and (2) a delivery vehicle.

[0336] In yet another aspect, the present disclosure provides a vector comprising a polynucleotide of the present disclosure. In some embodiments, the vector encodes a guide nucleic acid of the present disclosure. In some embodiments, the vector is a plasmid vector, a recombinant AAV (rAAV) vector (vector genome) or a recombinant lentiviral vector.

[0337] In another aspect, the present disclosure provides a recombinant AAV (rAAV) particle comprising the rAAV vector genome of the present disclosure. A brief introduction to AAV for delivery can be found in the "Adeno-associated virus (AAV) guide"

[0338] (addgene.org / guides / aav / ).

[0339] Adeno-associated virus (AAV), when engineered for delivery of, for example, a protein coding sequence of interest, may be referred to as an (r)AAV vector, (r)AAV vector particle, or (r)AAV particle, where "r" stands for "recombinant". And the genome packaged in an AAV vector for delivery may be referred to as an (r)AAV vector genome, vector genome, or simply vg, while the viral genome may refer to the original viral genome of native AAV.

[0340] The serotype of the rAAV particle capsid can be matched to the type of target cell. For example, Table 2 of WO 2018002719 A1 lists exemplary cell types that can be transduced by the indicated AAV serotypes (incorporated herein by reference).

[0341] In some embodiments, the rAAV particles include a capsid of a serotype suitable for delivery to an ear cell (e.g., inner hair cell). In some embodiments, the rAAV particles include a capsid of a serotype of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, or AAV.PHP.eB, AAV1-AAV13 to which any one belongs, or a functional variant thereof (e.g., a functional truncation), which encapsulates the rAAV vector genome. In some embodiments, the serotype of the capsid is AAV9 or a functional variant thereof.

[0342] The general principles of rAAV particle production are known in the art. In some embodiments, rAAV particles can be produced using the triple transfection method (described in detail in US Pat. No. 6,001,650).

[0343] Vector titer is usually expressed as vector genome / ml (vg / ml). In some embodiments, the vector titer is greater than 1×10 9 , higher than 5×10 10 , higher than 1×10 11 , higher than 5×10 11 , higher than 1×10 12 , higher than 5×10 12or higher than 1×10 13 vg / ml.

[0344] As an alternative to packaging single-stranded (ss) DNA sequences as vector genomes of rAAV particles, systems and methods for packaging RNA sequences as vector genomes into rAAV particles have recently been developed and can be applied herein. See PCT / CN2022 / 075366, which is incorporated herein by reference in its entirety.

[0345] When the vector genome is RNA, such as in PCT / CN2022 / 075366, for the sake of simplicity of description and declaration, the sequence elements for the DNA vector genome described herein, when present in the RNA vector genome, should generally be considered to be applicable to the RNA vector genome, except that the deoxyribonucleotides in the DNA sequence are the corresponding ribonucleotides in the RNA sequence (e.g., dT is equivalent to U, and dA is equivalent to A) and / or the elements in the DNA sequence are replaced with corresponding elements having corresponding functions in the RNA sequence, or are omitted because their functions are not required in the RNA sequence, and / or additional elements necessary for the RNA vector genome are introduced.

[0346] As used herein, a coding sequence (e.g., as a sequence element of an rAAV vector genome herein) is interpreted, understood, and considered to cover and encompass both DNA coding sequences and RNA coding sequences. When it is a DNA coding sequence, an RNA sequence can be transcribed from the DNA coding sequence, and optionally a protein can be further translated from the transcribed RNA sequence as needed. When it is an RNA coding sequence, the RNA coding sequence itself can be a functional RNA sequence for use, or an RNA sequence can be generated from the RNA coding sequence (e.g., by RNA processing), or a protein can be translated from the RNA coding sequence.

[0347] For example, a base editor coding sequence encoding a base editor covers a base editor DNA coding sequence from which the base editor is expressed (indirectly via transcription and translation) or a base editor RNA coding sequence from which the base editor is translated (directly).

[0348] For example, a gRNA coding sequence encoding a gRNA covers a gRNA DNA coding sequence from which the gRNA is transcribed or a gRNA RNA coding sequence that (1) is itself a functional gRNA for use or (2) from which the gRNA is generated (e.g., by RNA processing).

[0349] In some embodiments of the rAAV RNA vector genome, the 5'-ITR and / or 3'-ITR may not be necessary as DNA packaging signals and may be at least partially omitted, but RNA packaging signals may be introduced.

[0350] In some embodiments of the rAAV RNA vector genome, a promoter driving transcription of a DNA sequence may not be necessary and may be at least partially omitted.

[0351] In some embodiments of the rAAV RNA vector genome, the sequence encoding the polyA signal may be unnecessary and may be at least partially omitted, but a polyA tail may be introduced.

[0352] Similarly, other DNA elements of the rAAV DNA vector genome can be omitted or replaced with corresponding RNA elements and / or additional RNA elements can be introduced to accommodate strategies for delivering the RNA vector genome by rAAV particles.

[0353] In yet another aspect, the present disclosure provides a ribonucleoprotein (RNP) comprising a base editor of the present disclosure and a guide nucleic acid of the present disclosure.

[0354] In yet another aspect, the present disclosure provides a lipid nanoparticle (LNP) comprising an RNA (e.g., mRNA) encoding a base editor of the present disclosure and a guide nucleic acid of the present disclosure.

[0355] Modification method

[0356] The disclosed system comprising the disclosed base editor has multiple utilities, including modifying (e.g., cutting, deleting, inserting, translocating, inactivating or activating) target DNA in multiple cell types. The system has a wide range of applications, such as drug screening, disease diagnosis and prognosis, and treatment of various genetic disorders, which require high cutting activity and small size.

[0357] The methods and / or systems disclosed herein can be used to modify target DNA, for example, to modify the translation and / or transcription of one or more genes of a cell. For example, the modification can result in an increase in the transcription / translation / expression of a gene. In other embodiments, the modification can result in a decrease in the transcription / translation / expression of a gene.

[0358] In yet another aspect, the present disclosure provides a method for modifying a target DNA, the method comprising contacting the target DNA with a system of the present disclosure, a vector of the present disclosure, a ribonucleoprotein of the present disclosure, or a lipid nanoparticle of the present disclosure, wherein the guide sequence is capable of hybridizing with a target sequence of the target DNA, wherein the target DNA is modified by the complex.

[0359] In some embodiments, the target DNA is intracellular.

[0360] In some embodiments, the modification comprises one or more of cleavage, base editing, repair, and insertion or integration of exogenous sequences into the target DNA.

[0361] cell

[0362] The methods of the present disclosure can be used to introduce the systems of the present disclosure into cells and cause the cells to alter the production of one or more cell products (such as antibodies, starch, ethanol or any other desired product). Such cells and their progeny are within the scope of the present disclosure.

[0363] In yet another aspect, the present disclosure provides a cell comprising the system of the present disclosure. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell.

[0364] In yet another aspect, the disclosure provides a cell modified by a system of the disclosure or a method of the disclosure. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is modified in vitro, in vivo, or ex vivo.

[0365] In some embodiments, the cell is a stem cell. In some embodiments, the cell is not a human embryonic stem cell. In some embodiments, the cell is not a human germ cell.

[0366] In some embodiments, the cell is a prokaryotic cell.

[0367] In some embodiments, the cell is a eukaryotic cell (e.g., an animal cell, a vertebrate cell, a mammalian cell, a non-human mammalian cell, a non-human primate cell, a rodent (e.g., a mouse or rat) cell, a human cell, a plant cell, or a yeast cell) or a prokaryotic cell (e.g., a bacterial cell).

[0368] In some embodiments, the cell is from a plant or animal.

[0369] In some embodiments, the plant is a dicot. In some embodiments, the dicot is selected from soybean, cabbage (e.g., Chinese cabbage), rapeseed, Brassica, watermelon, melon, potato, tomato, tobacco, eggplant, pepper, cucumber, cotton, alfalfa, eggplant, grape.

[0370] In some embodiments, the plant is a monocot. In some embodiments, the monocot is selected from rice, corn, wheat, barley, oats, sorghum, millet, grasses, Poaceae, Zizania, Avena, Coix, Hordeum, Oryza, Panicum (e.g., Panicum miliaceum), Secale, Setaria (e.g., Setaria italica), Sorghum, Triticum, Zea, Cymbopogon, Saccharum (e.g., Saccharum officinarum), Phyllostachys, Dendrocalamus, Bambusa, Yushania.

[0371] In some embodiments, the animal is selected from the group consisting of pigs, cows, sheep, goats, mice, rats, alpacas, monkeys, rabbits, chickens, ducks, geese, fish (eg, zebrafish).

[0372] In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell, including a human cell (primary human cell or an established human cell line). In some embodiments, the cell is a non-human mammalian cell, such as a cell from a non-human primate (e.g., monkey), a cow / bull / cow, a sheep, a goat, a pig, a horse, a dog, a cat, a rodent (such as a rabbit, a mouse, a rat, a hamster, etc.). In some embodiments, the cell is from a fish (such as a salmon), a bird (such as a bird, including a chicken, a duck, a goose), a reptile, a shellfish (e.g., an oyster, a clam, a lobster, a prawn), an insect, a worm, a yeast, etc. In some embodiments, the cell is from a plant, such as a monocot or a dicot. In certain embodiments, the plant is a food crop, such as barley, cassava, cotton, groundnut or peanut, maize, millet, oil palm fruit, potato, dry bean, rapeseed or canola, rice, rye, sorghum, soybean, sugarcane, beet, sunflower and wheat. In some embodiments, the plant is a cereal (barley, maize, millet, rice, rye, sorghum, and wheat). In some embodiments, the plant is a tuber (cassava and potato). In some embodiments, the plant is a sugar crop (sugar beet and sugar cane). In some embodiments, the plant is an oil crop (soybean, groundnut or peanut, rapeseed or canola, sunflower, and oil palm fruit). In some embodiments, the plant is a fiber crop (cotton). In some embodiments, the plant is a tree (such as a peach or nectarine tree, an apple or pear tree, a nut tree (such as an almond or walnut or pistachio tree), or a citrus tree (e.g., an orange, grapefruit, or lemon tree)), a grass, a vegetable, a fruit, or an algae. In certain embodiments, the plant is a Solanum; a Brassica; a Lactuca; a Spinacia; a Capsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.

[0373] Pharmaceutical composition

[0374] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising (1) the system of the present disclosure, the vector of the present disclosure, the ribonucleoprotein of the present disclosure, the lipid nanoparticle of the present disclosure, or the cell of the present disclosure; and (2) a pharmaceutically acceptable excipient.

[0375] In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration selected from about 1×10 10 vg / mL, 2×10 10 vg / mL, 3×10 10 vg / mL, 4×10 10 vg / mL, 5×1010 vg / mL, 6×10 10 vg / mL, 7×10 10 vg / mL, 8×10 10 vg / mL, 9×10 10 vg / mL, 1×10 11 vg / mL, 2×10 11 vg / mL, 3×10 11 vg / mL, 4×10 11 vg / mL, 5×10 11 vg / mL, 6×10 11 vg / mL, 7×10 11 vg / mL, 8×10 11 vg / mL, 9×10 11 vg / mL, 1×10 12 vg / mL, 2×10 12 vg / mL, 3×10 12 vg / mL, 4×10 12 vg / mL, 5×10 12 vg / mL, 6×10 12 vg / mL, 7×10 12 vg / mL, 8×10 12 vg / mL, 9×10 12 vg / mL, 1×10 13 vg / mL, or a concentration in a numerical range between any two of the foregoing values, such as about 9×10 10 vg / mL to about 8×10 11 The concentration of vg / mL.

[0376] In some embodiments, the pharmaceutical composition is an injectable solution.

[0377] In some embodiments, the volume of the injection solution is selected from about 1 microliter, 10 microliters, 50 microliters, 100 microliters, 150 microliters, 200 microliters, 250 microliters, 300 microliters, 350 microliters, 400 microliters, 450 microliters, 500 microliters, 550 microliters, 600 microliters, 650 microliters, 700 microliters, 750 microliters, 800 microliters, 850 microliters, 900 microliters, 950 microliters, 1000 microliters, and volumes in a numerical range between any two of the foregoing values, for example, a concentration of about 10 microliters to about 750 microliters.

[0378] Treatment

[0379] In yet another aspect, the present disclosure provides a method for diagnosing, preventing or treating a disease in a subject in need thereof, the method comprising administering to the subject (e.g., a therapeutically effective dose of) a system of the present disclosure, a vector of the present disclosure, a ribonucleoprotein of the present disclosure, a lipid nanoparticle of the present disclosure, a cell of the present disclosure or a pharmaceutical composition of the present disclosure, wherein the disease is associated with a target DNA, wherein the guide sequence is capable of hybridizing with a target sequence of the target DNA, wherein the target DNA is modified by the complex, and wherein modification of the target DNA diagnoses, prevents or treats the disease.

[0380] In some embodiments, the disease is selected from Angelman syndrome (AS), Alzheimer's disease (AD), transthyretin amyloidosis (ATTR), transthyretin amyloid cardiomyopathy (ATTR-CM), cystic fibrosis (CF), hereditary angioedema, diabetes, Duchenne muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), spinal muscular atrophy (SMA), alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease (HTT), fragile X syndrome, Friedreich's ataxia, myopathy Atrophic lateral sclerosis (ALS), frontotemporal dementia, inherited chronic kidney disease, hyperlipidemia, Leber congenital amaurosis (LCA), sickle cell disease, thalassemia (e.g., beta thalassemia), Parkinson's disease (PD), myelodysplastic syndrome (MDS), retinitis pigmentosa (RP), age-related macular degeneration (AMD), hepatitis B, non-alcoholic fatty liver disease (NAFLD), acquired immunodeficiency syndrome, corneal dystrophy (CD), hypercholesterolemia, familial hypercholesterolemia (FH), heart disease (e.g., hypertrophic cardiomyopathy (HCM)), and cancer.

[0381] In some embodiments, the target DNA encodes mRNA, tRNA, ribosomal RNA (rRNA), microRNA (miRNA), non-coding RNA, long non-coding (lnc) RNA, nuclear RNA, interfering RNA (iRNA), small interfering RNA (siRNA), ribozyme, riboswitch, satellite RNA, microswitch, yeast (microzyme) or viral RNA.

[0382] In some embodiments, the target DNA is eukaryotic DNA.

[0383] In some embodiments, the eukaryotic DNA is mammalian DNA (such as non-human mammalian DNA), non-human primate DNA, human DNA, plant DNA, insect DNA, bird DNA, reptile DNA, rodent (e.g., mouse, rat) DNA, fish DNA, nematode DNA, or yeast DNA.

[0384] In some embodiments, the target DNA is within a eukaryotic cell, such as a human cell, a non-human primate cell, or a mouse cell.

[0385] In some embodiments, administering comprises local administration or systemic administration.

[0386] In some embodiments, administration comprises intrathecal administration, intramuscular administration, intravenous administration, transdermal administration, intranasal administration, oral administration, mucosal administration, intraperitoneal administration, intracranial administration, intracerebroventricular administration, or stereotactic administration.

[0387] In some embodiments, administration is injection or infusion.

[0388] In some embodiments, the subject is a human, a non-human primate, or a mouse.

[0389] In some embodiments, the level of a transcript (e.g., mRNA) of a target DNA is reduced in a subject by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or more compared to the level of a transcript (e.g., mRNA) of the target DNA in the subject before administration.

[0390] In some embodiments, the level of a transcript (e.g., mRNA) of a target DNA is increased in a subject by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or more compared to the level of a transcript (e.g., mRNA) of the target DNA in the subject before administration.

[0391] In some embodiments, the level of an expression product (e.g., protein) of a target DNA in a subject is reduced by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or more compared to the level of an expression product (e.g., protein) of the target DNA in the subject before administration.

[0392] In some embodiments, the level of the expression product (e.g., protein) of the target DNA in the subject is increased by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or more in the subject compared to the level of the expression product (e.g., protein) of the target DNA in the subject before administration. In some embodiments, the expression product is a functional mutant of the expression product of the target DNA.

[0393] In some embodiments, the median survival of subjects who have the disease but receive the administration is 5 days, 10 days, 20 days, 30 days, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, 2 years, 2.5 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or longer than the median survival of a subject or population of subjects who have the disease and do not receive the administration.

[0394] The therapeutically effective dose can be via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose may vary greatly depending on a variety of factors, such as vector selection, target cells, organisms, tissues, the general condition of the subject to be treated, the degree of transformation / modification sought, route of administration, mode of administration, the type of transformation / modification sought, etc.

[0395] For example, a therapeutically effective dose of rAAV particles can be about 1.0E+8, 2.0E+8, 3.0E+8, 4.0E+8, 6.0E+8, 8.0E+8, 1.0E+9, 2.0E+9, 3.0E+9, 4.0E+9, 6.0E+9, 8.0E+9, 1.0E+10, 2.0E+10, 3.0E+10, 4.0E+10, 6.0E+10, 8.0E+10, 1.0E+11, 2.0E+11, 3.0E+11, 4.0E+11, 6.0E+11, 8.0E+11, 1.0E+12, 2.0E+12, 3.0E+12, 4.0E+12, 6.0E+13, 8.0E+13, + 15, 2.0E+15, 3.0E+15, 4.0E+15, 6.0E+15, 8.0E+15, 1.0E+16, 2.0E+16, 3.0E+16, 4.0E+16, 6.0E+16, 8.0E+16, or 1.0E+17 vg, or a range between any two of these point values. vg represents the vector genome of the rAAV particle used for administration.

[0396] Detection Methods

[0397] In yet another aspect, the present disclosure provides a method for detecting a target DNA, the method comprising contacting the target DNA with a system of the present disclosure, wherein the target DNA is modified by the complex, and wherein the modification detects the target DNA. In some embodiments, the modification generates a detectable signal, such as a fluorescent signal.

[0398] Reagent test kit

[0399] In another aspect, the present disclosure provides a kit comprising the base editor of the present disclosure, the system of the present disclosure, the polynucleotide of the present disclosure, the vector of the present disclosure, the RNP of the present disclosure, the LNP of the present disclosure, the delivery system of the present disclosure, the cell of the present disclosure, or the pharmaceutical composition of the present disclosure or any one, two or all of its components.

[0400] In some embodiments, the kit further includes instructions for using one or more components contained therein, and / or for combining with additional one or more components that may be obtained or required elsewhere.

[0401] In some embodiments, the kit further comprises one or more buffers, which can be used to dissolve any of the components contained therein, and / or provide suitable reaction conditions for one or more of the components. Such buffers can include one or more of the following: PBS, HEPES, Tris, MOPS, Na2CO3, NaHCO3, NaB or a combination thereof. In some embodiments, the reaction conditions include a suitable pH, such as an alkaline pH. In some embodiments, the pH is between 7-10.

[0402] In some embodiments, any one or more of the kit components can be stored in a suitable container or at a suitable temperature, such as 4 degrees Celsius.

[0403] Further embodiments are described in the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0404] Example

[0405] Materials and methods

[0406] Unless otherwise specified, the experimental procedures used in the examples are conventional.

[0407] Unless otherwise specified, the materials, reagents and the like used in the Examples are commercially available.

[0408] Unless otherwise specified, the following materials and experimental methods were used in the examples.

[0409] Molecular cloning

[0410] The base editor constructs used in this study were cloned into a mammalian expression plasmid backbone under the control of the EF1α promoter by standard molecular cloning techniques. KOD-Plus-Neo DNA polymerase (KOD-401, TOYOBO) was used to amplify the insert fragments, and NEBuilder HiFi DNA assembly premix (E2621L, New England Biolabs) was used to perform Gibson assembly of multiple DNA fragments. The Gibson reaction was then transformed into chemically competent Escherichia coli DH5α.

[0411] The wild-type human MPG sequence (297 amino acids long, SEQ ID NO: 2) without the N-terminal start methionine (M) was PCR amplified from the cDNA of HEK293T and fused to ABE8e in three different orientations relative to nCas9 (D10A) by the Gibson assembly method. and The fusion proteins were generated as prototype versions of adenine transversion base editors (AYBEs) for A to T and A to C editing. The MPG-N169S mutation was introduced via site-directed mutagenesis by PCR.

[0412] For disease-associated single nucleotide variant (SNV) transversion editing, four disease-associated mutations with upstream and downstream flanking sequences (50 bp) were constructed in tandem into a lentiviral vector. Human Polη sequences were PCR amplified from HEK293T cDNA. bpNLS-Polη-P2A-BFP driven by a CAG promoter was constructed by standard molecular cloning techniques.

[0413] Design and construction of MPG mutants

[0414] N169S was a constant mutation during two rounds of MPG mutant screening based on the human MGP-N169S mutant (SEQ ID NO: 3) without the N-terminal start methionine (M) in Example 2. The MPG mutagenesis library was designed and generated as previously described 16 . The amino acid sequence of position 78 to position 298 of the MPG-N169S mutant (numbered according to the human wild-type MPG with an N-terminal start methionine as shown in SEQ ID NO:1) was divided into 13 segments, each 17 aa long. Thirteen (13) mutants with BpiI were introduced by PCR site-directed mutagenesis. In the first round of screening, 52 mutants were designed, in which for each variant, 4 or 5 random mutation sites were distributed almost evenly in distance. All non-alanine amino acids were replaced by alanine (X>A). In order to cover all residues in the segments mentioned in this article, we also mutated alanine to valine (A>V). In the second round of screening, 221 mutants were designed (scanning proteins with consecutive arginine (X>R) substitutions), in which all arginine amino acids were replaced by lysine to cover all residues in the segments mentioned here, because the two have similar size and charge. The oligonucleotides encoding the mutants in both rounds of screening were annealed and ligated into the corresponding BpiI-digested backbone vectors. The MPG mutants are listed in Tables 2 and 3.

[0415] Cell culture, transfection, and flow cytometric analysis

[0416] HEK293T, Hela and U2OS cells were cultured with DMEM (11995065, Gibco) supplemented with 10% fetal bovine serum (04-001-1ACS, BI Worldwide) and 0.1 mM non-essential amino acids (11875-093, Gibco). K562 cells were cultured with RPMI-1640 (11875-093, Gibco) supplemented with 10% fetal bovine serum (04-001-1ACS, BI Worldwide), 1% penicillin-streptomycin (15070-063, Gibco) and 0.mM non-essential amino acids (11140-050, Gibco). The cells were grown in an incubator at 37°C, 5% CO2.

[0417] MPG mutant screening was performed in 48-well or 24-well plates. One day before transfection, 3 × 10 4 HEK293T cells were plated in 250 μL complete growth medium in a 48-well plate. After 12 h, 100 ng of AYBE plasmid and 200 ng of A to T reporter plasmid were co-transfected into the cells with 600 ng of polyethyleneimine (PEI) per well (DNA / PEI ratio of 1:2.5). 5 Cells were plated per well in 500 μL complete growth medium, and 150 ng of AYBE plasmid and 300 ng of reporter plasmid were co-transfected into HEK293T cells containing 900 ng of PEI.

[0418] Disease-associated SNV transversion editing was tested in a stable HEK293T cell line via lentivirus. For lentiviral packaging, plasmids (1.2 μg) with disease-associated mutations were co-transfected into HEK293T cells with packaging plasmids Pax2 (0.9 μg) and Vsvg (0.6 μg) using FuGENE HD transfection reagent (E2311, Promega). After 72 h, the lentivirus-containing culture medium was collected for infection and then filtered through a 0.45 μm low protein binding membrane (Millipore). For lentiviral infection, HEK293T cells were dissociated by trypsin-EDTA (25200-072, Gibco), and the suspension was diluted to 18 × 10 per well in a 6-well plate. 5 Cells were plated and incubated with 150 μl of lentivirus for 48 h. Then, the medium was replaced with fresh complete medium.

[0419] For cell transfection of HEK293T, Hela, U2OS, and K562 cells for FACS, 5 × 10 5Cells were plated in 12-well plates containing 1 ml of complete growth medium. After 14-16 h, 2 μg of AYBE-gRNA plasmid was transfected into the cells using PEI (DNA / PEI ratio of 1:2.5) or FuGENE HD transfection reagent (E2311, Promega) (DNA:FuGENE ratio of 1:3).

[0420] Orthogonal R-loop analysis was performed as previously described 17 Then, 1 μg of AYBE plasmid with a single guide RNA (sgRNA) targeting site 3 and 1 μg of dSaCas9 plasmid with corresponding sgRNAs targeting five OT sites were co-transfected into HEK293T cells in 12-well plates using PEI (DNA:PEI ratio of 1:2.5) to generate R-loops.

[0421] 48 h after transfection, the expression of mCherry, BFP, and EGFP fluorescence was analyzed by BD FACSAria III or Beckman CytoFLEX S. Flow cytometry results were analyzed using FlowJo version 10.5.3. The gating strategy for the evaluation of on-target editing efficiency in identifying mCherry+, BFP+, and EGFP+ cells is provided in Figure 1 c.

[0422] Targeted sequencing of endogenous loci

[0423] 72 h after transfection, 10,000 mCherry-positive cells were isolated by FACS. Genomic DNA was extracted by adding 40 μl lysis buffer and 1 μL proteinase K (PD101-01, Vazyme) directly to each tube of sorted cells. The genomic DNA / lysis buffer mixture was incubated at 55°C for 45 min, followed by a 95°C enzyme inactivation step for 10 min. The region of interest of the target site was amplified by PCR using site-specific primers. The PCR reaction using Phanta Max Super-Fidelity DNA Polymerase (P505-d3, Vazyme) was performed as follows: 5 min at 95°C, 28 cycles at 95°C for 15 s, 60°C for 15 s, 72°C for 30 s, and a final extension of 72°C for 5 min. PCR products were purified using a universal DNA purification kit (TIANGEN) according to the manufacturer's instructions and analyzed by Sanger sequencing (GENEWIZ). The amplicons were ligated to adapters and sequenced on the Illumina MiSeq platform. The protospacer sequences / guide sequences (SEQ ID NOs: 40-89) of the genomic loci tested are listed in Table 1.

[0424] Table 1

[0425]

[0426]

[0427]

[0428] Target sequencing data analysis.

[0429] First, the targeted amplicon sequencing reads were input into trim_galore (powered by Cutadapt 0.6.6) for quality trimming, and the reads with less than 30bp were filtered. Then the cleaned pairs were merged using FLASH version 1.2.11. The sequences from the amplification of the single target were separated using fastx_barcode_splitter.pl from fastx_toolkit (0.0.14). The sequences were separated by CRISPResso2 20 Further amplicon sequencing analysis was performed. A 10 bp window was used to quantify modifications centered around the middle of the 20 bp gRNA. Otherwise, default parameters were used for analysis. The output files "Quantification_window_nucleotide_frequency_table.txt" and "Quantification_window_modification_count_vectors.txt" were combined to calculate the base substitution and indel rate for each individual targeting. Briefly, the counts of nucleotide bases (A, C, G, and T) as well as deletions (-) and ambiguous bases (N) at each position in the sgRNA were extracted from "alleles_frequency_table_around_sgRNA_*.txt". When insertions occurred for some specific positions, the aligned sequence with the inserted base was assigned to the reference base. In order to give a global view of the modifications of individual positions of this reference, the indel rate from

[0430] The counts of insertions of "Quantification_window_modification_count_vectors.txt" were collected and used to verify the counts of reference bases by subtracting the counts of insertions from the counts of reference bases. The verified counts of nucleotide bases (A, C, G, and T) and indels were further used to calculate the base substitution and indel rates at each position of sgRNA.

[0431] Statistical analysis

[0432] Statistical tests included one-way ANOVA followed by two-tailed unpaired two-sample t-test or Dunnett's multiple comparisons test performed by GraphPad Prism 8. All values ​​are reported as mean ± sem.

[0433] Example 1 Evaluation of the transversion activity of prototype AYBE

[0434] Prototype ABYE Build

[0435] Without wishing to be bound by any particular theory, the applicants hypothesize that, in order to induce A to T and A to C transversion editing, ABE-induced excision of hypoxanthine from deoxyinosine may occur by triggering the base excision repair (BER) pathway in cells. 10,11 Achieving more universal base editing outcomes ( Figure 1 a). Three prototypes (MTC, TMC, and TCM) of adenine transversion base editors (AYBE, Y=C or T base) were developed by fusing ABE8e (SEQ ID NO:3) to wild-type human N-methylpurine DNA glycosylase protein (MPG; also known as alkyladenine DNA glycosylase (AAG)) without the N-terminal start methionine (M); SEQ ID NO:2, which can excise damaged DNA in different orientations relative to nCas9 (D10A) (SEQ ID NO:4). 12,13 Hypoxanthine (Hx) ( Figure 1 b and Figure 4 a).

[0436] The full-length amino acid sequence of wild-type human MPG is shown in SEQ ID NO: 1, which has an N-terminal start methionine (M) (corresponding to the start codon ATG), and the position numbering of any mutations of wild-type human MPG throughout the present disclosure is based on this sequence. In the case where wild-type human MPG (or its mutant) is fused to another element such as NLS or TadA via the N-terminus, the N-terminal start methionine (M) (corresponding to the start codon ATG) of the wild-type human MGP (or its mutant) of SEQ ID NO: 1 will be removed, resulting in a wild-type human MGP (or its mutant) without an N-terminal start methionine (M) as shown in SEQ ID NO: 2, hereinafter referred to as "MPG" (SEQ ID NO: 2), unless otherwise indicated.

[0437] The amino acid sequence of TadA8e for AYBE (without the N-terminal initiating methionine (M)) is shown in SEQ ID NO:3.

[0438] The amino acid sequence of nCas9 (SpCas9-D10A nickase, hereinafter referred to as "nCas9" unless otherwise indicated) used in AYBE (without the N-terminal start methionine (M), and the position of the D10A mutation is numbered according to the full-length SpCas9 with the N-terminal start methionine (M)) is shown in SEQ ID NO:4.

[0439] The prototype version MTC (SEQ ID NO: 5) has a configuration of N'-MPG-TadA8e-nCas9-C'. In particular, the prototype version MTC is composed of the following from N-terminus to C-terminus: methionine (M; corresponding to the start codon ATG), bpNLS1 (SEQ ID NO: 11), MPG (SEQ ID NO: 2), TadA8e (SEQ ID NO: 3), SpCas9-D10A nickase (SEQ ID NO: 4), and bpNLS2 (SEQ ID NO: 12).

[0440] The prototype version TMC (SEQ ID NO: 6) has a configuration of N'-TadA8e-MPG-nCas9-C'. In particular, the prototype version TMC is composed of the following from the N-terminus to the C-terminus: methionine (M; corresponding to the start codon ATG), bpNLS1 (SEQ ID NO: 11), TadA8e (SEQ ID NO: 3), MPG (SEQ ID NO: 2), SpCas9-D10A nickase (SEQ ID NO: 4), and bpNLS2 (SEQ ID NO: 12).

[0441] The prototype version TCM (SEQ ID NO: 7) has a configuration of N'-TadA8e-nCas9-MPG-C'. In particular, the prototype version TCM (also referred to as "AYBEv0.1") is composed of the following from N-terminus to C-terminus: methionine (M; corresponding to the start codon ATG), bpNLS1 (SEQ ID NO: 11), TadA8e (SEQ ID NO: 3), SpCas9-D10A nickase (SEQ ID NO: 4), bpNLS2 (SEQ ID NO: 12), MPG (SEQ ID NO: 2), and bpNLS2 (SEQ ID NO: 12).

[0442] In addition, an MPG mutant substantially lacking glycosylase activity (SEQ ID NO: 8; inactive MPG, dMPG, or inactive MPG) was constructed by introducing triple mutations of E125A, Y127A, H136A into MPG (SEQ ID NO: 2).

[0443] Based on dMPG, AYBE-dMPG (SEQ ID NO: 9) was constructed as a negative control, which consists of the following from N-terminus to C-terminus: methionine (M; corresponding to the start codon ATG), bpNLS1 (SEQ ID NO: 11), TadA8e (SEQ ID NO: 3), SpCas9-D10A nickase (SEQ ID NO: 4), bpNLS2 (SEQ ID NO: 12), dMPG (SEQ ID NO: 8), and bpNLS2 (SEQ ID NO: 12).

[0444] In addition, ABE8e (TadA8e-nCas9 adenine base editor; SEQ ID NO: 10) was used as a blank control, which consisted of the following from N-terminus to C-terminus: methionine (M; corresponding to the start codon ATG), bpNLS1 (SEQ ID NO: 11), TadA8e (SEQ ID NO: 3), SpCas9-D10A nickase (SEQ ID NO: 4), and bpNLS2 (SEQ ID NO: 12).

[0445] Plasmid construction

[0446] In order to conveniently evaluate the transversion activity of AYBE, a simple intron-break EGFP reporter system consisting of an expression plasmid and a reporter plasmid was designed ( Figure 1 b and Figure 5 a).

[0447] Expression plasmid (vector) Figure 1 b) comprises in the 5'-3' orientation a polynucleotide sequence encoding a base editor of the present disclosure (e.g., MTC, TMC, TCM (also referred to as "AYBEv0.1"), AYBE-dMPG (negative control), or ABE8e (blank), as described above) under the regulation of the human EF-1α promoter, followed by a sequence encoding the SV40 polyA signal, and an mCherry reporter system (a polynucleotide sequence encoding mCherry under the regulation of the CBH promoter, followed by a sequence encoding the bGH polyA signal), which indicates successful transfection and expression of the expression plasmid.

[0448] The reporter plasmid (vector) contains, in 5'-3' orientation, a polynucleotide sequence encoding BFP-P2A-activatable EGxxFP under the regulation of human CAG promoter from N-terminus to C-terminus, followed by a sequence encoding SV40 polyA signal, and a polynucleotide sequence encoding EGxxFP-targeting single guide RNA (sgRNA) under the regulation of human U6 promoter, which consists of

[0449] The EGxxFP targeting spacer sequence and the Cas9 scaffold sequence (SEQ ID NO: 13) are composed of.

[0450] SEQ ID NO: 13, gRNA scaffold of Cas9, 76 bp

[0451] gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc

[0452] The intron-breaking EGFP reporter system was engineered by inserting the last intron (86 bp long) of human ribosomal protein S5 (RPS5) between the K126 and G127 codons of the EGFP coding sequence. The 68th base (G to C) or the 70th base (T to C) in the intron sequence was modified by site-directed mutagenesis by PCR to introduce an artificial Cas9 protospacer adjacent motif (PAM) on the template strand, and the corresponding mutations at the splice acceptor site were performed to construct an A to T reporter system or an A to C reporter system. Mutations at the splice acceptor site result in non-spliced ​​EGFP transcripts producing inactive EGFP. Proper splicing of the EGxxFP coding sequence requires transversion correction in the A to T reporter system or the A to C reporter system. Correctly spliced ​​EGxxFP transcripts can produce active EGFP signals detectable by flow cytometry.

[0453] In the case of a reporter plasmid for A to T transversion assessment, the A to T insertion sequence of SEQ ID NO: 14 (target strand) was inserted into the EGFP coding sequence between the codon AAG (amino acid residue K at position 126) and the codon GGC (amino acid residue G at position 127) of the EGFP coding sequence.

[0454] SEQ ID NO: 14, A to T reporter system, A to T insertion sequence (target strand),

[0455]

[0456] It also refers to its reverse complement sequence on the non-target strand of the reporter plasmid (SEQ ID NO: 90)

[0457]

[0458] SpCas9 PAM in the insert sequence of the A to T reporter plasmid for designing the spacer sequence of the sgRNA The EGxxFP protospacer sequence immediately adjacent to the 5' region is shown in SEQ ID NO: 15.

[0459] 19 bp, SEQ ID NO: 15

[0460] Wherein the double underlined sequence of the protospacer sequence is part of the insertion sequence (SEQ ID NO:90), the capital letters GCC correspond to the codon GGC (amino acid residue G at position 127) of the EGFP coding sequence, and the double underlined capital letter A corresponds to the double underlined capital letter T of the insertion sequence, which indicates the target A on the reporter plasmid intended for A to T transversion.

[0461] The EGxxFP targeting spacer sequence of the EGxxFP targeting sgRNA for A to T transversion is shown in SEQ ID NO: 16,

[0462] 19 bp, SEQ ID NO: 16 (T represents U in the case of RNA sequences)

[0463] It is capable of hybridizing to the reverse complement of the protospacer sequence on the target strand (target sequence) and is identical in sequence to the protospacer sequence on the non-target strand, despite natural differences between the DNA sequence and the corresponding RNA sequence.

[0464] Four base editing outcomes can be generated by using EGxxFP targeting sgRNA to make A to T transversions, such as Figure 1 b, where the original spacer sequence was edited as follows:

[0465] (1) SEQ ID NO: 17;

[0466] (2) SEQ ID NO: 18;

[0467] (3) SEQ ID NO: 19; or

[0468] (4) SEQ ID NO:20.

[0469] For (1), the inserted intron can be properly spliced, resulting in EGFP expression and thus generating a green fluorescent signal. For (2) to (4), the inserted intron cannot be properly spliced, resulting in no or almost no EGFP expression and green fluorescent signal.

[0470] A similar design was applied to the evaluation of A to C transversions ( Figure 5 ), wherein the inserted sequence is shown in SEQ ID NO:21 (target strand).

[0471] SEQ ID NO:21, A to C reporter system, A to C insert sequence

[0472]

[0473] It also refers to its reverse complement sequence on the non-target strand of the reporter plasmid (SEQ ID NO: 91)

[0474]

[0475] SpCas9 PAM in the insert sequence of the A to C reporter plasmid for designing the spacer sequence of the sgRNA The EGxxFP protospacer sequence immediately adjacent to the 5' region is shown in SEQ ID NO: 22.

[0476] 20 bp, SEQ ID NO: 22

[0477] Wherein the double underlined sequence of the protospacer sequence is part of the insertion sequence (SEQ ID NO:91), the capital letters ATGCC correspond to the codon GGC (amino acid residue G at position 127) and part of the codon AT (belonging to the codon ATC; amino acid residue I at position 128) of the EGFP coding sequence, and the double underlined capital letter A corresponds to the double underlined capital letter T of the insertion sequence, which indicates the target A on the reporter plasmid intended for A to C transversion.

[0478] The EGxxFP targeting spacer sequence of the EGxxFP targeting sgRNA for A to C transversion is shown in SEQ ID NO: 23,

[0479] 20 bp, SEQ ID NO: 23 (T represents U in the case of RNA sequences)

[0480] It is capable of hybridizing to the reverse complement of the protospacer sequence on the target strand (target sequence) and is identical in sequence to the protospacer sequence on the non-target strand, despite natural differences between the DNA sequence and the corresponding RNA sequence.

[0481] Four base editing outcomes can be generated by using EGxxFP targeting sgRNA to make A to C transversions, such as Figure 5 As shown, the protospacer sequence was edited as follows:

[0482] (1) SEQ ID NO: 24;

[0483] (2) SEQ ID NO: 25;

[0484] (3) SEQ ID NO:26; or

[0485] (4) SEQ ID NO:27.

[0486] For (1), the inserted intron can be properly spliced, resulting in EGFP expression and thus generating a green fluorescent signal. For (2) to (4), the inserted intron cannot be properly spliced, resulting in no or almost no EGFP expression and green fluorescent signal.

[0487] result

[0488] For the three prototype AYBEs (TCM, MTC, and TMC), their A to T transversions were evaluated using the A to T reporter system. After co-transfection with the A to T reporter vector (in which the sgRNA targets an intronic mis-splicing mutation), the prototype TCM (TCM, hereinafter referred to as AYBEv0.1) fused with MPG at the C-terminus in HEK293T cells showed the highest transversion activity (67.17%), compared with the prototype MTC fused with MPG at the N-terminus and the prototype TMC fused with MPG internally between TadA8e and nCas9, which showed 63.27% and 59.03% transversion activities, respectively ( Figure 4 b). Therefore, AYBEv0.1 was used in the subsequent experiments.

[0489] In addition, A to T transversion and A to C transversion of AYBEv0.1 were evaluated by using A to T and A to C reporter systems, respectively. The results showed that AYBEv0.1 achieved 56.6% A to T transversion, while ABE8e without MPG (blank) and AYBEv0.1 with a non-targeting spacer sequence (sgNT, negative control) achieved 2.10% and 0% A to T transversion, respectively. ( Figure 1 c). In additional experiments, AYBEv0.1 achieved 62.37% A to T transversions, while ABE8e without MPG (blank) and AYBE with inactivated MPG (negative control) achieved 2.34% and 2.25% A to T transversions, respectively ( Figure 1 d).

[0490] On the other hand, AYBEv0.1 achieved 7.32% A to C transversions, while ABE8e without MPG (blank) and AYBE with inactive MPG (negative control) achieved 0.32% and 0.29% A to T transversions, respectively ( Figure 5 b).

[0491] Therefore, it was demonstrated that AYBEv0.1 can achieve significant A to T and A to C transversions compared to the blank control and negative control.

[0492] Example 2 Protein Engineering of MPG to Improve the Transversion Activity of AYBEv0.1

[0493] To improve the transversion activity of AYBEv0.1, rational mutagenesis of MPG was performed to generate hundreds of AYBE variants for screening and to evaluate their transversion editing activity by using an A to T reporter system.

[0494] First, N169S, a mutation that enhances the hypoxanthine excision activity of MPG, was 14 ) was introduced into the MPG domain of AYBEv0.1, thereby generating AYBEv0.2 (SEQ ID NO: 29) containing MPG-N169S (SEQ ID NO: 28). It was observed that AYBEv0.2 increased EGFP expression compared to AYBEv0.1. + The percentage of cells (up to 83.60%, Figure 1 d) and MFI (mean fluorescence intensity) (2.74-fold increase; Figure 1 e), indicating that the transversion activity was enhanced by introducing the MPG mutation N169S.

[0495] In addition, two parallel rounds of mutagenesis and screening were performed based on AYBEv0.2 to further improve the transversion activity. Figure 4 c) and biochemical characterization, the non-conserved N-terminal region (1-79aa) had no effect on the base excision or DNA binding activity of the enzyme 13,15 , so using a recently developed strategy 16 The 78-298aa region of MPG-N169S was evenly divided into 13 segments (F1-F13, 17 amino acids each).

[0496] As shown in Table 2, in the first round of mutagenesis and screening, 52 mutants were designed and generated, which had 4 or 5 random amino acid substitutions in each segment (all non-alanines were replaced by alanine (X to A) and alanine was replaced by valine (A to V)), which were distributed nearly evenly in distance.

[0497] Table 2. AYBE variants with MPG variants in round 1 and their normalized transversion activities relative to MPG-N169S

[0498]

[0499]

[0500] In the second round of mutagenesis and screening, the MPG-N169S protein was scanned with consecutive arginine substitutions (X to R) or R to K substitutions, aiming to enhance the interaction of MPG with the substrate DNA ( Figure 1 f).

[0501] Table 3. AYBE variants with MPG variants in round 2 and their normalized transversion activities relative to MPG-N169S

[0502]

[0503]

[0504]

[0505] Among others, AYBE variant v1 (AYBEv1, SEQ ID NO:31) containing MPG-F8V1 (MPGv1, MPG-N169S+S198A+K202A+G203A+S206A+K210A, SEQ ID NO:30) from round 1 and AYBE variant v2 (AYBEv2, SEQ ID NO:33) containing MPG-G163R+N169S (MPGv2, SEQ ID NO:32) from round 2 showed the highest transversion activity in each round ( Figure 1 gh). Compared with AYBEv0.2, AYBEv1 and AYBEv2 showed 1.24-fold and 2.10-fold increases in transversion activity, respectively ( Figure 1 gh), showing 2.83-fold and 3.83-fold increases in transversion activity, respectively, compared with AYBEv0.1 ( Figure 1 i).

[0506] To explore the additive effect of the MPG mutations in MPGv1 and MPGv2, the mutations in MPGv1 and MPGv2 were combined in round 3 into MPGv3 (MPG-G163R+N169S+S198A+K202A+G203A+S206A+K210A, SEQ ID NO:34) to construct AYBEv3 (SEQ ID NO:35) containing MPGv3. Surprisingly, a 4.78-fold synergistic improvement in transversion activity was achieved compared to AYBEv0.1, while AYBEv1 alone achieved a 2.83-fold improvement, and AYBEv2 alone achieved a 3.83-fold improvement (compared to AYBEv0.1) ( Figure 1 i).

[0507] The enhanced transversion activity of the progressive AYBE variant was confirmed at the endogenous genomic locus using fluorescence-activated cell sorting (FACS). Figure 6a and 6b).

[0508] Similar to the synergistic increase in transversion activity (from 5.88% to 15.49% for A to T transversions and from 14.42% to 30.98% for A to C transversions), a synergistic decrease in indel (insertion and / or deletion) frequency (indicating undesired DNA cleavage) was also observed for AYBEv3 (from 34.28% to 11.64%) ( Figure 6 c), indicating improved safety. Without wishing to be bound by theory, it is believed that the MPG mutation in AYBEv3 may promote specific substrate selection or modulate the DNA binding activity of the MPG protein ( Figure 6 d).

[0509] In summary, the above results show that AYBE is effectively optimized towards high activity for both A to T and A to C transversion editing.

[0510] Example 3 Evaluation of on-target editing profile of AYBEv3

[0511] The editing profile of AYBEv3 was further characterized by targeting dozens of endogenous genomic loci. Efficient A to C or A to T editing was observed with AYBEv3, but almost no A to Y (A to C or A to T) transversion editing was observed at any of the 26 loci tested with ABE8e ( Figure 7-10 The top 12 efficient editing sites include five sites with A7 and seven sites with A8 ( Figure 2 a and Figure 7 ), with A to C editing as the major product (average editing frequencies ranging from 34.14% to 70%, with purity up to 70% at site 35), and A to T editing with an average editing frequency ranging from 16.29% to 39.09% (with purity up to 39.09% at site 12) ( Figure 2 bd), indicating that AYBEv3 exhibited high editing efficiencies for A to Y transversions at protospacer positions 7 and 8 (average editing frequencies ranging from 8% to 72%), including 3% to 53% editing efficiencies for A to C transversions and 3% to 32% editing efficiencies for A to T transversions ( Figure 2 a and Figure 7 ).

[0512] Overall, the results suggest that the editing window of AYBEv3 exists in positions 3 to 10 or preferably 5 to 9 of the protospacer, and that indels are distributed throughout the protospacer ( Fig.11 a), CAA and CAG are the two most preferred editing motifs ( Fig.11 b). Note that the average indel frequencies (percentage of alleles containing an insertion or deletion throughout the protospacer) induced by AYBEv3 ranged from 1.63% to 40.68% ( Fig.11 a). In addition, analysis of allelic composition showed that AYBEv3 induced less bystander editing than ABE8e ( Fig.12 ). In addition, in three different human cell lines (HeLa, U2OS, and K562 cells), AYBEv3 also exhibited efficient A-to-C and A-to-T transversion editing activities at protospacer positions 7 and 8, with A-to-C editing as the major product ( Fig.13 and Fig.14 ).

[0513] Example 4 Evaluation of off-target editing of AYBEv3

[0514] To explore the off-target effects of AYBE, the gRNA-dependent off-target (OT) activity of AYBEv3 was analyzed at two previously reported gRNA-dependent off-target (OT) sites ( Figure 2 f), and by 17 The ability of AYBEv3 to mediate gRNA-independent off-target DNA editing was characterized using an orthogonal R-loop assay ( Figure 2 g). When comparing AYBEv3 to ABE8e, decreased editing was observed for all six gRNA-dependent off-target sites and all five guide-independent off-target sites ( Figure 2 f and 2g and Fig.15 ).

[0515] Example 5 Evaluation of the therapeutic potential of AYBEv3

[0516] In addition, a proof-of-concept (POC) study was conducted to explore the therapeutic potential of AYBEv3 to correct disease-associated transversion mutations. By testing two nonsense mutations and two splice acceptor site mutations with AYBEv3 in a stable HEK293T cell line generated via lentiviral transduction, approximately 36% and 44% correction frequencies of A to C editing at the DMD and SLC26A4 nonsense mutation sites (spacer sequences of SEQ ID NOs: 75 and 77, respectively) and approximately 11% and 20% correction frequencies of A to T editing at the ATM and TTN splice acceptor site mutations (spacer sequences of SEQ ID NOs: 76 and 78, respectively) were observed, respectively. Figure 2 h and Fig.16 ), indicating that AYBE has promising potential in both basic research and therapeutic applications.

[0517] Example 6 Improving transversion purity by introducing TLS polymerase for more precise editing

[0518] During AYBE-mediated transversion editing, the activity of hypoxanthine excision repair proteins following adenine deamination directs the cellular DNA repair machinery to favor the base excision repair (BER) pathway.

[0519] Human Polη (SEQ ID NO: 36) is a translesion synthesis (TLS) polymerase that preferentially incorporates dA over AP sites 18 , co-expressed with AYBEv3 to increase the percentage or purity of A to T editing ( Figure 2 ik, Fig.17 After co-transfection of plasmids encoding AYBEv3 and Polη, the purity of A to T editing results increased significantly and reached 66% ( Figure 2 k). Polη is expressed using a plasmid comprising a polynucleotide encoding Polη under the regulation of a CAG promoter followed by a sequence encoding a bGH polyA signal.

[0520] AYBEv3 (referred to as AYBEmax) was also tested with the less processive deaminase TadA7.10 from ABEmax and found that AYBEmax did not produce more significant A>T or A>C results ( Fig.18 ).

[0521] Example 7 Alternative Nucleic Acid Programmable DNA Binding Domain (napDNAbd)

[0522] In order to expand the napDNAbd selection range of the AYBE disclosed herein, the Cas9 nickase (SpCas9-D10A, SEQ ID NO: 4) in AYBEv0.2 and AYBEv3 was replaced with inactivated Cas9 (SpCas9-D10A+H840A, SEQ ID NO: 37) or Cas12i nickase (nCas12imax (SiCas12i-N243R+E336R), SEQ ID NO: 38; the corresponding scaffold sequence of SEQ ID NO: 39) to evaluate the transversion activity in the A to T reporter system.

[0523] Observe that Fig.19 ) A fairly high transversion activity of about 90% was obtained by replacing the Cas9 nickase with an inactivated Cas9, which indicates that the AYBE of the present disclosure can use not only the Cas nickase but also the inactivated Cas (inactive) that essentially lacks nuclease activity.

[0524] It was also observed that ( Fig. 20) A significant transversion activity of about 14% was obtained by replacing the Cas9 nickase with the Cas12i nickase, indicating that the AYBE of the present disclosure is not limited to a specific type of Cas (i.e., Cas9), but can also utilize additional effector proteins that can act as nucleic acid-programmable DNA binding domains, such as Cas12, IscB.

[0525] References

[0526] 1. Porto, EM, Komor, AC, Slaymaker, IM & Yeo, GWBase editing: advances and therapeutic opportunities. Nat Rev Drug Discov 19, 839-859 (2020).

[0527] 2. Rees, HA&Liu, DRBase editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet 19, 770-788 (2018).

[0528] 3.Gaudelli,NMet al.Programmable base editing of A*T to G*C ingenomic DNA without DNA cleavage.Nature 551,464-471(2017).

[0529] 4.Komor,AC,Kim,YB,Packer,MS,Zuris,JA&Liu,DRProgrammable editing of a target base in genomic DNA without double-stranded DNAcleavage.Nature 533,420-424(2016).

[0530] 5. Zhao, D. et al. Glycosylase base editors enable C-to-A and C-to-G basechanges. Nat Biotechnol 39, 35-40 (2021).

[0531] 6.Kurt,I.C.et al.CRISPR C-to-G base editors for inducing targeted DNAtransversions in human cells.Nat Biotechnol 39,41-46(2021).

[0532] 7.Koblan,L.W.et al.Efficient C*G-to-G*C base editors developed usingCRISPRi screens,target-library analysis,and machine learning.Nat Biotechnol39,1414-1425(2021).

[0533] 8.Chen,L.et al.Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins.Nat Commun 12,1384(2021).

[0534] 9.Yuan,T.et al.Optimization of C-to-G base editors with sequencecontext preference predictable by machine learning methods.Nat Commun 12,4902(2021).

[0535] 10.Robertson,A.B.,Klungland,A.,Rognes,T.&Leiros,I.DNA repair inmammalian cells:Base excision repair:the long and short of it.Cell Mol LifeSci 66,981-993(2009).

[0536] 11.Hindi,N.N.,Elsakrmy,N.&Ramotar,D.The base excision repair process:comparison between higher and lower eukaryotes.Cell Mol Life Sci 78,7943-7965(2021).

[0537] 12.Saparbaev,M.&Laval,J.Excision of hypoxanthine from DNA containingdIMP residues by the Escherichia coli,yeast,rat,and human alkylpurine DNAglycosylases.Proc Natl Acad Sci U S A 91,5873-5877(1994).

[0538] 13.Lau,A.Y.,Scharer,O.D.,Samson,L.,Verdine,G.L.&Ellenberger,T.Crystalstructure of a human alkylbase-DNA repair enzyme complexed to DNA:mechanismsfor nucleotide flipping and base excision.Cell 95,249-258(1998).

[0539] 14.Connor,E.E.&Wyatt,M.D.Active-site clashes prevent the human 3-methyladenine DNA glycosylase from improperly removing bases.Chem Biol 9,1033-1041(2002).

[0540] 15.Vallur,A.C.,Maher,R.L.&Bloom,L.B.The efficiency of hypoxanthineexcision by alkyladenine DNA glycosylase is altered by changes in nearestneighbor bases.DNA Repair(Amst)4,1088-1098(2005).

[0541] 16.Tong,H.et al.High-fidelity Cas13variants for targeted RNAdegradation with minimal collateral effects.Nat Biotechnol(2022).

[0542] 17.Richter,M.F.et al.Phage-assisted evolution of an adenine baseeditor with improved Cas domain compatibility and activity.Nat Biotechnol 38,883-891(2020).

[0543] 18.Choi,J.Y.,Lim,S.,Kim,E.J.,Jo,A.&Guengerich,F.P.Translesionsynthesis across abasic lesions by human B-family and Y-family DNApolymerases alpha,delta,eta,iota,kappa,and REV1.J Mol Biol 404,34-44(2010).

[0544] 19.Thompson,P.S.&Cortez,D.New insights into abasic site repair andtolerance.DNA Repair(Amst)90,102866(2020).

[0545] 20. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol 37, 224-226 (2019).

[0546] ***

[0547] Without departing from the scope and spirit of the present disclosure, various modifications and variations of the products, methods and uses of the present disclosure will be clear to those skilled in the art. Although the present disclosure has been described in conjunction with specific embodiments, it will be understood that further modifications can be made, and the disclosure claimed for protection should not be unduly limited to such specific embodiments. In fact, various modifications of the described manner for performing the present disclosure that are obvious to those skilled in the art are intended to fall within the scope of the present disclosure. The present application is intended to cover any changes, uses or adaptations of the present disclosure that generally follow the principles of the present disclosure, and includes such deviations from the present disclosure: it is within the known customary operations in the field to which the present disclosure belongs and is applicable to the basic features shown heretofore.

[0548] sequence

[0549] SEQ ID NO: 1, wild-type human MPG with N-terminal start methionine (M), full length, 298aa MVTPALQMKKPKQFCRRMGQKKQRPARAGQPHSSSDAAQAPAEQPHSSSDAAQAPCPRERCLGPPTTPGPYRSIYFSSPKGHLTRLGLEFFDQPAVPLARAFLGQVLVRRLPNGTELRGRIVETEAYLGPEDEAAHSRGGRQTPRNRGMFMKPGTLYVYIIYGMYFCMNISSQGDGACVLLRALEPLEGLETMRQLRSTLRKGTASRVLKDRELCSGPSKLCQALAINKSFDQRDLAQDEAVWLERGPLEPSEPAVVAAARVGVGHAGEWARKPLRFYVRGSPWVSVVDRVAEQDTQA

[0550] SEQ ID NO: 2, wild-type human MPG without the N-terminal initiating methionine (M) (abbreviated as "MPG"), 297aa

[0551] VTPALQMKKPKQFCRRMGQKKQRPARAGQPHSSSDAAQAPAEQPHSSSDAAQAPCPRERCLGPPTTPGPYRSIYFSSPKGHLTRLGLEFFDQPAVPLARAFLGQVLVRRLPNGTELRGRIVETEAYLGPEDEAAHSRGGRQTPRNRGM FMKPGTLYVYIIYGMYFCMNISSQGDGACVLLRALEPLEGLETMRQLRSTLRKGTASRVLKDRELCSGPSKLCQALAINKSFDQRDLAQDEAVWLERGPLEPSEPAVVAAARVGVGHAGEWARKPLRFYVRGSPWVSVVDRVAEQDTQA

[0552] SEQ ID NO: 3, TadA8e without the N-terminal start methionine (M)

[0553] SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN

[0554] SEQ ID NO:4, SpCas9-D10A, Cas9 nickase, nCas9, without N-terminal start methionine (M)

[0555]

[0556]

[0557] SEQ ID NO:5, prototype AYBE, MTC, full length, with N-terminal start methionine (M)

[0558]

[0559]

[0560] SEQ ID NO:6, prototype AYBE, TMC, full length, with N-terminal start methionine (M)

[0561]

[0562] SEQ ID NO:7, prototype AYBE, TCM (AYBEv0.1), full length, with N-terminal start methionine (M)

[0563]

[0564]

[0565] SEQ ID NO:8, dMPG (inactive MPG, inactive MPG-E125A, Y127A, H136A mutant)

[0566]

[0567] SEQ ID NO:9, AYBE-dMPG (negative control)

[0568]

[0569]

[0570] SEQ ID NO: 10, ABE8e (blank control)

[0571]

[0572]

[0573] SEQ ID NO:11, bpNLS 1

[0574] KRTADGSEFESPKKKRKV

[0575] SEQ ID NO:12, bpNLS 2

[0576] KRTADGSEFEPKKKRKV

[0577] SEQ ID NO: 28, MPG-N169S (without the N-terminal start methionine (M); N169S is numbered according to the full-length wild-type human MPG of SEQ ID NO: 1)

[0578]

[0579] SEQ ID NO:29, AYBEv0.2

[0580]

[0581]

[0582] SEQ ID NO:30, MPG-N169S+S198A+K202A+G203A+S206A+K210A (MPGv1), 6 mutations, no N-terminal start methionine (M)

[0583]

[0584] SEQ ID NO:31, AYBEv1, full length, with N-terminal start methionine (M)

[0585] SEQ ID NO:32, MPGv2 (MPG-G163R+N169S), 2 mutations, no N-terminal start methionine (M)

[0586]

[0587] SEQ ID NO:33, AYBEv2, full length, with N-terminal start methionine (M)

[0588]

[0589] SEQ ID NO:34, MPGv3 (MPG-G163R+N169S+S198A+K202A+G203A+S206A+K210A), 7 mutations, no N-terminal start methionine (M)

[0590]

[0591]

[0592] SEQ ID NO:35, AYBEv3, full length, with N-terminal start methionine (M)

[0593]

[0594] SEQ ID NO:36, human Polη

[0595] MATGQDRVVALVDMDCFFVQVEQRQNPHLRNKPCAVVQYKSWKGGGIIAVSYEARAF

[0596] GVTRSMWADDAKKLCPDLLLAQVRESRGKANLTKYREASVEVMEIMSRFAVIERASIDE

[0597] AYVDLTSAVQERLQKLQGQPISADLLPSTYIEGLPQGPTTAEETVQKEGMRKQGLFQWL

[0598] DSLQIDNLTSPDLQLTVGAVIVEEMRAAIERETGFQCSAGISHNKVLAKLACGLNKPNRQ

[0599] TLVSHGSVPQLFSQMPIRKIRSLGGKLGASVIEILGIEYMGELTQFTESQLQSHFGEKNGS

[0600] WLYAMCRGIEHDPVKPRQLPKTIGCSKNFPGKTALATREQVQWWLLQLAQELEERLTK

[0601] DRNDNDRVATQLVVSIRVQGDKRLSSLRRCCALTRYDAHKMSHDAFTVIKNCNTSGIQTEWSPPLTMLFLCATKFSASAPSSSTDITSFLSSDPSSLPKVPVTSSEAKTQGSGPAVTATKKATTSLESFFQKAAERQKVKEASLSSLTAPTQAPMSNSPSKPSLPFQTSQSTGTEPFFKQK SLLLKQKQLNNSSVSSPQQNPWSNCKALPNSLPTEYPGCVPVCEGVSKLEESSKATPAEMDLAHNSQSMHASSASKSVLEVTQKATPNPSLLAAEDQVPCEKCGSLVPVWDMPEHMDYHFALELQKSFLQPHSSNPQVVSAVSHQGKRNPKSPLACTNKRPRPEGMQTLESFFKPLTHSEQ ID NO:37, SpCas9-D10A+H840A, inactivated Cas9, dCas9, no N-terminal start methionine (M)

[0602]

[0603] SEQ ID NO:38, nCas12imax (SiCas12i-N243R+E336R) with N-terminal start methionine (M)

[0604]

[0605]

[0606] SEQ ID NO: 39, scaffold sequence of nCas12imax (direct repeat (DR) sequence) AGAAATGTGTCCCCAGTTGACAC

[0607] Table A

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617] Table B

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

Claims

1. A base editor, comprising: (1) a nucleic acid programmable DNA binding domain (napDNAbd) capable of binding to a target dsDNA comprising a target A:T base pair, wherein the target A:T base pair comprises a target deoxyadenosine (dA) (first deoxyribonucleotide) in a protospacer sequence on a non-target strand (editing strand) of the target dsDNA and a deoxythymidine (dT) (second deoxyribonucleotide) in a target sequence on a target strand (non-editing strand) of the target dsDNA, wherein the protospacer sequence is completely reverse complementary to the target sequence; (2) an adenine deaminase domain capable of deaminating adenine of the target deoxyadenosine to hypoxanthine; and (3) a hypoxanthine excision domain capable of excising the hypoxanthine.

2. A system comprising: (1) A base editor or a polynucleotide encoding the base editor, wherein the base editor comprises: (a) a nucleic acid programmable DNA binding domain (napDNAbd) capable of binding to a target dsDNA comprising a target A:T base pair, wherein the target A:T base pair comprises a target deoxyadenosine (dA) (first deoxyribonucleotide) in a protospacer sequence on a non-target strand (editing strand) of the target dsDNA and a deoxythymidine (dT) (second deoxyribonucleotide) in a target sequence on a target strand (non-editing strand) of the target dsDNA, wherein the protospacer sequence is completely reverse complementary to the target sequence; (b) an adenine deaminase domain capable of deaminating adenine of the target deoxyadenosine to hypoxanthine; and (c) a hypoxanthine excision domain capable of excising said hypoxanthine; and (2) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, wherein the guide nucleic acid comprises: (a) a scaffold sequence capable of forming a complex with the napDNAbd; and (b) is capable of hybridizing to a target sequence on a target strand of the target dsDNA, thereby directing the complex to a guide sequence of the target dsDNA.

3. A method for modifying a target dsDNA, the method comprising contacting the target dsDNA with a system, the target dsDNA comprising a target A:T base pair, the target A:T base pair comprising a target deoxyadenosine (dA) (first deoxyribonucleotide) in a protospacer sequence on a non-target strand (editing strand) of the target dsDNA and a deoxythymidine (dT) (second deoxyribonucleotide) in a target sequence on a target strand (non-editing strand) of the target dsDNA, wherein the protospacer sequence is completely reverse complementary to the target sequence; The system comprises: (1) A base editor or a polynucleotide encoding the base editor, wherein the base editor comprises: (a) a nucleic acid programmable DNA binding domain (napDNAbd) capable of binding to the target dsDNA; (b) an adenine deaminase domain capable of deaminating adenine of the target deoxyadenosine to hypoxanthine; and (c) a hypoxanthine excision domain capable of excising said hypoxanthine; and (2) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, wherein the guide nucleic acid comprises: (1) a scaffold sequence capable of forming a complex with the napDNAbd; and (2) capable of hybridizing to a target sequence on the target strand of the target dsDNA, thereby directing the complex to a guide sequence of the target dsDNA.

4. A base editor, system or method as described in any preceding claim, wherein the target dsDNA is wild-type.

5. The base editor, system or method of any preceding claim, wherein the target deoxyadenosine is native to the target dsDNA.

6. A base editor, system or method as described in any preceding claim, wherein the target deoxyadenosine is a mutation in the target dsDNA.

7. A base editor, system or method as described in any preceding claim, wherein the target deoxyadenosine is a pathogenic mutation in the target dsDNA.

8. A base editor, system or method as described in any preceding claim, wherein the target dsDNA is a target gene.

9. A base editor, system or method as described in any preceding claim, wherein the target deoxyadenosine (first deoxyribonucleotide) is replaced by the base editor with a fourth deoxyribonucleotide different from the target deoxyadenosine (first deoxyribonucleotide).

10. The base editor, system or method of any preceding claim, wherein adenine of the target deoxyadenosine is deaminated by the adenine deaminase domain to form hypoxanthine in situ.

11. The base editor, system or method of any preceding claim, wherein the hypoxanthine excision domain is substantially capable of excising hypoxanthine formed in situ by the adenine deaminase domain.

12. A base editor, system or method as described in any preceding claim, wherein the hypoxanthine excision domain is substantially capable of cleaving or hydrolyzing the glycosidic bond connecting the in situ formed hypoxanthine and the deoxyribose of the target deoxyadenosine.

13. A base editor, system or method as described in any preceding claim, wherein excision of the in situ formed hypoxanthine converts the target deoxyadenosine in the original spacer sequence into an abasic site having the sugar-phosphate backbone of the target deoxyadenosine.

14. A base editor, system or method as described in any preceding claim, wherein a nick is created in the target strand by the napDNAbd.

15. A base editor, system or method as described in any preceding claim, wherein the nick on the target strand induces a deletion in the target strand.

16. The base editor, system or method of any preceding claim, wherein the dsDNA is in a target cell.

17. A base editor, system or method as described in any preceding claim, wherein the deletion on the target strand is repaired, for example, by trans-lesion synthesis (TLS) in the target cell using a protospacer sequence containing the abasic site as a repair template.

18. A base editor, system or method as described in any preceding claim, wherein during repair of the target strand, a third deoxyribonucleotide (e.g., dG, dA) different from deoxythymidine (dT) (the second deoxyribonucleotide) is formed at a site in the target sequence opposite to the abasic site in the original spacer sequence serving as a repair template.

19. A base editor, system or method as described in any preceding claim, wherein the sugar-phosphate backbone of the target deoxyadenosine at the abasic site is removed, for example by an enzyme in the target cell.

20. A base editor, system or method as described in any preceding claim, wherein after removing the sugar-phosphate backbone of the target deoxyadenosine at the abasic site, a fourth deoxyribonucleotide (e.g., dC, dT) is formed at the abasic site in the original spacer sequence to base pair with the third deoxyribonucleotide (e.g., dG, dA) in the target sequence, such that the target deoxyadenosine in the original spacer sequence is replaced with a fourth deoxyribonucleotide (e.g., dA to dC, dA to dT).

21. The base editor, system or method of any preceding claim, wherein the third deoxyribonucleotide is dA, dC or dG.

22. The base editor, system or method of any preceding claim, wherein the fourth deoxyribonucleotide is dT, dC or dG.

23. The base editor, system or method of any preceding claim, wherein the substitution of the target deoxyadenosine to the fourth deoxyribonucleotide is a dA to dC, dA to dT, or dA to dG substitution.

24. The base editor, system or method of any preceding claim, wherein the replacement converts a stop codon to a non-stop codon or converts a non-stop codon to a stop codon.

25. The base editor, system or method of any preceding claim, wherein the stop codon is on the sense strand of the dsDNA.

26. The base editor, system or method of any preceding claim, wherein the replacement occurs on the sense strand or the nonsense strand of the dsDNA.

27. A base editor, system or method as described in any preceding claim, wherein the replacement occurs on the sense strand of the dsDNA, converting a stop codon on the sense strand to a non-stop codon or converting a non-stop codon on the sense strand to a stop codon.

28. A base editor, system or method as described in any preceding claim, wherein the replacement occurs on the nonsense strand of the dsDNA, converting a stop codon on the sense strand to a non-stop codon or converting a non-stop codon on the sense strand to a stop codon.

29. A base editor, system or method as described in any preceding claim, wherein the replacement occurs in a splice site (e.g., splice donor, splice acceptor) of the target dsDNA.

30. A base editor, system or method as described in any preceding claim, wherein the substitution occurring in the splice site (e.g., splice donor, splice acceptor) increases or decreases the translation of a transcript transcribed from the target dsDNA.

31. The base editor, system or method of any preceding claim, wherein the base editor is a fusion protein.

32. The base editor, system or method of any preceding claim, wherein the base editor comprises, from N-terminus to C-terminus: (1) the adenine deaminase domain, the napDNAbd and the hypoxanthine excision domain; (2) the hypoxanthine excision domain, the adenine deaminase domain and the napDNAbd; or (3) The adenine deaminase domain, the hypoxanthine excision domain and the napDNAbd; Optionally, any two adjacent domains of (1), (2) or (3) are fused with or without a linker.

33. A base editor, system or method as described in any preceding claim, wherein the base editor comprises one, two, three or more hypoxanthine excision domains.

34. A base editor, system or method as described in any preceding claim, wherein the hypoxanthine excision domain is substantially capable of or has been engineered to be substantially capable of excising the hypoxanthine.

35. The base editor, system or method of any preceding claim, wherein the hypoxanthine excision domain comprises a glycosylase or a variant thereof.

36. A base editor, system or method as described in any preceding claim, wherein the glycosylase or variant thereof is substantially capable of or has been engineered to be substantially capable of removing the hypoxanthine.

37. A base editor, system or method as described in any preceding claim, wherein the glycosylase is selected from N-methylpurine DNA glycosylase (MPG), 8-oxoguanine DNA glycosylase (OGG1), methyl-CpG binding domain 4, DNA glycosylase (MBD4), thymine DNA glycosylase (TDG), uracil DNA glycosylase (UNG), single-stranded selective monofunctional uracil-DNA glycosylase 1 (SMUG1), mutY DNA glycosylase (MUTYH), nth-like DNA glycosylase 1 (NTHL1), nei-like DNA glycosylase 1 (NEIL1), nei-like DNA glycosylase 2 (NEIL2), nei-like DNA glycosylase 3 (NEIL3) and mutants or variants, which are capable of excising the hypoxanthine.

38. The base editor, system or method of any preceding claim, wherein the hypoxanthine excision domain comprises an N-methylpurine DNA glycosylase protein (MPG).

39. A base editor, system or method as described in any preceding claim, wherein the MPG is substantially capable of or has been engineered to be substantially capable of excising the hypoxanthine.

40. The base editor, system or method of any preceding claim, wherein the MPG substantially possesses or has been engineered to substantially possess N-methylpurine DNA glycosylase activity.

41. The base editor, system or method of any preceding claim, wherein the MPG comprises the motif GxxYxxxxYGxxxxxN.

42. A base editor, system or method as described in any preceding claim, wherein the MPG is obtained from a species selected from Table A.

43. A base editor, system or method as described in any preceding claim, wherein the MPG is a variant of MPG obtained from a species selected from Table A.

44. A base editor, system or method as described in any preceding claim, wherein the MPG is a variant of human MPG (SEQ ID NO: 1 or 2) or any MPG shown in Table B.

45. A base editor, system or method as described in any preceding claim, wherein the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to any MPG shown in SEQ ID NO: 1 or 2, or Table B.

46. ​​A base editor, system or method as described in any preceding claim, wherein the MPG is contained in SEQ ID NO:2 position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 , 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 1 45、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, and / or 297, or amino acid substitutions at corresponding positions in MPG of another species other than human (e.g., a species other than human selected from Table A), wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:

1.

47. A base editor, system or method as described in any preceding claim, wherein the MPG comprises an amino acid substitution at position N169 of SEQ ID NO:2, or at a corresponding position in MPG of another species other than human (e.g., a species other than human selected from Table A), wherein the position is numbered according to the amino acid sequence of SEQ ID NO:

1.

48. The base editor, system or method of any preceding claim, wherein the amino acid substitution is with alanine (Ala / A) or serine (Ser / S).

49. A base editor, system or method as described in any preceding claim, wherein the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO: 28; optionally, wherein the MPG comprises the amino acid sequence of SEQ ID NO:

28.

50. A base editor, system or method as described in any preceding claim, wherein the MPG further comprises an amino acid substitution at position S198, K202, G203, S206 and / or K210 of SEQ ID NO:28, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:

1.

51. The base editor, system or method of any preceding claim, wherein the amino acid substitution is a substitution with alanine (Ala / A).

52. A base editor, system or method as described in any preceding claim, wherein the MPG further comprises an amino acid substitution selected from S198A, K202A, G203A, S206A and K210A relative to SEQ ID NO:28, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:

1.

53. A base editor, system or method as described in any preceding claim, wherein the MPG comprises amino acid substitutions N169S, S198A, K202A, G203A, S206A and K210A relative to SEQ ID NO:2, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:

1.

54. A base editor, system or method as described in any preceding claim, wherein the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO: 30; optionally, wherein the MPG comprises the amino acid sequence of SEQ ID NO:

30.

55. The base editor, system or method of any preceding claim, wherein the MPG further comprises at positions S78, P79, K80, G81, R110, T115, E116, R120, R138, G163, Q173, G174, D175, A177, E185, L187, E188, L190, E191, T192, Q195, S198, T199, R201, K202, V208, K210, R212, S213, 6. amino acid substitutions at K220, A226, N228, K229, S230, Q238, E240, A241, R246, L249, P251, E253, P254, A255, R272, P274, V279, R280, G281, V291, Q294, D295, T296, Q297 and / or A298, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:

1.

56. The base editor, system or method of any preceding claim, wherein the amino acid substitution is with arginine (Arg / R) or lysine (Lys / K).

57. The base editor, system or method of any preceding claim, wherein the MPG further comprises a residue selected from the group consisting of S78R, P79R, K80R, G81R, R110K, T115R, E116R, R120K, R138K, G163R, Q173R, G174R, D175R, A177R, E185R, L187R, E188R, L190R, E191R, T192R, Q195R, S198R, T199R, R201K, K202R, V208R, K210R, R212K, Amino acid substitutions of S216R, K220R, A226R, N228R, K229R, S230R, Q238R, E240R, A241R, R246K, L249R, P251R, E253R, P254R, A255R, R272K, P274R, V279R, R280K, G281R, V291R, Q294R, D295R, T296R, Q297R and A298R, wherein the positions are numbered according to SEQ ID NO:

1.

58. A base editor, system or method as described in any preceding claim, wherein the MPG comprises amino acid substitutions N169S and G163R relative to SEQ ID NO:2, wherein the positions are numbered according to SEQ ID NO:

1.

59. A base editor, system or method as described in any preceding claim, wherein the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO: 32; optionally, wherein the MPG comprises the amino acid sequence of SEQ ID NO:

32.

60. A base editor, system or method as described in any preceding claim, wherein the MPG comprises amino acid substitutions N169S, G163R, S198A, K202A, G203A, S206A and K210A relative to SEQ ID NO:2, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:

1.

61. A base editor, system or method as described in any preceding claim, wherein the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO: 34; optionally, wherein the MPG comprises the amino acid sequence of SEQ ID NO:

34.

62. A base editor, system or method as described in any preceding claim, wherein the adenine deaminase domain comprises tRNA adenosine deaminase (TadA) or a functional variant or fragment thereof, such as TadA8e (SEQ ID NO: 3), TadA8.17, TadA8.20, TadA9, TadA8E V106W 、TadA8E V106W+D108Q TadA-CDa, TadA-CDb, TadA-CDc, TadA-CDd, TadA-CDe, TadA-dual, T AD AC-1.2, T AD AC-1.14, T AD AC-1.17, T AD AC-1.19, T AD AC-2.5, T AD AC-2.6, T AD AC-2.9, T AD AC-2.19, T AD AC-2.23, TadA8e-N46L, TadA8e-N46P.

63. A base editor, system or method as described in any preceding claim, wherein the adenine deaminase domain comprises an apolipoprotein B mRNA editing complex (APOBEC) family deaminase, activation-induced deaminase (AID), cytidine deaminase 1 from sea lamprey (pmCDA1), or a functional variant or fragment thereof, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H.

64. The base editor, system or method of any preceding claim, wherein the adenine deaminase domain comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO: 3; Optionally, wherein the adenine deaminase domain comprises the amino acid sequence of SEQ ID NO:

3.

65. The base editor, system or method of any preceding claim, wherein the napDNAbd substantially lacks dsDNA cleavage activity.

66. The base editor, system or method of any preceding claim, wherein the napDNAbd substantially lacks dsDNA cleavage activity and nickase activity.

67. A base editor, system or method as described in any preceding claim, wherein the napDNAbd has nickase activity.

68. A base editor, system or method as described in any preceding claim, wherein the napDNAbd has nickase activity to nick the target strand.

69. The base editor, system or method of any preceding claim, wherein the napDNAbd comprises a Cas nickase or an inactivated Cas of a Cas protein; Optionally, the Cas protein is selected from Cas9 protein (e.g., SpCas9, SaCas9, GeoCas9, CjCas9, Cas9-KKH, circularly permuted Cas9, Argonaute (Ago), SmacCas9, Spy-macCas9, xCas9, SpCas9-NG); Cas12 protein (e.g., Cas12a, AsCas12a, LbCas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f (Cas14), Cas12g, Cas12h, Cas12i, xC as12i, Cas12Max, hfCas12Max, Cas12j, Cas12k, Cas12l, Cas12m, Cas12n, Cas12o, Cas12p, Cas12q, Cas12r, Cas12s, Cas12t, Cas12u, Cas12v, Cas12w, Cas12x, Cas12y, Cas12z); Cas13 protein (e.g., Cas13a, Cas13b, Cas13c, Cas13d, Cas13e, Cas13f, Cas13x, Cas13y); Csn2; and mutants thereof; Optionally, the Cas nickase is a Cas9 nickase (nCas9), such as a SpCas9 nickase (SpCas9-D10A); optionally, the napDNAbd comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO: 4; optionally, the napDNAbd comprises the amino acid sequence of SEQ ID NO: 4; Optionally, the inactivated Cas is an inactivated Cas9 (dCas9), such as an inactivated SpCas9 (SpCas9-D10A+H840A); optionally wherein the napDNAbd comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO: 37; optionally wherein the napDNAbd comprises the amino acid sequence of SEQ ID NO: 37; Optionally, the Cas nickase is a Cas12i nickase (nCas12i) or an inactivated Cas12i (dCas12i), such as an inactivated Cas12i of an xCas12i polypeptide; optionally, the napDNAbd comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO: 38; optionally, the napDNAbd comprises the amino acid sequence of SEQ ID NO:

38.

70. A base editor, system or method as described in any preceding claim, wherein the napDNAbd comprises an IscB nickase (nIscB) or an inactivated IscB (dIscB) of an IscB protein (e.g., OgeuIscB).

71. A base editor, system or method as described in any preceding claim, wherein the napDNAbd comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO:4, 37 or 38; optionally wherein the napDNAbd comprises the amino acid sequence of SEQ ID NO:4, 37 or 38.

72. A base editor, system or method as described in any preceding claim, wherein the napDNAbd comprises a TnpB nickase or an inactivated TnpB of a TnpB protein.

73. A base editor, system or method as described in any preceding claim, wherein the base editor comprises an NLS at the N-terminus and / or C-terminus of the napDNAbp.

74. A base editor, system or method as described in any preceding claim, wherein the base editor comprises an NLS at the N-terminus and / or C-terminus of the hypoxanthine excision domain. 75.I A base editor, system or method as described in any preceding claim, wherein the base editor comprises an NLS at the N-terminus and / or C-terminus of the adenine deaminase domain.

76. A base editor, system or method as described in any preceding claim, wherein the NLS is SV40 NLS, bpSV40 NLS (e.g., SEQ ID NO: 11 or 12), or NP NLS (Xenopus laevis nucleoplasmin NLS, nucleoplasmin NLS).

77. A base editor, system or method as described in any preceding claim, wherein the base editor comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to any one of SEQ ID NOs: 5, 6, 7, 29, 31, 33, and 35; optionally wherein the base editor comprises the amino acid sequence of any one of SEQ ID NOs: 5, 6, 7, 29, 31, 33, and 35.

78. A base editor, system or method as described in any preceding claim, wherein the target deoxyadenosine is at a position of the protospacer sequence selected from the following: position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, and combinations thereof.

79. A base editor, system or method as described in any preceding claim, wherein the target deoxyadenosine is at a position of the protospacer sequence selected from the following: position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and combinations thereof.

80. A base editor, system or method as described in any preceding claim, wherein the target deoxyadenosine is at a position of the protospacer sequence selected from the following: position 5, position 6, position 7, position 8, position 9, and combinations thereof.

81. A base editor, system or method as described in any preceding claim, wherein the target deoxyadenosine is at position 7 or 8 of the protospacer sequence.

82. A base editor, system or method as described in any preceding claim, wherein the target deoxyadenosine is the N2 nucleotide in a N1N2N3 motif, wherein N1, N2, or N3 is A, T, G or C; optionally wherein the target deoxyadenosine is deoxyadenosine (dA) in a CAA or CAG motif.

83. The base editor, system or method of any preceding claim, wherein the protospacer sequence comprises about or at least about 16 consecutive nucleotides of the target dsDNA, such as about or at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 6, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more consecutive nucleotides, or in a numerical range between any two of the aforementioned values, such as about 16 to about 50 or about 17 to about 22 consecutive nucleotides of the target dsDNA; optionally, wherein the protospacer sequence comprises about 20 consecutive nucleotides of the target dsDNA.

84. The base editor, system or method of any preceding claim, wherein the protospacer sequence is immediately 5' or 3' to a protospacer adjacent motif (PAM) comprising the sequence 5'-NN-3', 5'-NNN-3', 5'-NNNN-3', 5'-NNNNN-3' or 5'-NNNNNN-3', wherein N is A, T, G or C; Optionally wherein the protospacer sequence is immediately 5' to a protospacer adjacent motif (PAM) comprising the sequence 5'-NGG-3', wherein N is A, T, G or C; or Optionally wherein the protospacer sequence is immediately 3' to a protospacer adjacent motif (PAM) comprising the sequence 5'-TTN-3', wherein N is A, T, G or C.

85. The base editor, system or method of any preceding claim, wherein the guide sequence is about or at least about 16 nucleotides in length, for example, about or at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 in length. , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more nucleotides, or a range of lengths between any two of the aforementioned values, for example, a length of about 16 to about 50 nucleotides or about 17 to about 22 nucleotides; optionally, wherein the spacer sequence has a length of about 20 nucleotides.

86. A base editor, system or method as described in any preceding claim, wherein (1) the guide sequence is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (perfectly) reverse complementary to the target sequence, optionally about 100% (perfectly) reverse complementary; (2) the guide sequence contains no more than 5, 4, 3, 2 or 1 mismatches with the target sequence or no mismatches; or (3) the guide sequence is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 30, 31, 32, 33, 34, 87. A base editor, system or method as described in any preceding claim, wherein the guide sequence comprises a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of any one of SEQ ID NOs: 40-89; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide differences, whether contiguous or not, compared to the sequence of any one of SEQ ID NOs: 40-89; optionally wherein the guide sequence comprises the polynucleotide sequence of any one of SEQ ID NOs: 40-89.

88. The base editor, system or method of any preceding claim, wherein the scaffold sequence is 5' or 3' to the guide sequence.

89. A base editor, system or method as described in any preceding claim, wherein the scaffold sequence is compatible with the napDNAbp.

90. The base editor, system or method of any preceding claim, wherein the scaffold sequence has a secondary structure substantially identical to the secondary structure of SEQ ID NO: 13 or 39; Optionally, the scaffold sequence comprises a polynucleotide sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 13 or 39; optionally, the scaffold sequence comprises a polynucleotide sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 13 or 39; 91. The base editor, system or method of any preceding claim, wherein the base editor or system further comprises a translesion synthesis (TLS) polymerase or a recruitment domain capable of recruiting a TLS polymerase optionally fused to the base editor, or a coding sequence thereof; Optionally wherein the TLS polymerase is selected from the group consisting of Pol α (α), Pol β (β), Pol δ (δ) (PCNA), Pol γ (γ), Pol η (η), Pol ι (ι), Pol κ (κ), Pol λ (λ), Pol μ (μ), Pol ν (ν), Pol θ (θ), and REV1; Optionally, the TLS polymerase comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO: 36; optionally, the TLS polymerase comprises an amino acid sequence of SEQ ID NO:

36. Optionally wherein the base editor or system further comprising a translesion synthesis (TLS) polymerase or a recruitment domain capable of recruiting a TLS polymerase causes the target deoxyadenosine (the first deoxyribonucleotide) to be replaced by dC, dT or dG.

92. The base editor, system or method of any preceding claim, wherein the base editor or system further comprises a cytidine deaminase domain.

93. The base editor, system or method of any preceding claim, wherein the base editor or system further comprising the cytidine deaminase domain causes the target deoxyadenosine (first deoxyribonucleotide) to be replaced by dT.

94. The base editor, system or method of any preceding claim, wherein the cytidine deaminase domain promotes conversion of a fourth deoxyribonucleotide of dC to dT.

95. A polynucleotide encoding a base editor as claimed in any preceding claim and optionally a guide nucleic acid as defined in any preceding claim.

96. A vector comprising the polynucleotide of any preceding claim.

97. A complex comprising a base editor as claimed in any preceding claim and a guide nucleic acid as defined in any preceding claim.

98. A cell comprising the base editor or system of any preceding claim, the polynucleotide of any preceding claim, the vector of any preceding claim, or the complex of any preceding claim.

99. A pharmaceutical composition comprising: (i) a base editor or system as claimed in any preceding claim, a polynucleotide as claimed in any preceding claim, a vector as claimed in any preceding claim, a complex as claimed in any preceding claim, or a cell as claimed in any preceding claim; and (ii) a pharmaceutically acceptable excipient.

100. A method for treating a subject having or at risk for a disease associated with a target deoxyadenosine of a target dsDNA, the method comprising administering to the subject (e.g., an effective amount) of the system of any preceding claim, wherein the target deoxyadenosine is modified by the system, and the modification treats or prevents the disease.

101. An MPG which is or has been engineered to be substantially capable of cleaving said hypoxanthine.

102. The MPG of any preceding claim, wherein the MPG is not wild-type human MPG (hMPG; SEQ ID NO: 1), hMPG-N169A, hMPG-N169S, hMPG-N169D, hMPG-N169H, or a variant thereof lacking an N-terminal starting methionine (M) (e.g., SEQ ID NO: 2).

103. The MPG of any preceding claim, wherein the MPG substantially possesses or has been engineered to substantially possess N-methylpurine DNA glycosylase activity.

104. The MPG of any preceding claim, wherein the MPG comprises the motif GxxYxxxxYGxxxxxN.

105. The MPG of any preceding claim, wherein the MPG is obtained from a species selected from Table A.

106. The MPG of any preceding claim, wherein the MPG is a variant of an MPG obtained from a species selected from Table A.

107. The MPG of any preceding claim, wherein the MPG is a variant of human MPG (SEQ ID NO: 1 or 2) or any MPG shown in Table B.

108. The MPG of any preceding claim, wherein the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to any of the MPGs set forth in SEQ ID NO: 1 or 2, or Table B.

109. The MPG of any preceding claim, wherein the MPG is contained in SEQ ID 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81 ,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146 6, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206 , 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, and / or 297, or amino acid substitutions at corresponding positions in MPG of another species other than human (e.g., a species other than human selected from Table A), wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:

1.

110. The MPG of any preceding claim, wherein the MPG comprises an amino acid substitution at position N169 of SEQ ID NO:2, or at a corresponding position in MPG of another species other than human (e.g., a species other than human selected from Table A), wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:

1.

111. The MPG of any preceding claim, wherein the amino acid substitution is with alanine (Ala / A) or serine (Ser / S).

112. The MPG of any preceding claim, wherein the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO:28; optionally, wherein the MPG comprises the amino acid sequence of SEQ ID NO:

28.

113. The MPG of any preceding claim, wherein the MPG further comprises an amino acid substitution at position S198, K202, G203, S206 and / or K210 of SEQ ID NO:28, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:

1.

114. The MPG of any preceding claim, wherein the amino acid substitution is with alanine (Ala / A).

115. The MPG of any preceding claim, wherein the MPG further comprises an amino acid substitution selected from the group consisting of S198A, K202A, G203A, S206A and K210A relative to SEQ ID NO:28, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:

1.

116. The MPG of any preceding claim, wherein the MPG comprises amino acid substitutions N169S, S198A, K202A, G203A, S206A and K210A relative to SEQ ID NO:2, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:

1.

117. The MPG of any preceding claim, wherein the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO:30; optionally, wherein the MPG comprises the amino acid sequence of SEQ ID NO:

30.

118. The MPG of any preceding claim, wherein the MPG further comprises at positions S78, P79, K80, G81, R110, T115, E116, R120, R138, G163, Q173, G174, D175, A177, E185, L187, E188, L190, E191, T192, Q195, S198, T199, R201, K202, V208, K210, R212, S216, , K220, A226, N228, K229, S230, Q238, E240, A241, R246, L249, P251, E253, P254, A255, R272, P274, V279, R280, G281, V291, Q294, D295, T296, Q297 and / or A298, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:

1.

119. The MPG of any preceding claim, wherein the amino acid substitution is with arginine (Arg / R) or lysine (Lys / K).

120. The MPG of any preceding claim, wherein the MPG further comprises a residue selected from the group consisting of S78R, P79R, K80R, G81R, R110K, T115R, E116R, R120K, R138K, G163R, Q173R, G174R, D175R, A177R, E185R, L187R, E188R, L190R, E191R, T192R, Q195R, S198R, T199R, R201K, K202R, V208R, K210R, R212K, Amino acid substitutions of S216R, K220R, A226R, N228R, K229R, S230R, Q238R, E240R, A241R, R246K, L249R, P251R, E253R, P254R, A255R, R272K, P274R, V279R, R280K, G281R, V291R, Q294R, D295R, T296R, Q297R and A298R, wherein the positions are numbered according to SEQ ID NO:

1.

121. The MPG of any preceding claim, wherein the MPG comprises amino acid substitutions N169S and G163R relative to SEQ ID NO:2, wherein the positions are numbered according to SEQ ID NO:

1.

122. The MPG of any preceding claim, wherein the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO:32; optionally, wherein the MPG comprises the amino acid sequence of SEQ ID NO:

32.

123. The MPG of any preceding claim, wherein the MPG comprises amino acid substitutions N169S, G163R, S198A, K202A, G203A, S206A and K210A relative to SEQ ID NO:2, wherein the positions are numbered according to the amino acid sequence of SEQ ID NO:

1.

124. The MPG of any preceding claim, wherein the MPG comprises an amino acid sequence having at least about 60% (e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) sequence identity to SEQ ID NO:34; optionally, wherein the MPG comprises the amino acid sequence of SEQ ID NO:34.

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