CN117751133A

CN117751133A - Deaminase mutants, compositions and methods for modifying mitochondrial DNA

Info

Publication number: CN117751133A
Application number: CN202380013022.9A
Authority: CN
Inventors: 伊成器; 雷芷芯; 孟浩巍; 刘璐璐; 赵华男
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2022-04-29
Filing date: 2023-04-13
Publication date: 2024-03-22
Also published as: WO2023207607A1

Abstract

A double-stranded DNA deaminase mutant for constructing mitochondrial DNA base editor, a base editor for mitochondrial DNA base editing and a base editing method.

Description

Deaminase mutants, compositions and methods for modifying mitochondrial DNA

Technical Field

The present application relates to the field of base editing (in particular mitochondrial base editing). Specifically, the present application relates to a double-stranded DNA deaminase mutant useful for constructing a mitochondrial base editor, a base editor for mitochondrial DNA base editing, and a base editing method.

Background

The human genome is largely divided into two parts, the first part being the nuclear genome in the nucleus, the DNA and histones being organized together in chromatin or chromosome form; the second part is circular DNA in mitochondria. Genomic DNA (gDNA) located in the nucleus exists in the form of chromosome, and the total length of the haplotype is about 3.1Gbp, and 23 pairs are total; the length of circular mitochondrial DNA (mtDNA) within mitochondria is about 16Kbp.

In normal human cells, there are about 1000 to 10000 mitochondria, and within each mitochondria, there are about 2 to 10 groups of mitochondria, each mtDNA containing 16569 base pairs in total. mtDNA is generally circular unlike nuclear genomic gDNA. A single mtDNA encodes a total of 37 genes, including 13 genes encoding proteins, 22 tRNA genes, and two rRNA genes. Mutations in the mitochondrial genome can lead to severe disease (Stewart, J.B.et al The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease. Nat Rev Genet,2015.16 (9): p.530-42.), so that it is important to correct mtDNA harboring pathogenic mutations by means of gene editing.

In 2020, joseph Mougous team and David Liu team developed based on TALE System that could target mitochondrial DNA and achieve site-directed C-to-T base editingMitochondrial base editor DdBE (Mok, B.Y., et al, A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base edition. Nature,2020.583 (7817): p.631-637.). Joseph Mougous team and David Liu team first developed a deaminase bacterial toxin DddA in one Burkholderia (Burkholderia cenocepacia) that specifically catalyzes DNA duplex by bioinformatics means, and a bacterial immune protein DddI that antagonizes DddA activity was found in another Burkholderia strain lacking the dddA gene _A . The fragment of the polypeptide near the C-terminus of DddA protein was the core region of the catalytic double-stranded DNA, and therefore this fragment was also named DddA as judged by structural biology _tox . Due to full length DddA _tox Can catalyze double-stranded DNA sequences, so DddA must be added _tox The division is carried out to ensure the normal culture in a conventional culture system such as escherichia coli and the like according to DddA _tox The difference of the division sites can be divided into two schemes G1333 and G1397, wherein G represents that the amino acid sequence of the division site is glycine Gly. Referring to related work by mitoTALEN, joseph mobile team and David Liu team are respectively associated with dda via TALE elements _tox The N end or the C end after the segmentation is connected; a mitochondrial localization signal MTS (mitochondrial targeting signal) which promotes the entry of proteins into mitochondria is serially connected at the N-terminus of TALE; with simultaneous reference to the previously developed cytosine base editor CBE based on CRISPR-Cas9 system, 1 UGI is serially connected at the C-terminus of the overall TALE element for inhibiting uracil glycosylase (UDG) activity in mitochondria, preventing from DddA _tox After catalytic deamination of C to dU, the dU is cleaved by intramitochondrial UDG to form a nick (nick). By changing the sequence of TALE, the N-terminal and C-terminal of the constructed element can be precisely targeted to specific areas of mitochondrial genome, and then the DddA is segmented _tox The components with complete deamination activity can be formed in the region, the accurate C-to-T editing in the nearby region is completed, double strand breaks of DNA are not generated in the editing process, and the degradation of mitochondrial DNA is prevented.

Disclosure of Invention

The treatment of mitochondrial genetic diseases is related to the physical health of human beings, and a reported mitochondrial base editor DdBE of Joseph mouous team and David Liu team in 2020 can edit mitochondrial single bases in real cells, thereby bringing possibility for accurately treating mitochondrial genetic diseases. However, off-target at the mitochondrial level is caused by non-specific binding of DdBE to mitochondrial DNA (Mok, B.Y., et al, A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base edition. Nature,2020.583 (7817): p.631-637.). Meanwhile, the present inventors have found from detection of Detect-seq that the current mitochondrial base editor dcbe, although already comprising the MTS sequence, still has a part of the dcbe incorrectly positioned in the nucleus and can cause severe off-target editing in the nucleus. Off-target caused by mitochondrial DNA level and nuclear genome level brings safety problems for the accurate treatment of mitochondrial genetic diseases.

In order to reduce off-target editing of existing dcbe in mitochondria and/or nuclei, the inventors have made extensive studies to provide double-stranded DNA deaminase mutants useful for constructing mitochondrial base editors with reduced off-target editing, which can be advantageously applied to the construction of mitochondrial base editors, and mitochondrial base editors constructed therefrom can effectively reduce off-target editing in mitochondria and/or nuclei while maintaining comparable target site editing efficiency. In addition, base editing compositions and methods with reduced off-target editing in the mitochondrial nucleus and/or nucleus are provided.

Accordingly, in a first aspect, the present application provides a polypeptide having double stranded DNA deaminase activity or a mutant thereof, said polypeptide or mutant thereof comprising amino acid residues in a wild-type double stranded DNA deaminase at positions corresponding to positions 1290-1427 of SEQ ID NO. 1; and, the polypeptide or mutant thereof has the following mutation compared with the amino acid residues at positions corresponding to positions 1290-1427 of SEQ ID NO. 1 in a wild-type double-stranded DNA deaminase:

(1) Replacement of an amino acid residue at a position corresponding to position 1308 of SEQ ID NO. 1 with an alanine residue or an amino acid residue which is a conservative substitution with respect to an alanine residue (e.g., glycine residue, leucine residue, isoleucine residue, valine residue); or,

(2) Replacement of an amino acid residue at a position corresponding to position 1310 of SEQ ID NO. 1 with an alanine residue or an amino acid residue which is a conservative substitution with respect to an alanine residue (e.g., glycine residue, leucine residue, isoleucine residue, valine residue);

wherein the mutant has at least 90%, e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the polypeptide having double-stranded DNA deaminase activity; alternatively, substitutions (preferably conservative substitutions), additions or deletions of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acids; and, has double-stranded DNA deaminase activity; and, in addition, the processing unit,

the amino acid residues at the positions corresponding to 1309, 1367 and 1368 of SEQ ID NO. 1 in the polypeptide or the mutant thereof are not mutated.

In certain embodiments, the wild-type double-stranded DNA deaminase has the amino acid sequence depicted as SEQ ID NO. 1.

In certain embodiments, the polypeptide having double stranded DNA deaminase activity has an amino acid sequence as shown in SEQ ID NO. 3 or 5.

In a second aspect, the present application provides a mutant double-stranded DNA deaminase or variant thereof having the following mutation compared to a wild-type double-stranded DNA deaminase:

wherein the variant has at least 90%, e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity compared to the mutant double-stranded DNA deaminase; alternatively, substitutions (preferably conservative substitutions), additions or deletions of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acids; and, has double-stranded DNA deaminase activity; and is also provided with

The mutated double-stranded DNA deaminase or variant thereof has NO mutation of the amino acid residues at positions corresponding to positions 1309, 1367 and 1368 of SEQ ID NO. 1.

In certain embodiments, the amino acid sequence of the mutant double stranded DNA deaminase or variant thereof at positions corresponding to positions 1290-1427 of SEQ ID NO. 1 is the amino acid sequence of a polypeptide or mutant thereof as described above.

In a third aspect, the present application provides a polypeptide polymer comprising a first polypeptide and a second polypeptide, wherein:

the first polypeptide comprises an N-terminal fragment and the second polypeptide comprises a C-terminal fragment;

the amino acid sequences of the N-terminal fragment and the C-terminal fragment are the amino acid sequences of the N-terminal fragment and the C-terminal fragment, respectively, formed by cleavage of the polypeptide according to the first aspect or a mutant thereof at the cleavage site;

wherein said polypeptide polymer is polymerized from said N-terminal fragment and said C-terminal fragment. For example, the polypeptide polymer is a dimer formed by the N-terminal fragment and the C-terminal fragment.

In certain embodiments, the N-terminal fragment and the C-terminal fragment, when each is present alone, do not possess double-stranded DNA deaminase activity, or possess significantly reduced deaminase activity (e.g., up to 40%, up to 30%, up to 20%, up to 10%, up to 5%, or up to 1% of the activity of a double-stranded DNA deaminase of a polypeptide as described above).

In certain embodiments, the polymer possesses double-stranded DNA deaminase activity (e.g., at least 70%, at least 80%, at least 90%, or at least 95% of the double-stranded DNA deaminase activity of the polypeptide) when the N-terminal fragment and the C-terminal fragment are polymerized.

In certain embodiments, the cleavage site is located in the polypeptide having double-stranded DNA deaminase activity or mutant thereof, immediately following the amino acid residue at position corresponding to position 1333 of SEQ ID NO. 1.

In certain embodiments, the N-terminal fragment has the amino acid sequence set forth in SEQ ID NO 104 or 106.

In certain embodiments, the C-terminal fragment has the amino acid sequence set forth in SEQ ID NO. 55.

In certain embodiments, the cleavage site is located in the polypeptide having double-stranded DNA deaminase activity or mutant thereof, immediately following the amino acid residue at position corresponding to position 1397 of SEQ ID NO. 1.

In certain embodiments, the N-terminal fragment has the amino acid sequence set forth in SEQ ID NO. 35 or 37.

In certain embodiments, the C-terminal fragment has the amino acid sequence set forth in SEQ ID NO. 15.

In certain embodiments, the first polypeptide further comprises a first DNA binding protein linked to the N-terminal fragment, and/or the second polypeptide further comprises a second DNA binding protein linked to the C-terminal fragment.

In certain embodiments, the first DNA-binding protein and/or the second DNA-binding protein are each independently a programmable DNA-binding protein.

In certain embodiments, each of the first polypeptide and the second polypeptide independently further comprises a Mitochondrial Targeting Sequence (MTS), and/or a Uracil Glycosylase Inhibitor (UGI) domain.

In certain embodiments, the first polypeptide comprises the following structure: a first Mitochondrial Targeting Sequence (MTS), the first DNA binding protein, the N-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain; the second polypeptide comprises the following structure: a second Mitochondrial Targeting Sequence (MTS), the second DNA binding protein, the C-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain.

In certain embodiments, each of the adjacent structures in the first and second polypeptides are independently linked directly or through a linker (e.g., a peptide linker, such as a flexible peptide comprising one or more glycine (G) and/or serine (S)).

In certain embodiments, the first Mitochondrial Targeting Sequence (MTS) is located N-terminal to the first polypeptide and/or the second Mitochondrial Targeting Sequence (MTS) is located N-terminal to the second polypeptide.

In certain embodiments, the first polypeptide comprises, in order from the N-terminus to the C-terminus: the first Mitochondrial Targeting Sequence (MTS), the first DNA binding protein, the N-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain; the second polypeptide comprises, in order from the N-terminal to the C-terminal: the second Mitochondrial Targeting Sequence (MTS), the second DNA binding protein, the C-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain.

In certain embodiments, the first mitochondrial targeting sequence or the second mitochondrial targeting sequence are each independently selected from mitochondrial targeting sequences derived from COX8 (cytochrome C oxidase 8A subunit), ATP5G2 (ATP synthase F0 complex C2 subunit), SOD2 (superoxide dismutase 2), COQ8A (mitochondrial atypical kinase COQ 8A).

In certain embodiments, the first mitochondrial targeting sequence is the same or different from the second mitochondrial targeting sequence.

In certain embodiments, the first mitochondrial targeting sequence is a mitochondrial targeting sequence derived from SOD 2. In certain embodiments, the first mitochondrial targeting sequence has the amino acid sequence shown in SEQ ID NO. 9.

In certain embodiments, the second mitochondrial targeting sequence is a mitochondrial targeting sequence derived from COX 8. In certain embodiments, the second mitochondrial targeting sequence has the amino acid sequence shown in SEQ ID NO. 19.

In certain embodiments, the first DNA-binding protein or the second DNA-binding protein is each independently selected from the group consisting of: TALE (transcription activator-like effector) proteins, zinc finger proteins, and Cas proteins. In certain embodiments, the first DNA-binding protein or the second DNA-binding protein is each independently a TALE (transcription activator-like effector) protein or a zinc finger protein.

In certain embodiments, the first DNA-binding protein is the same as or different from the second DNA-binding protein.

In certain embodiments, the first DNA-binding protein and the second DNA-binding protein are both TALE proteins.

In certain embodiments, the first polypeptide and/or the second polypeptide each independently further comprises a Nuclear Export Signal (NES) sequence.

In certain embodiments, the NES sequence is linked to other domains in the first polypeptide or the second polypeptide directly or through a linker (e.g., a peptide linker, such as a flexible peptide comprising one or more glycine (G) and/or serine (S)).

In certain embodiments, the first polypeptide comprises a first NES sequence and/or the second polypeptide comprises a second NES sequence.

In certain embodiments, the first NES sequence is located at the C-terminus of the first DNA binding protein.

In certain embodiments, the second NES sequence is located at the C-terminus of the second DNA binding protein.

In certain embodiments, the first NES sequence is the same as or different from the second NES sequence.

In certain embodiments, the first NES sequence or the second NES sequence is each independently selected from the group consisting of Rev protein (HIV regulator of virion) from HIV virus, mitogen-activated protein kinase (MAPK, mitogen-activated protein kinase), cellular tumor antigen protein P53 (cellular tumor antigen P53), and ribosome transport protein NMD3 (60S ribosomal export protein NMD3). In certain embodiments, the first NES sequence or the second NES sequence has the amino acid sequence shown as SEQ ID NO. 47 or 56, respectively.

In some embodiments of the present invention, in some embodiments,

the first polypeptide comprises, in order from the N-terminal to the C-terminal:

(i) The first Mitochondrial Targeting Sequence (MTS), the first DNA binding protein, the first NES sequence, the N-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain;

(ii) The first Mitochondrial Targeting Sequence (MTS), the first DNA binding protein, the N-terminal fragment, the first NES sequence, and the Uracil Glycosylase Inhibitor (UGI) domain;

or,

(iii) The first Mitochondrial Targeting Sequence (MTS), the first DNA binding protein, the N-terminal fragment, the Uracil Glycosylase Inhibitor (UGI) domain, and the first NES sequence;

and/or the number of the groups of groups,

the second polypeptide comprises, in order from the N-terminal to the C-terminal:

(i) The second Mitochondrial Targeting Sequence (MTS), the second DNA binding protein, the second NES sequence, the C-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain;

(ii) The second Mitochondrial Targeting Sequence (MTS), the second DNA binding protein, the C-terminal fragment, the second NES sequence, and the Uracil Glycosylase Inhibitor (UGI) domain; or,

(iii) The second Mitochondrial Targeting Sequence (MTS), the second DNA binding protein, the C-terminal fragment, the Uracil Glycosylase Inhibitor (UGI) domain, and the second NES sequence.

In certain embodiments, the first polypeptide comprises, in order from the N-terminus to the C-terminus: the first Mitochondrial Targeting Sequence (MTS), the first DNA binding protein, the N-terminal fragment, the Uracil Glycosylase Inhibitor (UGI) domain, and the first NES sequence; and/or, the second polypeptide comprises, in order from the N-terminal to the C-terminal: the second Mitochondrial Targeting Sequence (MTS), the second DNA binding protein, the C-terminal fragment, the Uracil Glycosylase Inhibitor (UGI) domain, and the second NES sequence.

In a fourth aspect, the present application provides a polypeptide polymer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first Mitochondrial Targeting Sequence (MTS), the first DNA binding protein, an N-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain; the second polypeptide comprises: a second Mitochondrial Targeting Sequence (MTS), the second DNA binding protein, a C-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain;

The amino acid sequences of the N-terminal fragment and the C-terminal fragment are the amino acid sequences of the N-terminal fragment and the C-terminal fragment formed by cleavage of a polypeptide having double-stranded DNA deaminase activity at the cleavage site; the polypeptide with double-stranded DNA deaminase activity comprises amino acid residues at positions corresponding to 1290-1427 of SEQ ID NO. 1 in wild-type double-stranded DNA deaminase;

wherein said polypeptide polymer is polymerized from said N-terminal fragment and said C-terminal fragment.

In certain embodiments, the first polypeptide further comprises a first Nuclear Export Signal (NES) sequence; and/or, the second polypeptide further comprises a second Nuclear Export Signal (NES) sequence.

In certain embodiments, the polypeptide having double stranded DNA deaminase activity has the amino acid sequence depicted as SEQ ID NO. 2.

In certain embodiments, the N-terminal fragment and the C-terminal fragment, when each is present alone, do not possess double-stranded DNA deaminase activity, or possess significantly reduced deaminase activity (e.g., up to 40%, up to 30%, up to 20%, up to 10%, up to 5%, or up to 1% of the activity of double-stranded DNA deaminase of a polypeptide having the double-stranded DNA deaminase activity).

In certain embodiments, the polymer possesses double-stranded DNA deaminase activity (e.g., at least 70%, at least 80%, at least 90%, or at least 95% of the double-stranded DNA deaminase activity of the polypeptide having double-stranded DNA deaminase activity) when the N-terminal fragment and the C-terminal fragment are polymerized.

In certain embodiments, the cleavage site is located in the polypeptide having double-stranded DNA deaminase activity at a peptide bond immediately following the amino acid residue at the position corresponding to position 1333 of SEQ ID NO. 1.

In certain embodiments, the N-terminal fragment has the amino acid sequence set forth in SEQ ID NO. 54.

In certain embodiments, the cleavage site is located in the polypeptide having double-stranded DNA deaminase activity at a peptide bond immediately following the amino acid residue at position corresponding to position 1397 of SEQ ID NO. 1.

In certain embodiments, the N-terminal fragment has the amino acid sequence set forth in SEQ ID NO. 14.

In certain embodiments, the first DNA-binding protein and/or the second DNA-binding protein is a programmable DNA-binding protein.

In certain embodiments, the structures are optionally linked by a linker (e.g., a peptide linker, such as a flexible peptide comprising one or more glycine (G) and/or serine (S)).

In certain embodiments, the first mitochondrial targeting sequence is a mitochondrial targeting sequence derived from SOD 2.

In certain embodiments, the second mitochondrial targeting sequence is a mitochondrial targeting sequence derived from COX 8.

In certain embodiments, the first NES sequence or the second NES sequence is each independently selected from the group consisting of Rev protein (HIV regulator of virion) from HIV virus, mitogen-activated protein kinase (MAPK, mitogen-activated protein kinase), cellular tumor antigen protein P53 (cellular tumor antigen P53), and ribosome transport protein NMD3 (60S ribosomal export protein NMD3).

In some embodiments of the present invention, in some embodiments,

or,

and/or the number of the groups of groups,

In a fifth aspect, the present application also provides an isolated nucleic acid molecule encoding a polypeptide having double-stranded DNA deaminase activity as described in the first aspect or a mutant thereof, a mutant double-stranded DNA deaminase as described in the second aspect or a variant thereof, a first polypeptide or a second polypeptide as described in the third aspect or the fourth aspect or a combination thereof.

In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the first polypeptide of the third aspect of the invention and a second nucleotide sequence encoding the second polypeptide of the third aspect, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecule. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecules of the invention comprise a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the first polypeptide of the fourth aspect of the invention and a second nucleotide sequence encoding the second polypeptide of the fourth aspect, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecule. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecules of the invention comprise a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

In a sixth aspect, the present application provides a vector comprising an isolated nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

In certain embodiments, the vector comprises a first nucleotide sequence encoding the first polypeptide of the third aspect of the invention and a second nucleotide sequence encoding the second polypeptide of the third aspect, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

In certain embodiments, the vector comprises a first nucleotide sequence encoding the first polypeptide of the fourth aspect of the invention and a second nucleotide sequence encoding the second polypeptide of the fourth aspect, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

In a seventh aspect, the present application also provides a host cell comprising a nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells, such as bacterial cells (e.g., E.coli cells), and eukaryotic cells, such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.).

In an eighth aspect, the present application also provides a method of preparing a polypeptide having double-stranded DNA deaminase activity as described in the first aspect or a mutant thereof, a mutant double-stranded DNA deaminase as described in the second aspect or a variant thereof, a first polypeptide or a second polypeptide as described in the third aspect or a first polypeptide or a second polypeptide as described in the fourth aspect, comprising culturing a host cell as described above under conditions allowing expression of the protein, and recovering the polypeptide having double-stranded DNA deaminase activity or a mutant thereof, a mutant double-stranded DNA deaminase or a variant thereof, the first polypeptide or the second polypeptide from the cultured host cell culture.

In certain embodiments, the first polypeptide and the second polypeptide are not co-expressed in the same host cell.

In a ninth aspect, the present application also provides a composition comprising a first component and a second component separated from each other, the first component comprising:

(i) The first polypeptide of the third aspect or a first polynucleotide encoding the first polypeptide; the second component comprises: the second polypeptide of the third aspect or a second polynucleotide encoding the second polypeptide;

or,

(ii) The first polypeptide of the fourth aspect or a first polynucleotide encoding the first polypeptide; the second component comprises: the second polypeptide of the fourth aspect or a second polynucleotide encoding the second polypeptide.

In certain embodiments, the combination further comprises a third component comprising a fusion protein or a third polynucleotide encoding the fusion protein; wherein the fusion protein comprises one or more Nuclear Localization Signal (NLS) sequences and a polypeptide capable of inhibiting double stranded DNA deaminase activity.

In certain embodiments, the fusion protein is capable of inhibiting double-stranded DNA deaminase activity of a polypeptide polymer formed by the first polypeptide and the second polypeptide.

In certain embodiments, the NLS sequence is located at the N-terminus and/or C-terminus of the polypeptide capable of inhibiting double-stranded DNA deaminase activity.

In certain embodiments, the NLS sequence is linked to the polypeptide capable of inhibiting double-stranded DNA deaminase activity directly or through a linker (e.g., a peptide linker, such as a flexible peptide comprising one or more glycine (G) and/or serine (S)).

In certain embodiments, the NLS sequence is selected from the group consisting of NLS sequences derived from simian cavitation virus 40 (SV 40), testis determinant (SRY), nuclear cytoplasmic protein (nucelopsmin), common bipartite NLS, bpNLS.

In certain embodiments, the fusion protein has one NLS sequence attached to each of the N-and C-termini of the polypeptide capable of inhibiting double stranded DNA deaminase activity. In certain embodiments, the polypeptide capable of inhibiting double-stranded DNA deaminase activity is linked at its N-terminus to a first NLS sequence and the polypeptide capable of inhibiting double-stranded DNA deaminase activity is linked at its N-terminus to a second NLS sequence.

In certain embodiments, the first NLS sequence has the amino acid sequence set forth in SEQ ID NO. 62. In certain embodiments, the second NLS sequence has the amino acid sequence set forth in SEQ ID NO. 63.

In certain embodiments, the first NLS sequence and the second NLS sequence have the amino acid sequence set forth in SEQ ID NO. 61.

In certain embodiments, the polypeptide capable of inhibiting double stranded DNA deaminase activity comprises Dddi _A A minimal active domain of a protein. In certain embodiments, the polypeptide capable of inhibiting double stranded DNA deaminase activity has the amino acid sequence depicted as SEQ ID NO. 60.

In certain embodiments, the fusion protein has an amino acid sequence as set forth in SEQ ID NO. 109 or 110.

In certain embodiments, the fusion protein has an amino acid sequence as set forth in SEQ ID NO. 64 or 65.

In a tenth aspect, the present application also provides a method of editing a target nucleotide sequence extracellular comprising contacting the target nucleotide sequence with a polypeptide polymer or composition as described above under conditions suitable for target nucleic acid editing, thereby inducing deamination of a target base in the target nucleotide sequence.

In certain embodiments, the target base is cytosine.

In certain embodiments, the method comprises contacting a target nucleotide sequence with a composition as described above, and the composition comprises a first component and a second component separated from each other, the first component comprising a first polypeptide as described above; the second component comprises a second polypeptide as described above; alternatively, the method comprises contacting a target nucleotide sequence with a polypeptide polymer as described above, the polypeptide polymer comprising a first polypeptide as described above and a second polypeptide as described above.

In certain embodiments, the first polypeptide comprises a first DNA binding protein and the second polypeptide comprises a second DNA binding protein.

In certain embodiments, the first DNA-binding protein targets a first nucleotide sequence flanking one of the target bases and the second DNA-binding protein targets a second nucleotide sequence flanking the other of the target bases; whereby said first polypeptide and said second polypeptide are capable of forming a polypeptide polymer, thereby inducing deamination of said target base.

In certain embodiments, the first polypeptide comprises, in order from the N-terminus to the C-terminus:

or,

and/or the number of the groups of groups,

In an eleventh aspect, the present application also provides a method of editing a target nucleotide sequence in a cell comprising delivering a polypeptide polymer or composition as described above into a cell comprising the target nucleotide sequence, thereby inducing deamination of a target base at a target site.

In certain embodiments, the target base is cytosine.

In certain embodiments, the method comprises delivering a composition as described above into a cell containing the target nucleotide sequence.

In certain embodiments, the composition comprises a first component and a second component separated from each other, the first component comprising a first polypeptide as described above; and, the second component comprises a second polypeptide as described above; alternatively, the first component comprises a first polynucleotide encoding the first polypeptide; and, the second component comprises a second polynucleotide encoding the second polypeptide.

In certain embodiments, the first component comprises a first polypeptide as described above; and, the second component comprises a second polypeptide as described above. After the first and second components are delivered into a cell, the first and second polypeptides are capable of forming a polypeptide polymer, thereby inducing deamination of the target base.

In certain embodiments, the first component comprises a first polynucleotide encoding the first polypeptide; and, the second component comprises a second polynucleotide encoding the second polypeptide. After the first and second components are delivered into a cell, a first polypeptide encoded by the first polynucleotide and a second polypeptide encoded by the second polynucleotide are capable of forming a polypeptide polymer, thereby inducing deamination of the target base.

In certain embodiments, the method comprises delivering a composition as described above that further comprises the third component into a cell containing the target nucleotide sequence; whereby said fusion protein in the composition or the fusion protein encoded by said third polynucleotide in the composition is located in the nucleus by virtue of its containing NLS sequence, reducing the double stranded DNA deaminase activity of said first polypeptide, or said second polypeptide, or a combination of said first and said second polypeptide, located in the nucleus.

In certain embodiments, the method comprises delivering a polypeptide polymer as described above into a cell containing the target nucleotide sequence.

In certain embodiments, the polypeptide polymer comprises a first polypeptide as described above and a second polypeptide as described above, wherein the first polypeptide comprises a first DNA binding protein and the second polypeptide comprises a second DNA binding protein.

In certain embodiments, the first DNA-binding protein targets a first nucleotide sequence flanking one of the target bases and the second DNA-binding protein targets a second nucleotide sequence flanking the other of the target bases; thereby, deamination of the target base is induced.

In certain embodiments, the method further comprises delivering a fusion protein or polynucleotide encoding the fusion protein as described above into a cell containing the target nucleotide sequence.

or,

and/or the number of the groups of groups,

In a twelfth aspect, the present application also provides a kit comprising a polypeptide having double-stranded DNA deaminase activity as described in the first aspect or a mutant thereof, a mutant double-stranded DNA deaminase as described in the second aspect or a variant thereof, a polypeptide polymer as described in the third or fourth aspect, an isolated nucleic acid molecule as described in the fifth aspect, a vector as described in the sixth aspect, a host cell as described in the seventh aspect, or a composition as described in the ninth aspect.

In certain embodiments, the kit comprises a polypeptide polymer as described in the third aspect. In certain embodiments, the kit further comprises a fusion protein as described above or a polynucleotide encoding the fusion protein.

In certain embodiments, the kit comprises a composition as described in the ninth aspect.

In a thirteenth aspect, the present application also provides the use of a polypeptide having double-stranded DNA deaminase activity as described in the first aspect or a mutant thereof, a mutant double-stranded DNA deaminase as described in the second aspect or a variant thereof, a polypeptide polymer as described in the third aspect or the fourth aspect, a first polypeptide as described in the third aspect or the fourth aspect, a second polypeptide as described in the third aspect or the fourth aspect, an isolated nucleic acid molecule as described in the fifth aspect, a vector as described in the sixth aspect, a host cell as described in the seventh aspect, or a composition as described in the ninth aspect for the preparation of a kit for editing a target nucleotide sequence or for editing a target nucleotide sequence.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the virology, biochemistry, immunology laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

When used herein, the terms "for example," such as, "" including, "" comprising, "or variations thereof, are not to be construed as limiting terms, but rather as meaning" but not limited to "or" not limited to.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

As used herein, the term "base editing" refers to a genomic editing technique that involves converting a particular nucleobase into another nucleobase at a genomic site of interest (e.g., included in mtDNA). In certain embodiments, this may be accomplished without the need for double-stranded DNA breaks (DSBs) or single-stranded breaks (immediate traces).

As used herein, the term "deaminase" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine (or adenine) deaminase that catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine to inosine in deoxyribonucleic acid (DNA). In certain embodiments, the deaminase is a cytidine (or cytosine) deaminase that catalyzes the hydrolytic deamination of cytidine or cytosine. In certain embodiments, the deaminase is a double-stranded DNA deaminase, or is modified to evolve or otherwise change to be able to utilize double-stranded DNA as a substrate for deamination. In certain embodiments, the deaminase is a cytidine (or cytosine) deaminase that catalyzes the hydrolytic deamination of cytidine or cytosine directly with double stranded DNA as a substrate for deamination.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in a first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in a second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions/total number of positions x 100%). In certain embodiments, the two sequences are the same length.

Determination of percent identity between two sequences can also be accomplished using mathematical algorithms. One non-limiting example of a mathematical algorithm for comparison of two sequences is the algorithm of Karlin and Altschul, 1990, proc.Natl. Acad. Sci.U.S. A.87:2264-2268, as modified in Karlin and Altschul,1993, proc.Natl. Acad. Sci.U.S. A.90:5873-5877. Such an algorithm was integrated into the NBLAST and XBLAST programs of Altschul et al, 1990, J.mol. Biol. 215:403.

As used herein, the term "variant", in the context of polypeptides (including polypeptides), also refers to polypeptides or peptides comprising an amino acid sequence that has been altered by the introduction of amino acid residue substitutions, deletions or additions. In some instances, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not by way of limitation, the polypeptide may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to a cell ligand or other protein, and the like. The derivatized polypeptide or peptide may be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the variants have similar, identical or improved functions as the polypeptide or peptide from which they are derived.

In certain embodiments, the programmable DNA binding protein may be selected from TALEs, ZFs, casx, casy, cpf1, C2, C2C3, argonaute proteins, or derived forms thereof. In certain embodiments, the programmable DNA binding protein has no nuclease activity. In certain embodiments, the programmable DNA binding protein cleaves only one strand of a nucleic acid duplex. In certain embodiments, the programmable DNA binding protein does not have the activity of forming a nucleic acid double strand break nick.

As used herein, the term "transcription activator-like effector protein" or "TALE protein" is a natural protein secreted by a bacterium of the genus Xanthomonas (Xanthomonas) as a plant pathogen when invading a host, and plays an important role in the invasion of plants by the bacterium of the genus Xanthomonas. Bacteria of the genus Xanthomonas inject the TALE protein into plant cells via the Type III secretion system, and the TALE protein can specifically bind to sites upstream of certain gene expression regions after entering host cells, and regulate the expression of these genes to aid in infection of the plant host by the bacteria. The natural TALE protein consists of an N-terminal transport signal (Translocation signal) domain, a C-terminal nuclear localization signal (nuclear localization signal, NLS) and a transcriptional activation domain (activation domain, AD), an intermediate highly repetitive sequence domain responsible for DNA recognition. The highly repetitive domain of the TALE protein takes 33-35 amino acids as basic units and has 12-25 repeats. In each basic unit, the other amino acids are highly conserved except for the two adjacent amino acids at positions 12 and 13, so that these two amino acids at positions 12, 13 are also referred to as RVDs (repeat variable di-residues, RVDs). Different RVDs can specifically recognize specific DNA bases, such as NI recognition A base, HD recognition C base, NG recognition T base and NN recognition G base, and the TALE basic units with different RVDs are repeatedly expressed in series, so that recombinant TALE proteins recognizing any DNA can be generated.

As used herein, a "zinc finger protein" or "ZFs" is a protein or domain that binds DNA in a sequence-specific manner by one or more zinc fingers, the structure of which is stabilized by coordination of zinc ions.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

Advantageous effects of the invention

The double-stranded DNA deaminase mutant provided by the application can be favorably applied to the construction of a mitochondrial base editor, and the mitochondrial base editor constructed by the double-stranded DNA deaminase mutant can effectively reduce off-target editing in mitochondria and/or nuclei while maintaining equivalent editing efficiency of target sites.

In addition, base editing compositions and methods with reduced off-target editing in the mitochondrial nucleus and/or nucleus are provided.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

FIG. 1 shows a number of off-target edits made by DdBE identified by the Detect-seq technique within the nuclear genome, with the outer circles labeled as chromosome numbers and the dots representing off-target edits made by the corresponding DdBE within the nuclear genome.

FIG. 2 shows a schematic component diagram of a tool for constructing TALE element deletions in DdBE using ND6-L1397N as an example.

FIG. 3 shows a schematic representation of the reduction of DdBE off-target activity by mutating DdDA (FIG. 3 a); and, schematic representation of DdBE structure after mutagenesis (FIG. 3 b).

FIG. 4 shows the editing efficiency of mitochondrial targeting sites after containing different DddA point mutations in ND6-L1397N (FIG. 4 a); mutations in Q1310A can significantly reduce mitochondrial off-target levels (fig. 4 b); and, Q1310A mutation may decrease the intensity of the Detect-seq signal at some off-target site of the nuclear genome (fig. 4 c).

FIG. 5 shows the results of detection of the level of editing of N1308A-ND6, Q1310A-ND6 at known 8 nuclear genome off-target sites using targeted amplicon sequencing.

FIG. 6 shows a schematic representation of the fusion positions of NES and DdBE.

FIG. 7 shows the results of testing the strategy for fusing NES sequences at different positions using ND 5.1-L1397N; FIG. 7a. Editing efficiency of mitochondrial targeting sites; figure 7b.8 off-target editing efficiency of off-target sites of known nuclear genomes.

FIG. 8 shows off-target editing efficiency of UGI-NES mode fusion versus control without NES fusion; FIG. 8a. Mitochondrial off-target levels slightly decreased after UGI-NES mode fusion at ND 6-L1397N; FIG. 8b. UGI-NES mode fusion the detection-seq signal at the nuclear genome off-target site was significantly reduced after ND 6-L1397N.

FIG. 9 shows the use of ND5.1-L1333N for testing the strategy of fusion of mapk-NES sequences; FIG. 9a. Editing efficiency of mitochondrial targeting sites; FIG. 9b.8 off-target editing efficiency of off-target sites of known nuclear genomes.

FIG. 10 shows the use of ND6-L1397N for fusion of HIV-NES and simultaneous use of DddA-Q1310A mutation strategy for testing; FIG. 10a. Editing efficiency of mitochondrial targeting sites; FIG. 10b.8 off-target editing efficiency of off-target sites of known nuclear genomes.

FIG. 11 shows the use of Dddi in combination _A Reducing the off-target effect of DdBE is schematically shown.

FIG. 12 shows Dddi _A Schematic of the tool configuration for use with DdBE.

FIG. 13 shows a different Dddi _A Effect of dose on ND6-L1397N mitochondrial targeting site editing efficiency (fig. 13 a); optimum Dddi _A Mitochondrial off-target levels were significantly reduced after dose co-transfection (fig. 13 b); optimal Dddi _A The detection-seq signal at the nuclear genome off-target site was significantly decreased after dose co-transfection (fig. 13 c).

FIG. 14 shows a different Dddi _A Effect of dose on ND6-L1397N mitochondrial target genome off-target site editing efficiency.

FIG. 15 shows the co-transfection of SV 40-NLS-DdI using ND6-L1397N pair _A Testing strategies; FIG. 15a. Editing efficiency of mitochondrial targeting sites; figure 15b.8 off-target editing efficiency of off-target sites of known nuclear genomes.

FIG. 16 shows the combination of ND6-L1397N pair Q1310A mutant with DdI _A Testing strategies; FIG. 16a editing efficiency of mitochondrial targeting sites; figure 16b.8 off-target editing efficiency of off-target sites of known nuclear genomes.

FIG. 17 shows that Q1310A-ND6-L1397N are respectively combined with Dddi _A NES, or Q1310A-ND6-L1397N in combination with NES and Dddi _A Off-target editing efficiency at 8 known nuclear genome off-target sites.

Figure 18 shows a performance comparison of all strategies. The value is "average on target editing efficiency/average off target editing efficiency" under a certain treatment, and the larger the value is, the better the off target effect is reduced. Wherein "canonic" indicates a traditional DdBE constructed from wild-type DddAtox, "hivNES", "TALE-hivNES", "DddA-hivNES" indicates a DdBE constructed from wild-type DddAtox and a NES derived from hiv, "SV40-NLS-DddIA" indicates a traditional DdBE constructed from wild-type DddAtox in combination with NLS-DdIA, "hivNES+Q1310A" indicates a DddBE constructed from DddAtox mutant Q1310A and a NES derived from hiv, "Q1310A+DddIA" indicates that DdBE constructed by DddAtox mutant Q1310A is combined with NLS-DdIA, "hivNES+Q1310 A+DdIA" indicates that DdBE constructed by DddAtox mutant Q1310A and NES derived from hiv is combined with NLS-DdIA, "UGI-hivNES" indicates that DdBE constructed by wild-type DddAtox, UGI and NES derived from hiv, and "mapKNES" indicates that DdBE constructed by wild-type DddAtox and NES derived from mapK.

Sequence information

A description of the sequences to which the present application relates is provided in the following table.

Table 1: sequence information

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, molecular biology experimental methods and immunoassays used in the present invention are basically described in j.sambrook et al, molecular cloning: laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, fine-compiled guidelines for molecular biology experiments, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the manufacturer of the product. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1: evaluation of existing DdBE off-target level in nuclear genome

Since DdBE is subjected to base editing, similar to the process of Cytosine Base Editor (CBE) generation editing developed by David Liu subject group, C at a target position is firstly deaminated into deoxyuracil dU by deaminase, dU is then recognized as T by a DNA replication or repair mechanism, and the conversion of C-to-T is finally completed. Thus, successful DdBE editing and off-target editing by DdBE will all produce the intermediate state product dU. So dU can be captured using the CBE off-target detection technique that has been successfully developed in this laboratory, detect-seq solutions out-of-protospacer editing and target-strand editing by cytosine base editors. Nat Methods,2021.18 (6): p.643-651.) to evaluate off-target conditions of existing DdBE on genomic DNA.

1.1 Experimental methods

Using G1397DddA _tox Two DdBE (L1397N and L1397C) of split construction mitochondrial gene editing was performed on four human mitochondrial sites of ND4, ND5.1, ND5.3 and ND6 reported in HEK293T cell line, mok, B.Y., et al, A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base coding. Nature,2020.583 (7817): p.631-637. Wherein the shorthand of "1397" in L1397N and L1397C means DddA _tox The cleavage site is G1397; "L" means Left-TALE; "N", "C" means DddA _tox -the N-or C-terminus of split. Thus, L1397N is abbreviated as: dddA _tox The division site is G1397, and the Left-TALE is DddA _tox -splitIs the N-terminal of (c).

In this example, ddBE of ND4-L1397N, ND5.1-L1397N, ND6-L1397N, ND4-L1397C and ND5.3-L1397C was used for editing in HEK293T cell line. The constituent elements of each DdBE are shown in Table 2.

TABLE 2 constituent elements of DdBE

1.1.1 editing with DdBE

Cell culture: HEK293T cell line was placed in 1% Glutamax supplemented with 10% FBS (PAA fetal bovine serum, cat# A15-151/101) ^TM (Gibco ^TM Cargo number: 35050061 0.5% penicillin/streptomyin (Gibco) ^TM Cargo number: 15140122 In 5% CO) ₂ Culturing at 37℃in a constant temperature incubator at a concentration, and passaging when the cell density reaches about 80%.

Transfection: will be 1.6X10 ⁶ Passage of individual HEK293T cells to 24 well plate cell culture plates (CORNING)Cargo number: 3524 For 16h. A pair of plasmids encoding DdBE, each 840ng, were then transfected into adherent cells using Lipofectamine LTX (ThermoFisher, cat# 15338100) according to their instructions. Transfected cells were collected by centrifugation after further culturing for 72h, genomic DNA was extracted from the collected cell pellet with Universal Genomic DNA Kit (CWBIO, cat# CW 2298M), finally eluted with 10mM Tris-HCl (pH 8.0) and the concentration was determined by Nanodrop spectrophotometry.

1.1.2 off-target assessment of DdBE using the Detect-seq technique

See Lei, Z., et al, detection-seq solutions out-of-protospacer editing and target-strand editing by cytosine base edition. Nat Methods,2021.18 (6): p.643-651.

1.1.3 target amplicon sequencing

The amplification primers of each point to be measured are shown in Table 3:

TABLE 3 primer sequences

For each site to be assayed, the reaction system of the first round of PCR is as shown in table 4:

TABLE 4 first round PCR reaction System

PCR amplification procedure was followed The instruction set of high-fidelity DNA polymerase sets the amplification cycle number to 10.

After the end of the first round of PCR, 5. Mu.L of the product of the PCR reaction system using the same genome but different amplification primers was mixed together, purified using 0.9X Agencourt AMPure XP (BECKMAN, cat# A63882), and then the DNA on XP beads was eluted using 18. Mu.L of nuclease-free water, and the eluted DNA was used for the second round of PCR reaction. The second round PCR reaction was as follows in Table 5:

TABLE 5 second round PCR reaction System

The amplification procedure of the second round PCR reaction was followedThe instruction set of high-fidelity DNA polymerase sets the amplification cycle number to 15.

After the second round of PCR reaction, the PCR reaction products with different index can be mixed together, purified by using 0.8X Agencourt AMPure XP, and eluted by using 20 mu L of water without nuclease to obtain the target amplicon sequencing library. 1. Mu.L of eluted DNA was taken and the concentration was determined using a Qubit 2.0 fluorometer; 1 μl of eluted DNA was used to detect library quality using an Agilent 4150 fragment analyzer.

The target amplicon sequencing library was sequenced using a Hua Dazhi MGI sequencing platform.

1.2 experimental results

The evaluation results showed that ND4-L1397N, ND5.1-L1397N, ND6-L1397N, ND4-L1397C and ND5.3-L1397C can result in 100, 652, 697, 91 and 610 off-target sites, respectively, at the nuclear genome level (FIG. 1).

To explore the mechanism of DdBE off-target in the nucleus, single-side deletion and total deletion operations are performed on the Left-TALE and Right-TALE of three DdBE types ND4-L1397N, ND5.1-L1397N and ND 6-L1397N. Taking ND6-L1397N as an example, after single-side deletion or total deletion of Left-TALE and Right-TALE, the composition of the DdBE editing tool is shown in FIG. 2. Subsequently, HEK293T cells transfected with DdBE corresponding to the deletion element described above were subjected to detection-seq pool sequencing. By deleting single-sided TALE elements, deleting all TALE elements and comparing the Detect-seq data of the complete DdCBE, two nuclear genome off-target types are defined: TAS-dependent (TAS-dependent) and TAS-independent (TAS-independent), where TAS is an abbreviation for TALE array sequence.

Example 2: reduction of DdBE off-target effects by mutating DddAtox

For DddA _tox Selective point mutation, mutation position and mutation type are: N1308A (SEQ ID NO: 3), G1309A (SEQ ID NO: 4), Q1310A (SEQ ID NO: 5), N1367A (SEQ ID NO: 6) and N1368A (SEQ ID NO: 7). Use of the mutated ddA _tox -split substitution of wild-type dda _tox The structural schematic diagram of the DdBE after modification of split construction is shown in FIG. 3b, and the tool constituent elements are shown in Table 6; the reduction of DdBE off-target activity by mutating DddA is schematically shown in FIG. 3 a.

TABLE 6 constituent elements of DdBE

After the target amplicon verification of the mutated ND6-L1397N DdBE, the DdBE constructed by the mutant N1308A and the mutant Q1310A is still edited at the mitochondrial targeting point, and other mutations can cause the great reduction of the mitochondrial targeting editing of the DdBE (figure 4 a). Off-target edits caused by DdBE at the mitochondrial level and the nuclear genome level in HEK293T cells were compared using the ATAC-seq and the Detect-seq for Q1310A-ND6, respectively. Q1310A can significantly reduce off-target editing of DdBE at mitochondrial levels to one third of the original DdBE compared to original DdBE (WT-ND 6) (FIG. 4 b); meanwhile, Q1310A may slightly reduce off-target editing intensity caused by dcbe on the nuclear genome compared to the original dcbe (fig. 4 c).

Subsequently, targeted amplicon sequencing of N1308A-ND6 and Q1310A-ND6 at known 8 nuclear genome off-target sites revealed that Q1310A-ND6 was more able to reduce the editing level of these 8 off-target sites than N1308A-ND6 (FIG. 5).

Example 3: reduction of DdBE off-target effects by increasing Nuclear Export Signal (NES)

The out-nuclear signal (nuclear export signal, NES) is a short peptide rich in water-transporting amino acids. Proteins with NES signals can be relocated to the cytoplasm by nuclear pore transporters (Azmi, A.S., M.H.Uddin, and R.M.Mohammad, the nuclear export protein XPO1-from biology to targeted therapy. Nat Rev Clin Oncol,2021.18 (3): p.152-169.). The currently known out-of-core signals generally conform to the amino acid sequence characteristics of Φ1-X3- Φ2-X2- Φ3-X- Φ4, wherein Φn represents n hydrophobic amino acids (e.g., leu, val, ile, phe, or Met) in tandem, and Xn represents any of the n amino acids in tandem. Currently, in eukaryotic cell systems, the two NES sequences most commonly used are the NES sequence extracted from the HIV virus (HIV-NES, amino acid sequence shown as SEQ ID NO: 47) and the NES sequence present in the cellular kinase pathway (mapk-NES, amino acid sequence shown as SEQ ID NO: 56).

In particular, 3 different fusion positions, namely the TALE element C-terminus, ddBE-split element C-terminus and UGI element C-terminus of DddA-split element (as shown in FIG. 6) can be selected for NES fusion. The three fusion modes can be respectively called TALE-NES-DdBE, dddA-NES-DdBE and UGI-NES-DdBE.

In combination with the above, the scheme exemplarily selects various combinations of 2 NES sequences, 3 NES fusion positions, different mitochondrial targeting sites and the like to reconstruct and compare the original DdBE. The constituent elements of each DdBE tool used are shown in Table 7:

TABLE 7 constituent elements of DdBE

TALE-NES-DdBE, dddA-NES-DdBE and UGI-NES-DdBE were first constructed using HIV-NES. By comparing the three NES fusion positions, it was found that the fusion positions of the different NES had little effect on the editing efficiency of the mitochondrial targeting site (FIG. 7 a). Further, 8 known nuclear genome off-target sites of ND5.1-L1397N were evaluated using targeted amplicon sequencing, and it was found that fusion of NES at the UGI-C terminus could maximally reduce editing efficiency of these 8 nuclear genome off-target sites (FIG. 7 b).

Off-target edits caused by DdBE at the mitochondrial level and the nuclear genome level in HEK293T cells were compared using ATAC-seq and Detect-seq for UGI-NES-ND6-L1397N, respectively. UGI-NES did not significantly reduce off-target editing of DdBE at mitochondrial levels compared to original DdBE (WT-ND 6) (FIG. 8 a); however, UGI-NES can significantly reduce the off-target editing intensity caused by DdCBE on the nuclear genome compared to the original DdCBE (fig. 8 b).

In order to verify that increasing NES to DdBE can reduce nuclear off-target as a general strategy, the NES sequence in UGI-NES was replaced, and the HIV-NES was replaced with mapk-NES sequence. And then, 8 known off-target sites are subjected to off-target detection on the mapk-UGI-NES-ND6-L1397N by using a targeted amplicon sequencing technology, and the DdBE fused with the mapk-NES sequence at the UGI-C end is found to still reduce off-target editing in the nucleus (figure 9), so that the universality of the fused NES strategy is proved.

Given that Q1310A can significantly reduce off-target editing of DdCBE at the mitochondrial level, increasing NES signaling to DdCBE can significantly reduce the signal intensity at nuclear genome off-target sites. Thus, Q1310A can be added to DdA while NES sequence is added at UGI-C end, and off-target editing of DdBE at mitochondrial level as well as nuclear genome level can be reduced simultaneously by combining two strategies.

Targeting amplicon sequencing of the Q1310A-ND6-L1397N-UGI-NES samples at the known 8 nuclear genome off-target sites revealed that Q1310A-ND6-L1397N-UGI-NES did significantly reduce the editing efficiency of the 8 nuclear genome off-target sites compared to the original DdBE (WT-ND 6) (FIG. 10).

Example 4: by Dddi _A Reducing off-target effect of DdBE

Joseph Mougous team and David Liu team excavated deaminase bacterial toxin DddA in Burkholderia (Burkholderia cenocepacia) which specifically catalyzes DNA double strand, and a bacterial immune protein DddI which antagonizes DddA activity was found in another Burkholderia strain lacking dddA gene _A . In order to ensure the editing activity of DdBE in mitochondria while reducing off-target editing thereof in the nucleus, the inventors have found, through extensive studies, that it is possible to increase DddA of Nuclear Localization Signal (NLS) by cotransfection when editing using DdBE _tox Activity-inhibiting protein Dddi _A Thereby reducing the catalytic activity of DdBE in the nucleus. By using Dddi simultaneously _A The schematic of reducing the off-target effect of DdBE is shown in FIG. 11, the tool configuration of the relevant configuration is shown in FIG. 12, and the constituent elements of the tool of the relevant configuration are shown in Table 8.

TABLE 8 constituent elements of each tool

In the simultaneous use of DdI with NLS _A In order to inhibit the catalytic activity of DdBE in the nucleus, it is necessary to determine the activity of DdBE and DdDI _A Dose ratio of molar mass of (c). Thus, the DdBE: ddDI was first conducted using ND6-L1397N _A Co-transfection conditions with different dose gradients of molar mass ratios of 1:0 to 1:1.5 were tested in HEK293T cells. The results indicate that for the mitochondrial targeting site, ddI was co-transfected _A There was little effect on targeted editing of mitochondria (fig. 13 a).

9 known nuclear genome off-target sites of ND6-L1397N were assessed using targeted amplicon sequencing and found to follow DdBE: dddi _A The ratio of molar masses increases and the efficiency of editing of nuclear genome off-target sites decreases (fig. 14). After comprehensive consideration of editing of the targeting site, ddBE is considered to be Dddi _A The ratio of molar masses was 1:1.2 as the optimal co-transfection ratio.

In DdBE and DdDI _A The off-target edits caused by dcbe at the mitochondrial and nuclear genome levels in HEK293T cells were compared using ATAC-seq and Detect-seq, respectively, with the molar mass ratio being the optimal co-transfection ratio. Co-transfection of DdDI compared to original DdBE _A Off-target editing of DdCBE at mitochondrial level can be significantly reduced to quarter level of original DdCBE (fig. 13 b); at the same time, co-transfection of DdDI compared to the original DdBE _A The off-target editing intensity caused by DdBE on the nuclear genome can be significantly reduced, and the detection-seq signal intensity of some off-target sites can be reduced to the background level (FIG. 13 c).

After replacing the bpNLS sequence with another common SV40-NLS sequence, targeted amplicon sequencing is performed on a plurality of known nuclear genome off-target sites.

Sequencing results showed that Dddi even if replaced by SV40-NLS sequence _A After cotransfection with DdBE, the editing efficiency of off-target sites on the nuclear genome can be obviously reduced under the condition that the editing level of the targeted mitochondria is not affected, and the universality of the strategy is proved (figure 15).

Example 5: Q1310A-ND6 and Dddi _A Evaluation of strategy combined off-target level

Considering that Q1310A can significantly reduce off-target editing of DdBE at the mitochondrial level, co-transfection of DdDI _A Entry into the nucleus hardly affects on-target editing of DdCBE at the mitochondrial level, but can significantly reduce the signal intensity at nuclear genome off-target sites. Thus, the Q1310A mutant can be added to DddA while co-transfecting DddI _A Off-target editing of dcbe at the mitochondrial level and at the nuclear genome level was simultaneously reduced by combining both strategies.

Co-transfection of Q1310A-ND6-L1397N with Dddi _A The sample is subjected to targeted amplicon sequencing at the known 8 nuclear genome off-target sites, and the result shows that compared with the original DdBE, the combined strategy can indeed remarkably reduce the editing efficiency of the 8 nuclear genome off-target sites, and the effectiveness of the strategy is proved (figure 16).

Example 6: Q1310A-ND6, NES and DdI added _A Assessment of off-target level with combination of three strategies

Q1310A mutant at ND6-L1397N and NES, ddI _A We found that the combination of the three strategies works best and the combination of the two strategies works less well (fig. 17, 18). The result shows that compared with the original DdBE, the strategy combined use can obviously reduce the editing efficiency of the 8 nuclear genome off-target sites, and the three strategy combined use effects are better than the double strategy combined use. Meanwhile, in the statistical results of fig. 18, all the exemplary optimization strategies proposed in the present application can significantly improve the off-target effect of the dcbe tool. The main structure of each construct corresponding to FIG. 18 is shown in Table 9:

TABLE 9 major constituent elements of the constructs

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

A polypeptide having double-stranded DNA deaminase activity or a mutant thereof, the polypeptide or mutant thereof comprising an amino acid residue at a position corresponding to positions 1290-1427 of SEQ ID No. 1 in a wild-type double-stranded DNA deaminase; and, the polypeptide or mutant thereof has the following mutation compared with the amino acid residues at positions corresponding to positions 1290-1427 of SEQ ID NO. 1 in a wild-type double-stranded DNA deaminase:

(1) Replacement of an amino acid residue at a position corresponding to position 1308 of SEQ ID NO. 1 with an alanine residue or an amino acid residue which is a conservative substitution with respect to an alanine residue (e.g., glycine residue, leucine residue, isoleucine residue, valine residue); or,

(2) Replacement of an amino acid residue at a position corresponding to position 1310 of SEQ ID NO. 1 with an alanine residue or an amino acid residue which is a conservative substitution with respect to an alanine residue (e.g., glycine residue, leucine residue, isoleucine residue, valine residue);

wherein the mutant has at least 90%, e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the polypeptide having double-stranded DNA deaminase activity; alternatively, substitutions (preferably conservative substitutions), additions or deletions of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acids; and, has double-stranded DNA deaminase activity; and, in addition, the processing unit,

the amino acid residues at the positions corresponding to 1309, 1367 and 1368 of SEQ ID NO. 1 in the polypeptide or the mutant thereof are not mutated;

preferably, the wild type double stranded DNA deaminase has an amino acid sequence as shown in SEQ ID NO. 1;

Preferably, the polypeptide having double-stranded DNA deaminase activity has an amino acid sequence as shown in SEQ ID NO. 3 or 5.
A mutant double-stranded DNA deaminase or variant thereof having the following mutation compared to a wild-type double-stranded DNA deaminase:

(1) Replacement of an amino acid residue at a position corresponding to position 1308 of SEQ ID NO. 1 with an alanine residue or an amino acid residue which is a conservative substitution with respect to an alanine residue (e.g., glycine residue, leucine residue, isoleucine residue, valine residue); or,

(2) Replacement of an amino acid residue at a position corresponding to position 1310 of SEQ ID NO. 1 with an alanine residue or an amino acid residue which is a conservative substitution with respect to an alanine residue (e.g., glycine residue, leucine residue, isoleucine residue, valine residue);

wherein the variant has at least 90%, e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity compared to the mutant double-stranded DNA deaminase; alternatively, substitutions (preferably conservative substitutions), additions or deletions of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acids; and, has double-stranded DNA deaminase activity; and is also provided with

The mutated double-stranded DNA deaminase or variant thereof has NO mutation of the amino acid residues at positions corresponding to positions 1309, 1367 and 1368 of SEQ ID No. 1;

preferably, the amino acid sequence of the mutant double stranded DNA deaminase or variant thereof at positions corresponding to positions 1290-1427 of SEQ ID No. 1 is the amino acid sequence of the polypeptide of claim 1 or a mutant thereof;

preferably, the wild type double stranded DNA deaminase has the amino acid sequence as shown in SEQ ID NO. 1.
A polypeptide polymer comprising a first polypeptide and a second polypeptide, wherein:

the first polypeptide comprises an N-terminal fragment and the second polypeptide comprises a C-terminal fragment;

the amino acid sequences of the N-terminal fragment and the C-terminal fragment are the amino acid sequences of the N-terminal fragment and the C-terminal fragment, respectively, formed by cleavage of the polypeptide of claim 1 or a mutant thereof at the cleavage site;

wherein said polypeptide polymer is polymerized from said N-terminal fragment and said C-terminal fragment;

preferably, the N-terminal fragment and the C-terminal fragment, when each is present alone, do not possess double-stranded DNA deaminase activity, or possess significantly reduced deaminase activity (e.g., up to 40%, up to 30%, up to 20%, up to 10%, up to 5% or up to 1% of the activity of the double-stranded DNA deaminase of the polypeptide of claim 1);

Preferably, the polymer possesses double-stranded DNA deaminase activity (e.g., at least 70%, at least 80%, at least 90% or at least 95% of the double-stranded DNA deaminase activity of the polypeptide of claim 1) when the N-terminal fragment is polymerized with the C-terminal fragment.
The polypeptide polymer of claim 3, wherein the cleavage site is located in the polypeptide having double-stranded DNA deaminase activity or a mutant thereof, a peptide bond immediately following the amino acid residue at the position corresponding to position 1333 of SEQ ID No. 1;

preferably, the N-terminal fragment has the amino acid sequence shown as SEQ ID NO 104 or 106;

preferably, the C-terminal fragment has the amino acid sequence shown as SEQ ID NO. 55.
The polypeptide polymer of claim 3, wherein the cleavage site is located in the polypeptide having double-stranded DNA deaminase activity or a mutant thereof, a peptide bond immediately following the amino acid residue at the position corresponding to position 1397 of SEQ ID No. 1;

preferably, the N-terminal fragment has the amino acid sequence shown as SEQ ID NO. 35 or 37;

preferably, the C-terminal fragment has the amino acid sequence shown in SEQ ID NO. 15.
The polypeptide polymer of claim 3, wherein the first polypeptide further comprises a first DNA binding protein linked to the N-terminal fragment, and/or the second polypeptide further comprises a second DNA binding protein linked to the C-terminal fragment;

Preferably, the first DNA-binding protein and/or the second DNA-binding protein are each independently a programmable DNA-binding protein.
The polypeptide polymer of claim 6, wherein each of said first polypeptide and said second polypeptide independently further comprises a Mitochondrial Targeting Sequence (MTS), and/or a Uracil Glycosylase Inhibitor (UGI) domain;

preferably, the first polypeptide comprises the following structure: a first Mitochondrial Targeting Sequence (MTS), the first DNA binding protein, the N-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain; the second polypeptide comprises the following structure: a second Mitochondrial Targeting Sequence (MTS), the second DNA binding protein, the C-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain;

preferably, adjacent structures in the first and second polypeptides are each independently linked directly or through a linker (e.g., a peptide linker, e.g., a flexible peptide comprising one or more glycine (G) and/or serine (S));

preferably, the first Mitochondrial Targeting Sequence (MTS) is located at the N-terminus of the first polypeptide and/or the second Mitochondrial Targeting Sequence (MTS) is located at the N-terminus of the second polypeptide;

Preferably, the first polypeptide comprises, in order from the N-terminus to the C-terminus: the first Mitochondrial Targeting Sequence (MTS), the first DNA binding protein, the N-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain; the second polypeptide comprises, in order from the N-terminal to the C-terminal: the second Mitochondrial Targeting Sequence (MTS), the second DNA binding protein, the C-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain.
The polypeptide polymer of any one of claims 3-7, wherein the first mitochondrial targeting sequence or the second mitochondrial targeting sequence is each independently selected from a mitochondrial targeting sequence derived from COX8 (cytochrome C oxidase 8A subunit), ATP5G2 (ATP synthase F0 complex C2 subunit), SOD2 (superoxide dismutase 2), COQ8A (mitochondrial atypical kinase COQ 8A);

preferably, the first mitochondrial targeting sequence is the same or different from the second mitochondrial targeting sequence;

preferably, the first mitochondrial targeting sequence is a mitochondrial targeting sequence derived from SOD 2; preferably, the first mitochondrial targeting sequence has the amino acid sequence shown as SEQ ID NO. 9;

Preferably, the second mitochondrial targeting sequence is a mitochondrial targeting sequence derived from COX 8; preferably, the second mitochondrial targeting sequence has the amino acid sequence shown as SEQ ID NO. 19.
The polypeptide polymer of any one of claims 3-8, wherein the first DNA-binding protein or the second DNA-binding protein is each independently selected from the group consisting of: TALE (transcription activator-like effector) proteins, zinc finger proteins, and Cas proteins;

preferably, the first DNA-binding protein is the same as or different from the second DNA-binding protein;

preferably, the first DNA-binding protein and the second DNA-binding protein are both TALE proteins.
The polypeptide polymer of any one of claims 3-9, wherein the first polypeptide and/or the second polypeptide each independently further comprises a Nuclear Export Signal (NES) sequence;

preferably, the NES sequence is linked to the other domains in the first or second polypeptide directly or through a linker (e.g., a peptide linker, e.g., a flexible peptide comprising one or more glycine (G) and/or serine (S));

preferably, the first polypeptide comprises a first NES sequence and/or the second polypeptide comprises a second NES sequence;

Preferably, the first NES sequence is located at the C-terminus of the first DNA-binding protein;

preferably, the second NES sequence is located at the C-terminus of the second DNA binding protein;

preferably, the first NES sequence is the same as or different from the second NES sequence;

preferably, the first NES sequence or the second NES sequence is each independently selected from the group consisting of Rev protein (HIV regulator of virion) derived from HIV virus, mitogen-activated protein kinase (MAPK, mitogen-activated protein kinase), cellular tumor antigen protein P53 (cellular tumor antigen P53), ribosomal transporter NMD3 (60S ribosomal export protein NMD3); for example, the first NES sequence or the second NES sequence has the amino acid sequence shown as SEQ ID NO. 47 or 56, respectively.
The polypeptide polymer of claim 10, wherein,

the first polypeptide comprises, in order from the N-terminal to the C-terminal:

(i) The first Mitochondrial Targeting Sequence (MTS), the first DNA binding protein, the first NES sequence, the N-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain;

(ii) The first Mitochondrial Targeting Sequence (MTS), the first DNA binding protein, the N-terminal fragment, the first NES sequence, and the Uracil Glycosylase Inhibitor (UGI) domain;

Or,

(iii) The first Mitochondrial Targeting Sequence (MTS), the first DNA binding protein, the N-terminal fragment, the Uracil Glycosylase Inhibitor (UGI) domain, and the first NES sequence;

and/or the number of the groups of groups,

the second polypeptide comprises, in order from the N-terminal to the C-terminal:

(i) The second Mitochondrial Targeting Sequence (MTS), the second DNA binding protein, the second NES sequence, the C-terminal fragment, and the Uracil Glycosylase Inhibitor (UGI) domain;

(ii) The second Mitochondrial Targeting Sequence (MTS), the second DNA binding protein, the C-terminal fragment, the second NES sequence, and the Uracil Glycosylase Inhibitor (UGI) domain; or,

(iii) The second Mitochondrial Targeting Sequence (MTS), the second DNA binding protein, the C-terminal fragment, the Uracil Glycosylase Inhibitor (UGI) domain, and the second NES sequence;

preferably, the first polypeptide comprises, in order from the N-terminus to the C-terminus: the first Mitochondrial Targeting Sequence (MTS), the first DNA binding protein, the N-terminal fragment, the Uracil Glycosylase Inhibitor (UGI) domain, and the first NES sequence; and/or, the second polypeptide comprises, in order from the N-terminal to the C-terminal: the second Mitochondrial Targeting Sequence (MTS), the second DNA binding protein, the C-terminal fragment, the Uracil Glycosylase Inhibitor (UGI) domain, and the second NES sequence.
An isolated nucleic acid molecule encoding the polypeptide having double-stranded DNA deaminase activity of claim 1 or a mutant thereof, the mutated double-stranded DNA deaminase of claim 2 or a variant thereof, the first polypeptide as defined in any of claims 3-11 or the second polypeptide as defined in any of claims 3-11 or a combination thereof.
A vector comprising the isolated nucleic acid molecule of claim 12; preferably, the vector is a cloning vector or an expression vector.
A host cell comprising the nucleic acid molecule of claim 12 or the vector of claim 13.
A method of preparing the polypeptide having double-stranded DNA deaminase activity of claim 1 or a mutant thereof, the mutant double-stranded DNA deaminase of claim 2 or a variant thereof, the first polypeptide as defined in any of claims 3-11 or the second polypeptide as defined in any of claims 3-11, comprising culturing the host cell of claim 14 under conditions allowing expression of the protein, and recovering the polypeptide having double-stranded DNA deaminase activity or a mutant thereof, the mutant double-stranded DNA deaminase or a variant thereof, the first polypeptide or the second polypeptide from the cultured host cell culture;

Preferably, the first polypeptide and the second polypeptide are not co-expressed in the same host cell.
A composition comprising a first component and a second component separated from each other, the first component comprising: a first polypeptide as defined in any one of claims 3 to 11 or a first polynucleotide encoding said first polypeptide;

the second component comprises: a second polypeptide as defined in any one of claims 3 to 11 or a second polynucleotide encoding said second polypeptide.
The composition of claim 16, wherein the combination further comprises a third component comprising a fusion protein or a third polynucleotide encoding the fusion protein; wherein the fusion protein comprises one or more Nuclear Localization Signal (NLS) sequences and a polypeptide capable of inhibiting double stranded DNA deaminase activity;

preferably, the fusion protein is capable of inhibiting double-stranded DNA deaminase activity of a polypeptide polymer formed by the first polypeptide and the second polypeptide;

preferably, the NLS sequence is located at the N-terminus and/or C-terminus of the polypeptide capable of inhibiting double-stranded DNA deaminase activity;

preferably, the NLS sequence is linked directly or through a linker (e.g., a peptide linker, e.g., a flexible peptide comprising one or more glycine (G) and/or serine (S)) to the polypeptide capable of inhibiting double-stranded DNA deaminase activity;

Preferably, the NLS sequence is selected from the group consisting of NLS sequences derived from simian cavitation virus 40 (SV 40), testis determinant (SRY), nuclear cytoplasmic protein (Nuceloplasmin), common bipartite NLS (bpNLS);

preferably, the polypeptide capable of inhibiting double-stranded DNA deaminase activity comprises Dddi _A A minimum active domain of a protein; for example, the inhibitor can inhibit double strandThe polypeptide with DNA deaminase activity has an amino acid sequence shown as SEQ ID NO. 60;

preferably, the fusion protein has the amino acid sequence shown as SEQ ID NO. 109 or 110.
A method of editing a target nucleotide sequence extracellularly, comprising contacting the target nucleotide sequence with the polypeptide polymer of any one of claims 3-11 or the composition of claim 16 under conditions suitable for target nucleic acid editing, thereby inducing deamination of a target base in the target nucleotide sequence;

preferably, the target base is cytosine;

preferably, the method comprises contacting the target nucleotide sequence with a composition according to claim 16, and the composition comprises a first component and a second component separated from each other, the first component comprising a first polypeptide as defined in any one of claims 3-11; the second component comprises a second polypeptide as defined in any one of claims 3-11; alternatively, the method comprises contacting the target nucleotide sequence with a polypeptide polymer according to any one of claims 3-11, said polypeptide polymer comprising a first polypeptide as defined in any one of claims 3-11 and a second polypeptide as defined in any one of claims 3-11;

Preferably, the first polypeptide comprises a first DNA binding protein and the second polypeptide comprises a second DNA binding protein;

preferably, the first DNA-binding protein targets a first nucleotide sequence flanking one of the target bases and the second DNA-binding protein targets a second nucleotide sequence flanking the other of the target bases; whereby said first polypeptide and said second polypeptide are capable of forming a polypeptide polymer, thereby inducing deamination of said target base.
A method of editing a target nucleotide sequence in a cell comprising delivering the polypeptide polymer of any one of claims 3-11 or the composition of claim 16 or 17 into a cell containing the target nucleotide sequence, thereby inducing deamination of a target base at a target site;

preferably, the target base is cytosine.
The method of claim 19, wherein the method comprises delivering the composition of claim 16 or 17 into a cell containing the target nucleotide sequence;

preferably, the composition comprises a first component and a second component separated from each other, the first component comprising a first polypeptide as defined in any one of claims 3-11; and, the second component comprises a second polypeptide as defined in any one of claims 3-11; alternatively, the first component comprises a first polynucleotide encoding the first polypeptide; and, the second component comprises a second polynucleotide encoding the second polypeptide;

Preferably, the first polypeptide comprises a first DNA binding protein and the second polypeptide comprises a second DNA binding protein;

preferably, the first DNA-binding protein targets a first nucleotide sequence flanking one of the target bases and the second DNA-binding protein targets a second nucleotide sequence flanking the other of the target bases; whereby said first polypeptide and said second polypeptide are capable of forming a polypeptide polymer, thereby inducing deamination of said target base;

preferably, the composition further comprises a third component, the third component being as defined in claim 17.
The method of claim 19, wherein the method comprises delivering the polypeptide polymer of any one of claims 3-11 into a cell containing the target nucleotide sequence;

preferably, the polypeptide polymer comprises a first polypeptide as defined in any one of claims 3-11 and a second polypeptide as defined in any one of claims 3-11, wherein the first polypeptide comprises a first DNA binding protein and the second polypeptide comprises a second DNA binding protein;

preferably, the first DNA-binding protein targets a first nucleotide sequence flanking one of the target bases and the second DNA-binding protein targets a second nucleotide sequence flanking the other of the target bases; thereby inducing deamination of the target base;

Preferably, the method further comprises delivering a fusion protein or a polynucleotide encoding the fusion protein into a cell containing the target nucleotide sequence; wherein the fusion protein is as defined in claim 17.
A kit comprising the polypeptide having double-stranded DNA deaminase activity of claim 1 or a mutant thereof, the mutant double-stranded DNA deaminase of claim 2 or a variant thereof, the polypeptide polymer of any of claims 3-11, the isolated nucleic acid molecule of claim 12, the vector of claim 13, the host cell of claim 14, or the composition of claim 16 or 17;

preferably, the kit comprises a polypeptide polymer according to any one of claims 3-11; preferably, the kit further comprises a fusion protein or a polynucleotide encoding said fusion protein, wherein said fusion protein is as defined in claim 17.

Preferably, the kit comprises a composition according to claim 16 or 17.
Use of the polypeptide having double-stranded DNA deaminase activity of claim 1 or a mutant thereof, the mutant double-stranded DNA deaminase of claim 2 or a variant thereof, the polypeptide polymer of any of claims 3-11, the first polypeptide as defined in any of claims 3-11, the second polypeptide as defined in any of claims 3-11, the isolated nucleic acid molecule of claim 12, the vector of claim 13, the host cell of claim 14, or the composition of claim 16 or 17 for preparing a kit for editing a target nucleotide sequence or for editing a target nucleotide sequence.