WO2023207607A1

WO2023207607A1 - Deaminase mutant, composition, and method for modifying mitochondrial dna

Info

Publication number: WO2023207607A1
Application number: PCT/CN2023/088008
Authority: WO
Inventors: 伊成器; 雷芷芯; 孟浩巍; 刘璐璐; 赵华男
Original assignee: 北京大学
Priority date: 2022-04-29
Filing date: 2023-04-13
Publication date: 2023-11-02
Also published as: CN117751133A

Abstract

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

Description

Deaminase mutants, compositions and methods for modifying mitochondrial DNA

Technical field

The present application relates to the technical field of base editing (especially mitochondrial base editing). Specifically, the present application relates to a double-stranded DNA deaminase mutant that can be used to construct a mitochondrial base editor, a base editor for mitochondrial DNA base editing, and a base editing method.

Background technique

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

There are approximately 1,000 to 10,000 mitochondria in normal human cells, and within each mitochondria, there are approximately 2 to 10 groups of mitochondria. Each mtDNA contains a total of 16,569 base pairs. mtDNA is different from nuclear genomic gDNA and is generally circular. A single mtDNA encodes a total of 37 genes, including 13 protein-coding genes, 22 tRNA genes and two rRNA genes. Mutations in the mitochondrial genome may cause serious diseases (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.), therefore It is of great significance to correct mtDNA with disease-causing mutations through gene editing.

In 2020, Joseph Mougous's team and David Liu's team developed DdCBE, a mitochondrial base editor that can target mitochondrial DNA and achieve site-specific C-to-T base editing based on the TALE system (Mok, BY, et al., A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature, 2020.583(7817):p.631-637.). Joseph Mougous's team and David Liu's team first used bioinformatics methods to discover the deaminase bacterial toxin DddA in a species of Burkholderia cenocepacia that can specifically catalyze DNA double strands, and in another A bacterial immune protein, DddI _A , that antagonizes DddA activity was discovered in a Burkholderia strain lacking the dddA gene. Judging from structural biology, the polypeptide fragment near the C-terminus of the DddA protein is the core region of catalytic double-stranded DNA, so this fragment is also named DddA _tox . Since the full-length DddA _tox can catalyze double-stranded DNA sequences, DddA _tox must be split to ensure normal culture in conventional culture systems such as E. coli. According to the different split sites of DddA _tox , it can be divided into Two schemes are G1333 and G1397, where G indicates that the amino acid sequence of the split site is glycine Gly. Referring to the related work of mitoTALEN, Joseph Mougous's team and David Liu's team connected to the N-terminal or C-terminal of DddA _tox through TALE elements respectively; the N-terminal of TALE was connected in series with the mitochondrial targeting signal MTS (mitochondrial targeting signal) that can promote the protein to enter mitochondria. ); At the same time, referring to the previously developed cytosine base editor CBE based on the CRISPR-Cas9 system, a UGI is connected in series to the C-terminus of the overall TALE element to inhibit the activity of uracil glycosylase (UDG) in mitochondria and prevent After DddA _tox catalyzes the deamination of C into dU, dU is cleaved by UDG in the mitochondria to form a nick. By replacing the sequence of TALE, the N-terminal and C-terminal ends of the constructed element can be precisely targeted to a specific region of the mitochondrial genome. The subsequently divided DddA _tox will be combined in this region to form an element with complete deamination activity, completing precise targeting of nearby regions. C-to-T editing does not produce DNA double-strand breaks during the editing process, preventing the degradation of mitochondrial DNA.

Contents of the invention

The treatment of mitochondrial genetic diseases is related to human health. In 2020, Joseph Mougous's team and David Liu's team reported that the mitochondrial base editor DdCBE can edit mitochondrial single bases in cells, bringing new insights to the precise treatment of mitochondrial genetic diseases. possible. However, due to the non-specific binding of DdCBE to mitochondrial DNA, it causes off-targeting at the mitochondrial level (Mok, B.Y., et al., A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature, 2020.583(7817): p.631-637 .). At the same time, the inventor of the present application found based on Detect-seq detection that although the current mitochondrial base editor DdCBE already contains the MTS sequence, some DdCBE will still be mislocalized into the nucleus and can cause serious off-targeting in the nucleus. edit. Off-targets caused at the mitochondrial DNA level and nuclear genome level bring safety issues to the precise treatment of mitochondrial genetic diseases.

In order to reduce the off-target editing of existing DdCBE in mitochondria and/or cell nuclei, the inventors have conducted extensive research and provided a double-stranded DNA deaminase mutant that can be used to construct a mitochondrial base editor with reduced off-target editing. Double-stranded DNA deaminase mutants can be advantageously used in the construction of mitochondrial base editors. The mitochondrial base editor constructed by them can effectively reduce the amount of DNA in mitochondria and/or the nucleus while maintaining considerable target site editing efficiency. Off-target editing within. In addition, the present application also provides base editing compositions and methods with reduced off-target editing in the mitochondrial nucleus/or cell nucleus.

Therefore, in a first aspect, the application provides a polypeptide or a mutant thereof having double-stranded DNA deaminase activity, the polypeptide or a mutant thereof comprising wild-type double-stranded DNA deaminase and SEQ ID NO: The amino acid residues at positions corresponding to positions 1290-1427 of 1; and, the polypeptide or its mutant is identical to SEQ in wild-type double-stranded DNA deaminase Compared with the amino acid residues corresponding to positions 1290-1427 of ID NO:1, it has the following mutations:

(1) The amino acid residue at the position corresponding to position 1308 of SEQ ID NO:1 is replaced by an alanine residue or an amino acid residue that is conservatively substituted relative to an alanine residue (for example, a glycine residue, a leucine residue) amino acid residue, isoleucine residue, valine residue) substitution; or,

(2) The amino acid residue at the position corresponding to position 1310 of SEQ ID NO:1 is an alanine residue or an amino acid residue that is conservatively substituted relative to an alanine residue (for example, a glycine residue, a leucine residue amino acid residues, isoleucine residues, valine residues) substitution;

Wherein, the mutant has at least 90%, for example, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity compared to the polypeptide with double-stranded DNA deaminase activity. or, having one or several (for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or Deleted; and, has double-stranded DNA deaminase activity; and,

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

In certain embodiments, the wild-type double-stranded DNA deaminase has the amino acid sequence set forth in 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 application provides a mutant double-stranded DNA deaminase or a variant thereof, which has the following mutations compared with a wild-type double-stranded DNA deaminase:

Wherein, the variant has at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity compared to the mutated double-stranded DNA deaminase; Alternatively, having one or several (for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9) substitutions (preferably conservative substitutions), additions or deletions of amino acids; And, has double-stranded DNA deaminase activity; and

The amino acid residues at positions corresponding to positions 1309, 1367 and 1368 of SEQ ID NO: 1 in the mutated double-stranded DNA deaminase or its variants are not mutated.

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

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

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

The amino acid sequences of the N-terminal fragment and the C-terminal fragment are respectively the amino acid sequences of the N-terminal fragment and the C-terminal fragment formed by cleavage of the polypeptide or its mutant at the cleavage site as described in the first aspect. ;

Wherein, the polypeptide polymer is formed by polymerizing the N-terminal fragment and the C-terminal fragment. For example, the polypeptide polymer is a dimer formed from the N-terminal fragment and the C-terminal fragment.

In certain embodiments, the N-terminal fragment and the C-terminal fragment each have no double-stranded DNA deaminase activity when present alone, or have significantly reduced deaminase activity (e.g., as described above At most 40%, at most 30%, at most 20%, at most 10%, at most 5% or at most 1% of the double-stranded DNA deaminase activity of the polypeptide.

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

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

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 a polypeptide having double-stranded DNA deaminase activity or a mutant thereof immediately following the amino acid residue at position 1397 of SEQ ID NO: 1 key.

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. White, 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, the first polypeptide and the second polypeptide each independently further comprise 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 sugar basalase 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, adjacent structures in the first polypeptide and the second polypeptide are each independently connected directly or through a linker (e.g., a peptide linker, for example, including one or more glycine (G) and / or serine (S) flexible peptide) linkage.

In certain embodiments, 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 second polypeptide. N-terminus of the peptide.

In certain embodiments, the first polypeptide comprises, in order from N-terminus to 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 sequentially includes from the N end to the C end: 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 the group consisting of COX8 (cytochrome c oxidase 8A subunit), ATP5G2 (ATP synthase F0 complex Mitochondrial targeting sequences of body C2 subunit), SOD2 (superoxide dismutase 2), and COQ8A (mitochondrial atypical kinase COQ8A).

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 SOD2. In certain embodiments, the first mitochondrial targeting sequence has the amino acid sequence set forth in SEQ ID NO: 9.

In certain embodiments, the second mitochondrial targeting sequence is a mitochondrial targeting sequence derived from COX8. In certain embodiments, the second mitochondrial targeting sequence has the amino acid sequence set forth in SEQ ID NO: 19.

In certain embodiments, the first DNA binding protein or the second DNA binding protein each independently Selected from: TALE (transcription activator-like effector) protein, zinc finger protein and Cas protein. In certain embodiments, the first DNA binding protein or the second DNA binding protein are each independently a TALE (transcription activator-like effector) protein or a zinc finger protein.

In certain embodiments, the first DNA binding protein and the second DNA binding protein are the same or different.

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 comprise a nuclear exit signal (NES) sequence.

In certain embodiments, the NES sequence is associated with the first polypeptide or the first polypeptide, either 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)). other domains in the second polypeptide.

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 C-terminal to the first DNA binding protein.

In certain embodiments, the second NES sequence is located C-terminal to the second DNA binding protein.

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

In certain embodiments, the first NES sequence or the second NES sequence are each independently selected from the group consisting of Rev protein (HIV regulator of virion), mitogen-activated protein kinase (MAPK, mitogen- activated protein kinase), cellular tumor antigen protein P53 (cellular tumor antigen p53), 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 set forth in SEQ ID NO: 47 or 56, respectively.

In certain embodiments,

The first polypeptide sequentially includes from N-terminus to C-terminus:

(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, First NES sequence;

and / or,

The second polypeptide sequentially includes from N-terminus to C-terminus:

(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, Second NES sequence.

In certain embodiments, the first polypeptide comprises, in order from N-terminus to C-terminus: the first mitochondrial targeting sequence (MTS), the first DNA binding protein, the N-terminal fragment, the the uracil glycosylase inhibitor (UGI) domain, and the first NES sequence; and/or the second polypeptide sequentially comprising from the N-terminus to the C-terminus: 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 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 includes: a second mitochondrial targeting sequence (MTS), the second DNA binding protein , 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 with double-stranded DNA deaminase activity at the cleavage site; the The polypeptide having double-stranded DNA deaminase activity includes the amino acid residues at positions corresponding to positions 1290-1427 of SEQ ID NO: 1 in the wild-type double-stranded DNA deaminase;

Wherein, the polypeptide polymer is formed by polymerizing the N-terminal fragment and the 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 wild-type double-stranded DNA deaminase has the amino acid sequence set forth in 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: 2.

In certain embodiments, the N-terminal fragment and the C-terminal fragment each have no double-stranded DNA deaminase activity when present alone, or have significantly reduced deaminase activity (e.g., have the At most 40%, at most 30%, at most 20%, at most 10%, at most 5% or at most 1% of the double-stranded DNA deaminase activity of the polypeptide with double-stranded DNA deaminase activity.

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

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 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 second DNA binding protein is a programmable DNA binding protein.

In certain embodiments, the structures are optionally connected by a linker (eg, 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 SOD2.

In certain embodiments, the second mitochondrial targeting sequence is a mitochondrial targeting sequence derived from COX8.

In certain embodiments, the first DNA binding protein or the second DNA binding protein are 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 are each independently a TALE (transcription activator-like effector) protein or a zinc finger protein.

In certain embodiments, the first NES sequence or the second NES sequence are each independently selected from the group consisting of Rev protein (HIV regulator of virion), mitogen-activated protein kinase (MAPK, mitogen- activated protein kinase), cellular tumor antigen protein P53 (cellular tumor antigen p53), ribosome transport protein NMD3 (60S ribosomal export protein NMD3).

In certain embodiments,

The first polypeptide sequentially includes from N-terminus to C-terminus:

or,

and / or,

The second polypeptide sequentially includes from N-terminus to C-terminus:

(ii) the second mitochondrial targeting sequence (MTS), the second DNA binding protein, the C-terminal patch segment, the second NES sequence, and, the uracil glycosylase inhibitor (UGI) domain; or,

In a fifth aspect, the 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 mutated double-stranded protein as described in the second aspect DNA deaminase or a variant thereof, the first polypeptide or the second polypeptide as described in the third or 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. Nucleotide sequences, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule of the present invention includes a third nucleotide sequence containing the first nucleotide sequence. a nucleic acid molecule and a second nucleic acid molecule containing said 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. Nucleotide sequences, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule of the present invention includes a third nucleotide sequence containing the first nucleotide sequence. a nucleic acid molecule and a second nucleic acid molecule containing said second nucleotide sequence.

In a sixth aspect, the 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 a first polypeptide of the third aspect of the invention and a second nucleotide sequence encoding a 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 When the sequences exist on different vectors, the vector of the present invention includes a first vector containing the first nucleotide sequence and a second vector containing the second nucleotide sequence.

In certain embodiments, the vector comprises a first nucleotide sequence encoding a first polypeptide of the fourth aspect of the invention and a second nucleotide sequence encoding a 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 exist on different vectors, the vector of the present invention includes a first vector containing the first nucleotide sequence and a vector containing the A second vector for a second nucleotide sequence.

In a seventh aspect, the present application also provides a host cell comprising the 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., small mouse cells, human cells, etc.).

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

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

In a ninth aspect, the application further provides a composition comprising a first component and a second component that are separated from each other, the first component comprising:

(i) The first polypeptide as described in the third aspect or the first polynucleotide encoding the first polypeptide; the second component includes: the second polypeptide as described in the third aspect or a second polynucleotide encoding said second polypeptide;

or,

(ii) The first polypeptide as described in the fourth aspect or the first polynucleotide encoding the first polypeptide; the second component includes: the second polypeptide as described in the fourth aspect or A second polynucleotide encoding said 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) sequence and polypeptides 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 coupled 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)) to the said NLS sequence capable of inhibiting double-stranded DNA detachment. Polypeptide linkage for ammonia enzyme activity.

In certain embodiments, the NLS sequence is selected from the group consisting of simian vacuolating virus 40 (SV40), testis determinant (SRY), nucleoplasmin (Nuceloplasmin), and commonly used bipartite NLS (bpNLS). NLS sequence.

In certain embodiments, the N-terminus and the C-terminus of the polypeptide capable of inhibiting double-stranded DNA deaminase activity in the fusion protein are each connected to an NLS sequence. In certain embodiments, the N-terminus of the polypeptide capable of inhibiting double-stranded DNA deaminase activity is connected to a first NLS sequence, and the N-terminus of the polypeptide capable of inhibiting double-stranded DNA deaminase activity is connected to a second NLS sequence. 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 the minimal active domain of DddI _A protein. In certain embodiments, the polypeptide capable of inhibiting double-stranded DNA deaminase activity has an amino acid sequence as shown in SEQ ID NO: 60.

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

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

In a tenth aspect, the present application also provides a method for editing a target nucleotide sequence extracellularly, which includes polymerizing the target nucleotide sequence with a polypeptide as described above under conditions suitable for target nucleic acid editing. Contact with a substance or composition thereby inducing deamination of the target base in the target nucleotide sequence.

In certain embodiments, the target base is cytosine.

In certain embodiments, the method includes contacting a target nucleotide sequence with a composition as described above, and, The composition includes a first component and a second component that are separated from each other, the first component includes a first polypeptide as described above; the second component includes a second polypeptide as described above; Alternatively, the method includes contacting the 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 first nucleotide sequence flanking the other target base. dinucleotide sequence; thus, the first polypeptide and the second polypeptide can form a polypeptide polymer, thereby inducing deamination of the target base.

In certain embodiments, the first polypeptide, sequentially from N-terminus to C-terminus, includes:

or,

and / or,

The second polypeptide sequentially includes from N-terminus to C-terminus:

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

In certain embodiments, the target base is cytosine.

In certain embodiments, the methods include 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 that are separate from each other, the first component comprising a first polypeptide as described above; and, the second component comprising A second polypeptide as described above; alternatively, the first component includes a first polynucleotide encoding the first polypeptide; and, the second component includes a first polynucleotide encoding the second polypeptide. Second polynucleotide.

In certain embodiments, the first component includes a first polypeptide as described above; and, the second component includes a second polypeptide as described above. After the first component and the second component are delivered into a cell, the first polypeptide and the second polypeptide 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. glycosides. After the first component and the second component are delivered into the cell, the first polypeptide encoded by the first polynucleotide and the second polypeptide encoded by the second polynucleotide Polypeptide polymers can be formed, thereby inducing deamination of the target base.

In certain embodiments, the method includes delivering a composition as described above further comprising the third component into a cell containing the target nucleotide sequence; thereby, the fusion protein in the composition Or the fusion protein encoded by the third polynucleotide in the composition is located in the cell nucleus through the NLS sequence it contains, reducing the first polypeptide, or the second polypeptide, or the Double-stranded DNA deaminase activity of the combination of the first polypeptide and the second polypeptide.

In certain embodiments, the methods include 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 The polypeptide includes 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 first nucleotide sequence flanking the other target base. dinucleotide sequence; thereby, deamination of the target base is induced.

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

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

or,

and / or,

The second polypeptide sequentially includes from N-terminus to C-terminus:

In certain embodiments, the first polypeptide comprises, in order from N-terminus to C-terminus: the first mitochondrial targeting sequence (MTS), the first DNA binding protein, the N-terminal fragment, the the uracil glycosylase inhibitor (UGI) domain, and the first NES sequence; and/or the second polypeptide sequentially comprising from the N-terminus to the C-terminus: the second mitochondrial targeting sequence (MTS), the second DNA binding protein, the C-terminal fragment, the the uracil glycosylase inhibitor (UGI) domain, and the second NES sequence.

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, and a mutated double-stranded DNA as described in the second aspect. Deaminase 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, as described in the seventh aspect The host cell, or the 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 includes a composition as described in the ninth aspect.

In the thirteenth aspect, the application also provides a polypeptide having double-stranded DNA deaminase activity as described in the first aspect or a mutant thereof, a mutated double-stranded DNA deaminase or a mutant thereof as described in the second aspect. Variant, a polypeptide polymer as described in the third or fourth aspect, a first polypeptide as described in the third or fourth aspect, a second polypeptide as described in the third or fourth aspect, The isolated nucleic acid molecule as described in the fifth aspect, the vector as described in the sixth aspect, the host cell as described in the seventh aspect, or the composition as described in the ninth aspect is used for preparing editing target nucleotide sequence Kits or uses for editing target nucleotide sequences.

Definition of Terms

In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the virology, biochemistry, and immunology laboratory procedures used in this article are routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

When the terms "such as," "such as," "such as," "including," "including," or variations thereof are used herein, these terms will not be considered limiting terms and will instead be interpreted to mean "without limitation ” or “without limitation.”

Unless otherwise indicated herein or clearly contradicted by context, the terms "a" and "an" as well as "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 Singular and plural.

As used herein, the term "base editing" refers to genome editing techniques involving the conversion of a specific nucleic acid base into another nucleic acid base at a target genomic site (eg, included in mtDNA). In certain embodiments, this can be accomplished without the need for double-stranded DNA breaks (DSBs) or single-stranded breaks (i.e., nicking).

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 which catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, 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, evolved or otherwise altered 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 using double-stranded DNA directly 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 may be introduced in the first amino acid sequence or nucleic acid sequence to best match the second amino acid or nucleic acid sequence). Good comparison). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. Molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (ie, percent identity = number of identical overlapping positions/total number of positions x 100%). In certain embodiments, both 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. Improved in .Acad.Sci.U.S.A.90:5873-5877. Such algorithms were 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 a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing substitutions, deletions, or additions of amino acid residues. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (ie, by covalently linking any type of molecule to the polypeptide or peptide). For example, and without limitation, polypeptides may be modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, Attached to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can 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. Furthermore, a variant has a similar, identical or improved function to the polypeptide or peptide from which it is derived.

In certain embodiments, the programmable DNA binding protein can be selected from TALEs, ZFs, Casx, Casy, Cpf1, C2c1, C2c2, C2c3, Argonaute proteins, or derivatives thereof. In certain embodiments, the The programmable DNA binding protein does not have nuclease activity. In certain embodiments, the programmable DNA binding protein can only cleave one strand of a nucleic acid duplex. In certain embodiments, the programmable DNA binding protein does not have the activity to form nucleic acid double-strand break nicks.

As used herein, the term "transcription activator-like effector protein" or "TALE protein" is a type of natural protein secreted by plant pathogenic bacteria of the genus Xanthomonas when they invade the host. Bacteria of the genus Bacillus play an important role in invading plants. Xanthomonas bacteria inject TALE proteins into plant cells through the Type III secretion system. After entering the host cells, the TALE proteins can specifically bind to the upstream sites of certain gene expression regions and regulate the expression of these genes to assist bacterial response. Infection of plant hosts. Natural TALE proteins consist of an N-terminal Translocation signal domain, a C-terminal nuclear localization signal (NLS) and a transcription activation domain (AD), and a highly repetitive sequence structure in the middle responsible for recognizing DNA. Domain composition. The highly repetitive domain of TALE protein has 33-35 amino acids as the basic unit, with a total of 12-25 repeats. In each basic unit, except for the two adjacent amino acid positions at positions 12 and 13, the other amino acids are highly conserved, so the two amino acids at positions 12 and 13 are also called RVD ( repeat variable di-residue,RVD). Different RVDs can specifically recognize specific DNA bases. For example, NI recognizes A base, HD recognizes C base, NG recognizes T base, and NN recognizes G base. These TALE basic units with different RVDs are connected in series. Repeated expression can produce recombinant TALE proteins that recognize any DNA.

As used herein, "zinc finger proteins" or "ZFs" are proteins or domains that bind DNA in a sequence-specific manner through 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 delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40). A vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication site.

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

Beneficial effects of the invention

The double-stranded DNA deaminase mutant provided by the present application can be advantageously used in the construction of mitochondrial base editors. The mitochondrial base editor constructed thereby can effectively reduce the mitochondrial DNA content while maintaining considerable target site editing efficiency. and/or off-target editing within the nucleus.

In addition, the present application also provides base editing compositions and methods with reduced off-target editing in the mitochondrial nucleus/or cell nucleus.

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and do not limit 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 accompanying drawings and the following detailed description of preferred embodiments.

Description of the drawings

Figure 1 shows a large number of off-target edits produced by DdCBE identified within the nuclear genome through Detect-seq technology. The outer circle in the figure is marked with the chromosome number, and the dots represent the off-target edits caused by the corresponding DdCBE in the nuclear genome.

Figure 2 shows a schematic diagram of the component composition of a tool for deleting TALE components in DdCBE, taking ND6-L1397N as an example.

Figure 3 shows a schematic diagram of reducing the off-target activity of DdCBE by mutating DddA (Fig. 3a); and a schematic diagram of the structure of DdCBE after mutation modification (Fig. 3b).

Figure 4 shows the editing efficiency of mitochondrial targeting sites after ND6-L1397N contains different DddA point mutations (Figure 4a); the Q1310A mutation can significantly reduce the level of mitochondrial off-targets (Figure 4b); and, the Q1310A mutation can cause certain changes in the nuclear genome. The Detect-seq signal intensity of some off-target sites decreased (Figure 4c).

Figure 5 shows the results of using targeted amplicon sequencing to detect the editing levels of N1308A-ND6 and Q1310A-ND6 at eight known nuclear genome off-target sites.

Figure 6 shows a schematic diagram of the fusion position of NES and DdCBE.

Figure 7 shows the results of testing the strategy of fusing NES sequences at different positions using ND5.1-L1397N; Figure 7a. Editing efficiency of mitochondrial target sites; Figure 7b. Off-target editing efficiency of 8 known nuclear genome off-target sites.

Figure 8 shows the off-target editing efficiency of UGI-NES fusion compared to the control group without NES fusion; Figure 8a. Mitochondrial off-target editing levels slightly decreased after UGI-NES fusion in ND6-L1397N; Figure 8b. UGI-NES fusion in The Detect-seq signal of off-target sites in the nuclear genome decreased significantly after ND6-L1397N.

Figure 9 shows the test of the strategy of fusing mapk-NES sequences using ND5.1-L1333N; Figure 9a. Editing efficiency of mitochondrial targeting sites; Figure 9b. Off-target editing efficiency of 8 known nuclear genome off-target sites.

Figure 10 shows the testing of fused HIV-NES using ND6-L1397N and simultaneously using the DddA-Q1310A mutation strategy; Figure 10a. Editing efficiency of mitochondrial targeting sites; Figure 10b. Off-targeting of 8 known nuclear genome off-target sites Editing efficiency.

Figure 11 shows a schematic diagram of the off-target effects of reducing DdCBE by concurrent use of DddI _A.

Figure 12 shows a schematic diagram of the tool structure used simultaneously with DddI _A and DdCBE.

Figure 13 shows the impact of different DddI _A doses on the editing efficiency of ND6-L1397N mitochondrial targeting sites (Figure 13a); the mitochondrial off-target level was significantly reduced after co-transfection with the optimal DddI _A dose (Figure 13b); and, the optimal DddI A dose The Detect-seq signal of off-target sites in the nuclear genome significantly decreased after dose _A co-transfection (Figure 13c).

Figure 14 shows the effect of different DddI _A doses on the editing efficiency of ND6-L1397N mitochondrial target nuclear genome off-target sites.

Figure 15 shows the test of the co-transfection SV40-NLS-DddI _A strategy using ND6-L1397N; Figure 15a. Editing efficiency of mitochondrial targeted sites; Figure 15b. Off-target editing efficiency of 8 known nuclear genome off-target sites .

Figure 16 shows the use of ND6-L1397N to test the Q1310A mutant combined with the DddI _A strategy; Figure 16a. Editing efficiency of mitochondrial target sites; Figure 16b. Off-target editing efficiency of 8 known nuclear genome off-target sites.

Figure 17 shows the off-target editing efficiency of Q1310A-ND6-L1397N combined with DddI _A and NES respectively, or Q1310A-ND6-L1397N combined with NES and DddI _A at eight known nuclear genome off-target sites.

Figure 18 shows the performance comparison of all strategies. The value is the "average on-target editing efficiency/average off-target editing efficiency" under a certain treatment. The larger the value, the better the reduction of off-target effects. Among them, "canonical" indicates the traditional DdCBE constructed from wild-type DddAtox, "hivNES", "TALE-hivNES", and "DddA-hivNES" indicate DdCBE constructed from wild-type DddAtox and HIV-derived NES, and "SV40-NLS- "DddIA" indicates the traditional DdCBE constructed from wild-type DddAtox combined with NLS-DddIA, "hivNES+Q1310A" indicates the DdCBE constructed from the DddAtox mutant Q1310A and hiv-derived NES, "Q1310A+DddIA" indicates the DddCBE constructed from the DddAtox mutant Q1310A DdCBE combined with NLS-DddIA, "hivNES+Q1310A+DddIA" indicates the DdCBE combination constructed from the DddAtox mutant Q1310A and HIV-derived NES NLS-DddIA, "UGI-hivNES" indicates DdCBE constructed from wild-type DddAtox, UGI, and hiv-derived NES, and "mapKNES" indicates DdCBE constructed from wild-type DddAtox and mapK-derived NES.

sequence information

A description of the sequences covered by this application is provided in the table below.

Table 1: Sequence information

Detailed ways

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

Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method was carried out according to the method described in F.M. Ausubel et al., Compiled Experimental Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the product manufacturer. 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 DdCBE at nuclear genome off-target levels

Since the base editing process of DdCBE is similar to the editing process of the cytosine base editor (CBE) previously developed by David Liu's research group, deaminase is first used to deaminate the C at the target position into deoxygenation. Uracil dU is then recognized as T through the DNA replication or repair mechanism, ultimately completing the C-to-T conversion. Therefore, both successful DdCBE editing and off-target editing by DdCBE will produce the intermediate product dU. Therefore, the CBE off-target detection technology Detect-seq that has been successfully developed by our laboratory is used (see Lei, Z., et al., Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors. Nat Methods, 2021.18(6):p.643-651.) dU can be captured to evaluate the off-target status of existing DdCBE on genomic DNA.

1.1 Experimental methods

Mok, BY, et al., A bacterial cytidine deaminase toxin _enables CRISPR-free mitochondrial base editing. Nature, 2020.583(7817): The four human mitochondrial sites ND4, ND5.1, ND5.3 and ND6 reported in p.631-637 are used for mitochondrial gene editing. Among them, the abbreviation of "1397" in L1397N and L1397C means that the DddA _tox split site is G1397; "L" represents Left-TALE; "N" and "C" represent the N-terminal or C-terminal of DddA _tox -split. Therefore, the abbreviation of L1397N means: the DddA _tox split site is G1397, and the Left-TALE carries the N-terminus of DddA _tox -split.

In this example, DdCBE of ND4-L1397N, ND5.1-L1397N, ND6-L1397N, ND4-L1397C and ND5.3-L1397C was specifically used for editing in the HEK293T cell line. The components of each DdCBE are shown in Table 2.

Table 2 Components of each DdCBE

1.1.1 Editing with DdCBE

Cell culture: HEK293T cell line was placed in a culture medium supplemented with 10% FBS (PAA fetal bovine serum, Cat. No.: A15-151/101), 1% GlutaMAX ^™ (Gibco ^™ , Cat. No.: 35050061) and 0.5% penicillin/streptomycin (Gibco ^™ , Catalog No.: 15140122), culture in a 37°C constant-temperature incubator at a concentration of 5% _CO2 , and passage when the cell density reaches about 80%.

Transfection: Passage 1.6×10 ⁶ HEK293T cells into a 24-well cell culture plate (CORNING No.: 3524) for 16 hours. Then use Lipofectamine LTX (ThermoFisher, Cat. No.: 15338100) according to its instructions, 840ng of each pair of plasmids encoding DdCBE were transfected into adherent cells. After continuing to culture for 72 hours, the transfected cells were collected by centrifugation, and genomic DNA was extracted from the collected cell pellet using Universal Genomic DNA Kit (CWBIO, Cat. No.: CW2298M). Finally, it was eluted with 10mM Tris-HCl (pH 8.0) and spectrophotometered by Nanodrop. Photometer determines concentration.

1.1.2 Use Detect-seq technology for off-target assessment of DdCBE

For specific experimental steps, see Lei, Z., et al., Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors. Nat Methods, 2021.18(6): p.643-651.

1.1.3 Target amplicon sequencing

The amplification primers for each test point are shown in Table 3:

Table 3 Primer sequences

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

Table 4 First round PCR reaction system

The PCR amplification procedure follows According to the instruction manual of high-fidelity DNA polymerase, the number of amplification cycles is set to 10.

After the first round of PCR, mix 5 μL of the products of the PCR reaction systems using the same genome and different amplification primers together, purify using 0.9×Agencourt AMPure XP (BECKMAN, Cat. No.: A63882), and then use 18 μL of nuclease-free water. The DNA on the XP beads is eluted and the eluted DNA is used in the second round of PCR reaction. The second round PCR reaction system is shown in Table 5:

Table 5 Second round PCR reaction system

The amplification procedure for the second round of PCR reaction is as follows: Instructions for setting up high-fidelity DNA polymerase, The number of amplification cycles was set to 15.

After the second round of PCR reaction, the PCR reaction products with different indexes can be mixed together, purified using 0.8×Agencourt AMPure XP, and then eluted with 20 μL nuclease-free water to obtain the target amplicon sequencing library. Take 1 μL of eluted DNA and use a Qubit 2.0 fluorometer to measure the concentration; take 1 μL of eluted DNA and use an Agilent 4150 fragment analyzer to check the library quality.

The target amplicon sequencing library was sequenced using the MGI sequencing platform manufactured by MGI.

1.2 Experimental results

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

In order to explore the off-target mechanism of DdCBE in the nucleus, the Left-TALE and Right-TALE of three DdCBEs, ND4-L1397N, ND5.1-L1397N, and ND6-L1397N, were deleted unilaterally or completely. Taking ND6-L1397N as an example, after deleting Left-TALE and Right-TALE unilaterally or completely, the schematic diagram of the DdCBE editing tool is shown in Figure 2. Subsequently, Detect-seq library construction and sequencing was performed on HEK293T cells transfected with DdCBE corresponding to the above-mentioned deleted elements. By comparing the Detect-seq data of deleting unilateral TALE elements, deleting all TALE elements and complete DdCBE, two types of nuclear genome off-targets were defined: TAS-dependent (TAS-dependent) and TAS-independent (TAS-independent). Among them, TAS is the abbreviation of TALE array sequence.

Example 2: Reducing the off-target effects of DdCBE by mutating DddAtox

For DddA _tox selective point mutations, the mutation positions and mutation types 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). The mutated DddA _tox -split was used to replace the wild-type DddA _tox -split to construct the modified DdCBE. The structural diagram of the DdCBE after the mutation is shown in Figure 3b, and the components of each tool are shown in Table 6; by mutating DddA The schematic diagram of reducing the off-target activity of DdCBE is shown in Figure 3a.

Table 6 Components of each DdCBE

After verifying the targeted amplicon of the above-mentioned mutated ND6-L1397N DdCBE, it was found that the DdCBE constructed with the mutants N1308A and Q1310A still had editing at the mitochondrial targeting site. Other mutations would cause a substantial increase in mitochondrial targeting editing of DdCBE. decreased (Fig. 4a). ATAC-seq and Detect-seq were used to compare the off-target editing caused by DdCBE in HEK293T cells at the mitochondrial level and nuclear genome level for Q1310A-ND6. Compared with original DdCBE (WT-ND6), Q1310A can significantly reduce the off-target editing of DdCBE at the mitochondrial level to one-third of the level of original DdCBE (Figure 4b); at the same time, compared with original DdCBE, Q1310A can slightly reduce DdCBE The resulting off-target editing intensity on the nuclear genome (Figure 4c).

Subsequently, targeted amplicon sequencing was performed on N1308A-ND6 and Q1310A-ND6 at eight known nuclear genome off-target sites. The results showed that Q1310A-ND6 can reduce the editing level of these eight off-target sites better than N1308A-ND6. (Figure 5).

Example 3: Reduce the off-target effects of DdCBE by increasing nuclear exit signal (NES)

The nuclear export signal (NES) is a short peptide rich in water-transporting amino acids. Proteins with NES signals can be relocated to the cytoplasm through 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 nuclear exit signals generally conform to the amino acid sequence characteristics of Φ1-X3-Φ2-X2-Φ3-X-Φ4, where Φn represents n hydrophobic amino acids in series (such as Leu, Val, Ile, Phe, or Met) , Xn represents any amino acid of n series. Currently in eukaryotic cell systems, the two most commonly used NES sequences are the NES sequence extracted from the HIV virus (HIV-NES, amino acid sequence such as SEQ ID NO: 47) and the NES sequence that exists in the cellular kinase pathway (mapk -NES, amino acid sequence such as SEQ ID NO:56).

When specifically fusing NES, three different fusion positions can be selected, namely the C-terminal of the TALE element of DdCBE, the C-terminal of the DddA-split element and the C-terminal of the UGI element (as shown in Figure 6). The above three fusion methods can be abbreviated as TALE-NES-DdCBE, DddA-NES-DdCBE and UGI-NES-DdCBE respectively.

Combining the above contents, this case exemplarily selected two kinds of NES sequences, three kinds of NES fusion positions, different mitochondrial targeting sites and other combinations to transform and compare the original DdCBE. The components of each DdCBE tool used are shown in Table 7:

Table 7 Components of each DdCBE

HIV-NES was first used to construct TALE-NES-DdCBE, DddA-NES-DdCBE and UGI-NES-DdCBE. By comparing the fusion positions of these three NESs, it was found that the fusion positions of different NESs had little impact on the editing efficiency of mitochondrial targeting sites (Figure 7a). Furthermore, targeted amplicon sequencing was used to evaluate eight known nuclear genome off-target sites of ND5.1-L1397N, and found that fusion of NES at the UGI-C terminus could improve the editing efficiency of these eight nuclear genome off-target sites. decreased the most (Fig. 7b).

ATAC-seq and Detect-seq were used to compare the off-target editing caused by DdCBE in HEK293T cells at the mitochondrial level and nuclear genome level for UGI-NES-ND6-L1397N. Compared with the original DdCBE (WT-ND6), UGI-NES cannot significantly reduce the off-target editing of DdCBE at the mitochondrial level (Figure 8a); but compared with the original DdCBE, UGI-NES can significantly reduce the off-target editing caused by DdCBE on the nuclear genome. Off-target editing intensity (Figure 8b).

In order to verify that adding NES to DdCBE can reduce off-target in the nucleus as a general strategy, the NES sequence in UGI-NES was replaced, and the HIV-NES was replaced with the mapk-NES sequence. Subsequently, targeted amplicon sequencing technology was used to conduct off-target detection of mapk-UGI-NES-ND6-L1397N at eight known off-target sites. It was found that DdCBE, which is fused with the mapk-NES sequence at the UGI-C terminus, can still reduce the number of nuclear Internal off-target editing (Figure 9) proves the versatility of this fusion NES strategy.

Considering that Q1310A can significantly reduce the off-target editing of DdCBE at the mitochondrial level, increasing the NES signal to DdCBE can significantly reduce the signal intensity of off-target sites in the nuclear genome. Therefore, Q1310A can be added to DddA and the NES sequence can be added to the UGI-C terminus to simultaneously reduce off-target editing of DdCBE at the mitochondrial level and nuclear genome level by combining the two strategies.

Targeted amplicon sequencing of the Q1310A-ND6-L1397N-UGI-NES sample at 8 known nuclear genome off-target sites showed that compared with the original DdCBE (WT-ND6), Q1310A-ND6-L1397N-UGI -NES can indeed significantly reduce the editing efficiency of these eight nuclear genome off-target sites (Figure 10).

Example 4: Reducing the off-target effects of DdCBE through DddI _A

Joseph Mougous's team and David Liu's team discovered the deaminase bacterial toxin DddA in Burkholderia cenocepacia that can specifically catalyze DNA double strands. At the same time, they discovered another Burkholderia cenocepacia lacking the dddA gene. A bacterial immune protein DddI _A that can antagonize the activity of DddA was discovered in Holder's strain. In order to ensure the editing activity of DdCBE in mitochondria and reduce its off-target editing in the nucleus, after extensive research, the inventors found that co-transfection can increase the nuclear localization signal (NLS) when using DdCBE for editing. DddA _tox activity inhibits the protein DddI _A , thereby reducing the catalytic activity of DdCBE in the nucleus. The schematic diagram of reducing the off-target effect of DdCBE by simultaneously using DddI _A is shown in Figure 11. The structure of the related tool is shown in Figure 12. The components of the related tool are shown in Table 8.

Table 8 Components of Each Tool

When simultaneously using DddI _A with NLS to inhibit the catalytic activity of DdCBE in the nucleus, it is necessary to determine the dose ratio of the molar masses of DdCBE and DddI _A. Therefore, ND6-L1397N was first used to test co-transfection conditions in HEK293T cells at different dose gradients with DdCBE:DddI _A molar mass ratios ranging from 1:0 to 1:1.5. The results showed that for mitochondrial targeting sites, co-transfection of DddI _A had little effect on mitochondrial targeting position editing (Figure 13a).

Using targeted amplicon sequencing, nine known nuclear genome off-target sites of ND6-L1397N were evaluated and found that as the molar mass ratio of DdCBE:DddI _A increases, the editing efficiency of nuclear genome off-target sites will also increase. decline (Figure 14). After comprehensive consideration of the editing of the targeted site, it is believed that the molar mass ratio of DdCBE:DddI _A is 1:1.2 as the optimal co-transfection ratio.

Under the condition that the molar mass ratio of DdCBE and DddI _A was the optimal co-transfection ratio, ATAC-seq and Detect-seq were used to detect off-target effects caused by DdCBE at the mitochondrial level and nuclear genome level in HEK293T cells, respectively. Edit for comparison. Compared with original DdCBE, co-transfection of DddI _A can significantly reduce the off-target editing of DdCBE at the mitochondrial level to a quarter of the level of original DdCBE (Figure 13b); at the same time, compared with original DdCBE, co-transfection of DddI _A can significantly reduce the off-target editing of DdCBE at the mitochondrial level (Figure 13b). The off-target editing intensity caused by DdCBE on the nuclear genome is significantly reduced, and the Detect-seq signal intensity of some off-target sites can be reduced to the background level (Figure 13c).

After replacing the above bpNLS sequence with another common SV40-NLS sequence, targeted amplicon sequencing was performed on several known nuclear genome off-target sites.

The sequencing results show that even if the SV40-NLS sequence is replaced, co-transfection of DddI _A and DdCBE can still significantly reduce the editing efficiency of off-target sites on the nuclear genome without affecting the level of targeted mitochondrial editing, proving that This demonstrates the versatility of this strategy (Figure 15).

Example 5: Evaluation of the off-target level of Q1310A-ND6 combined with DddI _A strategy

Considering that Q1310A can significantly reduce the off-target editing of DdCBE at the mitochondrial level, co-transfection of DddI _A into the nucleus has almost no effect on the on-target editing of DdCBE at the mitochondrial level, but can significantly reduce the signal intensity of off-target sites in the nuclear genome. Therefore, the Q1310A mutant can be added to DddA and co-transfected with DddI _A to simultaneously reduce off-target editing of DdCBE at the mitochondrial level and nuclear genome level by combining the two strategies.

Targeted amplicon sequencing of Q1310A-ND6-L1397N co-transfected DddI _A samples at 8 known nuclear genome off-target sites showed that compared with the original DdCBE, the combined strategy can indeed significantly reduce these 8 nuclear genome off-target sites. The editing efficiency of off-target sites proves the effectiveness of the strategy (Figure 16).

Example 6: Evaluation of the off-target level of the three strategies of Q1310A-ND6, increasing NES and DddI _A

In the strategy combination of the Q1310A mutant of ND6-L1397N with NES and DddI _A , we found that the combination of the three strategies had the best effect, followed by the combination of the two strategies (Figures 17 and 18). The results show that the combined use of strategies can significantly reduce the editing efficiency of these eight nuclear genome off-target sites compared with the original DdCBE, and the combined use of three strategies is better than the combined use of two strategies. At the same time, in the statistical results of Figure 18, all exemplary optimization strategies proposed in this application can significantly improve the off-target impact of the DdCBE tool. The main structures of each construct corresponding to Figure 18 are shown in Table 9:

Table 9 Main components of each construct

Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all teachings that have been published, and these changes are within the protection scope of the present invention. . The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

A polypeptide with double-stranded DNA deaminase activity or a mutant thereof, the polypeptide or a mutant thereof comprising wild-type double-stranded DNA deaminase at positions corresponding to positions 1290-1427 of SEQ ID NO:1 Amino acid residues; and, compared with the amino acid residues at positions corresponding to positions 1290-1427 of SEQ ID NO:1 in the wild-type double-stranded DNA deaminase, the polypeptide or its mutant has the following mutations:

(1) The amino acid residue at the position corresponding to position 1308 of SEQ ID NO:1 is replaced by an alanine residue or an amino acid residue that is conservatively substituted relative to an alanine residue (for example, a glycine residue, a leucine residue) amino acid residue, isoleucine residue, valine residue) substitution; or,

(2) The amino acid residue at the position corresponding to position 1310 of SEQ ID NO:1 is an alanine residue or an amino acid residue that is conservatively substituted relative to an alanine residue (for example, a glycine residue, a leucine residue amino acid residues, isoleucine residues, valine residues) substitution;

Wherein, the mutant has at least 90%, for example, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity compared to the polypeptide with double-stranded DNA deaminase activity. or, having one or several (for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or Deleted; and, has double-stranded DNA deaminase activity; and,

The amino acid residues at positions corresponding to positions 1309, 1367 and 1368 of SEQ ID NO: 1 in the polypeptide or its mutants are not mutated;

Preferably, the wild-type double-stranded DNA deaminase has the amino acid sequence shown in SEQ ID NO: 1;

Preferably, the polypeptide with double-stranded DNA deaminase activity has an amino acid sequence as shown in SEQ ID NO: 3 or 5.
A mutated double-stranded DNA deaminase or variant thereof, which has the following mutations compared with wild-type double-stranded DNA deaminase:

(1) The amino acid residue at the position corresponding to position 1308 of SEQ ID NO:1 is replaced by an alanine residue or an amino acid residue that is conservatively substituted relative to an alanine residue (for example, a glycine residue, a leucine residue) amino acid residue, isoleucine residue, valine residue) substitution; or,

(2) The amino acid residue at the position corresponding to position 1310 of SEQ ID NO: 1 is conservatively replaced by an alanine residue or an amino acid residue relative to an alanine residue (for example, a glycine residue, a leucine residue Amino acid residues, isoleucine residues, valine residue) substitution;

Wherein, the variant has at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity compared to the mutated double-stranded DNA deaminase; Alternatively, having one or several (for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9) substitutions (preferably conservative substitutions), additions or deletions of amino acids; And, has double-stranded DNA deaminase activity; and

The amino acid residues at positions corresponding to positions 1309, 1367 and 1368 of SEQ ID NO: 1 in the mutated double-stranded DNA deaminase or its variants are not mutated;

Preferably, the amino acid sequence of the mutated double-stranded DNA deaminase or its variant at the position corresponding to positions 1290-1427 of SEQ ID NO: 1 is the amino acid sequence of the polypeptide of claim 1 or its mutant;

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

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

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

Wherein, the polypeptide polymer is formed by the polymerization of the N-terminal fragment and the C-terminal fragment;

Preferably, the N-terminal fragment and the C-terminal fragment do not have double-stranded DNA deaminase activity when each exists alone, or have significantly reduced deaminase activity (for example, the polypeptide of claim 1 At most 40%, at most 30%, at most 20%, at most 10%, at most 5% or at most 1% of the double-stranded DNA deaminase activity);

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

Preferably, the N-terminal fragment has the amino acid sequence shown in SEQ ID NO: 104 or 106;

Preferably, the C-terminal fragment has the amino acid sequence shown in SEQ ID NO:55.
The polypeptide polymer of claim 3, wherein the cleavage site is located in the polypeptide with double-stranded DNA deaminase activity or a mutant thereof immediately adjacent to the amino acid residue at the position corresponding to position 1397 of SEQ ID NO:1 subsequent peptide bonds;

Preferably, the N-terminal fragment has the amino acid sequence shown in 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 first DNA binding protein linked to the C-terminal fragment. a second DNA-binding protein connected to the terminal fragments;

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 the first polypeptide and the second polypeptide each independently further comprise a mitochondrial targeting sequence (MTS), and/or, a uracil glycosylase inhibitor (UGI). ) domain;

Preferably, said first polypeptide comprises the following structure: a first mitochondrial targeting sequence (MTS), said first DNA binding protein, said N-terminal fragment, and, said 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 glycosylation Enzyme inhibitor (UGI) domain;

Preferably, adjacent structures in the first polypeptide and the second polypeptide are each independently connected directly or through a linker (such as a peptide linker, such as one or more glycine (G) and/or serine (S)). ) flexible peptide) connection;

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 includes, in order from N-terminus to C-terminus: the first mitochondrial targeting sequence (MTS), the first DNA-binding protein, the N-terminal fragment, and the urinary protein. Pyrimidine glycosylase inhibitor (UGI) domain; the second polypeptide sequentially includes from the N-terminus to the C-terminus: 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 line Mitochondrial targeting sequences are each independently selected from COX8 (cytochrome C oxidase 8A subunit), ATP5G2 (ATP synthase F0 complex C2 subunit), SOD2 (superoxide dismutase 2), COQ8A (mitochondrial Atypical kinase COQ8A) mitochondrial targeting sequence;

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

Preferably, the first mitochondrial targeting sequence is a mitochondrial targeting sequence derived from SOD2; Preferably, the first mitochondrial targeting sequence has the amino acid sequence shown in SEQ ID NO: 9;

Preferably, the second mitochondrial targeting sequence is a mitochondrial targeting sequence derived from COX8; preferably, the second mitochondrial targeting sequence has the amino acid sequence shown in 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: TALE (transcription activator-like effector) protein, zinc finger protein and Cas proteins;

Preferably, the first DNA binding protein and the second DNA binding protein are the same or different;

Preferably, both the first DNA-binding protein and the second DNA-binding protein are 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 exit signal (NES) sequence;

Preferably, said NES sequence is linked to said first polypeptide or said second polypeptide directly or via a linker (eg a peptide linker, for example a flexible peptide comprising one or more glycine (G) and/or serine (S)). other domains in the peptide are connected;

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 and the second NES sequence are the same or different;

Preferably, the first NES sequence or the second NES sequence are each independently selected from the group consisting of Rev protein (HIV regulator of virion) and mitogen-activated protein kinase (MAPK) derived from HIV virus. , cellular tumor antigen p53 (cellular tumor antigen p53), ribosome transport protein NMD3 (60S ribosomal export protein NMD3); for example, the first NES sequence or the second NES sequence respectively has SEQ ID NO: 47 or The amino acid sequence shown in 56.
The polypeptide polymer of claim 10, wherein,

The first polypeptide sequentially includes from N-terminus to C-terminus:

(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, First NES sequence;

and / or,

The second polypeptide sequentially includes from N-terminus to C-terminus:

(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, second NES sequence;

Preferably, the first polypeptide includes in order from N-terminus to C-terminus: the first mitochondrial targeting sequence (MTS), the first DNA binding protein, the N-terminal fragment, the uracil sugar basalase inhibitor (UGI) domain, and, the first NES sequence; and/or, the second polypeptide sequentially includes from the N-terminus to the C-terminus: 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.
Isolated nucleic acid molecule encoding the polypeptide with 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, any one of claims 3-11 The first polypeptide defined in claim 3 or the second polypeptide defined in any one 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.
Preparation of 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, as defined in any one of claims 3-11 A method for a first polypeptide or a second polypeptide as defined in any one of claims 3 to 11, comprising culturing the host cell of claim 14 under conditions allowing protein expression, and from the cultured Recovering the polypeptide having double-stranded DNA deaminase activity or its mutant, the mutated double-stranded DNA deaminase or its variant, the first polypeptide or the second polypeptide from the host cell culture;

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

The second component comprises: a second polypeptide as defined in any one of claims 3-11 or a second polynucleotide encoding the 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 a or multiple nuclear localization signal (NLS) sequences and polypeptides capable of inhibiting double-stranded DNA deaminase activity;

Preferably, the fusion protein is capable of inhibiting the double-stranded DNA deaminase activity of the 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 directly or through a linker (such as a peptide linker, such as a flexible peptide containing one or more glycine (G) and/or serine (S)) with the enzyme capable of inhibiting double-stranded DNA deaminase activity. polypeptide linkage;

Preferably, the NLS sequence is selected from NLS sequences derived from simian vacuolating virus 40 (SV40), testis determinant (SRY), nucleoplasmin (Nuceloplasmin), and commonly used bipartite NLS (bpNLS);

Preferably, the polypeptide capable of inhibiting double-stranded DNA deaminase activity includes the minimal active domain of DddI A protein; for example, the polypeptide capable of inhibiting double-stranded DNA deaminase activity has the structure shown in SEQ ID NO: 60 of amino acid sequence;

Preferably, the fusion protein has the amino acid sequence shown in SEQ ID NO: 109 or 110.
A method for editing a target nucleotide sequence extracellularly, which includes, under conditions suitable for target nucleic acid editing, polymerizing the target nucleotide sequence with the polypeptide polymer of any one of claims 3-11 or claim 16 Contact with a composition to induce deamination of a target base in a target nucleotide sequence;

Preferably, the target base is cytosine;

Preferably, the method comprises contacting the target nucleotide sequence with the composition of claim 16, and the composition comprises a first component and a second component that are separate from each other, the first component comprising e.g. The 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; or, the method comprises converting the target The nucleotide sequence is contacted with the polypeptide polymer of any one of claims 3-11, said polypeptide polymer comprising a first polypeptide as defined in any one of claims 3-11 and a polypeptide as defined in any one of claims 3-11 a second polypeptide as defined in a paragraph;

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 side of the target base, and the second DNA-binding protein targets a second nucleotide sequence flanking the other side of the target base. sequence; thus, the first polypeptide and the second polypeptide can form a polypeptide polymer, thereby inducing deamination of the target base.
A method for editing a target nucleotide sequence in a cell, comprising delivering the polypeptide polymer of any one of claims 3-11 or the composition of claims 16 or 17 into a cell containing the target nucleotide sequence, thereby inducing deamination of the target base at the 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 that are 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 side of the target base, and the second DNA-binding protein targets a second nucleotide sequence flanking the other side of the target base. sequence; thereby, the first polypeptide and the second polypeptide can form a polypeptide polymer, thereby inducing deamination of the target base;

Preferably, the composition further comprises a third component 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 said first polypeptide is as defined in any one of claims 3-11. a polypeptide comprising a first DNA binding protein, the second polypeptide comprising a second DNA binding protein;

Preferably, the first DNA-binding protein targets a first nucleotide sequence flanking one side of the target base, and the second DNA-binding protein targets a second nucleotide sequence flanking the other side of the target base. sequence; 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 with 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, any one of claims 3-11 The polypeptide polymer, 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 the polypeptide polymer of any one of claims 3-11; Preferably, the kit further comprises a fusion protein or a polynucleotide encoding the fusion protein, wherein the fusion protein is such as as defined in claim 17.

Preferably, the kit comprises a composition according to claim 16 or 17.
The polypeptide having double-stranded DNA deaminase activity according to claim 1 or a mutant thereof, the mutated polypeptide according to claim 2 Double-stranded DNA deaminase or variant thereof, the polypeptide polymer of any one of claims 3-11, the first polypeptide as defined in any one of claims 3-11, as defined in any one of claims 3-11 The second polypeptide as defined in claim 12, 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 use in preparing an editing target nucleotide sequence Kits or uses for editing target nucleotide sequences.