Gene editing method and system based on strand displacement and application of gene editing method and system

Abstract

The invention relates to the field of gene engineering. The invention relates to a gene editing method and system based on strand displacement and application of the gene editing method and system. Specifically, the invention relates to a gene editing method based on Twinkle protein mediated strand displacement, a gene editing system based on strand displacement and application of the gene editing system.

Description

[0001] Priority and related applications

[0002] This invention claims priority to Chinese Patent Application No. 2024117535977, filed on December 1, 2024, entitled "Gene Editing Method, System and Use Thereof Based on Stroke Replacement". The entire contents of the above-cited patent application are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of genetic engineering. Specifically, this invention relates to a gene editing method based on Twinkle protein-mediated strand substitution, as well as a gene editing system utilizing said strand substitution and its applications. Background Technology

[0004] Precise editing of target nucleotide regions is an important research topic in the field of genetic engineering, and it has very promising application value for the development and application of genetics, especially in biomedical research and plant genetic improvement.

[0005] Currently, an ideal genome editing technology should have the following four characteristics: 1) high gene editing efficiency; 2) extremely low off-target activity; 3) broad editing capabilities at almost any site in the genome; and 4) the ability to perform different types of gene editing (base substitution, DNA insertion, DNA deletion, DNA replacement, etc.).

[0006] Early genome engineering tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR effector proteins (CRISPR / Cas), achieved gene editing by specifically cutting nucleotide sequences and causing DNA mutations at the cleavage site through non-homologous end joining (NHEJ) or by replacing the DNA around the cleavage site through targeted repair (HDR).

[0007] With technological advancements, base editing systems have emerged that utilize the deamination action of nucleoside deaminases to precisely edit target bases. Based on the type of bases targeted, base editing systems can be categorized into cytosine base editors (CBE) and adenine base editors (ABE). CBE editing converts CG base pairs in double-stranded DNA to TA base pairs; ABE editing converts AT base pairs in double-stranded DNA to GC base pairs.

[0008] However, the application of Base Editor is limited by factors such as the limited types of base substitutions, the "bystander effect" on non-target bases, and the fact that the editing site can only be 15±2 nucleotides upstream of the PAM sequence. In 2019, a guided editing system (Prime Editor) was developed. This system includes a fusion protein containing reverse transcriptase and Cas protein. It utilizes reverse transcriptase to reverse transcribe the template sequence, enabling all 12 possible base substitution types in the cellular genome. Nevertheless, the Prime Editor system still has problems with editing efficiency, limiting its application prospects in disease treatment, animal model establishment, and plant genetic breeding. Summary of the Invention

[0009] This invention proposes a strand substitution gene editing system (TwinkleEditor) based on the mitochondrial DNA helicase Twinkle. In human cells, Twinkle is a helicase belonging to the SF4 superfamily. Its basic biological function is as a helicase component in the mitochondrial DNA replication complex, participating in the replication and maintenance of mitochondrial DNA, and typically functioning in a multimeric form (e.g., hexamer). The biological processes involved in Twinkle in cells also include: translocation on DNA; DNA replication; DNA strand exchange; and DNA single-strand annealing. Under the action of Twinkle, this invention introduces a foreign invading DNA strand into the target nucleotide region through a series of strand substitution reactions, thereby achieving precise DNA editing.

[0010] The present invention provides the following specific technical solutions:

[0011] [1]. A method for performing strand substitution gene editing in a DNA sequence, the method comprising:

[0012] The double-stranded DNA sequence is brought into contact with a sequence-specific nuclease, thereby creating a nick in the double-stranded DNA sequence;

[0013] The DNA is digested along the cut using exonuclease, thereby producing free single-stranded DNA.

[0014] The free single-stranded DNA is contacted with invader DNA and the human mitochondrial DNA helicase Twinkle; wherein the invader DNA is single-stranded or double-stranded DNA, and at least one strand of the invader DNA contains a glue point complementary to the free single-stranded DNA and a displacement template containing the desired nucleotide changes.

[0015] This allows the adhesion sites of the invading DNA to pair complementaryly with free single-stranded DNA;

[0016] This causes the template of the invading DNA to undergo a strand displacement reaction with the endogenous DNA strand near the nick site;

[0017] DNA repair / replication, thereby introducing the desired nucleotide changes into the double-stranded DNA sequence.

[0018] [2]. According to the method described in [1], the DNA repair process includes: (1) performing DNA repair using a replacement template as a template to form desired nucleotide change products on two DNA strands; and (2) repairing DNA nicks.

[0019] [3]. According to the method of [1], the DNA repair process further includes, after the strand displacement reaction, using a structure-specific nuclease to remove the endogenous DNA strand adjacent to the nick site; wherein the structure-specific nuclease is preferably FEN1.

[0020] [4]. According to the method of [1], the sequence-specific nuclease is selected from CRISPR-related protein (Cas) polypeptides, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), broad-spectrum nucleases, sequence-specific endonucleases or their variants, fragments or combinations thereof that have cleavage enzyme activity.

[0021] [5]. According to the method of [4], wherein the Cas polypeptide is selected from Cas3, Cas4, Cas5, Cas5e, Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cast10d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1, Cse2, Cse3, Cse4, C sc1, Csc2, Csa5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, TranC and their variants, fragments or any combination thereof.

[0022] [6]. According to the method of [1], the sequence-specific nuclease comprises a mutant corresponding to the D10A mutation or H840A mutation of the amino acid sequence shown in SEQ ID NO: 1.

[0023] [7]. The method according to [1], wherein the Twinkle enzyme is a natural or truncated Twinkle enzyme; the Twinkle enzyme contains a carboxyl-terminal domain (CTD) of the amino acid sequence shown in SEQ ID NO: 2.

[0024] [8]. The method according to [7], wherein the Twinkle enzyme removes the mitochondrial localization signal and, after removal, fuses a nuclear localization signal (NLS) at its amino acid or / and carboxyl terminus.

[0025] [9]. According to the method of [1], the exonuclease is a 3' exonuclease or a 5' exonuclease or a combination thereof; wherein the 3' exonuclease is preferably Trex2; and the 5' exonuclease is preferably mExoI or T5 exonuclease.

[0026]

[10] . According to the method of [1], the desired nucleotide change includes substitution, insertion or deletion of a single or multiple nucleotides.

[0027]

[11] . A strand substitution gene editing system comprising components of a strand substitution editing system and / or an expression construct containing nucleotide sequences encoding components of the strand substitution editing system, the components of the strand substitution gene editing system comprising:

[0028] i) A sequence-specific nuclease or its domain thereof, wherein the sequence-specific nuclease or its domain thereof is guided to target DNA by a guide RNA (sgRNA) sequence;

[0029] ii) Exonucleases or their domains;

[0030] iii) Twinkle enzyme, a human mitochondrial DNA helicase, or its domain;

[0031] iv) Single-stranded or double-stranded invading DNA, wherein at least one strand of the invading DNA contains a glue point complementary to free single-stranded DNA produced by nicking enzymes and exonucleases, and a displacement template containing the desired nucleotide changes.

[0032] The components are independent of each other, or at least two components are connected by a connector to form a fusion protein.

[0033]

[12] . The strand substitution gene editing system according to

[11] , wherein the components of the strand substitution gene editing system further include:

[0034] v) Structure-specific nucleases;

[0035] The structure-specific nuclease is preferably selected from FEN1.

[0036]

[13] . According to the chain substitution gene editing system described in

[11] , wherein the sequence-specific nuclease is selected from CRISPR-associated protein (Cas) polypeptides, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), broad-spectrum nucleases, sequence-specific endonucleases or their variants, fragments or combinations thereof that have nuclease activity.

[0037]

[14] . According to the chain substitution gene editing system described in

[11] , wherein the sequence-specific nuclease is a CRISPR-associated protein (Cas) polypeptide with cleavage enzyme activity; and the components of the chain substitution gene editing system further include: vi) guide RNA that guides the corresponding Cas polypeptide to the target nucleotide region.

[0038]

[15] . According to the chain substitution gene editing system described in

[14] , wherein the Cas polypeptide is selected from Cas3, Cas4, Cas5, Cas5e, Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cast10d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1, Cse2, Cse3, Cs e4, Csc1, Csc2, Csa5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, TranC and their variants, fragments or any combination thereof.

[0039]

[16] . According to the chain substitution gene editing system of

[11] , wherein the sequence-specific nuclease comprises a mutant corresponding to the D10A mutation or the H840A mutation of the amino acid sequence shown in SEQ ID NO: 1.

[0040]

[17] . The chain substitution gene editing system according to

[11] , wherein the Twinkle enzyme is a natural or truncated Twinkle enzyme; the Twinkle enzyme contains a carboxyl-terminal domain (CTD) of the amino acid sequence shown in SEQ ID NO: 2.

[0041]

[18] . According to the strand substitution gene editing system described in

[11] , the exonuclease is a 3' exonuclease or a 5' exonuclease or a combination thereof; wherein the 3' exonuclease is preferably Trex2; and the 5' exonuclease is preferably mExoI or T5 exonuclease.

[0042]

[19] . A chain substitution gene editing system according to any one of

[11] -

[18] , wherein at least one of the components has an amino or carboxyl terminus fused with a nuclear localization signal (NLS).

[0043]

[20] . A chain substitution gene editing system according to any one of

[11] -

[18] , wherein the linker comprises an amino acid sequence (GGGS)n, (GGGGS)n, (G)n, (EAAAK)n, (GGS)n, (SGGS)n, SGSETPGTSESATPES, or (XP)n motif or a combination thereof, wherein n is independently an integer from 1 to 30, and wherein X is any amino acid.

[0044]

[21] . A chain substitution gene editing system according to any one of

[11] -

[18] , wherein the components of the system are recruited by a recruitment system selected from the MCP-MS2 system or the GCN4-ScFv system.

[0045]

[22] . A host cell comprising any one of the chain substitution gene editing systems described in

[11] -

[21] .

[0046]

[23] . A method for producing at least one genetically modified cell, wherein the method comprises editing at least one of the cells using any one of [1-10], or introducing a strand substitution gene editing system of any one of

[11] -

[21] into at least one of the cells, thereby causing substitution, insertion or deletion of one or more nucleotides in a target nucleotide editing region in at least one of the cells.

[0047]

[24] . The method according to

[23] further includes the step of screening cells from the at least one cell for cells having one or more desired nucleotide substitutions.

[0048]

[25] . The method according to any one of

[23] -

[24] , wherein the cells are derived from prokaryotes such as bacteria; eukaryotes such as plants, fungi or vertebrates.

[0049] 26. The method according to

[25] is characterized in that the vertebrate is a mammal such as a human, mouse, rat, monkey, dog, pig, sheep, cow, or cat.

[0050]

[27] . The method according to

[25] is characterized in that the plant is a crop plant, such as wheat, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava or potato.

[0051]

[28] . Use of a method for strand substitution gene editing in a DNA sequence as described in any one of [1]-

[10] or a strand substitution gene editing system as described in any one of

[11] -

[21] , wherein the use includes:

[0052] a) Gene or genome editing;

[0053] b) Targeted nuclear nucleotide detection and / or diagnosis;

[0054] c) Editing target nucleotide sequences to modify biological or non-human organisms;

[0055] d) Treatment of diseases.

[0056]

[29] . A kit comprising the chain substitution gene editing system described in any one of

[11] -

[21] or the host cell described in

[22] .

[0057] The effects of the invention

[0058] Compared to the Prime Editor system, the Twinkle Editor directly replaces exogenous DNA sequences without reverse transcription, thereby improving gene editing efficiency and reducing inaccurate editing caused by errors in reverse transcription. Compared to the BaseEditor system, the Twinkle Editor can perform more types of editing, including all 12 types of base substitution, DNA insertion, and DNA deletion. In addition, the invading DNA strand in the Twinkle Editor contains a glue point complementary to the upstream sequence of the editing region. The strand substitution reaction only occurs when the glue point is complementary to the target nucleotide. That is, the Twinkle Editor contains two sets of recognition sequences that recognize the target nucleotide region, thus exhibiting lower off-target activity compared to existing gene editing systems. Attached Figure Description

[0059] Figure 1 : Schematic diagram of a chain substitution gene editing system;

[0060] Figure 2 : Schematic diagram of the Twinkle Editor system component structure;

[0061] Figure 3 The Twinkle Editor system's editing efficiency in base substitution;

[0062] Figure 4 Editing efficiency of the Twinkle Editor system recruited via MCP-MS2 in precise DNA insertion;

[0063] Figure 5 The editing efficiency of the Twinkle Editor system for DNA deletion in double-stranded invading DNA templates. Detailed Implementation

[0064] To enrich the toolbox of gene editing systems, a gene editing method based on Twinkle protein-mediated strand substitution is presented, along with a gene editing system utilizing this strand substitution and its applications. This editing system enables efficient and precise editing of target nucleotide sequences.

[0065] In one aspect of the present invention, a method for strand substitution gene editing in a DNA sequence is provided, the method comprising:

[0066] The double-stranded DNA sequence is brought into contact with a sequence-specific nuclease, thereby creating a nick in the double-stranded DNA sequence;

[0067] The DNA is digested along the cut using exonuclease, thereby producing free single-stranded DNA.

[0068] The free single-stranded DNA is contacted with invader DNA and human mitochondrial DNA helicase Twinkle; wherein the invader DNA is single-stranded or double-stranded DNA, and at least one strand of the invader DNA contains a glue point complementary to the free single-stranded DNA and a displacement template containing the desired nucleotide changes.

[0069] This allows the adhesion sites of the invading DNA to pair complementaryly with free single-stranded DNA;

[0070] This causes the template of the invading DNA to undergo a strand displacement reaction with the endogenous DNA strand near the nick site;

[0071] DNA repair / replication, thereby introducing the desired nucleotide changes into the double-stranded DNA sequence.

[0072] In some embodiments, the DNA repair process of the present invention includes: (1) performing DNA repair using a replacement template as a template to form desired nucleotide change products on both DNA strands; and (2) repairing DNA nicks.

[0073] In some embodiments, the DNA repair process of the present invention further includes, after the strand displacement reaction, using a structure-specific nuclease to excise the endogenous DNA strand adjacent to the nick site; wherein the structure-specific nuclease is preferably FEN1.

[0074] In some embodiments, the sequence-specific nuclease of the present invention is selected from CRISPR-associated protein (Cas) polypeptides, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), broad-spectrum nucleases, sequence-specific endonucleases, or variants, fragments, and combinations thereof that have cleavage enzyme activity.

[0075] In some embodiments, the Cas peptides of the present invention are selected from Cas3, Cas4, Cas5, Cas5e, Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cast10d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1, Cse2, Cse3, Cse4, and Cs. c1, Csc2, Csa5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, TranC and their variants, fragments or any combination thereof.

[0076] In some embodiments, the sequence-specific nuclease of the present invention comprises a mutant of the D10A mutation or the H840A mutation corresponding to the amino acid sequence of SEQ ID NO: 1.

[0077] In some embodiments, the Twinkle enzyme of the present invention is a natural or truncated Twinkle enzyme; said Twinkle enzyme comprises a carboxyl-terminal domain (CTD) of the amino acid sequence of SEQ ID NO: 2.

[0078] In some embodiments, the Twinkle enzyme of the present invention removes the mitochondrial localization signal and, upon removal, fuses a nuclear localization signal (NLS) at its amino acid and / or carboxyl terminus.

[0079] In some embodiments, the exonuclease of the present invention is a 3' exonuclease or a 5' exonuclease or a combination thereof; wherein the 3' exonuclease is preferably Trex2; and the 5' exonuclease is preferably mExoI or T5 exonuclease.

[0080] In some embodiments, the desired nucleotide changes of the present invention include substitution, insertion, or deletion of a single or multiple nucleotides.

[0081] In another aspect, the present invention provides a strand substitution gene editing system comprising components of a strand substitution editing system and / or an expression construct containing nucleotide sequences encoding components of the strand substitution editing system, wherein the components of the strand substitution gene editing system include:

[0082] i) Sequence-specific nucleases or their domains;

[0083] ii) Exonucleases or their domains;

[0084] iii) Human mitochondrial DNA helicase Twinkle or its domain;

[0085] iv) Single-stranded or double-stranded invading DNA, wherein at least one strand of the invading DNA contains a glue point complementary to the free single-stranded DNA produced by the action of nicking enzymes and exonucleases, and a displacement template containing the desired nucleotide changes.

[0086] The components are independent of each other, or at least two components are connected by a connector to form a fusion protein.

[0087] In some embodiments, the components of the strand displacement gene editing system of the present invention further include:

[0088] v) Structure-specific nucleases;

[0089] The structure-specific nuclease is preferably selected from FEN1.

[0090] In some embodiments, the sequence-specific nuclease of the present invention is selected from CRISPR-associated protein (Cas) polypeptides, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), broad-spectrum nucleases, sequence-specific endonucleases, or variants, fragments, and combinations thereof that have cleavage enzyme activity.

[0091] In some embodiments, the sequence-specific nuclease of the present invention is a CRISPR-associated protein (Cas) polypeptide with nickase activity; and the components of the strand substitution gene editing system further include: vi) guide RNA that guides the corresponding Cas polypeptide to the target nucleotide region.

[0092] In some embodiments, the Cas peptides of the present invention are selected from Cas3, Cas4, Cas5, Cas5e, Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cast10d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1, Cse2, Cse3, Cse4, and Cs. c1, Csc2, Csa5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, TranC and their variants, fragments or any combination thereof.

[0093] In some embodiments, the sequence-specific nuclease of the present invention comprises a mutant of the D10A mutation or the H840A mutation corresponding to the amino acid sequence of SEQ ID NO: 1.

[0094] In some embodiments, the Twinkle enzyme of the present invention is a natural or truncated Twinkle enzyme; said Twinkle enzyme comprises a carboxyl-terminal domain (CTD) of the amino acid sequence of SEQ ID NO: 2.

[0095] In some embodiments, the exonuclease of the present invention is a 3' exonuclease or a 5' exonuclease or a combination thereof; wherein the 3' exonuclease is preferably Trex2; and the 5' exonuclease is preferably mExoI or T5 exonuclease.

[0096] In some embodiments, at least one component of the present invention has a nuclear localization signal (NLS) fused to its amino or carboxyl terminus. In some embodiments, the NLS comprises the amino acid sequence KKRKV (SEQ ID NO: 16), PKKKRKV (SEQ ID NO: 17), KRPAATKKAGQAKKKK (SEQ ID NO: 18), KRTADGSEFESPKKKRKV (SEQ ID NO: 19), or DSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 20).

[0097] In some embodiments, the linker of the present invention comprises an amino acid sequence (GGGS)n (SEQ ID NO: 21), (GGGGS)n (SEQ ID NO: 22), (G)n, (EAAAK)n (SEQ ID NO: 23), (GGS)n, (SGGS)n (SEQ ID NO: 24), SGSETPGTSESATPES (SEQ ID NO: 25), or (XP)n motif or a combination thereof, wherein n is independently an integer from 1 to 30, and wherein X is any amino acid.

[0098] In some embodiments, the components of the system of the present invention are recruited by a recruitment system selected from the MCP-MS2 system or the GCN4-ScFv system.

[0099] In another aspect of the invention, a host cell is provided, wherein the host cell comprises the strand substitution gene editing system of the invention.

[0100] In another aspect of the invention, the invention provides a method for generating at least one genetically modified cell, wherein the method comprises editing at least one cell using the method of the invention, or introducing the strand substitution gene editing system of the invention into at least one cell, thereby causing substitution, insertion or deletion of one or more nucleotides in a target nucleotide editing region in at least one cell.

[0101] In some embodiments, the method of the present invention further includes the step of screening cells from at least one cell for cells having one or more desired nucleotide substitutions.

[0102] In some embodiments, the cells of the present invention are derived from prokaryotes such as bacteria; eukaryotes such as plants, fungi, or vertebrates.

[0103] In some embodiments, the vertebrates of the present invention are mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, and cats.

[0104] In some embodiments, the plant of the present invention is a crop plant, such as wheat, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava, or potato.

[0105] In another aspect of the present invention, a method for performing strand substitution gene editing in a DNA sequence or a strand substitution gene editing system of the present invention is provided for use, wherein the use includes:

[0106] a) Gene or genome editing;

[0107] b) Targeted nuclear nucleotide detection and / or diagnosis;

[0108] c) Editing target nucleotide sequences to modify biological or non-human organisms;

[0109] d) Treatment of the disease.

[0110] In another aspect of the invention, a kit is provided, wherein the kit comprises the strand displacement gene editing system of the invention or the host cell of the invention.

[0111] The gene editing system and method for producing genetically modified cells of the present invention are particularly suitable for genetic modification of plants. Preferably, the plant is a crop plant, including but not limited to wheat, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava, and potato. More preferably, the plant is rice.

[0112] In another aspect, the present invention provides a method for producing genetically modified plants, comprising introducing the gene editing system of the present invention into at least one of the plants, thereby causing substitution of one or more nucleotides in a target nucleic acid region in the genome of the at least one plant.

[0113] In some embodiments, the method further includes screening from the at least one plant for plants having one or more desired nucleotide substitutions.

[0114] In the method of this invention, the gene-editing composition can be introduced into plants using various methods well known to those skilled in the art. Methods for introducing the gene-editing system of this invention into plants include, but are not limited to: gene gun method, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube pathway method, and ovary injection method. Preferably, the gene-editing composition is introduced into plants via transient transformation.

[0115] In the method of this invention, modification of the target sequence can be achieved simply by introducing or generating the gene-editing fusion protein and guide RNA in plant cells, and the modification can be stably inherited without the need for stable transformation of plants with exogenous polynucleotides encoding components of the gene-editing system. This avoids the potential off-target effects of a stably existing (continuously generated) gene-editing composition and also avoids the integration of exogenous nucleotide sequences into the plant genome, thus providing higher biosafety.

[0116] In some preferred embodiments, the introduction is performed in the absence of selection pressure, thereby avoiding the integration of exogenous nucleotide sequences into the plant genome.

[0117] In some embodiments, the introduction includes converting the gene editing system of the present invention into isolated plant cells or tissues, and then regenerating the converted plant cells or tissues into complete plants. Preferably, the regeneration is performed without selection pressure, that is, without using any selection agents targeting the selection genes carried on the expression vector during tissue culture. Not using selection agents can improve the regeneration efficiency of the plants, resulting in modified plants free of exogenous nucleotide sequences.

[0118] In other embodiments, the gene-editing system of the present invention can be transformed into specific parts of a whole plant, such as leaves, shoot tips, pollen tubes, young spikelets, or hypocotyls. This is particularly suitable for the transformation of plants that are difficult to regenerate through tissue culture.

[0119] In some embodiments of the present invention, in vitro expressed proteins and / or in vitro transcribed RNA molecules (e.g., the expression construct is an in vitro transcribed RNA molecule) are directly transformed into the plant. The proteins and / or RNA molecules enable gene editing in plant cells and are subsequently degraded by the cells, avoiding the integration of exogenous nucleotide sequences into the plant genome.

[0120] Therefore, in some embodiments, using the methods of the present invention to genetically modify and breed plants can yield plants whose genomes are free of foreign polynucleotide integration, i.e., non-transgene-free modified plants.

[0121] In some embodiments of the invention, the modified target nucleic acid region is associated with plant traits such as agronomic traits, whereby the substitution of one or more nucleotides results in the plant having altered (preferably improved) traits, such as agronomic traits, relative to the wild-type plant.

[0122] In some embodiments, the method further includes the step of screening plants having one or more desired nucleotide substitutions and / or desired traits such as agronomic traits.

[0123] In some embodiments of the invention, the method further includes obtaining offspring of the genetically modified plant. Preferably, the genetically modified plant or its offspring has one or more desired nucleotide substitutions and / or desired traits such as agronomic traits.

[0124] In another aspect, the present invention also provides genetically modified plants or their offspring or portions thereof, wherein said plants are obtained by the methods described above. In some embodiments, the genetically modified plants or their offspring or portions thereof are non-GMO. Preferably, the genetically modified plants or their offspring have the desired genetic modification and / or desired traits such as agronomic traits.

[0125] In another aspect, the present invention also provides a plant breeding method, comprising crossing a genetically modified first plant, obtained by the method described above, containing one or more nucleotide substitutions in a target nucleic acid region, with a second plant not containing the one or more nucleotide substitutions, thereby introducing the one or more nucleotide substitutions into the second plant. Preferably, the genetically modified first plant has desired traits such as agronomic traits.

[0126] This invention also covers the application of the gene editing system of this invention in disease treatment.

[0127] By modifying disease-related genes using the gene editing system of this invention, it is possible to achieve upregulation, downregulation, inactivation, activation, or mutation correction of disease-related genes, thereby achieving disease prevention and / or treatment. For example, the target nucleic acid region described in this invention can be located within the protein-coding region of the disease-related gene, or, for example, within gene expression regulatory regions such as promoter regions or enhancer regions, thereby enabling modification of the function or expression of the disease-related gene. Therefore, the modification of disease-related genes described herein includes modification of the disease-related gene itself (e.g., protein-coding region), as well as modification of its expression regulatory regions (e.g., promoters, enhancers, introns, etc.).

[0128] "Disease-associated" genes are any genes that produce transcriptional or translational products at abnormal levels or in abnormal forms in cells derived from tissues affected by a disease, compared to tissues or cells from non-disease control groups. In cases where altered expression is associated with the onset and / or progression of the disease, it can be a gene expressed at abnormally high levels; it can also be a gene expressed at abnormally low levels. Disease-associated genes also refer to genes with one or more mutations or genetic variations that are directly responsible for or linked to one or more genes responsible for the etiology of the disease in disequilibrium. Such mutations or genetic variations are, for example, single nucleotide variants (SNVs). The transcribed or translated products can be known or unknown and can be at normal or abnormal levels.

[0129] Therefore, the present invention also provides a method for treating a disease in a subject of need, comprising delivering an effective amount of the gene editing system of the present invention to the subject to modify a gene associated with the disease (e.g., deamination of mitochondrial DNA via a fusion protein or multiple fusion proteins). The present invention also provides the use of the gene editing system in the preparation of a pharmaceutical composition for treating a disease in a subject of need, wherein the gene editing system is used to modify a gene associated with the disease. The present invention also provides a pharmaceutical composition for treating a disease in a subject of need, comprising the gene editing system of the present invention, and optionally a pharmaceutically acceptable vector, wherein the gene editing system is used to modify a gene associated with the disease.

[0130] In some embodiments, the fusion protein or gene editing system described in this invention is used to introduce point mutations into nucleic acids by deaminating a target nucleobase (e.g., an A residue). In some embodiments, the deamination of the target nucleobase results in the correction of a genetic defect, such as in the correction of a point mutation that results in loss of function in a gene product. In some embodiments, the genetic defect is associated with a disease or condition (e.g., lysosomal storage disease or metabolic disease, such as, for example, type 1 diabetes). In some embodiments, the methods provided herein can be used to introduce inactive point mutations into a gene or allele encoding a gene product associated with a disease or condition.

[0131] In some embodiments, the purpose of the schemes described in this invention is to restore the function of dysfunctional genes via genome editing. The nuclear gene-editing proteins provided herein are intended for use in vitro gene editing in human cells, such as correcting disease-related mutations in human cell cultures. The nuclear gene-editing proteins provided herein, such as fusion proteins containing nucleic acid-editable DNA proteins (e.g., CRISPR effector protein Cas9) and adenosine deaminase domains, can be used to correct any single-point G to A or C to T mutation. In the first case, mutant A is corrected through deamination, while in the latter case, A paired with mutant T is corrected through deamination and a subsequent round of replication.

[0132] In some embodiments, the purpose of the schemes described in this invention is to treat diseases associated with or caused by point mutations, which can be corrected by the DNA gene-editing fusion protein provided herein. In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is a neonatal disease. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a lysosomal storage disease.

[0133] In some embodiments, the purposes of the solutions described in this invention are for the treatment of mitochondrial diseases or disorders. As used herein, "mitochondrial disease" refers to diseases caused by abnormal mitochondria, such as mitochondrial gene mutations, enzyme pathways, etc. Examples of diseases include, but are not limited to: neurological disorders, loss of motor control, muscle weakness and pain, gastrointestinal disorders and dysphagia, poor growth, heart disease, liver disease, diabetes, respiratory complications, epilepsy, visual / hearing problems, lactic acidosis, developmental delay, and susceptibility to infection.

[0134] Examples of diseases described in this invention include, but are not limited to, genetic diseases, circulatory system diseases, muscle diseases, brain, central nervous system and immune system diseases, Alzheimer's disease, secretase disorders, amyotrophic lateral sclerosis (ALS), autism, trinucleotide repeat amplification disorders, hearing disorders, gene-targeted therapy of non-dividing cells (neurons, muscles), liver and kidney diseases, epithelial cell and lung diseases, cancer, Usher syndrome or retinitis pigmentosa-39, cystic fibrosis, HIV and AIDS, β-thalassemia, sickle cell disease, herpes simplex virus, autism, drug addiction, age-related macular degeneration, and schizophrenia. Other diseases that can be treated by correcting point mutations or introducing inactive mutations into disease-related genes are known to those skilled in the art, and therefore this disclosure is not limited in this respect. In addition to the diseases exemplarily described in this invention, other related diseases can also be treated with the strategies and fusion proteins provided by this invention, and this application will be apparent to those skilled in the art. The diseases or targets to which this invention can be applied are related to the gene editing systems listed in WO2015089465A1 (PCT / US2014 / 070135), WO2016205711A1 (PCT / US2016 / 038181), WO2018141835A1 (PCT / EP2018 / 052491), WO2020191234A1 (PCT / US2020 / 023713), WO2020191233A1 (PCT / US2020 / 023712), WO2019079347A1 (PCT / US2018 / 056146), and WO2021155065A1 (PCT / US2021 / 015580).

[0135] The administration of the gene-editing system or pharmaceutical composition of the present invention can be tailored to the patient's or subject's weight and species. The frequency of administration is within medically or veterinary limits. It depends on conventional factors including the patient's or subject's age, sex, general health condition, other conditions, and the specific symptom or condition being addressed.

[0136] The present invention also includes a kit for use with the methods of the present invention, the kit comprising the genome editing system of the present invention, and instructions for use. The kit generally includes a label indicating the intended use and / or method of use of the kit contents. Terminology labels include any written or documented material provided on or with the kit or otherwise accompanied by the kit.

[0137] definition

[0138] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. For example, the standard recombinant DNA and molecular cloning techniques used in this invention are well known to those skilled in the art and are described more fully in the following literature: Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook"). Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0139] The term "genome," as used in this article, encompasses not only chromosomal DNA located in the cell nucleus but also organelle DNA located in subcellular components of the cell, such as mitochondria and plastids.

[0140] As used herein, “organism” includes any organism suitable for genome editing, preferably eukaryotes. Examples of organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, and cats; poultry such as chickens, ducks, and geese; and plants including monocots and dicots such as rice, corn, wheat, sorghum, barley, soybeans, peanuts, and Arabidopsis thaliana.

[0141] "Genetically modified organism" or "genetically modified cell" refers to an organism or cell whose genome contains exogenous polynucleotides or modified genes or expression regulatory sequences. For example, exogenous polynucleotides can be stably integrated into the genome of an organism or cell and inherited across generations. Exogenous polynucleotides can be integrated into the genome alone or as part of a recombinant DNA construct. Modified genes or expression regulatory sequences are sequences in the genome of an organism or cell that contain single or multiple deoxynucleotide substitutions, deletions, and additions.

[0142] In relation to a sequence, “exogenous” means a sequence that originates from a foreign species, or, if from the same species, a sequence whose composition and / or loci have been significantly altered from its natural form through deliberate human intervention.

[0143] The terms “polynucleotide,” “nucleic acid sequence,” “nucleotide sequence,” or “nucleic acid fragment” are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or modified nucleotide bases. Nucleotides are designated by their single-letter names as follows: “A” for adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), “C” for cytidine or deoxycytidine, “G” for guanosine or deoxyguanosine, “U” for uridine, “T” for deoxythymidine, “R” for purine (A or G), “Y” for pyrimidine (C or T), “K” for G or T, “H” for A, C, or T, “I” for inosine, and “N” for any nucleotide.

[0144] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this invention to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0145] As used in this invention, "expression construct" refers to a vector, such as a recombinant vector, suitable for expressing a nucleotide sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, the expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (e.g., transcription to generate mRNA or functional RNA) and / or the translation of RNA into a precursor or mature protein.

[0146] The "expression construct" of the present invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, a translatable RNA (such as mRNA).

[0147] The "expression construct" of the present invention may contain regulatory sequences and nucleotide sequences of interest from different sources, or regulatory sequences and nucleotide sequences of interest from the same source but arranged in a manner different from those normally found in nature.

[0148] Example

[0149] To facilitate understanding of the present invention, a more complete description will be given below with reference to specific embodiments and accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0150] Example 1: Obtaining the Twinkle Editor gene editing system

[0151] The inventors constructed a Twinkle editor comprising the following protein elements: (1) a nickase nCas9: Cas9 (D10A / H840A) protein (SEQ ID NO: 3 or 4); (2) a human Twinkle protein (SEQ ID NO: 2) with 42 amino acids removed from its amino terminus (encoding a mitochondrial localization signal); (3) an exonuclease (SEQ ID NO: 5-7); and (4) a FEN1 protein (SEQ ID NO: 8). One to two nuclear localization signal (NLS) sequences were fused to the ends of each protein element. The encoding nucleotide sequences of each protein element were cloned into a pCMV vector suitable for expression in human cells for protein expression. Cas9 (H840A) was guided to the target DNA by sgRNA, and suitable invading DNA was synthesized in vitro according to the desired editing type. The invading DNA contained a glue point sequence complementary to the free single-stranded DNA of the target DNA and a displacement template sequence containing the desired nucleotide changes. The above components constitute the Twinkle Editor gene editing system of this invention. A schematic diagram of an exemplary free-state Twinkle Editor is shown below. Figure 2 As shown, its working principle is as follows Figure 1 As shown.

[0152] Example 2: Human Cell Genome Editing

[0153] Using the gene editing system obtained in Example 1, the protein elements selected were (1) Cas9 (H840A) protein (SEQ ID NO: 4); (2) human Twinkle protein (SEQ ID NO: 2) with 42 amino acids removed from its amino terminus (encoding a mitochondrial localization signal); (3) 3' exonuclease Trex2 (SEQ ID NO: 5); and (4) FEN1 protein (SEQ ID NO: 8), wherein the N-terminus and C-terminus of nCas9 and Twinkle proteins were fused with NLS sequences, and the N-terminus of Trex2 and FEN1 was fused with NLS sequences. An Invader DNA sequence was designed for editing the human HEK3 gene (HGNC ID: 10747), the target sequence for sgRNA was GGCCCAGACTGAGCACGTGATGG (SEQ ID NO: 26), and the Invader DNA sequence designed for TA base substitution was: (SEQ ID NO: 27), where lowercase letters represent the nucleotide sequences of the adhesive sites of the Invader DNA, uppercase letters represent the nucleotide sequences of the template replacement, and underlined letters represent the target editable nucleotide sequences to be introduced. The coding plasmids of each component of the gene editing system (pCMV-NLS-Cas9(H840A)-NLS; pCMV-NLS-hTwinkle) Δ1-42aa HEK293T cells were transfected with nCas9-NLS, pCMV-NLS-Trex2-NLS, and pCMV-NLS-FEN1-NLS, sgRNA, and Invader DNA. Template sequences containing only nCas9 and Invader DNA were selected as the control group for the nicking enzyme system. DNA was extracted after 72 hours, and analysis of the target site using next-generation sequencing revealed the expected gene editing event at the target site, indicating that the gene editing system described in this invention can achieve effective gene editing. Figure 3 ).

[0154] Example 3: Introduction of MS2-MCP to recruit Twinkle protein in human cell genome editing

[0155] This embodiment further optimizes the Twinkle system in Example 2 by introducing an MS2-MCP recruitment system to recruit each element in the Twinkle system near the target site. The difference between this and the Twinkle Editor system in Example 2 is that the MCP protein is fused between the Twinkle and its N-terminal NLS sequence, and two MS2 adaptor RNAs are introduced into the sgRNA. The sgRNA guides Cas9 (H840A) to the target DNA, and the MS2 on the sgRNA recruits the Twinkle protein fused with the MCP protein.

[0156] An Invader DNA sequence was designed for DNA insertion, specifically for editing the human RNF2 gene (HGNC ID: 10061). The target sequence for the sgRNA is GTCATCTTAGTCATTACCTGAGG. The designed Invader DNA sequence is as follows: (SEQ ID NO: 28), where lowercase letters represent the nucleotide sequence of the adhesive site of the Invader DNA, uppercase letters represent the nucleotide sequence of the template replacement, and underlined letters represent the target editing nucleotide sequence to be introduced. The coding plasmids of each component of the gene editing system and the Invader DNA were transfected into HEK293T cells, and a template sequence containing nCas9 and Invader DNA recruited by MS2-MCP was selected as the control group for the nicking enzyme system. DNA was extracted after 72 hours, and analysis of the target site using next-generation sequencing revealed that the expected gene editing event occurred at the target site, and the three bases CCT were successfully inserted at the target site, indicating that the gene editing system of this invention can achieve effective gene editing. Figure 4 ).

[0157] Example 4: Human cell genome editing using double-stranded DNA as Invader DNA

[0158] This embodiment further optimizes the Invader DNA. In this embodiment, the inventors tested using double-stranded DNA as the Invader DNA for Twinkle editor editing. The difference between this and the MS2-MCP-recruited Twinkle Editor system in Example 3 is that the Invader DNA used is a double-stranded Invader DNA sequence obtained through PCR amplification.

[0159] Designed for base deletion editing of the human RUNX1 gene (HGNC ID: 10471), the target sequence is... (SEQ ID NO: 29), where the underlined letters indicate sequences expected to be deleted by the Twinkle editor. The forward sequence of the amplified double-stranded Invader DNA is: CTAGAGGGGTGAGGCTGAAACAGTGACCTGTCTTGGTTTTCGCTCCGAAGGTAAAAGAAATCATTGAGTCCCCCGCCTTCAGAAGAGGGTGCATTTTCAGGAGGAAGGATGGCTTCAGACAGCATATTTGAGTCATTTCCTTCGTACCCACAGTGCTTCATGAGAGGTGAGTACATGCTGGTCTTGTAATATCTACTTTTGCTCAGCTTTGCCTGTAATGAAATGGCAGCTTGTTTCACCTCGGTGCAGAGATGCCTCGGTGCCTGCCAGTTCCCTGTCTTGTTTGT (SEQ ID NO: 29) NO: 30), complementary sequence is ACAAACAAGACAGGGAACTGGCAGGCACCGAGGCATCTCTGCACCGAGGTGAAACAAGCTGCCATTTCATTACAGGCAAAGCTGAGCAAAAGTAGATATTACAAGACCAGCATGTACTCACCTCTCATGAAGCACTGTGGGTACGAAGGAAATGACTCAAATATGCTGTCTGAAGCCATCCTTCCTCCTGAAAATGCACCCTCTTCTGAAGGCGGGGACTCAATGATTTCTTTTACCTTCGGAGCGAAAACCAAGACAGGTCACTGTTTCAGCCTCACCCCTCTAG (SEQ ID NO: 31). The coding plasmids and Invader DNA of each component of the gene editing system were transfected into HEK293T cells. After 72 hours, DNA was extracted, and the target site was analyzed using next-generation sequencing. The analysis revealed that the expected gene editing event occurred at the target site, and the target base C was successfully deleted at the target site, indicating that the gene editing system of the present invention can achieve effective gene editing. Figure 5 ).

[0160] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

[0161] Sequence List:

[0162] >SEQ ID NO:1 Cas9

[0163]

[0164] >SEQ ID NO:2 Twinkle-CTD(△1-42)

[0165] LPAWHKSIVSFRQLREEVLGELSNVEQAAGLRWSRFPDLNRILKGHRKGELTVFTGPTGSGKTTFISEYALDLCSQGVNTLWGSFEISNVRLARVMLTQFAEGRLEDQLDKYDHWADRFEDLPLYFMTFHGQQSIRTVIDTMQHAVYVYDICHVIIDNLQFMMGHEQLSTDRIAAQDYIIGVFRKFATDNNCHVTLVIHPRKEDDDKELQTASIFGSAKASQEADNVLILQDRKLVTGPGKRYLQVSKNRFDGDVGVFPLEFNKNSLTFSIPPKNKARLKKIKDDTGPVAKKPSSGKKGATTQNSEICSG QAPTPDQPDTSKRSK

[0166] > SEQ ID NO:3 nCas9(D10A)

[0167]

[0168] > SEQ ID NO:4 nCas9(H840A)

[0169]

[0170] > SEQ ID NO:5 Trex2

[0171] MSEPPRAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGSLVLPRVLDKLTLCMCPERPFTAKASEITGLSSESLMHCGKAGFNGAVVRTLQGFLSRQEGPICLVAHNGFDYDFPLLCTELQRLGAHLPQDTVCLDTLPALRGLDRAHSHGTRAQGRKSYSLASLFHRYFQAEPSAAHSAEGDVHTLLLIFLHRAPELLAWADEQARSWAHIEPMYVPPDGPSLEA

[0172] > SEQ ID NO:6 mExoI

[0173] MGIQGLLQFIQEASEPVNVKKYKGQAVAVDTYCWLHKGAIACAEKLAKGEPTDRYVGFCMKFVNMLLSYGVKPILIFDGCTLPSKKEVERSRRERRQSNLLKGKQLLREGKVSEARDCFARSINITHAMAHKVIKAARALGVDCLVAPYEADAQLAYLNKAGIVQAVITEDSDLLAFGCKKVILKMDQFGNGLEVDQARLGMCKQLGDVFTEEKFRYMCILSGCDYLASLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLRMNITVPEDYITGFIRANNTFLYQLVFDPIQRKLVPLNAYGDDVNPETLTYAGQYVGDSVALQIALGNRDVNTFEQIDDYSPDTMPAHSRSHSWNEKAGQKPPGTNSIWHKNYCPRLEVNSVSHAPQLKEKPSTLGLKQVISTKGLNLPRKSCVLKRPRNEALAEDDLLSQYSSVSKKIKENGCGDGTSPNSSKMSKSCPDSGTAHKTDAHTPSKMRNKFATFLQRRNEESGAVVVPGTRSRFFCSSQDFDNFIPKKESGQPLNETVATGKATTSLLGALDCPDTEGHKPVDANGTHNLSSQIPGNAAVSPEDEAQSSETSKLLGAMSPPSLGTLRSCFSWSGTLREFSRTPSPSASTTLQQFRRKSDPPACLPEASAVVTDRCDSKSEMLGETSQPLHELGCSSRSQESMDSSCGLNTSSLSQPSSRDSGSEESDCNNKSLDNQGEQNSKQHLPHFSKKDGLRRNKVPGLCRSSSMDSFSTTKIKPLVPARVSGLSKKSGSMQTRKHHDVENKPGLQTKISELWKNFGFKKDSEKLPSCKKPLSPVKDNIQLTPETEDEIFNKPECVRAQRAIFH

[0174] > SEQ ID NO:7 T5 Exonuclease

[0175] MSKSWGKFIEEEEAEMASRRNLMIVDGTNLGFRFKHNNSKKPFASSYVSTIQSLAKSYSARTTIVLGDKGKSVFRLEHLPEYKGNRDEKYAQRTEEEKALDEQFFEYLKDAFELCKTTFPTFTIRGVEADDMAAYIVKLIGHLYDHVWLISTDGDWDTLLTDKVSRFSFTTRREYHLRDMYEHHNVDDVEQFISLKAIMGDLGDNIRGVEGIGAKRGYNIIREFGNVLDIIDQLPLPGKQKYIQNLNASEELLFRNLILVDLPTYCVDAIAAVGQDVLDKFTKDILEIAEQ

[0176] > SEQ ID NO:8 FEN1

[0177] MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSIYQFLIAVRQGGDVLQNEEGETTSHLMGMFYRTIRMMENGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQQAQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGIPYLDAPSEAEASCAALVKAGKVYAAATEDMDCLTFGSPVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVDLCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPNEEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSLSSAKRKEPEPKGSTKKKAKTGAAGKFKRGK*

[0178] > SEQ ID NO:9 NLS

[0179] MKRTADGSEFESPKKKRKV

[0180] > SEQ ID NO:10 MS2 coat protein (MCP)

[0181] MASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

[0182] > SEQ ID NO:11 MS2

[0183] ACATGAGGATCACCCATGT

[0184] > SEQ ID NO:12 pCMV-NLS-Cas9(H840A)-NLS plasmid sequence:

[0185]

[0186] > SEQ ID NO:13 pCMV-NLS-hTwinkleΔ1-42aa-NLS plasmid sequence:

[0187]

[0188] > SEQ ID NO:14 pCMV-NLS-Trex2-NLS plasmid sequence:

[0189]

[0190] > SEQ ID NO:15 pCMV-NLS-FEN1-NLS plasmid sequence:

[0191]

Claims

1. A method for performing strand substitution gene editing in a DNA sequence, the method comprising: The double-stranded DNA sequence is brought into contact with a sequence-specific nuclease, thereby creating a nick in the double-stranded DNA sequence; The DNA is digested along the cut using exonuclease, thereby producing free single-stranded DNA. The free single-stranded DNA is contacted with invader DNA and the human mitochondrial DNA helicase Twinkle; wherein the invader DNA is single-stranded or double-stranded DNA, and at least one strand of the invader DNA contains a glue point complementary to the free single-stranded DNA and a displacement template containing the desired nucleotide changes. This allows the adhesion sites of the invading DNA to pair complementaryly with free single-stranded DNA; This causes the template of the invading DNA to undergo a strand displacement reaction with the endogenous DNA strand near the nick site; DNA repair / replication, thereby introducing the desired nucleotide changes into the double-stranded DNA sequence.

2. The method according to claim 1, wherein, The DNA repair process includes: (1) using a replacement template as a template to perform DNA repair, thereby forming the desired nucleotide change product on both DNA strands; and (2) repairing DNA nicks.

3. The method according to claim 1, wherein, The DNA repair process also includes, after the strand displacement reaction, using a structure-specific nuclease to excise the endogenous DNA strand adjacent to the nick site; The structure-specific nuclease is preferably selected from FEN1.

4. The method according to claim 1, wherein the sequence-specific nuclease is selected from CRISPR-associated protein (Cas) polypeptides, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), broad-spectrum nucleases, sequence-specific endonucleases, or variants, fragments, and combinations thereof that have cleavage enzyme activity.

5. The method according to claim 4, wherein the Cas polypeptide is selected from Cas3, Cas4, Cas5, Cas5e, Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cast10d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1, Cse2, Cse3, Cse4 Csc1, Csc2, Csa5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, TranC and their variants, fragments or any combination thereof.

6. The method according to claim 1, wherein the sequence-specific nuclease comprises a mutant corresponding to the D10A mutation or H840A mutation of the amino acid sequence shown in SEQ ID NO:

1.

7. The method according to claim 1, wherein the Twinkle enzyme is a natural or truncated Twinkle enzyme; the Twinkle enzyme comprises a carboxyl-terminal domain (CTD) of the amino acid sequence shown in SEQ ID NO:

2.

8. The method of claim 7, wherein the Twinkle enzyme removes the mitochondrial localization signal and, after removal, fuses a nuclear localization signal (NLS) at its amino acid and / or carboxyl terminus.

9. The method according to claim 1, wherein the exonuclease is a 3' exonuclease or a 5' exonuclease or a combination thereof; wherein the 3' exonuclease is preferably Trex2; and the 5' exonuclease is preferably mExoI or T5 exonuclease.

10. The method of claim 1, wherein the desired nucleotide change comprises substitution, insertion, or deletion of a single or multiple nucleotides.

11. A strand substitution gene editing system comprising components of a strand substitution editing system and / or an expression construct containing nucleotide sequences encoding components of the strand substitution editing system, the components of the strand substitution gene editing system comprising: i) A sequence-specific nuclease or its domain thereof, wherein the sequence-specific nuclease or its domain thereof is guided to target DNA by a guide RNA (sgRNA) sequence; ii) Exonucleases or their domains; iii) Twinkle enzyme, a human mitochondrial DNA helicase, or its domain; iv) Single-stranded or double-stranded invading DNA, wherein at least one strand of the invading DNA contains a glue point complementary to free single-stranded DNA produced by nicking enzymes and exonucleases, and a displacement template containing the desired nucleotide changes. The components are independent of each other, or at least two components are connected by a connector to form a fusion protein.

12. The strand substitution gene editing system according to claim 11, wherein, The components of the strand displacement gene editing system also include: v) Structure-specific nucleases; The structure-specific nuclease is preferably selected from FEN1.

13. The strand substitution gene editing system according to claim 11, wherein, The sequence-specific nuclease is selected from CRISPR-associated protein (Cas) peptides, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), broad-spectrum nucleases, sequence-specific endonucleases, or their variants, fragments, and combinations thereof that have cleavage enzyme activity.

14. The strand substitution gene editing system according to claim 11, wherein, The sequence-specific nuclease is a CRISPR-associated protein (Cas) polypeptide with nickase activity; Furthermore, the components of the chain substitution gene editing system also include: vi) guide RNA that guides the corresponding Cas polypeptide to the target nucleotide region.

15. The chain substitution gene editing system according to claim 14, wherein the Cas polypeptide is selected from Cas3, Cas4, Cas5, Cas5e, Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cast10d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1, Cse2, Cse3 Cse4, Csc1, Csc2, Csa5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, TranC and their variants, fragments or any combination thereof.

16. The chain substitution gene editing system of claim 11, wherein the sequence-specific nuclease comprises a mutant corresponding to the D10A mutation or the H840A mutation of the amino acid sequence shown in SEQ ID NO:

1.

17. The strand substitution gene editing system of claim 11, wherein the Twinkle enzyme is a natural or truncated Twinkle enzyme; the Twinkle enzyme comprises a carboxyl-terminal domain (CTD) of the amino acid sequence shown in SEQ ID NO:

2.

18. The strand substitution gene editing system according to claim 11, wherein the exonuclease is a 3' exonuclease or a 5' exonuclease or a combination thereof; wherein the 3' exonuclease is preferably Trex2; and the 5' exonuclease is preferably mExoI or T5 exonuclease.

19. The chain substitution gene editing system according to any one of claims 11-18, wherein at least one of the components has an amino or carboxyl terminus fused with a nuclear localization signal (NLS).

20. The chain substitution gene editing system according to any one of claims 11-18, wherein the linker comprises an amino acid sequence (GGGS)n, (GGGGS)n, (G)n, (EAAAK)n, (GGS)n, (SGGS)n, SGSETPGTSESATPES, or (XP)n motif or a combination thereof, wherein n is independently an integer from 1 to 30, and wherein X is any amino acid.

21. The chain displacement gene editing system according to any one of claims 11-18, wherein the components of the system are recruited by a recruitment system selected from the MCP-MS2 system or the GCN4-ScFv system.

22. A host cell, wherein, The host cell comprises the strand displacement gene editing system according to any one of claims 11-21.

23. A method for producing at least one genetically modified cell, wherein, The method includes editing at least one of the cells using the method of any one of claims 1-10, or introducing a strand substitution gene editing system of any one of claims 11-21 into at least one of the cells, thereby causing substitution, insertion, or deletion of one or more nucleotides in the target nucleotide editing region of at least one of the cells.

24. The method according to claim 23, wherein, The method further includes the step of screening cells from the at least one cell for cells having one or more desired nucleotide substitutions.

25. The method according to any one of claims 23-24, wherein, The cells are derived from prokaryotes such as bacteria; eukaryotes such as plants, fungi, or vertebrates.

26. The method according to claim 25, characterized in that, The vertebrates mentioned are mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, and cats.

27. The method according to claim 25, characterized in that, The plants mentioned are crop plants, such as wheat, rice, corn, soybeans, sunflowers, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomatoes, tobacco, cassava, or potatoes.

28. Use of a method for performing strand substitution gene editing in a DNA sequence according to any one of claims 1-10 or the strand substitution gene editing system according to any one of claims 11-21, wherein the use includes: a) Gene or genome editing; b) Targeted nuclear nucleotide detection and / or diagnosis; c) Editing target nucleotide sequences to modify biological or non-human organisms; d) Treatment of diseases.

29. A reagent kit, wherein, The kit comprises the strand displacement gene editing system of any one of claims 11-21 or the host cell of claim 22.

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