Exonuclease editor and gene editing method

Abstract

The invention provides an exonuclease gene editor and a gene editing system and application thereof, and particularly provides an exonuclease editor formed by fusing exonuclease and Cas9 or nicking enzyme, the exonuclease editor has very good gene editing activity, and compared with Cas9 nuclease, the exonuclease editor can remarkably improve accurate gene editing efficiency. The exonuclease editor provided by the invention can also be used for accurately deleting long-fragment genes.

Description

Technical Field

[0001] This invention relates to the field of gene editing. Specifically, this invention relates to exonuclease editors and gene editing methods. Background Technology

[0002] The existing gene editing tool CRISPR / Cas9 can efficiently edit any site on the genome, but there are still important problems to be solved in the field of gene editing. One of them is the low efficiency of homologous recombination and the high proportion of byproducts of random insertion or deletion.

[0003] In mammalian cells, DNA double-strand breaks are primarily repaired through two pathways: non-homologous end joining (NHEJ) and homology-directed repair (HDR). HDR, in the presence of a homologous template, enables template-dependent, precise repair by introducing a foreign gene into the template for accurate gene knock-in. HDR and NHEJ are in competition; in mammalian cells, DNA damage is typically repaired via the NHEJ pathway, resulting in lower efficiency for HDR-mediated precise repair. However, precise gene editing is often required in gene repair for genetic diseases, the creation of animal models, and plant breeding; therefore, improving the efficiency of precise gene editing remains crucial.

[0004] Therefore, there is a need in this field to develop a gene editor that improves gene editing efficiency. Summary of the Invention

[0005] The purpose of this invention is to provide a gene editor that improves gene editing efficiency.

[0006] In a first aspect of the present invention, an exonuclease editor for gene editing is provided, the exonuclease editor comprising:

[0007] A) Cas nuclease or its active fragment;

[0008] B) Exonuclease or its active fragment, wherein the exonuclease is a 5'-3' exonuclease.

[0009] In another preferred embodiment, the exonuclease editor has the following structure from N-terminus to C-terminus as shown in formula Ia or Ib:

[0010] CLE(Ia);

[0011] ELC(Ib);

[0012] In the formula, "-" represents a peptide bond;

[0013] C represents the Cas nuclease or its active fragment;

[0014] L represents an empty or linked peptide;

[0015] E stands for exonuclease.

[0016] In another preferred embodiment, the Cas nuclease is a Cas9 nuclease.

[0017] In another preferred embodiment, the Cas9 nuclease is a wild-type Cas9 nuclease or a mutant Cas9 nuclease.

[0018] In another preferred embodiment, the mutant Cas9 nuclease is a D10A mutant or an H840A mutant Cas9 nuclease.

[0019] In another preferred embodiment, the mutant Cas9 nuclease induces single-strand breaks in DNA.

[0020] In another preferred embodiment, the 5'-3' exonuclease hydrolyzes nucleotides one by one, starting from the 5' end of the polynucleotide chain.

[0021] In another preferred embodiment, the exonuclease acts on DNA.

[0022] In another preferred embodiment, the exonuclease active fragment is a fragment with catalytic activity and capable of binding DNA.

[0023] In another preferred embodiment, the exonuclease is selected from the group consisting of human exonuclease I (hEXO1), T5 exonuclease, or combinations thereof.

[0024] In another preferred embodiment, the exonuclease is hEXO1.

[0025] In another preferred embodiment, the exonuclease fragment is composed of amino acid residues from the N-terminus of hEXO1, positions 1-352.

[0026] In another preferred embodiment, L is an XTEN connector.

[0027] In a second aspect of the invention, a fusion protein is provided, the fusion protein comprising:

[0028] I) an exonuclease editor as described in the first aspect of the present invention; and

[0029] II) One or more functional structural domains.

[0030] In another preferred embodiment, the functional domain is selected from the group consisting of: localization signals, reporter proteins, tags that assist in expression or purification, and acquisition combinations.

[0031] In another preferred embodiment, the positioning signal is a nuclear positioning signal (NLS).

[0032] In another preferred embodiment, the sequence of the nuclear localization signal is located at the N-terminus or C-terminus of the exonuclease editor.

[0033] In a third aspect of the invention, an isolated polynucleotide is provided, said polynucleotide encoding an exonuclease editor as described in the first aspect of the invention or a fusion protein as described in the second aspect of the invention.

[0034] In another preferred embodiment, the polynucleotide is selected from the group consisting of genomic sequences, cDNA sequences, RNA sequences, or combinations thereof.

[0035] In another preferred embodiment, the polynucleotide is a polynucleotide whose codons have been optimized according to the codon preferences of the host cell.

[0036] In a fourth aspect of the invention, a guide RNA (gRNA) is provided, the guide RNA comprising a direct repeat sequence capable of binding to an exonuclease editor as described in the first aspect of the invention and a spacer sequence capable of targeting a target sequence.

[0037] In another preferred embodiment, the direct repeat sequence binds to the Cas nuclease portion of the exonuclease editor.

[0038] In another preferred embodiment, the target sequence comprises a cDNA sequence.

[0039] In another preferred embodiment, the target sequence includes single-stranded DNA and double-stranded DNA sequences.

[0040] In another preferred embodiment, the target sequence is present within the cell.

[0041] In another preferred embodiment, the target sequence is located in the cell nucleus or in the cytoplasm (e.g., organelles).

[0042] In a fifth aspect of the invention, a complex is provided, the complex comprising:

[0043] (i) Protein components selected from the group consisting of: exonuclease editors as described in the first aspect of the invention, fusion proteins as described in the second aspect of the invention, or combinations thereof; and

[0044] (ii) a nucleic acid component, wherein the nucleic acid component is the guide RNA as described in the fourth aspect of the present invention;

[0045] The protein component and the nucleic acid component combine to form a complex.

[0046] In another preferred embodiment, the nucleic acid component binds to the Cas nuclease portion of the protein component.

[0047] In a sixth aspect of the invention, a carrier is provided comprising the polynucleotide as described in the third aspect of the invention.

[0048] In another preferred embodiment, the vector further comprises a nucleic acid sequence encoding a guide RNA as described in the fourth aspect of the invention.

[0049] In another preferred embodiment, the vector further includes an expression regulatory element operatively linked to the polynucleotide.

[0050] In another preferred embodiment, the expression regulation element includes a promoter.

[0051] In another preferred embodiment, the promoter is selected from the group consisting of: constitutive promoters, tissue-specific promoters, inducible promoters, or strong promoters.

[0052] In another preferred embodiment, the vector includes plasmids and viral vectors.

[0053] In a seventh aspect of the invention, a CRISPR-Cas composition is provided, comprising:

[0054] (i) Selected from the first component of the group below: an exonuclease editor as described in the first aspect of the invention, a fusion protein as described in the second aspect of the invention, a polynucleotide as described in the third aspect of the invention, or a vector as described in the sixth aspect of the invention, or a combination thereof; and

[0055] (ii) A second component comprising one or more guide RNAs as described in the fourth aspect of the invention, or encoding a nucleotide sequence comprising one or more guide RNAs as described in the fourth aspect of the invention.

[0056] In another preferred embodiment, the CRISPR-Cas composition comprises two guide RNAs that bind to two target sequence fragments on the same target gene, respectively.

[0057] In another preferred embodiment, micro-homologous sequences exist near the two target sequence fragments.

[0058] In another preferred embodiment, the micro-homologous sequence is capable of mediating micro-homologous end recombination.

[0059] In an eighth aspect of the invention, a pharmaceutical composition is provided comprising a therapeutically effective amount of an exonuclease editor as described in the first aspect of the invention, a fusion protein as described in the second aspect of the invention, a polynucleotide as described in the third aspect of the invention, a complex as described in the fifth aspect of the invention, a carrier as described in the sixth aspect of the invention, a CRISPR-Cas composition as described in the seventh aspect of the invention, or a combination thereof; and a pharmaceutically acceptable carrier.

[0060] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of lyophilized formulations, liquid formulations, or combinations thereof.

[0061] In another preferred embodiment, the dosage form of the composition is a liquid formulation.

[0062] In another preferred embodiment, the composition is in the form of an injection.

[0063] In another preferred embodiment, the composition is a cell preparation.

[0064] In a ninth aspect of the invention, a kit is provided comprising one or more components selected from the following: an exonuclease editor as described in the first aspect of the invention, a fusion protein as described in the second aspect of the invention, a polynucleotide as described in the third aspect of the invention, a complex as described in the fifth aspect of the invention, a carrier as described in the sixth aspect of the invention, a CRISPR-Cas composition as described in the seventh aspect of the invention, a pharmaceutical composition as described in the eighth aspect of the invention, or a combination thereof.

[0065] In another preferred embodiment, the kit also includes a label or instructions.

[0066] In another preferred embodiment, the kit is used for gene or genome editing, disease treatment, targeting a gene, cutting a target gene or a non-target gene, or one or more other applications.

[0067] In a tenth aspect of the invention, a host cell is provided comprising an exonuclease editor as described in the first aspect of the invention, a fusion protein as described in the second aspect of the invention, a polynucleotide as described in the third aspect of the invention, a complex as described in the fifth aspect of the invention, a vector as described in the sixth aspect of the invention, and a composition as described in the seventh aspect of the invention.

[0068] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell, plant cell, or mammalian cell (including human and non-human mammals).

[0069] In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0070] In another preferred embodiment, the host cell's genes are edited by the exonuclease editor, complex, or composition.

[0071] In an eleventh aspect of the present invention, a method for targeting and editing or cutting a target gene is provided, comprising: contacting the target gene with an exonuclease editor as described in the first aspect of the present invention, or a fusion protein as described in the second aspect of the present invention, or a complex as described in the fifth aspect of the present invention, or a composition as described in the seventh aspect of the present invention, or a pharmaceutical composition as described in the eighth aspect of the present invention, or delivering it to a cell containing the target gene.

[0072] In another preferred embodiment, the target gene is present within the cell.

[0073] In another preferred embodiment, the cell is a prokaryotic cell.

[0074] In another preferred embodiment, the cell is a eukaryotic cell, such as a mammalian cell (e.g., a human cell) or a plant cell.

[0075] In another preferred embodiment, the target gene is present in an in vitro nucleic acid molecule (e.g., a plasmid).

[0076] In another preferred embodiment, the target gene comprises DNA.

[0077] In another preferred embodiment, the DNA includes single-stranded DNA and double-stranded DNA.

[0078] In another preferred embodiment, the edited target gene or cleaved target gene includes:

[0079] i) Breakage of the target sequence;

[0080] ii) Missing segments of the target sequence; and / or

[0081] iii) Insert the exogenous nucleic acid into the fracture.

[0082] In another preferred embodiment, the breakage of the target sequence is a double-strand break (DSB), a single-strand break (SSB), or a combination thereof.

[0083] In another preferred embodiment, the fragment deletion is a fragment cut mediated by micro-homogeneous end joining (MMEJ).

[0084] In another preferred embodiment, the number of missing fragments is 5-100 nt, more preferably 10-70 nt, and even more preferably 20-50 nt.

[0085] In a twelfth aspect of the invention, the use of an exonuclease editor as described in the first aspect of the invention, a fusion protein as described in the second aspect of the invention, a polynucleotide as described in the third aspect of the invention, a complex as described in the fifth aspect of the invention, a carrier as described in the sixth aspect of the invention, a CRISPR-Cas composition as described in the seventh aspect of the invention, and a pharmaceutical composition as described in the eighth aspect of the invention in the preparation of a medicament for the prevention and / or treatment of a disease are provided.

[0086] In another preferred embodiment, the disease benefits from gene editing therapy.

[0087] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0088] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0089] Figure 1 The structures of candidate exonucleases for enhancing HDR are shown. A. Schematic diagram of the exonuclease editor; B. Candidate catalytic domain of hEXO1; C. Crystal structure of hEXO1; D. Candidate catalytic domain of T5 exonuclease; E. Crystal structure of T5 exonuclease; F. Candidate catalytic domain of WRN exonuclease; G. Crystal structure of WRN exonuclease.

[0090] Figure 2 The results show the identification of the exonuclease structures that enhance HDR. A. Representative flow cytometry plot of HEK293 cells targeting and integrating EGFP at the Rosa26 site, showing the HDR-positive cell population. All experiments were performed in triplicate; B. Schematic diagrams of the structures of Cas9, CHE, and CWE; C. Bar graph showing the HDR efficiency at the Rosa26 site in HEK293 cells. HDR efficiency was quantified by the proportion of EGFP-positive cells. All experiments were performed in triplicate.

[0091] Figure 3 The effects of the exonuclease gene editor on NHEJ are shown. A. Experimental procedure for EGFP targeting; B. Flow cytometry plot of EGFP at the Rosa26 site in the HEK293 reporter cell line, showing the EGFP-negative cell population. All experiments were performed in triplicate.

[0092] Figure 4The effects of the exonuclease gene editor on NHEJ are shown. A. Bar charts show the NHEJ efficiency of the EGFP site in the HEK293 reporter cell line. NHEJ efficiency is quantified by the proportion of EGFP-negative cells. All experiments were performed in triplicate; B. Sanger sequencing data of the EGFP target site in the HEK293 reporter cell line; C. Sanger sequencing data of the hLMNA target site in the HEK293 cell line; D. Sanger sequencing data of the hTP53 target site in the HEK293 cell line; E. Sanger sequencing data of the hDMD target site in the HEK293 cell line; F. Targeting efficiency of the hDMD, hLMNA, and hTP53 sites in the corresponding experimental groups.

[0093] Figure 5 This study demonstrates how the use of CXE nickase can further improve the precision editing efficiency. A. HDR efficiency at the hAAVS1 and hRosa26 loci treated with different Cas9 and CXE nickases was measured by flow cytometry; B. Indel frequencies at the hAAVS1 and hRosa26 loci treated with different Cas9 and CXE nickases were quantified by high-throughput sequencing; C. The HDR / indel ratio at the hAAVS1 and hRosa26 loci was normalized relative to the Cas9 control group in HEK293 cells treated with different Cas9 and CXE nickases.

[0094] Figure 6This diagram illustrates gene modification patterns based on MMEJ and paired sgRNA, exonuclease editors. A. Schematic diagram of precise deletion mediated by CXE. The target DNA sequence contains two primitive spacer sequences on opposite strands. CXE targets each primitive spacer sequence and generates long 3' single-stranded DNA overhangs on both sides of the DSB. Micro-homological (MH) sequences flanking the overhangs anneal to each other. This process results in the deletion of one of the MH sequences and the non-homologous DNA wings in between. MH sequences are shown in yellow, and PAMs are shown in red; B. Frequency of NHEJ editing induced by CXE-assisted paired sgRNA at three endogenous genomic sites in HEK293 cells; C. MH:indel ratio at CFTR, Rosa26, and EMX1 sites in HEK293 cells; D. Proportion of MH sequences specified in MH reads generated by Cas9 and CXE at the three endogenous sites; E. Heatmap showing the frequency of MH reads of different lengths; F. Distribution of deletion size in total deletions induced by Cas9 and CXE at the three endogenous sites. Statistical significance was calculated using unpaired t-tests in B and C (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, not significant). Error bars represent standard deviations. Three independent experiments were conducted. Detailed Implementation

[0095] Through extensive and in-depth research, the inventors have developed, for the first time, an exonuclease editor and a gene editing method. This invention fuses the catalytic domain of human exonuclease I (hEXO1) with Cas9 nuclease, providing an exonuclease editor (EXO Editor). hEXO1 can generate long 3' single-stranded DNA (ssDNA), which is essential for homologous recombination. Furthermore, the 3' single strand can inhibit NHEJ. Compared to Cas9 nuclease, the EXO editor can improve HDR efficiency by approximately four times.

[0096] To expand the application scope of exonuclease gene editing systems, this invention also provides a nicking enzyme editor based on an exonuclease mutant of Cas9 nuclease, which significantly improves the precision gene editing efficiency (HDR:indel) compared to wild-type Cas9 nuclease. The exonuclease editor does not globally inhibit NHEJ, thus avoiding the adverse effects of global NHEJ inhibition. Furthermore, when microhomologous sequences are present near gRNAs, the exonuclease editor can be used for precise gene deletion.

[0097] The gene editing system of this invention can improve the efficiency of HDR-based precision editing while reducing NHEJ, providing a new option for precise gene editing. Based on this, this invention was completed.

[0098] the term

[0099] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0100] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0101] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0102] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0103] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0104] Exonuclease editor

[0105] As used in this invention, the term "exonuclease editor" refers to the fusion protein for gene editing provided in the first aspect of this invention. The exonuclease editor of this invention comprises:

[0106] A) Cas nuclease or its active fragment;

[0107] B) Exonuclease or its active fragment, wherein the exonuclease is a 5'-3' exonuclease.

[0108] In the exonuclease editor of the present invention, the Cas nuclease can be a Cas9 nuclease, including wild-type Cas9 nuclease or mutant Cas9 nuclease.

[0109] In this invention, the terms "Cas nuclease," "Cas protein," and "Cas enzyme" are used interchangeably. "Cas protein" is used in its broadest sense, encompassing wild-type Cas proteins, their derivatives or variants, analogs, and their functional fragments such as oligonucleotide-binding fragments.

[0110] In some embodiments, wild-type Cas9 nucleases can be used. "Wild-type Cas9 nuclease" refers to naturally occurring, unmodified Cas9 proteins. In some embodiments, to further improve the precision of editing, an EXO-nickase editor can be used. "EXO-nickase" refers to a class of mutant Cas9 nucleases that initiate single-strand cleavage of DNA. The EXO-nickase can be any Cas9 nuclease known in the art. In some embodiments, the mutant Cas9 nuclease can be a D10A mutant or an H840A mutant, both of which have amino acid sequences known in the art. Experiments have shown that the H840A mutant exonuclease editor achieves approximately 400 times higher precision editing efficiency than Cas9. The exonuclease-nickase editor does not produce DSBs after DNA cleavage, significantly reducing the generation of indels. This tool expands the use of exonuclease gene editing systems, providing a new option for precise gene editing.

[0111] Homologous recombination requires a long 3' single strand to invade the homologous template; therefore, promoting the generation of long 3' single strands may promote homologous recombination. Figure 1 As shown in A, during homologous recombination, digestion in the 5'-3' direction is conducive to the generation of long 3' single strands. The 5' end DNA competitively binds to the homologous template and is expressed through Cas9 fusion with an exonuclease. The exonuclease can digest the 5' end DNA, which may promote the generation of 3' single strands at the DSB end, which is conducive to the 3' single strand invading the homologous template.

[0112] The exonuclease used in this invention is a 5'-3' direction digestion exonuclease. In one embodiment, human exonuclease I (hEXO1) or its active fragment can be used. Human exonuclease I is a member of the Rad52 family and has 5' exonuclease activity, capable of continuously cleaving single nucleotides and DNA sequences with Flap structures. hEXO1 also participates in DNA repair, recombination, and replication processes. The N-terminal 1-352 amino acid residues of hEXO1 have catalytic activity and the ability to bind DNA.

[0113] In a preferred embodiment, the exonuclease editor of the present invention comprises a fused Cas9 nuclease and amino acid residues from the N-terminus of hEXO1, positions 1-352.

[0114] CRISPR system

[0115] The terms “regularly clustered short palindromic repeats (CRISPR)-associated (Cas) system”, “CRISPR-Cas system” or “CRISPR system” are used interchangeably and have the meaning commonly understood by those skilled in the art, which typically includes transcripts or other elements relating to the expression of CRISPR-associated (“Cas”) genes, or transcripts or other elements capable of directing the activity of said Cas genes.

[0116] CRISPR / Cas complex

[0117] The term "CRISPR / Cas complex" refers to a complex formed by the binding of gRNA (guide RNA) or mature crRNA (or guide RNA) to an exonuclease editor. This complex contains a guide sequence that hybridizes to the target sequence and binds to the Cas9 nuclease portion of the exonuclease editor. The complex is capable of recognizing and cleaving target nucleotides that hybridize with the guide RNA or mature crRNA.

[0118] Guide RNA (gRNA)

[0119] The terms “guide RNA (gRNA),” “mature crRNA,” “crRNA,” “guide sequence,” and “guide RNA” are used interchangeably and have the meanings commonly understood by those skilled in the art. Generally, a guide RNA may comprise a direct repeat (DR) sequence and a spacer sequence, or consist essentially of or composed of direct repeat sequences and spacer sequences.

[0120] In one aspect, the present invention provides a guide RNA comprising a direct repeat sequence capable of binding to the Cas nuclease portion of the exonuclease editor of the present invention and a spacer sequence capable of targeting a target sequence.

[0121] This invention can use a single guide RNA or multiple guide RNAs. In one embodiment, paired guide RNAs can be used, with the two guide RNAs respectively used to locate the start and end positions of the target DNA sequence, thereby guiding the Cas protein to cut between these two positions to achieve precise gene editing. When microhomologous sequences are present near the paired gRNA sites, the exonuclease editor of this invention can be used to achieve precise deletion.

[0122] target nucleic acid

[0123] In this invention, the terms "target nucleic acid" and "target sequence" are used interchangeably and refer to a specific nucleic acid containing a nucleic acid sequence that is wholly or partially complementary to the spacer sequence in the guide RNA. Hybridization between the target sequence and the spacer sequence will promote the formation of a CRISPR-Cas complex (including an exonuclease editor and the guide RNA). Perfect complementarity is not required, as long as sufficient complementarity exists to induce hybridization and promote the formation of a CRISPR-Cas complex. In some embodiments, the target nucleic acid contains a non-coding region (e.g., a promoter or terminator). In some embodiments, the target nucleic acid is single-stranded or double-stranded.

[0124] The target sequence can contain any polynucleotide, such as DNA. In some cases, the target sequence is located inside or outside the cell. In other cases, the target sequence is located in the cell nucleus, cytoplasm, or organelles (such as mitochondria or chloroplasts).

[0125] The target nucleic acid can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or useless DNA). In some cases, the target sequence should be associated with a protospacer adjacent motif (PAM).

[0126] Donor template

[0127] In this invention, the donor template nucleic acid or the donor template can be used interchangeably, meaning that after the exonuclease editor described herein alters the target nucleic acid, one or more cellular proteins can use it to change the structure of the target nucleic acid.

[0128] In some embodiments, the donor template nucleic acid is a double-stranded or single-stranded nucleic acid. In some embodiments, the donor template nucleic acid is linear or circular (e.g., a plasmid). In some instances, the donor template nucleic acid is a foreign nucleic acid molecule. In some instances, the donor template nucleic acid is an endogenous nucleic acid molecule (e.g., a chromosome). In some embodiments, gene recombination, specifically homologous recombination, can be achieved using the donor template.

[0129] The main advantages of this invention include:

[0130] (1) The exonuclease editor of the present invention integrates the catalytic domain of exonuclease I (hEXO1) with Cas9 nuclease, which can generate long 3' single-stranded DNA during cleavage and effectively improve the efficiency of homologous recombination.

[0131] (2) The exonuclease editor of the present invention can inhibit NHEJ and significantly reduce the production of byproduct indels.

[0132] (3) The exonuclease editor of the present invention can significantly improve the efficiency of accurate editing (HDR / indel).

[0133] (4) The exonuclease editing system of the present invention can be modified into a CHE cutase editor, which does not produce DSB, and at certain sites, the precise editing efficiency is about 400 times higher than that of Cas9.

[0134] (5) When micro-homologous sequences are present near gRNA sites, the exonuclease editor of this invention can be used for precise deletion.

[0135] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0136] Example 1: Screening for exonuclease domains that promote homologous recombination

[0137] In this embodiment, an exonuclease gene editor was constructed by fusing the N-terminal 1-352 amino acid residue domain of hEXO1 with the Cas9 protein for expression. Figure 1 B, C). The T5 exonuclease also possesses 5'-3' digestive activity and is composed of 270 amino acids (B, C). Figure 1 D, E). To compare with the 5'-3' digestion direction, this study also fused the WRN exonuclease catalytic domains of Cas9 digested in the 3'-5' direction (D, E). Figure 1 F, G). Next, we verified whether the Cas9 fusion protein with the exonuclease promoted homologous recombination. EGFP (without a promoter on the donor template) was knocked into the Rosa26 site in HEK293 cells. The results showed that the gene knock-in efficiency of Cas9 was 1.68%, and the gene knock-in efficiency of the Cas9-hEXO1 fusion protein (CHE) was 6.65%, significantly improving the homologous recombination efficiency. However, the Cas9-WRN exonuclease fusion protein (CWE) did not improve the gene knock-in efficiency. Figure 2 AC).

[0138] The results showed that the exonuclease editor obtained by fusing the catalytic domain of Cas9 nuclease with hEXO1 promoted homologous recombination and had higher editing efficiency than Cas9. Therefore, the fusion protein of Cas9 and hEXO1 (CHE) was selected for further research.

[0139] Example 2: Effect of the exonuclease gene editor on NHEJ

[0140] The generation of long 3' single strands is detrimental to the final step of DNA end joining in the non-homologous end joining (NHEJ) pathway. The NHEJ and HDR pathways are in competition. For homologous recombination, random insertions or deletions (indels) generated by the NHEJ pathway are byproducts. Therefore, it is necessary to verify the effect of the exonuclease gene editor on NHEJ to evaluate the efficiency of indel generation. First, the effect of the exonuclease editor on NHEJ was examined in the HEK293 reporter cell line (Rosa26-EGFP). This reporter cell line expresses green fluorescent protein. After transfecting the cells with nuclease and U6-gRNA targeting EGFP, the proportion of EGFP-negative cells was detected by flow cytometry to assess the efficiency of indel generation. Figure 3 A). The results showed that after Cas9 targeting EGFP, the proportion of EGFP-negative cells was approximately 78%, while after targeting the exonuclease editor CHE, the proportion of EGFP-negative cells was approximately 25%. Figure 3 B, Figure 4 A). Consistent with this, at the genomic level, Sanger sequencing of the EGFP target site clearly showed that Cas9 targeting resulted in higher heterogeneous peaks caused by indels, while CHE targeting resulted in lower heterogeneous peaks compared to the Cas9 group. Figure 4 B). Subsequently, targeting of the endogenous genes LMNA, DMD, and p53 was performed, and Sanger sequencing revealed that the proportion of indels in the CHE group was lower than that in the Cas9 group. Figure 4 CE). Indel efficiency was quantified using high-throughput sequencing, and it was also observed that the indel efficiency in the CHE group was lower than that in the Cas9 group ( Figure 4 F).

[0141] The above results indicate that the exonuclease editor inhibits NHEJ at multiple target sites, effectively preventing unwanted random editing events.

[0142] Example 3: Effect of the exonuclease-fused nicking enzyme editor

[0143] The D10A or H840A mutant nickases of Cas9 induce single-strand breaks (SSBs) instead of double-strand breaks (DSBs) and have the potential to universally avoid the NHEJ repair pathway. To achieve DSB-free HDR with high efficiency and minimal byproducts, attempts were made to fuse hExo1 with programmable nickases (Cas9D10A or Cas9H840A) to generate CXE nickase editors (CXE D10A and CXE H840A). Similarly, the efficiency of precise genome editing was measured using dsDNA donor plasmids at two different human safe loci (AAVS1 and Rosa26).

[0144] Flow cytometry results showed that at the tested loci, the HDR frequencies of CXE D10A and CXE H840A were higher than those of the Cas9 control group. Figure 5 A). The number of indels induced by both Cas9 nickase and CXE nickase is significantly less than that Cas9, while the frequency of indels induced by CXE nickase is lower than that Cas9 nickase. Figure 5 B). Notably, compared to Cas9, the HDR / indel ratios of CXE nickase at both hAAVS1 and hRosa26 loci were significantly increased, with CXEH840A increasing by 453-fold and 50-fold respectively, and CXE D10A increasing by 217-fold and 28-fold respectively. Figure 5 C).

[0145] The results show that the CXE nickase editor can moderately stimulate HDR and significantly reduce unwanted mutation events, thereby increasing the HDR / indel ratio and improving gene editing efficiency.

[0146] Example 4: Exonuclease editors can perform precise gene deletion by binding paired sgRNAs.

[0147] MMEJ is an error-prone DNA repair process that uses microhomological sequences flanking the DSB for end joining. This process results in the deletion of one of the microhomological sequences and the non-homologous DNA flaps in between. Long-distance recutting caused by hExo1 at the DNA break produces long 3' single-stranded DNA overhangs. Figure 6 A). The micro-homologous sequences exposed on the 3' protrusion anneal to each other, and the non-homologous 3' DNA wings are removed from the annealing intermediate, resulting in the precise deletion of the micro-homologous sequences and the DNA sequences in between. Figure 6 A). It is speculated that a pair of sgRNAs could be used to locate microhomologous sites, potentially enabling precise long-fragment deletions. Figure 6A). To test this hypothesis, HEK293 cells were transfected with paired sgRNAs targeting three endogenous sites (CFTR, Rosa26, and EMX1), while simultaneously transfected with either Cas9 or CXE. Four days post-transfection, genomic DNA was extracted from the cells, and the target regions were amplified by PCR. The PCR amplification products were then subjected to deep sequencing to quantify insertion and deletion efficiencies and to detect micro-homology editing. Results showed that Cas9-mediated indel efficiencies ranged from 48.8% to 82.3% at the three sites, while CXE significantly reduced the indel rate at endogenous sites, ranging from 3.0% to 23.5%. Figure 6 B).

[0148] MMEJ efficiency was determined by calculating the ratio of the number of reads with micro-homology deletions to the total number of reads with indels. Results showed that Cas9 generated precise deletion frequencies based on micro-homology ranging from 2.4% to 12.7%, while CXE significantly improved MMEJ efficiency at all three sites (7.2%–19.1%). Figure 6 C). In microhomological sequences, the number of nucleotides ranges from 2 to 6 base pairs. Figure 6 D). Compared with the Cas9 control group, CXE showed a higher proportion and frequency of microhomology lengths of 4 and 6 at Rosa26 and EMX1 sites, respectively. Figure 6 D, E).

[0149] Next, the size of the deletions produced by Cas9 and CXE at the target sites was analyzed. Most deletions were approximately 25-50 base pairs. ​ F).

[0150] The results clearly demonstrate that CXE can improve MMEJ efficiency and potentially induce predictable and precise deletions based on micro-homologous sequences.

[0151] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An exonuclease editor for gene editing, characterized in that, The exonuclease editor includes: A) Cas nuclease or its active fragment; B) Exonuclease or its active fragment, wherein the exonuclease is a 5'-3' exonuclease.

2. The exonuclease editor as described in claim 1, characterized in that, The exonuclease editor has the following structure from N-terminus to C-terminus, as shown in equation Ia or Ib: CLE(Ia); ELC(Ib); In the formula, "-" represents a peptide bond; C represents the Cas nuclease or its active fragment; L represents an empty or linked peptide; E stands for exonuclease.

3. The exonuclease editor as described in claim 1, characterized in that, The Cas nuclease mentioned is either wild-type Cas9 nuclease or mutant Cas9 nuclease.

4. The exonuclease editor as described in claim 3, characterized in that, The mutant Cas9 nuclease is either the D10A mutant or the H840A mutant Cas9 nuclease.

5. The exonuclease editor as described in claim 1, characterized in that, The exonuclease is selected from the following group: human exonuclease I (hEXO1), T5 exonuclease, or a combination thereof.

6. The exonuclease editor as described in claim 1, characterized in that, The exonuclease fragment is composed of amino acid residues from the N-terminus of hEXO1, positions 1-352.

7. A fusion protein, characterized in that, The fusion protein includes: I) the exonuclease editor as described in claim 1; and II) One or more functional structural domains.

8. The fusion protein as described in claim 7, characterized in that, The functional structural domain is the nuclear localization signal (NLS).

9. An isolated polynucleotide, characterized in that, The polynucleotide encodes the exonuclease editor as described in claim 1 or the fusion protein as described in claim 7.

10. A guide RNA (gRNA), characterized in that, The guide RNA includes a direct repeat (DR) sequence capable of binding to the exonuclease editor as described in claim 1 and a spacer sequence capable of targeting the target sequence.

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