CN115786304A - Cas12a protein mutant, base editor containing same and application - Google Patents

Abstract

The invention discloses a Cas12a protein mutant, a base editor containing the same and application. The Cas12a protein mutant has any one or combination of at least two of D156R, G532R and K538R combined mutation, G532R and K595R combined mutation or G532R, K538V and Y542R combined mutation on the basis of the dLbCas12a protein. According to the invention, the dLbCas12a is modified on the basis of obtaining the Cas12a protein mutant capable of identifying PAM range expansion, and the high-efficiency base editor capable of identifying PAM range expansion is constructed, and the base editor can realize high-efficiency C > T or A > G editing on classical PAM and non-classical PAM on the level of cells and embryos; and can realize simultaneous multi-site efficient C > T or A > G editing at the level of cells and embryos, thereby successfully enriching the existing base editing tool boxes.

Description

Cas12a protein mutant, base editor containing same and application

Technical Field

The invention belongs to the technical field of gene editing, and relates to a Cas12a protein mutant, a base editor containing the same and application.

Background

Thanks to the generation and development of gene editing technology, people are now in the era of flexible and precise gene manipulation, and can research gene functions, perform gene therapy, improve animal and plant traits and the like by modifying genes. The emergence of gene editing technology has greatly promoted the innovative development of human genetic diseases from the basic research of symptoms and heredity to the treatment of diseases.

With the discovery and development of CRISPR/Cas system, it has become a novel gene editing technology most commonly used by researchers due to the advantages of simple design, easy operation, high editing activity, low cost, etc. According to survey statistics, the largest group of human genetic diseases is caused by gene point mutation (also called single nucleotide polymorphism SNP), and accounts for about 58% of pathogenic genetic variation. Among them, C > T mutation accounted for 47% and A > G mutation accounted for 14% in diseases caused by gene point mutation. Before the advent of base editing tools, the way of base substitution was mainly homologous recombination (HDR), and although the emergence of CRISPR/Cas system significantly improved HDR efficiency, the efficiency was still low at present. Therefore, the development of a novel gene editing method capable of efficiently introducing a single-base mutation into a genome or correcting a single-base mutation is of great importance for the study of the mechanism of a disease associated with a point mutation and the treatment thereof.

The base editing technology is a newly developed gene editing technology and can meet the requirement of single base mutation. Cytosine Base Editors (CBE) which can induce the conversion of base C into T and Adenine Base Editors (ABE) which can induce the conversion of base A into G have been successfully constructed at present by fusing deaminase with CRISPR/Cas systems (KomorAC, kimYB, packer M S, et al. Programmable adjusting of a target base in genetic DNA with double-stranded DNA restriction [ J ]. Nature,2016,533 (7603): 420-424 Gaudelli N M, komor A C, rees HA, et al. Programmable base editing of A.T to G.C in genetic DNA restriction [ J ]. Nature, 201464, 551 (7681): 7681). However, most of the currently used ABEs and CBEs are constructed based on Cas9, which recognizes C/G-rich PAM (Protoplacer adjacent motif) sequences, thus limiting the targeting range of these base editors. Cas12a (earlier referred to as Cpf 1) can recognize PAM sequences rich in A/T, so that the existing Cas 9-based base editor can be well supplemented, and the targetable range of the base editing system is widened. In addition, cas12a has other advantages: generating cohesive ends after cutting the DNA double strand; has RNase activity, and can cut pre-crRNA to form multiple mature guide RNAs (gRNAs); higher specificity than Cas 9. Among them, the rnase activity of Cas12a is particularly important, and it can cleave pre-crRNA to form multiple mature guide RNAs, and target multiple gene loci simultaneously, thereby realizing simultaneous editing of multiple genes. This makes Cas12a have unique application value as an important gene editing tool. At present, there are reports of native Cas9 and Cas12a homologous proteins that, although recognizing different PAM sequences, can still have limited targetable range, especially some native proteins have greater PAM restriction or their activity in mammalian cells is poor. Therefore, modifying existing Cas9 and Cas12a proteins to obtain protein variants with less PAM restriction is an effective way to break through this limitation.

Currently, researchers have fused different Cas9 variants with deaminases, developed a series of PAM sequence-extended base editors, but few have reported studies to extend the targeting range of Cas12 a-derived base editors. Furthermore, attempts to perform multi-site simultaneous editing using a Cas12 a-derived base editor have a problem of being extremely inefficient. In conclusion, the base editor which has a wider target range and can efficiently edit multiple gene loci simultaneously is developed based on the Cas12a, and has important significance for the field of gene editing.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides a Cas12a protein mutant, a base editor containing the same and application thereof, the invention takes dLbCas12a as a basis, respectively introduces mutation, obtains the Cas12a protein mutant capable of identifying PAM (polyacrylamide) range expansion, can be effectively applied to gene editing, expands the gene editing target range, and simultaneously obtains an efficient base editor capable of simultaneously carrying out base editing on multiple gene loci by utilizing the RNA enzyme activity of the mutant.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a mutant of a Cas12a protein, wherein the mutant of the Cas12a protein has any one or a combination of at least two of D156R, G532R and K538R combined mutation, G532R and K595R combined mutation or G532R, K538V and Y542R combined mutation on the basis of the dLbCas12a protein, and the amino acid sequence of the dLbCas12a protein comprises a sequence shown as SEQ ID NO. 1.

In the invention, based on the problem that the existing Cas12a protein targeting range is limited, based on the dLbCas12a protein (catalytic dead Lachnospiraceae bacterium Cas12 a), the dLbCas12a protein is modified on the basis of the dLbCas12a protein, mutations D156R, G532R and K538R combined mutation (D156R/G532R/K538R), G532R and K595R combined mutation (G532R/K595R) or G532R, K538V and Y542R combined mutation (G532R/K538V/Y542R) are respectively introduced, the Cas12a protein mutant capable of identifying the expanded PAM range is obtained, and the dLbCas12a protein mutant can be effectively applied to gene editing and has an important significance for widening the genome targetable range of the existing gene editing system.

SEQ ID NO.1：

MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIARGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALADLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVAFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHGS。

In a second aspect, the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding a mutant Cas12a protein according to the first aspect.

In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the second aspect.

In a fourth aspect, the present invention provides a recombinant cell comprising a nucleic acid molecule according to the second aspect.

In a fifth aspect, the present invention provides a use of the Cas12a protein mutant of the first aspect in preparing a gene editing product.

In a sixth aspect, the invention provides a base editor, wherein the base editor is a fusion protein formed by fusing deaminase and the Cas12a protein mutant of the first aspect.

According to the invention, the Cas12a protein mutant capable of identifying PAM range expansion is obtained through genetic modification, and the mutant is fused with deaminase to further construct an efficient base editor capable of identifying PAM range expansion.

In the present invention, the deaminase can be fused to the N-terminus, C-terminus or an internal fusion site of the Cas12a protein mutant.

In the present invention, deaminases used in the art to construct base editors are suitable for use in the present invention.

In the present invention, the deaminase may be selected from cytosine deaminase or adenine deaminase.

In the present invention, the cytosine deaminase is selected from a human cytosine deaminase (hAPOBEC 3A, hA 3A).

In the present invention, it was found that the base editor obtained by fusing the cytosine deaminase hA3A and the Cas12a protein mutant of the present invention has higher editing efficiency than the base editor constructed using rat-derived cytosine deaminase (rAPOBEC 1), without being affected by the sequence environment.

In the invention, the amino acid sequence of hA3A comprises a sequence shown in SEQ ID NO.2 or SEQ ID NO. 3.

SEQ ID NO.2：

MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIFDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN。

SEQ ID NO.3：

MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHGQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN。

In the present invention, the adenine deaminase may be selected from TadA-8e (TadA x 8 e).

In the invention, the amino acid sequence of the TadA-8e comprises a sequence shown in SEQ ID NO. 4.

SEQ ID NO.4：

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN。

In the invention, cytosine deaminase and a mutant of the dLbCas12a protein of the invention are fused to prepare a cytosine base editor (named as dLbCas12 a-CBE), adenine deaminase and a mutant of the Cas12a protein of the invention are fused to prepare an adenine base editor (named as dLbCas12 a-ABE), the action principle schematic diagrams are respectively shown in figure 1 and figure 2, after the mutant of the dLbCas12a protein is combined with guide RNA to form a compound, a target site is searched on a genome, after the guide RNA is combined with the genome in a targeted way, the deaminase activity is used for deaminating bases in a window range, and the base editing is realized.

In addition, the Cas12a protein has rnase characteristics, can cut the pre-crRNA in series to form multiple mature guide RNAs, can form different base editor complexes respectively, and can realize simultaneous and efficient editing of multiple gene sites, specifically as shown in fig. 9, multiple guide RNAs can be connected in series at the downstream of a promoter, and can be processed into multiple mature guide RNAs by using the rnase characteristics of Cas12a after being transcribed to form pre-crRNA, a target site in a genome can be searched to realize gene editing after the Cas12a forms a complex with the guide RNA, multiple genome sites can be simultaneously edited at the level of cells and embryos, and the editing efficiency is the same as that of single-site editing.

In a seventh aspect, the present invention provides a Cas12a protein mutant according to the first aspect or a base editor according to the sixth aspect for use in gene editing.

Preferably, the gene editing may be simultaneous gene editing at multiple gene sites.

In an eighth aspect, the present invention provides a gene editing method comprising:

the CRISPR-Cas system is formed by the Cas12a protein mutant and the guide RNA for gene editing, or,

the CRISPR-Cas system consisting of the Cas12 protein mutant and guide RNA formed by processing tandem guide RNA is utilized to carry out multigene site simultaneous gene editing.

In a ninth aspect, the present invention provides a base editing method comprising:

the base editing is performed using the base editor described in the sixth aspect.

Preferably, the base editing method comprises:

the simultaneous base editing of multiple gene sites is carried out using the base editor and the tandem guide RNA described in the sixth aspect.

Preferably, the base editing method comprises the steps of:

any mutant of D156R/G532R/K538R, G532R/K595R or G532R/K538V/Y542R is introduced into dLbCas12a protein to obtain a Cas12a mutant capable of identifying non-classical PAM, the mutant is fused with deaminase to prepare a base editor with widened targeting range, the base editor and a target site guide RNA are together transferred into a cell to be edited, and the target site is edited by using a protein-RNA complex formed by the base editor.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, based on dLbCas12a, mutations D156R/G532R/K538R, G532R/K595R and G532R/K538V/Y542R are respectively introduced, and a Cas12a protein mutant capable of identifying PAM (polyacrylamide) range expansion is obtained: encAS12a, RR and RVR, and then fusing cytosine deaminase hA3A or adenine deaminase TadA-8e with the Cas12a protein mutant to construct a high-efficiency base editor capable of identifying PAM range broadening, wherein the base editor can realize high-efficiency C > T or A > G editing on both cell and embryo levels of classical PAM (TTTV) and non-classical PAM; meanwhile, by means of the RNase activity of the Cas12a, the system can realize simultaneous and efficient editing of multiple genome sites, and has proved that the system can realize simultaneous and efficient C > T or A > G editing of multiple genome sites at the level of cells and embryos, the editing efficiency is the same as that of single-site editing, and the existing base editing toolbox is successfully enriched.

Drawings

FIG. 1 is a schematic diagram of a cytosine base editor of the present invention;

FIG. 2 is a schematic diagram of an adenine base editor according to the present invention;

FIG. 3 is a schematic diagram of a cytosine base editor of the present invention;

FIG. 4 is a schematic diagram of an adenine base editor according to the present invention;

FIG. 5 is a graph of cytosine base editing efficiency in HEK293T cells;

FIG. 6 is a graph of adenine base editing efficiency in HEK293T cells;

FIG. 7 is a graph of cytosine base editing efficiency in embryos;

FIG. 8 is a graph of adenine base editing efficiency in embryos;

fig. 9 is a schematic diagram of multi-site simultaneous editing using rnase function of Cas12 a;

fig. 10A is a diagram of a result of implementing multi-site simultaneous editing by a cytosine base editor constructed by using a Cas12a protein mutant on a HEK293T cell;

fig. 10B is a diagram of the result of implementing multi-site simultaneous editing by using an adenine base editor constructed by a Cas12a protein mutant on HEK293T cells;

fig. 11 is a diagram of the result of implementing multi-site simultaneous editing by using a cytosine base editor constructed by a Cas12a protein mutant on a porcine embryo.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1

In this example, a Cas12a protein mutant is constructed, based on dLbCas12a (amino acid sequence is shown in SEQ ID No. 1), mutations D156R/G532R/K538R, G532R/K595R and G532R/K538V/Y542R are respectively introduced, so as to obtain 3 Cas12a protein mutants, which are respectively and correspondingly named as encAS12a, RR and RVR.

Example 2

This example constructs a base editor, exemplified by the fusion site being the N-terminus, three mutants of dlbccas 12a in example 1: n-terminal fusion cell pyrimidine deaminase hA3A and adenine deaminase TadA-8e of encAS12a, RR and RVR construct related base editors, and the structural schematic diagrams are shown in FIG. 3 and FIG. 4. FIG. 3 is a schematic diagram of a cytosine base editor, using two kinds of hA3A (one containing a Y130F mutation with a sequence of SEQ ID NO.2; and the other containing an N57G mutation with a sequence of SEQ ID NO. 3), respectively, in which UGI is a uracil glycosylase inhibitor, NLS is a nuclear localization signal, and dCas12a represents a wild-type dLbCas12a protein as a control. FIG. 4 is a schematic diagram of the adenine base editor using TadA-8e containing the V106W mutation and having the sequence SEQ ID NO.4, where dCas12a represents the wild-type dLbCas12a protein and bqNLS is biopartite SV40 NLS as a control.

Example 3

This example analyzes the editing efficiency of the base editor constructed in example 2 from the cellular and embryo levels, respectively.

The specific method comprises the following steps:

at the cellular level, the cell genome was extracted 72 hours later by transfecting the base editor and the plasmid encoding the guide RNA into cells to be transformed of appropriate confluence, PCR amplification of the proposed editing sites using specific primers, followed by Sanger sequencing. Sequencing results can be analyzed by the EditR on-line tool for editR efficiency assessment.

At the level of an embryo, a base editor and a guide RNA plasmid are subjected to in vitro transcription to obtain RNA, then the RNA is injected into an animal embryo by using a micromanipulation system, the embryo is cultured in vitro to a blastocyst stage, a single blastocyst is collected, genome acquisition is carried out, then a specific primer is used for carrying out PCR amplification on a quasi-editing site, and Sanger sequencing is carried out. Sequencing results can be analyzed by the EditR online tool to evaluate editing efficiency.

The results are shown in fig. 5-8, fig. 5 is a graph of cytosine base editing efficiency in HEK293T cells, and the dlbbs 12a-CBE constructed by enCas12a and RR can improve the editing efficiency from less than 10% to 40% and 8 times for TCCC which is a non-classical PAM; the dLbCas12a-CBE constructed by RR can improve the editing efficiency from 2% to nearly 20% and improve about 10 times for the non-classical PAM TCCA; the dLbCas12a-CBE constructed by the enCas12a and the RVR improves the editing efficiency of TATG, namely non-classical PAM, to more than 30 percent from almost nothing. FIG. 6 is a diagram of adenine base editing efficiency in HEK293T cells, which shows that dLbCas12a-ABE constructed by Cas12a mutant can realize effective deamination for non-classical PAM, for example, for TTCC, the dLbCas12a-ABE constructed by encAS12a and RR can improve the efficiency from 10% to more than 20%, and improve the efficiency by about 2 times; for TATAPAM, dLbCas12a-ABE constructed with RVR can improve the efficiency from less than 5% to 10%.

At the embryo level, fig. 7 is a diagram of cytosine base editing efficiency in embryos, the activity of a dlbbs 12a-CBE system constructed by a Cas12a mutant in a classical PAM TTTV is the same as that of an original dlbbs 12a-CBE, while the efficiency is greatly improved for some non-classical PAMs, for example, for TTCA, the efficiency of the dlbbs 12a-CBE constructed by enCas12a and RR can be improved from less than 5% to about 50%, and is improved by about 10 times; for TTCT, TCCC and TCCA which are non-classical PAM, the efficiency can be improved from nearly ineffectiveness to nearly 40%; and the dLbCas12a-CBE constructed by RR has certain improvement on ATTC, TATG and TGTA. As shown in FIG. 8, the dLbCas12a-ABE constructed from RR can improve the efficiency from less than 5% to more than 35% and reach 7 times of efficiency improvement at the position of PUM1-16 (TCCA); in the case of IGF1-41 (CTTC), the dLbCas12a-ABE constructed with en-Cas12a can raise the efficiency to over 40% with almost no efficiency.

Example 4

This example utilizes the base editor constructed in example 2 to achieve simultaneous multi-gene editing in HEK293T cells and porcine embryos.

As shown in fig. 10A and 10B, guide RNAs targeting DNMT3B, KLF4, TET1, PRR5L and CFTR were concatenated together at cellular level in the manner of fig. 9, transfected HEK293T cells with CBE or ABE constructed from Cas12a protein mutants in example 2, 72h later for cell genome extraction, sanger sequencing by specific primer PCR followed by editing efficiency evaluation using EditR online tool, where multiple guide RNAs mixed transfected cells with single guide RNA transfected cells served as control group. From the results of the efficiency statistics, it was found that the editing efficiency was not very different in single guide RNA transfection, multiple guide RNA mixed transfection, or multiple guide RNAs in tandem. Moreover, the results were similar whether CBE or ABE.

As shown in fig. 11, at the embryo level, guide RNAs targeting pig PUM1, GHR, HMGA2 and PUM2 are connected in series according to the method of fig. 9, CBE or ABE constructed with Cas12a variants are respectively transcribed into RNA in vitro and then injected into parthenogenetic embryo of pig by microinjection, genome extraction is performed after development to blastocyst, PCR amplification is performed by using specific primers and Sanger sequencing is performed, and the sequencing result is used for the evaluation of editing efficiency by using EditR online tool, wherein a plurality of guide RNAs are mixed and injected as a control group, and as can be seen from the statistical result of efficiency, there is no difference in editing efficiency of the mixture of the tandem guide RNA and the plurality of guide RNAs for PUM1, GHR and HMGA2 site; for the PUM2 site, the efficiency of editing the tandem guide RNA is significantly improved compared to the efficiency of mixing multiple guide RNAs.

In conclusion, the invention is modified on the basis of dLbCas12a to obtain a Cas12a protein mutant capable of identifying PAM range expansion, and deaminase is fused with the Cas12a protein mutant to construct an efficient base editor capable of identifying PAM range expansion, and can realize multipoint simultaneous efficient editing, can be realized on both cell and embryo level, well supplements the existing base editing tool kit, successfully expands the targeted genome range, and can also be applied to the aspects of disease animal model construction, disease gene therapy, variety improvement and the like.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims (10)

1. A mutant of Cas12a protein, wherein the mutant of Cas12a protein has any one or combination of at least two of D156R, G532R and K538R combined mutation, G532R and K595R combined mutation or G532R, K538V and Y542R combined mutation on the basis of dLbCas12a protein;

the amino acid sequence of the dLbCas12a protein comprises a sequence shown in SEQ ID NO. 1.

2. A nucleic acid molecule comprising a nucleic acid sequence encoding a mutant Cas12a protein of claim 1.

3. An expression vector comprising the nucleic acid molecule of claim 2.

4. Use of a Cas12a protein mutant according to claim 1 in the preparation of a gene editing product.

5. A base editor, wherein the base editor is a fusion protein formed by fusing a deaminase and the Cas12a protein mutant of claim 1.

6. The base editor of claim 5, wherein the deaminase is fused to the N-terminus, C-terminus, or an internal fusion site of the Cas12a protein mutant.

7. The base editor of claim 5 or 6 wherein said deaminase is selected from the group consisting of cytosine deaminase and adenine deaminase;

preferably, the cytosine deaminase is selected from hA3A;

preferably, the amino acid sequence of hA3A comprises the sequence shown in SEQ ID NO.2 or SEQ ID NO. 3;

preferably, the adenine deaminase is selected from TadA-8e;

preferably, the amino acid sequence of TadA-8e comprises the sequence shown in SEQ ID NO. 4.

8. Use of a Cas12a protein mutant as claimed in claim 1 or a base editor as claimed in any one of claims 5 to 7 in gene editing;

preferably, the gene editing comprises gene editing at multiple gene sites simultaneously.

9. A gene editing method comprising:

using the Cas12a protein mutant and the guide RNA of claim 1 to form a CRISPR-Cas system for gene editing, or,

the CRISPR-Cas system composed of Cas12 protein mutant as claimed in claim 1 and guide RNA formed by processing tandem guide RNA is used for multigene site simultaneous gene editing.

10. A base editing method comprising:

base editing using the base editor of any one of claims 5-7;

preferably, the base editing method comprises:

the simultaneous base editing of multiple gene sites using the base editor of any one of claims 5-7 and a tandem guide RNA.

Images