A cytosine base editor based on cynomolgus monkey APOBEC3A and its mutants

Abstract

The present invention discloses a cytosine base editor based on cynomolgus monkey APOBEC3A and its mutants, belonging to the field of genetic engineering technology. A series of CBE single-base editors constructed by the present invention mainly include sgRNA, mA3A or its mutants, nCas9 (D10A), red fluorescent protein, uracil DNA glycosylase inhibitor, high copy replication origin and ampicillin resistance screening marker gene. The editing window of the single-base editor constructed by the present invention is the C3-C14 site, the editing efficiency can reach up to 75%, and the product purity can reach up to 95%. The present invention uses APOBEC3A of non-human primate cynomolgus monkey as the cytosine deaminase of the CBE system for the first time, enriching the gene editing toolkit, and providing a good idea and direction for the optimization of cytosine base editors in the future.

Description

Technical Field

[0001] The present invention relates to a cytosine base editor based on cynomolgus monkey APOBEC3A and mutants thereof, belonging to the technical field of genetic engineering. Background Art

[0002] At present, gene editing technology is developing rapidly. By modifying specific genes, it can study gene functions, the pathogenesis of genetic diseases, develop new drugs, and be used for gene therapy and crop improvement. In recent years, various derivative technologies based on the CRISPR-Cas system have been widely used in the fields of life sciences and medicine. Among them, base editors (BE) have become an important part of gene editing technology. BE is designed based on the CRISPR-Cas9 system. The wild-type Cas9 protein is modified and connected to cytosine deaminase or adenine deaminase, and then guided by sgRNA (small guide RNA), a single base is directly edited without generating double stranded breaks (DSB) of DNA. BE is mainly divided into two categories: cytosine base editor (CBE) and adenine base editor (ABE). In 2016, the cytosine deaminase used in the CBE system developed by Dvid R Liu.'s team can convert the corresponding cytosine (C) in the non-complementary chain into uracil (U) through deamination. During DNA replication or repair, U is recognized as thymine (T), and the corresponding guanine (G) on the complementary chain will become adenine (A), ultimately achieving the conversion of C>T on the non-complementary chain and G>A on the complementary chain. In 2017, Dvid R Liu.'s team developed the ABE system, which has a similar principle to the CBE system, except that the cytosine deaminase is replaced by adenine deaminase, which can complete the editing of A>G on the non-complementary chain and T>C on the complementary chain, further supplementing the types of single-base editors.

[0003] The Cas9 protein in the BE system can be further optimized. One is dCas9 (Catalytically dead Cas9) with no endonuclease activity that can bind to the target gene but does not cut the target gene, and the other is nCas9 (Cas9 nickase) with single-strand DNA nickase activity. Both Cas9 proteins will not produce DBS, thus avoiding the mismatch of non-homologous end-joining (NHEJ) and the low efficiency of homologous recombination repair (HDR). Subsequently, uracilglycosylase inhibitor (UGI) was added to BE to inhibit the excision of the intermediate product U, thereby improving the editing efficiency of C>T on the DNA chain. At present, the BE system has developed rapidly. The CBE system has been optimized to the fourth generation BE4, and the ABE system has also been optimized to the ABE7.10 version. Even some dual-base editors that integrate the functions of the CBE and ABE systems have been developed, which can simultaneously achieve the conversion of C>T and A>G at the same target site.

[0004] The cytosine deaminase used in the traditional CBE system is rat Apobec1 (rA1). In 2018, Jason M Gehrke et al. tried to use human modified APOBEC3A (eA3A) to replace rA1 in the third-generation CBE system (BE3). They performed site-directed mutagenesis on human APOBEC3A (hA3A) and constructed a series of BE3 mutant editors carrying single amino acid mutations in hA3A, among which hA3A-N57G targeting ( HBB -28 site) editing efficiency is high and the adjacent ( HBB -25 The editing efficiency of hA3A-N57G was the lowest among all mutants. They then used BE3 carrying hA3A-N57G for the treatment of β-thalassemia, and improved its editing accuracy by nearly 40 times compared with BE3. HBB -28 The editing efficiency of the site is only about 30%.

[0005] Since most of the pathogenic mutations of single-gene genetic diseases are point mutations, the BE system can edit single bases in the DNA sequence, thereby correcting the pathogenic site mutations of genetic patients, achieving "molecular surgery" and curing genetic diseases on the basis of pathogenic substances. It is known that there are about 75,000 human genome site mutations related to genetic diseases, of which about 50% are estimated to be potential therapeutic targets for the CBE system. Therefore, the development and optimization of more efficient and accurate single-base editors are of great significance for the treatment of single-gene genetic diseases. Summary of the invention

[0006] Most of the cytosine deaminases used in existing CBE systems are rat Apobec1 (rA1), and a few are human APOBEC3A (hA3A) and APOBEC3G (hA3G). Currently, there is no application of cytosine deaminases from non-human primates, crab-eating macaques, in CBE systems.

[0007] In order to solve the above problems, the object of the present invention is to provide a gene editing tool with high editing efficiency, high product purity and flexible editing window.

[0008] The first object of the present invention is to provide a mutant of cynomolgus monkey-derived cytosine deaminase, wherein the mutant is based on the parent enzyme with an amino acid sequence as shown in SEQ ID NO: 1, with the phenylalanine at position 137 mutated to proline, the methionine at position 123 mutated to valine, the glutamic acid at position 111 mutated to glycine and / or the cysteine ​​at position 77 mutated to arginine.

[0009] The second object of the present invention is to provide a gene encoding the above cytosine deaminase mutant.

[0010] In one embodiment, the nucleotide sequence of the mutant is shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0011] The third object of the present invention is to provide an expression cassette, which comprises the gene encoding the cytosine deaminase mutant.

[0012] In one embodiment, the expression cassette further contains a promoter, nCas9 (D10A), a uracil DNA glycosylase inhibitor (UGI), a nuclear localization sequence NLS, and a termination sequence.

[0013] In one embodiment, the promoter drives the expression of the gene encoding the above-mentioned cytosine deaminase mutant, and the sequence of connection is the promoter, the cytosine deaminase mutant, nCas9 (D10A), uracil DNA glycosylase inhibitor (UGI), nuclear localization sequence NLS and termination sequence.

[0014] In one embodiment, the promoter includes but is not limited to CMV promoter and Amp promoter.

[0015] In one embodiment, the nucleotide sequence of the nCas9 (D10A) is shown as SEQ ID NO:6, the nucleotide sequence of UGI is shown as SEQ ID NO:7, the nucleotide sequence of the NLS is shown as SEQ ID NO:8, and the nucleotide sequence of the termination sequence is shown as SEQ ID NO:9.

[0016] The fourth object of the present invention is to provide a CBE single-base editing system, which comprises four parts. The first part is a transfection efficiency indicator part, which comprises a red fluorescent protein (dTomato) and a promoter; the second part is a sgRNA transcription unit, which comprises a carrying frame for inserting the sgRNA sequence and its promoter; the third part comprises the above-mentioned expression cassette, and the fourth part comprises a high-copy replication origin ori from the colicin factor (colE1) and an ampicillin resistance screening gene AmpR.

[0017] In one embodiment, the promoter includes a CMV promoter and a U6 promoter.

[0018] In one embodiment, the nucleotide sequence of the red fluorescent protein is as shown in SEQ ID NO:10.

[0019] In one embodiment, the nucleotide sequence of the sgRNA transcription unit is shown in SEQ ID NO:11.

[0020] The fifth object of the present invention is to provide the application of the above-mentioned CBE single-base editing system in the field of gene editing.

[0021] In one embodiment, an sgRNA sequence is designed according to the target gene and inserted into the sgRNA transcription unit of the above-mentioned CBE single-base editing system to obtain a CBE single-base editing system with a specific targeted gene. The CBE single-base editing system is introduced into a recipient cell to achieve a mutation of the target base C to T, thereby obtaining cells containing a single base mutation.

[0022] In one embodiment, the cell is a eukaryotic cell.

[0023] In one embodiment, the eukaryotic cell is a mammalian cell.

[0024] In one embodiment, the mammalian cell comprises a human embryonic kidney epithelial cell HEK293T.

[0025] The sixth object of the present invention is to provide a method for constructing the CBE single-base editing system, and the specific construction steps are as follows: (1) using the pSpCas9(BB)-2A-dTomato (PX458) plasmid as a template to amplify the red fluorescent protein dTomato gene and its CMV enhancer and promoter to obtain the vector skeleton Part 1;

[0026] (2) Using the BE3-rA1 plasmid as a template, amplify the carrier frame for inserting the sgRNA sequence and its U6 promoter to obtain the vector skeleton Part 2;

[0027] (3) Using the cDNA of cynomolgus macaque as a template, the nucleotide sequence of APOBEC3A (mA3A-B5) was amplified to obtain the vector backbone Part 3;

[0028] (4) Using plasmid BE4-rA1 as a template, amplification was performed to obtain the vector backbone Part 4 including nCas9 (D10A), UGI, NLS, termination sequence, and CMV enhancer and promoter gene sequences;

[0029] (5) Using plasmid BE4-rA1 as a template, amplify the vector backbone Part 5 containing the colE1 high-copy replication origin, the ampicillin resistance selection gene, and its ampicillin promoter sequence;

[0030] (6) Connect the five fragments in the order of Part 1, Part 2, Part 3, Part 4, and Part 5 to obtain the vector plasmid BE4-mA3A-B5.

[0031] (7) Using the vector plasmid BE4-mA3A-B5 constructed in step (6) as a template, the phenylalanine at position 137 of the amino acid sequence of mA3A-B5 was mutated to proline to obtain mA3A-V4V5, the methionine at position 123 of mA3A-V4V5 was mutated to valine to obtain mA3A-V3, the glutamic acid at position 111 of mA3A-V3 was mutated to glycine to obtain mA3A-V2, and the cysteine ​​at position 77 of mA3A-V2 was mutated to arginine to obtain mA3A-V1.

[0032] (8) Designing a sgRNA sequence that binds to the target gene and inserting it into the carrier framework constructed in step (2) to obtain a single base editor that can target the corresponding sites of different target genes.

[0033] In one embodiment, the pSpCas9(BB)-2A-dTomato (PX458) plasmid is constructed by replacing GFP with dTomato based on the plasmid pSpCas9(BB)-2A-GFP (PX458), and the nucleotide sequence of the dTomato is shown in SEQ ID NO:10.

[0034] The present invention also provides application of the mutant in the field of gene editing.

[0035] The present invention also provides application of the above expression cassette in the field of gene editing.

[0036] Beneficial effects of the present invention:

[0037] 1. A series of single-base editors BE4-mA3A constructed by the present invention contain sgRNA transcription units. When a series of BE4-mA3A editors are used for gene editing, only the BE4-mA3A single plasmid needs to be transfected into cells. The sgRNA targeting the target gene carried on the plasmid guides the fusion protein to bind to the target gene DNA region through the principle of base complementary pairing. Then mA3A-B5 binds to the single-stranded DNA (ssDNA) in the R-loop region formed by the nCas9 protein, sgRNA and target gene DNA, and deaminates cytosine (C) within a certain range of the ssDNA (20 bp original spacer sequence) into uracil (U), and then converts uracil (U) into thymine (T) through DNA replication or repair, ultimately achieving direct replacement of CG base pairs to TA base pairs. In addition, since nCas9 only has single-stranded DNA nickase activity, it will not cut the double-stranded DNA of the target gene between 2-3 bases upstream of the PAM (Protospacer-Adjacent Motif) sequence, thereby not forming DSB, which greatly reduces the off-target efficiency.

[0038] 2. The present invention successfully constructed a series of cynomolgus monkey-derived cytosine deaminase mA3A-B5 and its mutants, and used cytosine deaminase mA3A-B5 and its mutants to construct a series of cytosine base editors that are superior to those carrying rat-derived cytosine deaminase in editing efficiency, product purity and editing window size, and successfully applied them to site3, RNF2 and EMX1 C>T editing of target sites in genes. This provides a more flexible and controllable prototype and new ideas and directions for further optimization and transformation of CBE, enriches the toolkit of gene editing, and also provides a potential tool for gene therapy of genetic diseases. (Note: site3 is the name in the published article, and the gene name of site3 is LINC01509 ) BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 :pSpCas9(BB)-2A-dTomato (PX458) plasmid map;

[0040] Figure 2 :Plasmid map of cytosine base editor BE4-mA3A-B5;

[0041] Figure 3 : Part1 and Part2 of BE4-mA3A (BE4-mA3A-B5, BE4-mA3A-V1, BE4-mA3A-V2, BE4-mA3A-V3 and BE4-mA3A-V4V5) plasmid elements;

[0042] Figure 4 : Part 3 of the BE4-mA3A-B5 plasmid element, namely the APOBEC3A (mA3A-B5) fragment of cynomolgus monkey;

[0043] Figure 5 : Part4 and Part5 of BE4-mA3A (BE4-mA3A-B5, BE4-mA3A-V1, BE4-mA3A-V2, BE4-mA3A-V3 and BE4-mA3A-V4V5) plasmid elements;

[0044] Figure 6 :Sanger sequencing verification of RNA self-editing sites of APOBEC3A in cynomolgus monkeys;

[0045] Figure 7 :Sanger sequencing verified the editing efficiency of different cytosine single-base editors (BE3-rA1, BE4-rA1, BE4-mA3A-B5, BE4-mA3A-V1, BE4-mA3A-V2, BE4-mA3A-V3 and BE4-mA3A-V4V5) at the site3 gene targeting site (n=3);

[0046] Figure 8 :Sanger sequencing validates the expression of different cytosine base editors (BE3-rA1, BE4-rA1, BE4-mA3A-B5, BE4-mA3A-V1, BE4-mA3A-V2, BE4-mA3A-V3, and BE4-mA3A-V4V5) in RNF2 Editing efficiency of gene targeting sites (n=3);

[0047] Fig. 9:Sanger sequencing validates the expression of different cytosine base editors (BE3-rA1, BE4-rA1, BE4-mA3A-B5, BE4-mA3A-V1, BE4-mA3A-V2, BE4-mA3A-V3, and BE4-mA3A-V4V5) in EMX1 Editing efficiency of gene targeting sites (n=3);

[0048] Fig.10 : Comparison of product distribution of BE3-rA1, BE4-rA1, BE4-mA3A-B5, BE4-mA3A-V1, BE4-mA3A-V2, BE4-mA3A-V3, and BE4-mA3A-V4V5 editors at all targeted sites of the site3 gene (n=3);

[0049] Fig.11 : BE3-rA1, BE4-rA1, BE4-mA3A-B5, BE4-mA3A-V1, BE4-mA3A-V2, BE4-mA3A-V3 and BE4-mA3A-V4V5 editors are in RNF2 Comparison of product distribution at all targeted sites of the gene (n=3);

[0050] Fig.12 : BE3-rA1, BE4-rA1, BE4-mA3A-B5, BE4-mA3A-V1, BE4-mA3A-V2, BE4-mA3A-V3 and BE4-mA3A-V4V5 editors are in EMX1 Comparison of product distribution at all targeted sites of the gene (n=3);

[0051] Fig.13 :The average editing efficiency of Cs in three gene loci was sorted according to the position of cytosine C in the protospacer (PAM is located at positions 21-23). ​​Different shapes and grayscales represent different editors (n = 3, 6, 9). DETAILED DESCRIPTION

[0052] The plasmids involved in the following examples are:

[0053] BE4 Plasmid: Addgene Plasmid #100802.

[0054] pSpCas9(BB)-2A-dTomato (PX458) plasmid: This plasmid was constructed by chemically synthesizing dTomato (SEQ ID NO: 10) to replace GFP based on the pSpCas9(BB)-2A-GFP(PX458) (Addgene Plasmid #48138) plasmid. See the plasmid map for details. Figure 1 .

[0055] BE3-rA1 plasmid: donated by Professor Yang Hui, reported in the article Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. The BE3 in the article is BE3-rA1 in the present invention.

[0056] BE4-rA1 plasmid: constructed in our laboratory, based on BE4 plasmid (Addgene Plasmid #100802). Not The chemically synthesized dTomato gene sequence (SEQ ID NO: 10) and the U6 promoter of the BE3-rA1 plasmid + the sgRNA sequence carrying framework (sgRNA scaffold) + CMV enhancer + CMV promoter fragment were inserted into the Ⅰ restriction site in sequence.

[0057] The cynomolgus monkeys involved in the present invention were purchased from Guangzhou Xiangguan Biotechnology Co., Ltd. (Production License No.: SCXK (Yue) 2018-0043). They were in good health as confirmed by records and veterinary examinations before the experiment, and the animal facilities met the national laboratory animal standards (GB14925-2010). Subsequently, cynomolgus monkeys that had been treated with a high-sugar and high-fat diet for 19 months and had hypercholesterolemia and could recover to normal blood lipids on their own were selected for the research of the present invention.

[0058] Example 1: Construction of a cytosine single-base editor based on cynomolgus monkey APOBEC3A (mA3A-B5) and its mutants

[0059] (1) Acquisition of target genes (mA3A-B5)

[0060] The present invention uses a Paxgene tube to obtain a blood sample of a cynomolgus monkey, and uses a blood RNA extraction kit of TIANGEN to extract RNA from the cynomolgus monkey, and uses a reverse transcriptase kit of Takara to obtain a cDNA sequence of the cynomolgus monkey. Then, using the cDNA sequence as a template, PCR amplification is performed using a primer pair 5'-TATAGGGAGAGCCGCCACCATGGAAGCCAGCCCAG-3' and 5'-ACCAGAAGAACCACCAGAGTTTCCCTGATTCTGG-3' to obtain a mA3A-B5 gene fragment (i.e., Part 3, Figure 4 , SEQ ID NO: 1), and then purify the PCR product using TIANGEN's agarose gel DNA extraction kit. The PCR reaction system is shown in Table 1.

[0061] Table 1 PCR reaction system

[0062]

[0063] The reaction procedure was as follows: pre-denaturation at 95°C for 3 min; 35 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 25 s; extension at 72°C for 5 min, and cooling to 4°C to finally obtain mA3A-B5 (Part 3).

[0064] (2) Preparation of linearized plasmid vector (BE4)

[0065] Using BE4-rA1 plasmid as template, PCR amplification was performed with primers 5'-TCTGGTGGTTCTTCTGGTGGTTCTAGCGGC-3' and 5'-GGTGGCGGCTCTCCCTATAGTGAGTCGTAT-3' to obtain PCR product Part4, including nCas9 (D10A), UGI, NLS, bGH poly(A) signal BE4, and CMV enhancer and promoter gene sequences ( Figure 5 );

[0066] Using BE4-rA1 plasmid as template, PCR amplification was performed with primer pair 5'-CGGTGGCTTCGATAGCCCTACAGTTGCCT-3' and 5'-CTACTAGGACAGAATAGGCAACTGTAGGGC-3' to obtain PCR product Part5, including colE1 high copy replication origin and ampicillin resistance selection gene and its ampicillin promoter sequence.

[0067] PCR products Part 4 and Part 5 were purified by TIANGEN's agarose gel DNA extraction kit ( Figure 5 ).

[0068] (3) Obtaining the gene for red fluorescent protein (dTomato)

[0069] Using the pSpCas9(BB)-2A-dTomato (PX458) plasmid as a template, PCR amplification was performed using primers 5'-TAGAGATCCGCGCCACCATGGTGAGC-3' and 5'-GAAGGCACAGTTACTTGTACAGCTCG-3' to obtain the red fluorescent protein dTomato gene and its CMV enhancer and promoter, namely the vector backbone Part 1 ( Figure 3 ). Then purify the PCR product Part 1 using TIANGEN's agarose gel DNA extraction kit.

[0070] (4) Obtaining the gene carrying frame of sgRNA and U6 promoter

[0071] Using the BE3-rA1 plasmid as a template, PCR amplification was performed using the following primers: 5'-GCTCACATGTGAGGGCCTATTTCCC-3' and 5'-ATAGGCCCTCACATGTGAGCAAAAG-3' to obtain the carrier frame (sgRNAscaffold) for inserting the sgRNA sequence and its U6 promoter, i.e., the vector skeleton Part2 ( Figure 3 ). Then use TIANGEN's agarose gel DNA extraction kit to purify the PCR product Part 2.

[0072] (5) Construction of vector plasmid BE4-mA3A-B5 (homologous recombination method)

[0073] The five PCR fragments Part 1, Part 2, Part 3, Part 4 and Part 5 purified in steps (1) to (4) were ligated using the MultiS One Step Cloning Kit from Novozymes to obtain ligation products, which were transformed into Escherichia coli DH5α and spread on LB plates containing 0.05% Amp (ampicillin at a concentration of 100 μg / mL) resistance and cultured inverted at 37°C overnight.

[0074] Select 3 colonies on each cloning plate and inoculate them into liquid LB medium containing 0.1% Amp (100 μg / mL ampicillin) for more than 8 hours, and then send the bacterial solution to Genewise for Sanger sequencing verification. The clone vector with the fragment successfully inserted was expanded and cultured, and then the vector plasmid was obtained using the endotoxin-free plasmid extraction kit of Kangwei Century Company and named BE4-mA3A-B5, and stored at -20℃ for future use. The BE4-mA3A-B5 plasmid map is shown below: Figure 2 shown.

[0075] (6) Construction of vector plasmid containing mA3A mutant

[0076] The results of Sanger sequencing verification of RNA self-editing sites of cynomolgus monkey APOBEC3A are shown in Figure 6 A series of mA3A mutants were obtained by site-directed mutagenesis based on their RNA self-editing sites (see Table 2), among which the gene sequences of V1, V2, V3 and V4V5 are shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0077] Using the vector plasmid BE4-mA3A-B5 constructed in step (5) as a template, the mutant primer 5'-GATTACGATC CC CTGTATCAG-3′ and 5′-CAG GG GATCGTAATCATAGATG-3' was used for PCR to amplify the PCR product. Dpn The PCR product was digested with I (the digestion system is shown in Table 3), the template plasmid that was not amplified was removed, and the PCR product after digestion was directly transformed into competent Escherichia coli (DH5α), sequenced and verified, and the plasmid was extracted with a kit to finally obtain the BE4-mA3A-V4V5 vector plasmid.

[0078] Table 2: mA3A mutant construction table

[0079]

[0080] In the same way, for Figure 6 A series of mutant editors were obtained after site-directed mutagenesis of the RNA self-editing sites in the genome. The specific mutation sites are shown in Table 2:

[0081] Using BE4-mA3A-V4V5 as template, the mutant primer 5'-AGAACACAC A CGTGAGACTG-3' and 5'-CA C GTGTGTGTTCTCCTGAAG -3' was used for PCR amplification to obtain the BE4-mA3A-V3 vector plasmid.

[0082] Using BE4-mA3A-V3 as template, the mutant primer 5'-GCTGTGCCG G GCAAGTGCGT-3' and 5'-TTGC C CGGCACAGCCCCTCC-3' was amplified by PCR to obtain the BE4-mA3A-V2 vector plasmid.

[0083] Using BE4-mA3A-V2 as template, the mutant primer 5'-AGAACACAC A CGTGAGACTG-3' and 5'-GAAGC G CAGCTCCGCGTGGC-3' was amplified by PCR to obtain the BE4-mA3A-V1 vector plasmid.

[0084] Table 3 Dpn I restriction enzyme system

[0085]

[0086] Example 2: Construction of a cytosine single-base editor targeting a target gene

[0087] (1) Inserting sgRNA that specifically targets a gene

[0088] Since the single-base editor is based on the CRISPR-Cas9 system, its targeting specificity is composed of two parts, one is the base complementary pairing between the sgRNA and the target DNA sequence, and the other is determined by the Cas9 protein and the short DNA sequence at the 3' end of the target DNA sequence. The target DNA sequence is called the protospacer (the foreign DNA fragment is called the protospacer during the natural immune defense of bacteria), and the short DNA sequence at the 3' end of the target DNA sequence is called PAM (protospacer adjacent motif).

[0089] In order to better compare with existing studies, the sgRNA used is a sequence that has been reported in the literature, see Table 4 for details.

[0090] Table 4: sgRNA and its PAM sequence list

[0091]

[0092] 1) Utilize Bbs I respectively digested the BE3-rA1 plasmid, the BE4-rA1 plasmid, and a series of cytosine base editors (BE4-mA3A-B5, BE4-mA3A-V1, BE4-mA3A-V2, BE4-mA3A-V3, and BE4-mA3A-V4V5) constructed in Example 1 to obtain linearized vectors. The enzyme digestion system is shown in Table 5;

[0093] 2) Chemically synthesize the target site 3, RNF2 and EMX1 The cloning primers of the sgRNA were used, and the cloning primers were self-ligated into double-stranded oligonucleotide fragments by heat shock annealing (the sgRNA self-ligation system is shown in Table 7). The double-stranded oligonucleotide fragments were respectively connected to the linearized vectors in step 1) (the connection system is shown in Table 8), and the connection fragments were transformed into Escherichia coli DH5α competent cells. The plasmids were extracted and sequenced to obtain the target sites 3, RNF2 and EMX1 A series of cytosine single-base editors for genes.

[0094] Table 5 Bbs I restriction enzyme system

[0095]

[0096] Table 6 sgRNA cloning primers

[0097]

[0098] Table 7 sgRNA self-ligation system

[0099]

[0100] Table 8 Connection system

[0101]

[0102] (2) Cell transfection

[0103] Preparation of high-glucose DMEM complete medium: High-glucose DMEM medium contains 10% fetal bovine serum (FBS) and 1% triple antibody (penicillin-streptomycin-gentamicin).

[0104] 1) Cell culture: Take out the frozen HEK293T cells from the liquid nitrogen tank, thaw them quickly in a 37°C water bath, add the thawed cell suspension to 10 mL of high-glucose DMEM complete medium, collect the cell pellet by centrifugation, resuspend the cells in high-glucose DMEM complete medium and add them to a cell culture dish containing 10 mL of high-glucose DMEM complete medium, and culture them in a 37°C constant temperature cell culture incubator containing 5% CO2.

[0105] 2) Cell plating: When HEK293T cells have grown all over the culture dish, digest the cells with 0.25% trypsin for 1 min, terminate the digestion with high-glucose DMEM complete medium, centrifuge at 1500 rpm for 2 min, discard the supernatant, resuspend the cells with 1 mL high-glucose DMEM complete medium, and plate at 1×10 5 The cells were seeded in 6-well plates (each well contained 2 mL of high-glucose DMEM complete medium);

[0106] 3) Cell transfection: In a 1.5 mL EP tube, dilute 3.75 µL of transfection reagent Lipofectamine 3000 with 125 µL Opti-MEM medium and mix thoroughly to obtain mixed solution A. In another 1.5 mL EP tube, dilute 2.5 µg of the target site 3 constructed in step (1) with 125 µL Opti-MEM medium. RNF2 or EMX1The cytosine single base editor of the gene was added, and 5 µL of P3000 auxiliary transfection reagent was added and mixed thoroughly to obtain a mixture B; the mixture A and B were mixed in a ratio of 1:1, and incubated at room temperature for 15 min to obtain a mixture; the mixture was added to the 6-well plate where the cells had just been plated in step 2), and gently mixed to avoid damaging the cells; the 6-well plate was then returned to a 37°C, 5% CO2 incubator to incubate the cells for 72 h before use for subsequent experiments. During this period, the cells were observed and photographed using an inverted microscope every 24 h.

[0107] (3) Flow sorting

[0108] First, the cells transfected for 72 h were digested with 0.25% trypsin, and the digestion was terminated with high-glucose DMEM complete medium. The cell pellet was collected by balanced centrifugation at 1500 rpm for 2 min, and then 2 mL of PBS containing 2% fetal bovine serum (FBS) was added to resuspend and wash the cells. The cells were balanced centrifuged at 1500 rpm for 2 min. After repeated washing twice, the cells were resuspended in 1 mL of PBS containing 2% FBS, and then the cell suspension was collected through a flow cytometry tube with a filter membrane to remove larger cell debris aggregates and avoid clogging of the instrument during sorting.

[0109] The prepared cell suspension and the corresponding collection tube containing 2 mL of high-glucose DMEM complete medium were placed on ice, and then flow cytometry sorting was performed. Because the cytosine base editor constructed in the present invention carries red fluorescent protein (dTomato), dTomato-positive cells were directly sorted and collected according to the excitation wavelength (554 nm) and emission wavelength (581 nm). The BE3-rA1 plasmid carries green fluorescent protein (GFP), and GFP-positive cells were directly sorted and collected according to its excitation wavelength (488 nm) and emission wavelength (507 nm). At least 100,000 cells were obtained for each sample.

[0110] (4) Sanger sequencing verification

[0111] The dTomato-positive cells and GFP-positive cells collected in step (3) were balanced centrifuged at 1500 rpm for 2 min, and the precipitate was collected. Then, the cell DNA was extracted using the Novogene cell / tissue DNA extraction kit. Using DNA as a template, a high-fidelity enzyme was used to PCR amplify the specific sequence of the target gene (~400 bp), that is, the amplicon containing the 20 bp fragment targeted by sgRNA, to obtain the PCR product. The sequencing primers are shown in Table 9. Take 5 µL of the PCR product for agarose gel electrophoresis, and send the PCR product with the expected band size to Genewise for Sanger sequencing. The peak diagram of the Sanger sequencing was viewed using Chromas software, and the results of the Sanger sequencing were analyzed using SnapGene Viewer software to analyze the proportion of each base.

[0112] Table 9 Sanger sequencing primers for sgRNA

[0113]

[0114] Example 3: Application of cytosine single-base editors based on mA3A-B5 and its mutants

[0115] (1) Cytosine single base editor targeting site3 gene

[0116] A series of editors targeting the site3 gene (such as BE3-rA1, BE4-rA1, BE4-mA3A-B5, BE4-mA3A-V1, BE4-mA3A-V2, BE4-mA3A-V3 and BE4-mA3A-V4V5) were transfected into HEK293T cells respectively, and the successfully transfected cells were sorted out. The editing status of each editor at the same gene site was analyzed. The specific implementation method was the same as steps (2) to (4) of Example 2.

[0117] The editing efficiency of different editors at the site3 gene targeting site is compared in Figure 7 The results showed that compared with the original BE3-rA1 and BE4-rA1 carrying the rat-derived cytosine deaminase Apobec1, the editors carrying the cytosine deaminase mA3A-B5 or its mutants from the cynomolgus monkey increased the editing efficiency at C>T by nearly 1 times, and the editing window was relatively expanded. The editors carrying mA3A-B5 or its mutants also performed efficient editing at the C14 site ( Figure 7 , Fig.10 and Fig.13 In addition, compared with the original BE3-rA1 and BE4-rA1 plasmids, the editors carrying mA3A-B5 or its mutants had improved purity at the five targeted sites of the site3 gene ( Fig.10), the higher the proportion of C edited to T, the higher the product purity, that is, the proportion of non-T products produced is greatly reduced, especially at site3-C14. Compared with BE3-rA1 and BE4-rA1 plasmids, the purity of the editing product of the editor carrying mA3A-B5 or its mutants is increased by nearly 8 times.

[0118] (2) Targeting RNF2 Genetic cytosine single base editor

[0119] Will target RNF2 A series of gene editors (such as BE3-rA1, BE4-rA1, BE4-mA3A-B5, BE4-mA3A-V1, BE4-mA3A-V2, BE4-mA3A-V3 and BE4-mA3A-V4V5) were transfected into HEK293T cells respectively, and the successfully transfected cells were sorted out. The editing status of each editor at the same gene site was analyzed. The specific implementation method is the same as steps (2) to (4) of Example 2.

[0120] Different editors RNF2 Comparison of editing efficiency of gene targeting sites is shown in Figure 8 The results showed that the editing efficiency of editors carrying mA3A-B5 or its mutants was 2-3 times higher than that of BE3-rA1 and BE4-rA1 plasmids carrying rA1, which expanded the editing window and increased the editing efficiency of C>T at the RNF2-C12 site by nearly 20 times. In addition, the editing products of editors containing mA3A-B5 and its mutants were also more pure, especially at the RNF2-C6 site, where the purity was nearly 10 times higher than that of BE3-rA1 and BE4-rA1 plasmids (see Fig.11 ).

[0121] (3) Targeting EMX1 Genetic cytosine single base editor

[0122] Will target EMX1 A series of gene editors (such as BE3-rA1, BE4-rA1, BE4-mA3A-B5, BE4-mA3A-V1, BE4-mA3A-V2, BE4-mA3A-V3 and BE4-mA3A-V4V5) were transfected into HEK293T cells respectively, and the successfully transfected cells were sorted out. The editing status of each editor at the same gene site was analyzed. The specific implementation method is the same as steps (2) to (4) of Example 2.

[0123] Different editors EMX1 Comparison of editing efficiency of gene targeting sites is shown in Fig. 9The results showed that the editing efficiency of editors carrying mA3A-B5 or its mutants was more than 3 times higher than that of BE3-rA1 and BE4-rA1 plasmids carrying rA1 in C>T, which expanded the editing window and increased the editing efficiency of C>T at the EMX1-C10 site by 5 to 10 times. In addition, the editing products of editors carrying mA3A-B5 and its mutants were also more pure, especially at the EMX1-C6 and EMX1-C10 sites, where the purity was nearly 10 times higher than that of the original editors (see Fig.12 ).

[0124] Example 4: Comparison of editing windows of cytosine single-base editors

[0125] Integrate all tested genes (site3, RNF2 and EMX1 ) and the average editing efficiency of Cs (C bases) in these three gene loci were sorted out according to the position of the C base in the protospacer (PAM is located at positions 21-23). ​​It can be seen that the editing window of editors carrying mA3A-B5 or its mutants is enlarged and the overall editing efficiency is higher, but the difference between different variants is not large. Among them, BE3-rA1 and BE4-rA1 have the smallest editing window, and can only be effectively edited between C4-C6, while the editing window of a series of editors containing mA3A-B5 and its mutants is the C3-C14 site, and the editing efficiency is higher, up to 60%. In addition, the editing sites of a series of editors containing mA3A-B5 and its mutants have preferences, mainly concentrated in C5-C6 and C11-C14 (see Fig.13 ).

[0126] The present invention uses APOBEC3A of the non-human primate cynomolgus monkey as the cytosine deaminase of the CBE system for the first time, providing a good idea and direction for the future optimization of cytosine base editors. The series of BE4-mA3A editors constructed by the present invention enrich the gene editing toolkit.

[0127] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims. SEQUENCE LISTING <110> Jiangnan University <120> A cytosine base editor based on cynomolgus monkey APOBEC3A and its mutants <130> BAA210638A <160> 11 <170> PatentIn version 3.3 <210> 1 <211> 202 <212> PRT <213> Artificial Sequence <400> 1 Met Glu Ala Ser Pro Ala Ser Arg Pro Arg His Leu Met Asp Pro Asn 1 5 10 15 Thr Phe Thr Phe Asn Phe Asn Asn Asp Leu Ser Val Arg Gly Arg His 20 25 30 Gln Thr Tyr Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Trp 35 40 45 Val Pro Met Asp Glu Arg Arg Gly Phe Leu Cys Asn Lys Ala Lys Asn 50 55 60 Val Pro Cys Gly Asp Tyr Gly Cys His Ala Glu Leu Cys Phe Leu Gly 65 70 75 80 Glu Val Pro Ser Trp Gln Leu Asp Pro Ala Gln Thr Tyr Arg Val Thr 85 90 95 Trp Phe Ile Ser Trp Ser Pro Cys Phe Arg Arg Gly Cys Ala Glu Gln 100 105 110 Val Arg Ala Phe Leu Gln Glu Asn Thr His Met Arg Leu Arg Ile Phe 115 120 125 Ala Ala Arg Ile Tyr Asp Tyr Asp Phe Leu Tyr Gln Glu Ala Leu Arg 130 135 140 Thr Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Glu Glu 145 150 155 160 Phe Lys His Cys Trp Asp Thr Phe Val Asp Arg Gln Gly Arg Pro Phe 165 170 175 Gln Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg 180 185 190 Leu Arg Asp Ile Leu Gln Asn Gln Gly Asn 195 200 <210> 2 <211> 609 <212> DNA <213> Artificial Sequence <400> 2 atggaagcca gcccagcatc caggcccaga cacttgatgg atccaaacac gttcactttc 60 aactttaaca atgacctttc ggtccgtgga cggcaccaga cctacttgtg ctacgaggtg 120 gagcgcctgg acaatggcac ctgggtcccg atggacgagc gcaggggctt tctatgcaac 180 aaggctaaga atgttccctg tggtgattat ggctgccacg cggagctgcg cttcctgggc 240 gaggttcctt cttggcagtt ggacccggcc cagacgtaca gggtcacttg gttcatctcc 300 tggagcccct gcttcaggag gggctgtgcc gggcaagtgc gtgcgttcct tcaggagaac 360 acacacgtga gactgcgcat ctttgctgcc cgcatctatg attacgatcc cctgtatcag gaggcactgc gacgctgcg ggatgctggg gcccaagtct ccatcatgac ctacgagga 540. tttaagcact gctgggacac ctttgtggac cgccagggac gtcccttcca gccctgggat ggactagatg agcacagcca agccctgagt gggaggcttc gggacattct ccagaatcag ggaaactga 609 <210> 3 <211> 609 <212> DNA <213> The snowstorm <400> 3 60. atggaagcca gcccagcatc caggcccaga cacttgatgg atccaaacac gttcactttc 120. aactttaaca atgacctttc ggtccgtgga cggcaccaga cctacttgtg ctacgaggtg 180. gcgcctgg acaatggcac ctgggtcccg atggacgagc gcaggggctt tctatgcaac aaggctaaga atgttccctg tggtgattat ggctgccacg cggagctgtg cttcctgggc 240 gaggttcctt cttggcagtt ggacccggcc cagacgtaca gggtcacttg gttcatctcc 300 tggagcccct gcttcaggag gggctgtgcc gggcaagtgc gtgcgttcct tcaggagaac 360 acacacgtga gactgcgcat ctttgctgcc cgcatctatg attacgatcc cctgtatcag gaggcactgc gacgctgcg ggatgctggg gcccaagtct ccatcatgac ctacgagga 540. tttaagcact gctgggacac ctttgtggac cgccagggac gtcccttcca gccctgggat ggactagatg agcacagcca agccctgagt gggaggcttc gggacattct ccagaatcag ggaaactga 609 <210> 4 <211> 609 <212> DNA <213> The snowstorm <400> 4 60. atggaagcca gcccagcatc caggcccaga cacttgatgg atccaaacac gttcactttc 120. aactttaaca atgacctttc ggtccgtgga cggcaccaga cctacttgtg ctacgaggtg 180. gcgcctgg acaatggcac ctgggtcccg atggacgagc gcaggggctt tctatgcaac aaggctaaga atgttccctg tggtgattat ggctgccacg cggagctgtg cttcctgggc 240 gaggttcctt cttggcagtt ggacccggcc cagacgtaca gggtcacttg gttcatctcc 300 tggagcccct gcttcaggag gggctgtgcc ggcaagtgc gtgcgttcct tcaggagaac 360 acacacgtga gactgcgcat ctttgctgcc cgcatctatg attacgatcc cctgtatcag gaggcactgc gacgctgcg ggatgctggg gcccaagtct ccatcatgac ctacgagga 540. tttaagcact gctgggacac ctttgtggac cgccagggac gtcccttcca gccctgggat ggactagatg agcacagcca agccctgagt gggaggcttc gggacattct ccagaatcag ggaaactga 609 <210> 5 <211> 609 <212> DNA <213> The snowstorm <400> 5 60. atggaagcca gcccagcatc caggcccaga cacttgatgg atccaaacac gttcactttc 120. aactttaaca atgacctttc ggtccgtgga cggcaccaga cctacttgtg ctacgaggtg 180. gcgcctgg acaatggcac ctgggtcccg atggacgagc gcaggggctt tctatgcaac aaggctaaga atgttccctg tggtgattat ggctgccacg cggagctgtg cttcctgggc 240 gaggttcctt cttggcagtt ggacccggcc cagacgtaca gggtcacttg gttcatctcc 300 tggagcccct gcttcaggag gggctgtgcc ggcaagtgc gtgcgttcct tcaggagaac 360 acacacatga gactgcgcat ctttgctgcc cgcatctatg attacgatcc cctgtatcag 420 gaggcactgc gaacgctgcg ggatgctggg gcccaagtct ccatcatgac ctacgaggaa 480 tttaagcact gctgggacac ctttgtggac cgccagggac gtcccttcca gccctgggat 540 ggactagatg agcacagcca agccctgagt gggaggcttc gggacattct ccagaatcag 600 ggaaactga 609 <210> 6 <211> 4101 <212> DNA <213> Artificial Sequence <400> 6 gataaaaagt attctattgg tttagccatc ggcactaatt ccgttggatg ggctgtcata 60 accgatgaat acaaagtacc ttcaaagaaa tttaaggtgt tggggaacac agaccgtcat 120 tcgattaaaa agaatcttat cggtgccctc ctattcgata gtggcgaaac ggcagaggcg 180 actcgcctga aacgaaccgc tcggagaagg tatacacgtc gcaagaaccg aatatgttac 240 ttacaagaaa tttttagcaa tgagatggcc aaagttgacg attctttctt tcaccgtttg 300 gaagagtcct tccttgtcga agaggacaag aaacatgaac ggcaccccat ctttggaaac 360 atagtagatg aggtggcata tcatgaaaag tacccaacga tttatcacct cagaaaaaag 420 ctagttgact caactgataa agcggacctg aggttaatct acttggctct tgcccatatg 480 ataaagttcc gtgggcactt tctcattgag ggtgatctaa atccggacaa ctcggatgtc 540 gacaaactgt tcatccagtt agtacaaacc tataatcagt tgtttgaaga gaaccctata 600 aatgcaagtg gcgtggatgc gaaggctatt cttagcgccc gcctctctaa atcccgacgg 660 ctagaaaacc tgatcgcaca attacccgga gagaagaaaa atgggttgtt cggtaacctt 720 atagcgctct cactaggcct gacaccaaat tttaagtcga acttcgactt agctgaagat 780 gccaaattgc agcttagtaa ggacacgtac gatgacgatc tcgacaatct actggcacaa 840 attggagatc agtatgcgga cttatttttg gctgccaaaa accttagcga tgcaatcctc 900 ctatctgaca tactgagagt taatactgag attaccaagg cgccgttatc cgcttcaatg 960 atcaaaaggt acgatgaaca tcaccaagac ttgacacttc tcaaggccct agtccgtcag 1020 caactgcctg agaaataataa ggaaatattc tttgatcagt cgaaaaacgg gtacgcaggt 1080 tatattgacg gcggagcgag tcagaggaa ttctacaagt ttcaacc catattagag 1140 aagatgaatg ggacggaag gttgcttgta aactcaatc gcgaagagatct actgcgaag 1200 cagcggactt tcgacaacgg tagcattcca catcaatcc acttaggcga attgcatgct 1260 attacktagaa ggcaggagga ttttttaccg ttcctchcaag acatcgtga aaagattgag 1320 aaaatcctaa ccttcgcat accttact gtgggacccc tggcccgagg gaactctcgg 1380 ttcgcatgga tgacaagaaa gtccgaagaa acgattactc catggaattt tgaggaagtt 1440 gtcgataaag gtgcgtcagc tcaatcgttc atcgagagga tgaccaactt tgacaagaat 1500 ttaccgaacg aaaaagtatt gcctaagcac agttacttt acgagtattt cacagtgtac 1560 aatgaactca cgaaagttaa gtatgtcact gaggcatgc gtaaacccgc ctttctaagc 1620 ggagaacaga agaaagcaat agtagatctg ttattcaag ccaaccgcaa agtgacagtt 1680 aagcaattga aagaggacta ctttaagaaa attgaatgct tcgattctgt cgagatctcc 1740 ggggtagaag atcgatttaa tgcgtcactt ggtacgtatc atgaccctct aagataatt 1800 aaagataagg acttcctgga taacgaag aatgaagata tctttagaaga tatagtgttg 1860 actcttaccc tctttgaaga tcgggaaatg attgagaaa gactaaaaac atacgctcac 1920 ctgttcgacg ataaggttat gaaacagtta aagaggcgtc gctatacggg ctggggacga 1980 ttgtcgcgga aacttatcaa cgggataaga gacaagcaaa gtggtaaaac tattctcgat 2040 tttctaaaga gcgacggctt cgccaatagg aactttatgc agctgatcca tgatgactct 2100 ttaaccttca aagaggattat acaaaaggca caggtttccg gacaagggga ctcattgcac 2160 gaacatattg cgaatcttgc tggttcgcca gccatcaaaa agggcatact ccagacagtc 2220 aaagtagtgg atgagctagt taaggtcatg ggacgtcaca aaccggaaaa cattgtaatc 2280 gagatggcac gcgaaaatca aacgactcag aaggggcaaa aaaacagtcg agagcggatg 2340 aagagaatag aagagggtat taaagaactg ggcagccaga tcttaaagga gcatcctgtg 2400 gaaaataccc aattgcagaa cgagaaactt tacctctatt acctacaaaa tggaagggac 2460 atgtatgttg atcaggaact ggacataaac cgtttatctg attacgacgt cgatcacatt 2520 gtaccccaat cctttttgaa ggacgattca atcgacaata aagtgcttac acgctcggat 2580 aagaaccgag ggaaaagtga caatgttcca agcgaggaag tcgtaagaa aatgagaac 2640 tattggcggc agctcctaaa tgcgaactg ataacgcaaa gaagttcga taacttaact 2700 aaagctgaga ggggtggctt gtctgaactt vakaaggccg gatttatta acgtcagctc 2760 gtggaaaccc gccaaatcac aaagcatgtt gcacagatac tagattcccg atgaatacg 2820 aaatacgacg agaacgaata gctgattcgg gaagtcaag taatcacttt aaagtcaaaa 2880 ttggtgtcgg acttcagaaa ggattttcaa ttcttaag ttaggagat aaataactac 2940 caccatgcgc acgacgctta tcttaatgcc gtcgtaggga ccgcactcat windowaatac 3000 ccgaagctag aaagtgagtt tgtgtatggt gattacaaag ttatgacgt ccgtaagatg 3060 atcgcgaaaa gcgaacagga gataggcaag gctacagcca atactctt ttattctaac 3120 attatgaatt tctttagac ggaatcact ctggcaacg gagagatacg caacgacct 3180 ttaattgaaa ccaatgggga gandaggtgaa atcgtatggg ataagggccg ggactcgcg 3240 acggtgagaa aagttttgtc catgccccaa gtcaacatag taaagaaaac tgaggtgcag 3300 accggagggt tttcaaagga atcgattctt ccaaaaagga atagtgataa gctcatcgct 3360 cgtaaaaagg actgggaccc gaaaaagtac ggtggcttcg atagccctac agttgcctat 3420 tctgtcctag tagtggcaaa agttgagaag ggaaaatcca agaaactgaa gtcagtcaaa 3480 gaattattgg ggataacgat tatggagcgc tcgtctttg aaaagaaccc catcgacttc 3540 cttgaggcga aaggttacaa ggaagtaaaa aaggatctca taattaaact accaaagtat 3600 agtctgtttg agttagaaaa tggccgaaaa cggatgttgg ctagcgccgg agagcttcaa 3660 aaggggaacg aactcgcact accgtctaaa tacgtgaatt tcctgtattt agcgtcccat 3720 tacgagaagt tgaaaggttc acctgaagat aacgaacaga agcaactttt tgttgagcag 3780 cacaaacatt atctcgacga aatcatag caaatttcgg aattcagtaa gagagtcatc 3840 ctagctgatg ccaatctgga caaagtatta agcgcataca acaagcacag ggataaaccc 3900 atacgtgagc aggcggaaaa tattatccat ttgtttactc ttaccaacct cggcgctcca 3960 gccgcattca agtattttga cacaacgata gatcgcaaac gatacacttc taccaaggag 4020 gtgctagacg cgacactgat tcaccaatcc atcacgggat tatatgaaac tcggatagat 4080 ttgtcacagc ttgggggtga c 4101 <210> 7 <211> 249 <212> DNA <213> Artificial sequence <400> 7 actaatctgt cagatattat tgaaaaggag accggtaagc aactggttat ccaggaatcc 60 atcctcatgc tcccagagga ggtggaagaa gtcattggga acaagccgga aagcgatata 120 ctcgtgcaca ccgcctacga cgagagcacc gacgagaatg tcatgcttct gactagcgac 180 gcccctgaat acaagccttg ggctctggtc atacaggata gcaacggtga gaacaagatt 240 aagatgctc 249 <210> 8 <211> 21 <212> DNA <213> Artificial sequence <400> 8 cccaagaaga agaggaaagt c 21 <210> 9 <211> 225 <212> DNA <213> Artificial sequence <400> 9 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180 gggttgacaa tagcaggcat gctggggatg cggtgggctc tatgg 225 <210> 10 <211> 705 <212> DNA <213> Artificial Sequence <400> 10 atggtgagca agggcgagga ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag 60 ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag 120 ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac 180 atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc 240 cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc 300 gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc 360 tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaaa 420 accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc 480 gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc 540 atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg 600 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc 660 cgccaccacc tgttcctgta cggcatggac gagctgtaca agtaa 705 <210> 11 <211> 343 <212> DNA <213> Artificial Sequence <400> 11 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgc 343

Claims

1. A cytosine deaminase mutant, characterized in that: The mutant is a parent enzyme with an amino acid sequence as shown in SEQ ID NO: 1, and any of the following mutations are performed: (1) The phenylalanine at position 137 was mutated to proline, the methionine at position 123 was mutated to valine, the glutamic acid at position 111 was mutated to glycine, and the cysteine ​​at position 77 was mutated to arginine to obtain mutant V1; (2) mutating the phenylalanine at position 137 to proline, the methionine at position 123 to valine, and the glutamic acid at position 111 to glycine to obtain mutant V2; (3) Mutate phenylalanine at position 137 to proline and methionine at position 123 to valine to obtain mutant V3; (4) The phenylalanine at position 137 was mutated to proline to obtain the mutant V4V5.

2. A gene encoding the cytosine deaminase mutant according to claim 1.

3. An expression cassette, characterized in that Comprising the gene according to claim 2.

4. The expression cassette according to claim 3, characterized in that The expression cassette also contains a promoter, nCas9 (D10A), a uracil DNA glycosylase inhibitor, a nuclear localization sequence and a termination sequence; the promoter initiates the expression of the gene according to claim 2, and the sequence is, in order of connection, a promoter, the gene according to claim 2, nCas9 (D10A), a uracil DNA glycosylase inhibitor, a nuclear localization sequence and a termination sequence, the nucleotide sequence of the nCas9 (D10A) is shown in SEQ ID NO: 6, the nucleotide sequence of the uracil DNA glycosylase inhibitor is shown in SEQ ID NO: 7, the nucleotide sequence of the nuclear localization sequence is shown in SEQ ID NO: 8, and the nucleotide sequence of the termination sequence is shown in SEQ ID NO:

9.

5. A CBE single-base editing system based on cynomolgus monkey cytosine deaminase, characterized in that: The CBE single-base editing system comprises four parts. The first part is the transfection efficiency indicator part, which comprises a red fluorescent protein and a promoter; the second part is the sgRNA transcription unit, which comprises a carrying frame for inserting the sgRNA sequence and its promoter; the third part comprises the expression cassette described in claim 3 or 4; and the fourth part comprises the high-copy replication origin ori from the colicin factor and the ampicillin resistance screening gene AmpR.

6. The CBE single base editing system according to claim 5, characterized in that The nucleotide sequence of the red fluorescent protein is shown in SEQ ID NO:10, and the nucleotide sequence of the sgRNA transcription unit is shown in SEQ ID NO:

11.

7. Use of the CBE single-base editing system according to claim 5 or 6 in gene editing, characterized in that: The gene editing refers to the mutation of the target base cytosine to thymine.

8. The use according to claim 7, characterized in that: An sgRNA sequence is designed according to the target gene and inserted into the sgRNA transcription unit of the CBE single-base editing system according to claim 5 or 6 to obtain a CBE single-base editing system with a specific targeted gene, and then the CBE single-base editing system is introduced into a recipient cell to achieve a mutation of the target base cytosine to thymine, thereby obtaining cells containing a single base mutation.

9. Use of the cytosine deaminase mutant according to claim 1, the gene according to claim 2, or the expression cassette according to claim 3 or 4 in gene editing, characterized in that: The gene editing refers to the mutation of the target base cytosine to thymine.

Images