Cytosine deaminase mutant as well as base editing system and application thereof

Abstract

The invention discloses a cytosine deaminase mutant as well as a base editing system and application thereof, and relates to the technical field of molecular biology, the cytosine deaminase mutant is AncY16H, AncI50A, AncG149D, AncHA, AncHD or AncAD, the amino acid sequence is as shown in SEQ ID NO.4-9, and the amino acid sequence is as shown in SEQ ID NO.4-9. The cytosine base editing system comprises any one of the cytosine deaminase mutants, a Cas9 nick enzyme and a uracil-DNA (Deoxyribose Nucleic Acid) glycosylase inhibitor UGI (Uracil-Deoxyribose Nucleic Acid). The invention discloses a novel efficient cytosine deaminase variant without base preference, a novel accurate and efficient cytosine base editing system is established on the basis of the novel cytosine deaminase variant, and the cytosine deaminase variant has a good application prospect in plant genome adaptability transformation.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a cytosine deaminase mutant and a base editing system and application thereof. Background Art

[0002] With the rapid development of gene editing technology, base editing systems have been widely used in genetic improvement of plants and animals. The cytosine base editing system (CBE) consists of cytosine deaminase, Cas9 nickase (nCas9), and uracil-DNA glycosylase inhibitor (UGI). Under the guidance of sgRNA, nCas9 targets the DNA of the plant and animal genome and forms an R-loop structure. Subsequently, cytosine deaminase binds to the single-stranded DNA in the R-loop region, deaminates cytosine (C) at a specific position to uracil (U), and converts U to thymine (T) through mismatch repair (MMR) or DNA replication, thereby achieving base substitution from C·G to T·A.

[0003] In nature, most cytosine deaminases act on RNA, and only a few APOBEC family deaminases can act on single-stranded DNA. However, the activities of these deaminases in animal and plant genomes vary greatly, and even the same deaminase exhibits different deamination activities in different crops (such as rice and wheat). At present, only hAPOBEC3A (A3A) has been verified to have high deamination activity in the wheat genome. In order to broaden the non-NGG PAM genome targeting range, scientists replaced the wild-type Cas9 with variants such as Cas9-NG, SpG, and SpRY, but the applicant's previous studies found that different deaminases and SpG variants have compatibility issues, which to a certain extent reduces the editing efficiency of the base editing system in the wheat genome. Therefore, it is urgent to adapt the existing potential cytosine deaminases to the wheat genome to improve their editing efficiency and accuracy.

[0004] To date, most modifications of deaminases and other tool enzymes rely on prokaryotic systems, while there is still a lack of effective genome adaptive modification methods for eukaryotic cells, especially plant cells. Summary of the invention

[0005] The object of the present invention is to provide a cytosine deaminase mutant and its base editing system and application.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] As a first aspect of the present invention, a cytosine deaminase mutant is provided. The cytosine dehydrogenase mutants are AncY16H, AncI50A, AncG149D, AncHA, AncHD and AncAD, and the amino acid sequences are shown in SEQ ID NO.4-9.

[0008] A further improvement is that the cytosine deaminase mutants are AncHA, AncHD and AncAD, and the amino acid sequences are shown in SEQ ID NO.7-9.

[0009] A further improvement is that the cytosine deaminase mutant is AncAD, and the amino acid sequence of AncAD is shown in SEQ ID NO.9

[0010] As a second aspect of the present invention, a method for screening a cytosine deaminase mutant as described above is also provided, wherein the screening is performed by flow cytometry analysis, comprising the following steps:

[0011] (1) introducing random mutations into the gene of the cytosine deaminase to be modified, and then ligating the vector to obtain a mutant vector library;

[0012] (2) First, the mutant vector library constructed in step (1) is co-transfected with the fluorescent reporter system into plant protoplasts, and then the transformed plant protoplasts are sorted and enriched to obtain various mutants by flow cytometry. Finally, flow cytometric analysis and screening are performed according to the proportion of luminescent protoplasts of various mutants.

[0013] A further improvement is that the method for constructing the fluorescent reporter system in step (2) is: connecting the mutated BFP protein sequence mBFP as shown in SEQ ID NO.1 to the backbone vector to construct a fluorescent reporter system containing backbone vector-mBFP-ACAC, backbone vector-mBFP-ACCC, backbone vector-mBFP-ACGC, and backbone vector-mBFP-ACTC.

[0014] As a third aspect of the present invention, a cytosine base editing system is also provided, comprising a cytosine deaminase mutant as described above, a Cas9 nickase, and a uracil-DNA glycosylase inhibitor UGI.

[0015] A further improvement is that the Cas9 nickase is a wild-type nCas9 or a variant thereof, the amino acid sequence of the wild-type nCas9 is shown in SEQ ID NO.2, and the nCas9 variant is nSpG, the amino acid sequence of which is shown in SEQ ID NO.3.

[0016] As a fourth aspect of the present invention, there is also provided an application of a cytosine base editing system as described above in the adaptive modification of plant genomes.

[0017] A further improvement is that the plants include rice and wheat.

[0018] The present invention has the following beneficial effects:

[0019] The present invention first fuses the known deaminase and variants with the nCas9 variant (nSpG), and screens them through the wheat protoplast system to obtain the chassis cytosine deaminase (Anc656) adapted to nSpG, and uses error-prone PCR to introduce random mutations to the Anc656 deaminase protein, and replaces alanine one by one on its ssDNA binding domain according to structural analysis, thereby constructing the Anc656 random mutation library and its ssDNA binding domain saturated alanine mutation library. Flow cytometry sorting and second-generation sequencing are used to evaluate the changes in mutation frequencies of each site before and after sorting, and the positively regulated mutation sites are screened for mutation one by one, and then flow cytometry analysis is performed one by one with the saturated alanine mutation library variants to obtain efficient new variants of cytosine deaminase without base preference. Finally, a new system for precise and efficient base editing of wheat was established through wheat and rice protoplast testing and gene gun transformation of immature wheat embryos. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 To optimize the sheath fluid system and parameters for protoplast sorting;

[0021] Figure 2 Screening for chassis cytosine deaminases for adaptive modification of the wheat genome;

[0022] Figure 3 Construction of a random mutation library of Anc656 and a saturated alanine mutation library of its ssDNA binding domain;

[0023] Figure 4A , Figure 4B The results of flow sorting and identification of synergistic mutation sites in the Anc656 deaminase random mutation library;

[0024] Figure 5 Further screening of variants using a random mutation library for Anc656 based on flow cytometric analysis;

[0025] Figure 6 Screening of alanine-saturated variants of the Anc656 ssDNA binding domain based on flow cytometry analysis;

[0026] Figure 7 Information on base editing vectors constructed for different cytosine deaminases and iteratively optimized variants;

[0027] Figure 8 To compare the editing efficiency of different base editing systems on different endogenous targets;

[0028] Fig. 9 To compare the comprehensive editing efficiency of different base editing systems;

[0029] Fig.10 Compare the editing windows and efficiencies of different base editing systems;

[0030] Fig.11 is the mutation type of the wheat mutant plant;

[0031] Fig.12 Testing the compatibility of deaminase variants with nCas9. DETAILED DESCRIPTION

[0032] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0033] 1. Materials:

[0034] Unless otherwise specified, the methods used in the present invention are conventional methods known to those skilled in the art. If no specific conditions are specified, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0035] 2. Methods:

[0036] 1. Vector construction and plasmid extraction

[0037] The known rAPOBEC1, hAPOBEC3A, Anc656, and evoFERNY deaminases were fused to the N-terminus of the SpCas9 nickase variant (nSpG), and 2×UGI was fused to its C-terminus, and then the fusion protein gene fragments were introduced into the pKUC57 vector backbone (based on the addition of ZmUbi promoter and CaMV terminator to the known pUC57-Kan vector) to construct pKUC57-rAC1-nSpG, pKUC57-A3A-nSpG, pKUC57-Anc656-nSpG, and pKUC57-evoFERNY-nSpG editing vectors. The sequence of the SpCas9 nickase variant (nSpG) is shown in SEQ ID NO.3, and the sequence of 2×UGI is shown in SEQ ID NO.10;

[0038] Random point mutations were introduced into Anc656 by error-prone PCR and connected to the pKUC57 vector backbone by Gibson ligation to construct the pKUC57-mAnc656-nSpG mutation vector library.

[0039] Four synonymous and nonsynonymous mutations were performed on the T65 and F71 positions of the EBFP protein, and the amino acid sequence after mutation (mBFP) is shown in SEQ ID NO.1. Then, the mutants were ligated into the pKUC57 vector backbone through Gibson to construct the pKUC57-mBFP-ACAC (FR-ACAC), pKUC57-mBFP-ACCC (FR-ACCC), pKUC57-mBFP-ACGC (FR-ACGC), and pKUC57-mBFP-ACTC (FR-ACTC) fluorescence reporter systems.

[0040] Three endogenous targets were selected from three rice genes (OsAAT, OsACC, and OsALS) to construct the pOsU3-sgRNA vector, and 10 endogenous targets and four targets of the fluorescent reporter system were selected from six wheat genes (TaALS, TaACC, TaGASR7, TaGW2, TaHRC, and TaMYB10) to construct the pTaU3-sgRNA vector. The sequences of all the targeting sites are shown in Table 1. Design and synthesize target primers (Table 1), anneal to form oligo DNA with sticky ends; use T4 ligase to connect sgRNA oligos to the pOsU3-sgRNA2.0 and pTaU3-sg RNA2.0 vector backbones digested with BsaI, respectively.

[0041] Table 1. sgRNA targeting sites and sequences

[0042]

[0043]

[0044] Note: PAM sequence is shown in bold.

[0045] Construct vector heat shock transformation and positive clone identification: The constructed plasmid was mixed with Fast-T1 competent cells, heat-shocked at 42°C for 45 seconds, then placed on ice for 2 minutes, and then added with antibiotic-free LB medium for recovery culture. The plates were then coated with LB plates with corresponding resistance and cultured at 37°C overnight. Single colony clones were identified by colony PCR the next day, positive clones were picked for sequencing, and colonies carrying the correct construction were screened by Sanger sequencing.

[0046] Plasmid extraction: Refer to Promega's Wizard Plus Midipreps DNA Purification System kit, pick a monoclonal bacterial block and inoculate it into 100mL sterile LB medium, culture it in an incubator at 37℃ and 200rpm overnight, collect the bacterial block by centrifugation, add 3mL Cell Resuspention Solution, place it on a vortex oscillator to fully oscillate and suspend the bacterial cells, add 3mL Cell Lysis Solution, gently invert the bacteria to fully mix and lyse for 3-5 minutes, at which time the solution becomes transparent. Add 3mL Neutralization Solution, gently invert the liquid to mix, centrifuge at 12,000rpm for 10 minutes, and aspirate the supernatant into a new 50mL centrifuge tube. Suspend Resin, add 10mL Resin to the centrifuge tube, mix thoroughly, transfer to the preparation tube, and purify by negative pressure method. The recovery column was washed twice with Wash Buffer containing ethanol, each time using 15 mL. The column with resin was then cut off and placed in a new 2 mL centrifuge tube. The tube was centrifuged at 12,000 rpm for 2 minutes. The tube was replaced with a new 1.5 mL centrifuge tube. After drying, 200 μL of eluent was added and allowed to stand. The tube was centrifuged at 12,000 rpm for 2 minutes. The sample concentration was detected using a NanoDrop ultra-micro spectrophotometer.

[0047] 2. Protoplasts and transformation

[0048] The rice and wheat materials used for protoplast separation and transformation of the present invention are Zhonghua 11 and Annong 0711 respectively.

[0049] 2.1. Cultivation of etiolated rice seedlings

[0050] The Zhonghua 11 rice seeds were first rinsed with 75% ethanol for 1 minute, then treated with 4% sodium hypochlorite for 30 minutes, washed with sterile water for more than 5 times, and cultured on M6 medium for 3-4 weeks at 26°C in the dark.

[0051] 2.2. Rice protoplast isolation

[0052] (1) Cut off the stem tissue of the yellowed seedlings, cut the middle part into 0.5-1 mm silk with a blade, put it into 0.6M Mannitol solution and keep it away from light for 10 minutes, then filter it with a filter, put it into 50mL enzymatic solution (filtered with 0.45μm filter membrane), evacuate (pressure about 15Kpa) for 30 minutes, take it out and place it on a shaker (10rpm) for enzymatic hydrolysis at room temperature for 5 hours; (2) Add 30-50mL W5 to dilute the enzymatic hydrolysis product, filter the enzymatic hydrolysis solution with a 75μm nylon filter membrane and put it into a round-bottom centrifuge tube (50mL); (3) 23℃, 250g (rcf), up 3 times and down 3 times, centrifuge for 3 minutes, and discard the supernatant; (4) Gently suspend the cells with 20mL W5 and repeat step (3); (5) Add an appropriate amount of MMG to suspend and wait for transformation.

[0053] 2.3. Rice protoplast transformation

[0054] (1) Place the transformation vector in a 2m centrifuge tube and mix well. Use a sharpened pipette tip to draw up 200μL of protoplasts and flick to mix well. Add 220μL of PEG4000 solution and flick to mix well. Induce transformation at room temperature in the dark for 20-30min. (3) Add 880μL of W5 and gently invert to mix well. Centrifuge at 250g (rcf), up 3 times and down 3 times for 3 minutes and discard the supernatant. (3) Add 1mL of WI solution and gently invert to mix well. Incubate in the dark at 23℃ for 48h.

[0055] 2.4. Wheat seedling culture

[0056] The Annong 0711 wheat seeds were potted and planted in a culture room, and cultured for about 10 days at a temperature of 25±2°C, a light intensity of 1000Lx, and a light intensity of 14-16h / d.

[0057] 2.5. Wheat protoplast isolation

[0058] (1) Take young wheat leaves, cut the middle part into 0.5-1 mm silk with a blade, put them into 0.6M Mannitol solution and keep away from light for 10 minutes, then filter them with a filter, put them into 50mL enzymatic solution (filtered with 0.45μm filter membrane), evacuate (pressure about 15Kpa) for 30 minutes, take them out and place them on a shaker (10rpm) for enzymatic hydrolysis at room temperature for 5 hours; (2) Add 30-50mL W5 to dilute the enzymatic hydrolysis product, filter the enzymatic hydrolysis solution with a 75μm nylon filter membrane and place it in a round-bottom centrifuge tube (50mL); (3) 23℃, 100g (rcf), up 3 times and down 3 times, centrifuge for 3 minutes, and discard the supernatant; (4) Gently suspend with 10mL W5 and place on ice for 30 minutes; the protoplasts gradually settle and the supernatant is discarded; (5) Add an appropriate amount of MMG to suspend, place on ice, and wait for transformation.

[0059] 2.6. Wheat protoplast transformation

[0060] (1) Place the transformed plasmid in a 2 mL centrifuge tube and mix well. Use a sharpened pipette tip to draw up 200 μL of protoplasts and flick to mix well. Immediately add 250 μL of PEG 4000 solution and flick to mix well. Induce transformation at room temperature in the dark for 20-30 min. (2) Add 800 μL of W5 (room temperature) and gently invert to mix well. Centrifuge at 100 g (rcf), up 3 times and down 3 times for 3 minutes, and discard the supernatant. (3) Add 1 mL of W5 and gently invert to mix well. Incubate in the dark at 23°C for 48 h.

[0061] 3. Flow cytometry sorting / analysis

[0062] After 24 hours of dark culture, the transformed wheat protoplasts were transferred to flow cytometry tubes for flow cytometry sorting / analysis. In order to solve the problem of protoplast stability during sorting, the pH and concentration of the sheath fluid used in flow cytometry sorting were tested and analyzed. The results showed that wheat protoplasts were most stable in 1*PBS solution at pH 5.7. In addition, adjusting the sorting threshold using GFP-F71L as a positive control and BFP-F71L as a negative control can effectively improve the accuracy of flow sorting ( Figure 1 ).

[0063] The specific flow cytometry operation is as follows: (1) After turning on the instrument, open the BD FACS Software and perform instrument calibration and other operations. (2) Click "New Protocol" to create a suitable experimental plan. (3) Select "density plot" to draw an FSC / SSC scatter plot, and then draw a GFP / PE-Texas Red scatter plot. (4) Adjust the FSC / SSC voltage so that the cell population appears in the center of the scatter plot, and adjust the FL1 voltage so that the wild-type control protoplast population appears in the center of the scatter plot, and the GFP-positive protoplasts appear at a location with a stronger GFP fluorescence channel signal. (5) Set a gate to circle the GFP-positive population, and use the negative control to determine the boundaries of the gate. (6) Right-click the cell population to be sorted and select "left sort". Set the analysis conditions and sorting / analysis mode according to the experimental needs and the percentage of the target cells. (7) Load and sort / analyze the prepared protoplast samples cultured in the flow tube in turn, and record the relevant data. (8) Close the software and shut down the instrument.

[0064] 4. Protoplast DNA extraction and amplicon sequencing analysis

[0065] 4.1. Protoplast DNA extraction

[0066] The protoplasts were collected in 2 mL centrifuge tubes, and the protoplast DNA (~30 μL) was extracted using the CTAB method. The concentration (30-60 ng / μL) was determined using a NanoDrop ultra-micro spectrophotometer and stored at -20°C.

[0067] 4.2. Amplicon sequencing sample preparation and library construction

[0068] The extracted protoplast DNA was used as the first round PCR template and amplified using a high-fidelity PCR mix; the first round PCR product was diluted 10 times as a template for the second round of amplification (primers see Table 2), and the length of the PCR product was about 120-180 bp; the second round PCR products were mixed into a library in equal amounts according to the number of samples, and then the DNA was purified and recovered, and its concentration was measured using a NanoDrop ultra-micro spectrophotometer; finally, it was sent to Nanjing Painosen Gene Technology Co., Ltd. for library construction and sequencing.

[0069] 4.3 Analysis of amplicon sequencing results

[0070] Refer to the method of Gaudelli et al.: (1) Use the sequence of the second-round primer of the amplified target site to split each sample in each library to obtain all sequences with different treatments at different target sites; (2) Use the flash splicing tool with default parameters to splice the split sequences; (3) Compare the split sequences with the wild-type sequences, analyze the mutation type of each sequence, and use the perl script to analyze the mutation frequency and mutation type of each sequence.

[0071] Table 2. Information of first-round and second-round PCR primers used in amplicon library construction

[0072]

[0073]

[0074] Note: NGS primers are second-round PCR primers. Different treatments require the addition of different 6-base barcodes at the 5' end of the primers for data splitting.

[0075] 5. Genetic transformation of wheat mediated by gene gun

[0076] The base editing plasmids pKUC57-A3A-nSpG and pKUC57-AncAD-nSpG were co-delivered with the pTaU3-sgTaALS-T1 plasmid into Bobwhite wheat immature embryos by gene gun bombardment. The bombarded immature embryos were transferred to hypertonic culture for dark culture overnight, transferred to recovery medium for dark culture for 2 weeks to induce callus formation, and the callus tissue was transferred to regeneration medium for light induction for 3-5 weeks to induce the formation of shoots. The shoots were transferred to rooting medium and cultured under light conditions for 1 week to obtain wheat seedlings. No additional resistance screening was performed during the entire culture process.

[0077] 6. Wheat mutant detection

[0078] Positive mutant plants were identified using the PCR / T7EI method. Individual samples of the regenerated plants were taken in order and placed in 2 mL centrifuge tubes. After being sampled by a tissue grinder, wheat genomic DNA was extracted using the CTAB method. PCR amplification was performed using the AB D specific primers in Table 3. The 20 μL amplification system contained 10 μL 2×PCR Mix, 0.8 μL Forward primer (10 μM), 0.8 μL Reverse primer (10 μM), and 2 μL DNA template (~60 ng). Amplification conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 50-64°C annealing for 30 s, 72°C extension for 30 s, 37 cycles; 72°C full extension for 5 min, and storage at 12°C.

[0079] Take 2 μL of mutant and wild-type PCR amplification products, add 1 μL of 10×T7 Endonuclease I Reacti on Buffer, and make up to 9 μL with water; after mixing, perform annealing reaction in a plant PCR instrument (95℃, 5min; 95~85℃, -2℃ / sec; 85~25℃, -0.2℃ / sec; store at 4℃); add 1 μL of T7 Endonuc leaseI to the annealing product, incubate at 37℃ for 30min, and immediately perform detection on 2% agarose gel electrophoresis after completion. For the positive mutant plants identified, the first generation sequencing is continued to be used for verification, and the specific mutation type is obtained. For samples that cannot be typed, the monoclonal sequencing method is used for genotyping analysis.

[0080] Table 3. Specific primers for wheat mutant detection

[0081]

[0082]

[0083] 7. Statistical analysis

[0084] This study used the Student's t-test to evaluate the significance of differences between treatments, and the test method used was a two-tailed test, and the results were distinguished by P value. When P>0.05, it means that there is no significant difference between the two sample means (ns), when P<0.05, it means that the two sample means are significantly different (*), and when P<0.01, it means that the two sample means are extremely significantly different (**).

[0085] Example 2 Screening of cytosine deaminase mutants

[0086] The known rAPOBEC1, hAPOBEC3A, Anc656 and evoFERNY deaminases were fused with the SpCas9 variant (SpG) to construct a base editing system.

[0087] Through rice and wheat protoplast transformation, editing tests were performed on 10 endogenous target sites, including OsALS, OsAAT, OsACC, TaALS-T1, TaALS-T2, and TaACC. The analysis of the second-generation sequencing results showed that for the same deamination target sequence (OsALS and TaALS-T1), the deamination efficiency of these deaminases in the rice genome was about 5 times that of the wheat genome, indicating the existence of genomic differences; among these deaminases, Anc656 not only has a higher deamination efficiency, but also has a narrower deamination window ( Figure 2 ), therefore, it was also optimized as a chassis deaminase adapted to SpG for adaptive modification of the wheat genome.

[0088] The Anc656 deaminase protein was randomly mutated by error-prone PCR to construct the RM-Anc656 random mutation library ( Figure 3 ) and co-transfected wheat protoplasts with four fluorescent reporter systems, FR-ACAC, FR-ACCC, FR-ACGC, and FR-ACTC. After culturing for 24 h, flow cytometry was performed and 1×10 5Green fluorescent protoplasts were selected, and DNA was extracted for next-generation sequencing analysis. The results showed that 28 mutation sites such as P8L, L11V, and Y16H were significantly enriched before and after protoplast sorting (Figure 4), indicating that they may have a positive regulatory effect on the enhancement of Anc656 deamination activity. These screening sites were mutated one by one on the pKUC57-Anc656-nSpG vector, and saturated alanine mutation scanning was performed in the Anc656 ssDNA binding domain (41-80aa) region to construct a series of mAnc656 mutant vectors, which were co-transfected with FR-ACAC, FR-ACCC, FR-ACGC, and FR-ACTC fluorescent reporter systems into wheat protoplasts and analyzed by flow cytometry, and then the proportion of luminescent protoplasts of each variant was statistically compared.

[0089] The results showed that the eight variants, AncY16H, AncG24D, AncI50A, AncR55A, AncV81M, AncG141D, AncE144D, and AncG149D, performed better than the control Anc656 under the ACAC, ACCC, ACGC, and ACTC motifs, among which AncY16H, AncI50A, and AncG149D performed better than hAPOBEC3A, evoFER NY( Figure 5 and 6 ), the amino acid sequences of AncY16H, AncI50A, and AncG149D are shown in SEQ ID NO.4-6.

[0090] Example 2 Establishment of a new system for accurate and efficient base editing in wheat

[0091] Based on the single site mutations of AncY16H, AncI50A, and AncG149D, double mutants AncHA, AncHD, and AncAD were further constructed. The amino acid sequences of AncHA, AncHD, and AncAD are shown in SEQ ID NO.7-9. The above cytosine dehydrogenase variants were used to construct base editing systems AncY16H-nSpG, AncI50A-nSpG, AncG149D-nSpG, AncHA-nSpG, AncHD-nSpG, and AncAD-nSpG in sequence. In addition, the new system was tested and compared with wheat endogenous targets using A3A-nSpG and Anc656-nSpG as controls ( Figure 7 ).

[0092] The results showed that: Overall, the base editing efficiency of AncHA-nSpG (24.67%), AncHD-nSpG (25.92%), and AncAD-nSpG (25.93%) was significantly higher than that of AncY16H-nSpG (21.03%), AncI50A-nSpG (18.53%), and AncG149D-nSpG (20.04%), which was also significantly higher than that of the control Anc656-nSpG (14.32%), A3A-nSpG (15.47%) ( Figure 8 and 9 ).

[0093] Further evaluation of the editing window and efficiency revealed that AncY16H-nSpG, AncH A-nSpG, and AncHD-nSpG containing the Y16H mutation all exhibited wide-window editing properties similar to A3A-nSpG, while AncI50A-nSpG, AncY149D-nSpG, and AncAD-nSpG containing I50A and G149D maintained the narrow-window editing properties of Anc656-nSp G to a certain extent, but the editing efficiency of cytosine bases within the deamination window was significantly improved (the maximum multiple was more than 3 times) ( Fig.10 ).

[0094] Since the editing efficiency of A3A-nSpG on TaALS-T1 was low, TaALS-T1 was selected as the target site for testing and comparison of A3A-nSpG and AncAD-nSpG. Genetic transformation of wheat mediated by gene gun showed that the number of regenerated seedlings of A3A-nSpG was less than that of AncAD-nSpG (Table 4), which may be related to the cytotoxicity of A3A. In the absence of screening markers, AncAD-nSpG showed a higher editing efficiency (1.96%) than A3A-nSpG (Table 4), and the three mutants detected all produced base editing in the AABBDD genome of wheat allohexaploid ( Fig.11 ), which also illustrates the high efficiency of the new system.

[0095] Table 4. Comparison of base editing efficiency of endogenous wheat targets mediated by gene gun

[0096]

[0097] Example 3 Compatibility of the new system for precise and efficient base editing in wheat

[0098] The nSpG in the above base editing system was replaced with nCas9 to construct Anc656-BEmax, A3A-BEmax, AncY16H-BEmax, AncI50A-BEmax, AncG149D-BEmax, AncHA-BEmax, AncHD-BEmax, and AncAD-BEmax, and wheat protoplast transformation and second-generation sequencing analysis were performed to test the compatibility of the new variants with nCas9. The amino acid sequence of nCas9 is shown in SEQ ID NO.2. The results also showed that these variants can improve the efficiency of the base editing system constructed based on nCas9 ( Fig.12 ).

[0099] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A cytosine deaminase mutant, characterized in that: The cytosine deaminase mutant is AncY16H, AncI50A, AncG149D, AncHA, AncHD or AncAD, and the amino acid sequence is shown in SEQ ID NO.4-9.

2. A cytosine deaminase mutant according to claim 1, characterized in that: The cytosine deaminase mutant is AncHA, AncHD or AncAD, and the amino acid sequence is shown in SEQ ID NO.7-9.

3. A cytosine deaminase mutant according to claim 1, characterized in that: The cytosine deaminase mutant is AncAD, and the amino acid sequence of AncAD is shown in SEQ ID NO.

9.

4. A method for screening a cytosine deaminase mutant according to any one of claims 1 to 3, wherein the screening is performed by flow cytometry analysis, characterized in that: The following steps are involved: (1) introducing random mutations into the gene of the cytosine deaminase to be modified, and then ligating the vector to obtain a mutant vector library; (2) First, the mutant vector library constructed in step (1) is co-transfected with the fluorescent reporter system into plant protoplasts, and then the transformed plant protoplasts are sorted and enriched to obtain various mutants by flow cytometry. Finally, flow cytometric analysis and screening are performed according to the proportion of luminescent protoplasts of various mutants.

5. The method for screening cytosine deaminase mutants according to claim 4, characterized in that: The method for constructing the fluorescent reporter system in step (2) is: connecting the mutated BFP protein sequence mBFP as shown in SEQ ID NO.1 to the backbone vector to construct a fluorescent reporter system containing backbone vector-mBFP-ACAC, backbone vector-mBFP-ACCC, backbone vector-mBFP-ACGC, and backbone vector-mBFP-ACTC.

6. A cytosine base editing system, characterized in that: It comprises a cytosine deaminase mutant, a Cas9 nickase and a uracil-DNA glycosylase inhibitor UGI as described in any one of claims 1 to 3.

7. A cytosine base editing system according to claim 6, characterized in that: The Cas9 nickase is a wild-type nCas9 or a variant thereof, the amino acid sequence of the wild-type nCas9 is shown in SEQ ID NO.2, and the nCas9 variant is nSpG, and the amino acid sequence is shown in SEQ ID NO.

3.

8. An application of the cytosine base editing system as described in any one of claims 6-7 in the adaptive modification of plant genomes.

9. The use according to claim 7, characterized in that: The plants include rice and wheat.

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