A cytosine deaminase mutant, base editing system and application thereof

Abstract

This invention discloses a cytosine deaminase mutant, its base editing system, and its applications, relating to the field of molecular biology. The cytosine deaminase mutant is AncY16H, AncI50A, AncG149D, AncHA, AncHD, or AncAD, with amino acid sequences as shown in SEQ ID NO. 4-9. The cytosine base editing system includes any of the aforementioned cytosine deaminase mutants, a Cas9 nickase, and the uracil-DNA glycosylase repressor UGI. This invention discloses a novel, highly efficient, base-free cytosine deaminase variant and establishes a new, precise, and efficient cytosine base editing system, which shows promising application prospects in plant genome adaptation.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a cytosine deaminase mutant, its base editing system, and its applications. Background Technology

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

[0003] In nature, most cytosine deaminases act on RNA, with only a few APOBEC family deaminases acting on single-stranded DNA. However, the activities of these deaminases vary considerably in plant and animal genomes, and even the same deaminase exhibits different deaminase activities in different crops (such as rice and wheat). Currently, only hAPOBEC3A (A3A) has been verified to have high deaminase activity in the wheat genome. To broaden the target range of non-NGG PAM genomes, scientists have replaced wild-type Cas9 with variants such as Cas9-NG, SpG, and SpRY. However, previous research by the applicant revealed compatibility issues between different deaminases and SpG variants, which to some extent reduced the editing efficiency of the base editing system in the wheat genome. Therefore, there is an urgent need to adapt 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 effective methods for genome adaptation modification of eukaryotic cells, especially plant cells, are still lacking. Summary of the Invention

[0005] The purpose of this invention is to provide a cytosine deaminase mutant, its base editing system, and its applications.

[0006] The present invention achieves the above objectives through the following technical solutions:

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

[0008] A further improvement is that the cytosine deaminase mutants are AncHA, AncHD, and AncAD, with amino acid sequences as 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 cytosine deaminase mutants as described in any of the above-described methods is also provided, wherein the screening is performed by flow cytometry analysis, and includes the following steps:

[0011] (1) The gene of the cytosine deaminase to be modified was randomly mutated and then the mutant vector library was constructed by ligation.

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

[0013] A further improvement is that the method for constructing the fluorescent reporter system in step (2) is as follows: the mutated BFP protein sequence mBFP shown in SEQ ID NO.1 is linked 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 invention, a cytosine base editing system is also provided, comprising a cytosine deaminase mutant, a Cas9 nickase, and a uracil-DNA glycosylase repressor UGI as described in any of the above descriptions.

[0015] A further improvement is that the Cas9 nick enzyme is wild-type nCas9 or a variant thereof, the amino acid sequence of which 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 invention, the application of the cytosine base editing system as described above in adaptive modification of plant genomes is also provided.

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

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

[0019] This invention first fuses known deaminases and their variants with the nCas9 variant (nSpG), and then screens them using a wheat protoplast system to obtain a chassis cytosine deaminase (Anc656) adapted to nSpG. Random mutations are then introduced into the Anc656 deaminase protein using error-prone PCR. Simultaneously, based on structural analysis, alanine substitutions are performed sequentially in its ssDNA binding domain, thereby constructing a random mutant library of Anc656 and a ssDNA binding domain saturated alanine mutant library. Flow cytometry sorting and next-generation sequencing are used to assess the changes in mutation frequency at each site before and after sorting. Positively regulated mutation sites are screened and mutated sequentially. These mutations are then analyzed sequentially with the saturated alanine mutant library variants using flow cytometry to obtain highly efficient, base-biased new cytosine deaminase variants. Finally, through wheat and rice protoplast testing and gene gun transformation of immature wheat embryos, a new system for precise and efficient base editing in wheat is established. Attached Figure Description

[0020] Figure 1 Optimization of sheath fluid system and parameters for protoplast sorting;

[0021] Figure 2 Screening for chassis cytosine deaminases for wheat genome adaptation;

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

[0023] Figure 4A , Figure 4B Flow cytometry sorting results of enhancing mutation sites in the Anc656 deaminase random mutant library;

[0024] Figure 5 Further screening of variants for Anc656 random mutant libraries based on flow cytometry analysis;

[0025] Figure 6 For screening of Anc656 ssDNA binding domain saturated alanine variants based on flow cytometry analysis;

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

[0027] Figure 8 Comparison of editing efficiency of different base editing systems at different endogenous target sites;

[0028] Figure 9 A comparison of the overall editing efficiency of different base editing systems;

[0029] Figure 10 A comparison of editing windows and efficiency of different base editing systems;

[0030] Figure 11 The mutation type of the wheat mutant plant;

[0031] Figure 12 Compatibility testing of deaminase variants with nCas9. Detailed Implementation

[0032] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0033] I. Materials:

[0034] Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art. Where specific conditions are not specified, they shall be performed according to conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0035] II. Method:

[0036] 1. Vector construction and plasmid extraction

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

[0038] Anc656 was randomly mutated using error-prone PCR, and the pKUC57 vector backbone was ligated using Gibson to construct the pKUC57-mAnc656-nSpG mutant vector library.

[0039] Four synonymous and non-synonymous mutations were performed on the T65 and F71 positions of the EBFP protein, respectively. The resulting amino acid sequence (mBFP) is shown in SEQ ID NO.1. Subsequently, the EBFP was ligated into the pKUC57 vector backbone via 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) fluorescent reporter systems.

[0040] Three endogenous targets were selected from three rice genes (OsAAT, OsACC, and OsALS) to construct the pOsU3-sgRNA vector. Simultaneously, ten endogenous targets were selected from six wheat genes (TaALS, TaACC, TaGASR7, TaGW2, TaHRC, and TaMYB10) and four targets from a fluorescent reporter system to construct the pTaU3-sgRNA vector. All target site sequences are shown in Table 1. Target primers were designed and synthesized (Table 1), and annealed to form oligo DNA with sticky ends. Using T4 ligase, sgRNA oligos were ligated into the BsaI-digested pOsU3-sgRNA2.0 and pTaU3-sgRNA2.0 vector backbones, respectively.

[0041] Table 1. sgRNA Target Sites and Sequences

[0042]

[0043]

[0044] Note: PAM sequences are shown in bold.

[0045] Construction of vector heat shock transformation and identification of positive clones: The constructed plasmid was mixed with Fast-T1 competent cells, heat-shocked at 42℃ for 45s, then incubated on ice for 2 minutes, and then revived in LB medium without antibiotics. The plasmid was then plated on LB plates with the corresponding antibiotics and incubated overnight at 37℃. The next day, single colony clones were identified by colony PCR. Positive clones were selected for sequencing, and colonies carrying the correct construction were identified and screened by Sanger sequencing.

[0046] Plasmid extraction: Following the Promega Wizard Plus Midipreps DNA Purification System kit, single-clone bacterial blocks were picked and inoculated into 100 mL of sterile LB medium. The mixture was incubated overnight at 37°C and 200 rpm. After centrifugation, the bacterial blocks were collected, and 3 mL of Cell Resuspention Solution was added. The cells were vortexed thoroughly to resuspend the cells. Then, 3 mL of Cell Lysis Solution was added, and the mixture was gently inverted to thoroughly mix and lyse for 3-5 minutes, at which point the solution became clear. 3 mL of Neutralization Solution was added, and the mixture was gently inverted to mix. The mixture was then centrifuged at 12,000 rpm for 10 minutes, and the supernatant was transferred to a new 50 mL centrifuge tube. Resin was resuspended, and 10 mL of Resin was added to the centrifuge tube. The mixture was thoroughly mixed, transferred to a preparation tube, and purified using a negative pressure method. The recovery column was washed twice with 15 mL of ethanol-added Wash Buffer. The column containing Resin was then cut into a new 2 mL centrifuge tube and centrifuged at 12,000 rpm for 2 minutes. The tube was then replaced with a new 1.5 mL centrifuge tube, dried, and 200 μL of elution buffer was added. The tube was then centrifuged at 12,000 rpm for 2 minutes. The sample concentration was then measured using a NanoDrop micro spectrophotometer.

[0047] 2. Protoplasts and Transformation

[0048] The rice and wheat materials used in this invention for protoplast isolation and transformation are Zhonghua 11 and Anong 0711, respectively.

[0049] 2.1 Cultivation of Erythrogenic Rice Seedlings

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

[0051] 2.2 Isolation of rice protoplasts

[0052] (1) Cut off the stem tissue of the etiolated seedlings, cut the middle part into 0.5-1mm shreds with a blade, put them in 0.6M Mannitol solution and treat them in the dark for 10min, then filter them with a filter screen, put them into 50mL of enzyme hydrolysate (filtered with a 0.45μm filter membrane), vacuum (pressure about 15Kpa) for 30min, take them out and place them on a shaker (10rpm) at room temperature for 5h of enzymatic hydrolysis; (2) Add 30-50mL of W5 to dilute the enzyme hydrolysate, filter the enzyme hydrolysate into a round bottom centrifuge tube (50mL) with a 75μm nylon filter membrane; (3) Centrifuge at 23℃, 250g (rcf), 3°C, 3°C, 3°C, for 3min, and discard the supernatant; (4) Gently suspend the cells with 20mL of W5 and repeat step (3); (5) Add an appropriate amount of MMG to suspend the cells and wait for transformation.

[0053] 2.3 Rice protoplast transformation

[0054] (1) Place the transformation vector in a 2m centrifuge tube, mix well, then use a de-pointed pipette tip to take 200μL of protoplasts, gently tap to mix, add 220μL of PEG4000 solution, gently tap to mix, and induce transformation at room temperature in the dark for 20-30min; (3) Add 880μL of W5 and gently invert to mix, 250g (rcf), increase 3 and decrease 3, centrifuge for 3min, and discard the supernatant; (3) Add 1mL of WI solution, gently invert to mix, and incubate in the dark at 23℃ for 48h.

[0055] 2.4 Wheat Seedling Cultivation

[0056] Anong 0711 wheat seeds were planted in pots in a cultivation room and cultured for about 10 days under the conditions of temperature 25±2℃, light intensity 1000Lx, and light intensity 14~16h / d.

[0057] 2.5. Wheat protoplast isolation

[0058] (1) Take tender wheat leaves, cut the middle part into 0.5-1mm shreds with a blade, put them in 0.6M Mannitol solution and treat them in the dark for 10min, then filter them with a filter screen, put them into 50mL of enzyme hydrolysate (filtered with a 0.45μm filter membrane), vacuum (pressure about 15Kpa) for 30min, take them out and place them on a shaker (10rpm) at room temperature for 5h of enzymatic hydrolysis; (2) Add 30-50mL of W5 to dilute the enzyme hydrolysate, filter the enzyme hydrolysate into a round bottom centrifuge tube (50mL) with a 75μm nylon filter membrane; (3) Centrifuge at 23℃, 100g (rcf), 3°C, 3°C, 3°C, for 3min, and discard the supernatant; (4) Gently suspend them with 10mL of W5 and place them on ice for 30min; the protoplasts gradually settle, and discard the supernatant; (5) Add an appropriate amount of MMG to suspend them, place them on ice, and wait for transformation.

[0059] 2.6 Wheat protoplast transformation

[0060] (1) Place the transformed plasmid in a 2 mL centrifuge tube, mix well, then use a de-pointed pipette tip to take 200 μL of protoplasts, gently tap to mix, immediately add 250 μL of PEG 4000 solution, gently tap to mix, and induce transformation at room temperature in the dark for 20-30 min; (2) Add 800 μL of W5 (room temperature), gently invert to mix, add 100 g (rcf), increase 3 and decrease 3, centrifuge for 3 min, and discard the supernatant; (3) Add 1 mL of W5, gently invert to mix, and incubate at 23℃ in the dark for 48 h.

[0061] 3. Flow cytometry cell sorting / analysis

[0062] After transformation and dark culture for 24 h, wheat protoplasts were transferred to flow cytometry tubes for sorting / analysis. To address the stability of protoplasts during sorting, the pH and concentration of the sheath solution used in flow cytometry were tested and analyzed. The results showed that wheat protoplasts exhibited the best stability in 1*PBS solution at pH 5.7. Furthermore, using GFP-F71L as a positive control and BFP-F71L as a negative control to adjust the sorting threshold effectively improved the accuracy of flow cytometry sorting. Figure 1 ).

[0063] The specific flow cytometry operation is as follows: (1) After the instrument is turned on, open the BD FACS Software and perform instrument calibration and other operations. (2) Click "New Protocol" to create a suitable experimental protocol. (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, while the GFP-positive protoplasts appear in the position where the GFP fluorescence channel signal is stronger. (5) Set a gate to enclose the GFP-positive population, and use the negative control to determine the boundary of the gate. (6) Right-click on the cell population to be sorted, select "left sort", and set the analysis conditions and sorting / analysis mode according to the experimental needs and the percentage of target cells. (7) Load and sort / analyze the prepared protoplast samples cultured in the flow cytometer tubes in sequence, and record the relevant data. (8) Close the software and turn off the instrument.

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

[0065] 4.1 Protoplast DNA Extraction

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

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

[0068] The extracted protoplast DNA was used as the template for the first round of PCR, and amplification was performed using a high-fidelity PCR mix. The first-round PCR product was diluted 10-fold as a template for the second round of amplification (primers are shown in Table 2). The PCR product length was approximately 120–180 bp. The second-round PCR products were mixed in equal amounts according to the number of samples to form a library, and then the DNA was purified and recovered. The concentration was measured using a NanoDrop ultra-micro spectrophotometer. Finally, the samples were sent to Nanjing Panoson Gene Technology Co., Ltd. for library construction and sequencing.

[0069] 4.3 Analysis of Amplicon Sequencing Results

[0070] Referring to the method of Gaudelli et al.: (1) Using the sequence of the second round primers for amplifying the target site, each sample in each library was split to obtain all sequences of different treatments for different target sites; (2) The split sequences were spliced ​​using the flash splicing tool with default parameters; (3) The split sequences were compared with the wild-type sequences to analyze the mutation type of each sequence, and the mutation frequency and mutation type of each sequence were analyzed using a Perl script.

[0071] Table 2. Information on the first and second rounds of PCR primers used for amplicon library construction.

[0072]

[0073]

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

[0075] 5. Gene gun-mediated genetic transformation of wheat

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

[0077] 6. Detection of wheat mutants

[0078] Positive mutant plants were identified using the PCR / T7EI method. Individual samples were taken from regenerated plants sequentially, placed in 2 mL centrifuge tubes, and homogenized using a tissue homogenizer. Wheat genomic DNA was extracted using the CTAB method. PCR amplification was performed using the A, B, and D specific primers listed in Table 3. A 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℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 50-64℃ annealing for 30 s, 72℃ extension for 30 s, 37 cycles; 72℃ final extension for 5 min; storage at 12℃.

[0079] Take 2 μL of PCR amplification products from both the mutant and wild-type plants, add 1 μL of 10×T7 Endonuclease I Reactin Buffer, and bring the volume to 9 μL with water. After mixing, perform annealing reactions in a plant PCR instrument (95℃, 5 min; 95–85℃, -2℃ / sec; 85–25℃, -0.2℃ / sec; store at 4℃). Add 1 μL of T7 Endonuclease I to the annealed products and incubate at 37℃ for 30 min. Immediately after incubation, perform detection by 2% agarose gel electrophoresis. Positive mutant plants were further validated using first-generation sequencing to determine the specific mutation type. For samples that could not be genotyped, single-clone sequencing was used for genotyping.

[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 assess the significance of differences between treatments, employing a two-tailed test. Results were differentiated by p-value. A p-value > 0.05 indicated no significant difference between the two sample means (ns), a p-value < 0.05 indicated a significant difference (*), and a p-value < 0.01 indicated an extremely significant difference (**).

[0085] Example 2: Screening of cytosine deaminase mutants

[0086] Base editing systems were constructed by fusing known rAPOBEC1, hAPOBEC3A, Anc656, and evoFERNY deaminases with SpCas9 variants (SpG).

[0087] Editing tests were conducted on 10 endogenous target sites, including OsALS, OsAAT, OsACC, TaALS-T1, TaALS-T2, and TaACC, through protoplast transformation in rice and wheat. Next-generation sequencing analysis showed that, for the same deamination target sequences (OsALS and TaALS-T1), these deamination enzymes exhibited approximately five times the deamination efficiency in the rice genome compared to the wheat genome, indicating genomic differences. Among these deamination enzymes, Anc656 not only demonstrated higher deamination efficiency but also a narrower deamination window. Figure 2 Therefore, it has also been used as a chassis deaminase adapted to SpG for wheat genome adaptation and optimization.

[0088] Error-prone PCR was used to randomly introduce mutations into the Anc656 deaminase protein, and an RM-Anc656 random mutant library was constructed. Figure 3 Wheat protoplasts were co-transformed with four fluorescent reporter systems (FR-ACAC, FR-ACCC, FR-ACGC, and FR-ACTC) and cultured for 24 hours. Flow cytometry was then used for sorting, and 1×10⁶ cells were collected. 5DNA was extracted from green fluorescent protoplasts and analyzed using next-generation sequencing. The results showed that 28 mutation sites, including P8L, L11V, and Y16H, were significantly enriched before and after protoplast sorting (Figure 4), indicating that they may have a positive regulatory effect on enhancing the deamination activity of Anc656. These screening sites were mutated amino acid-by-amino acid in the pKUC57-Anc656-nSpG vector, and alanine saturation mutation scanning was performed in the Anc656 ssDNA binding domain (41-80 aa) to construct a series of mAnc656 mutant vectors. These vectors were co-transformed into wheat protoplasts using FR-ACAC, FR-ACCC, FR-ACGC, and FR-ACTC fluorescent reporter systems, and analyzed by flow cytometry. The proportion of luminescent protoplasts in each variant was then statistically analyzed and compared.

[0089] The results showed that the eight variants AncY16H, AncG24D, AncI50A, AncR55A, AncV81M, AncG141D, AncE144D, and AncG149D all outperformed the control Anc656 under the ACAC, ACCC, ACGC, and ACTC motifs. Among them, AncY16H, AncI50A, and AncG149D outperformed hAPOBEC3A and 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 Precise and Efficient Base Editing in Wheat

[0091] Based on single-site mutations at 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 then used to construct base editing systems AncY16H-nSpG, AncI50A-nSpG, AncG149D-nSpG, AncHA-nSpG, AncHD-nSpG, and AncAD-nSpG. Furthermore, using A3A-nSpG and Anc656-nSpG as controls, the new systems were compared using wheat endogenous target assays. 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%). The latter was also significantly higher than the controls Anc656-nSpG (14.32%) and 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 exhibited wide-window editing characteristics similar to A3A-nSpG, while AncI50A-nSpG, AncY149D-nSpG, and AncAD-nSpG containing I50A and G149D maintained, to some extent, the narrow-window editing characteristics of Anc656-nSpG, but the editing efficiency of cytosine bases within the deamination window was significantly improved (up to more than 3 times). Figure 10 ).

[0094] Because A3A-nSpG has low editing efficiency against TaALS-T1, TaALS-T1 was selected as the target for testing and comparison of A3A-nSpG and AncAD-nSpG. Wheat genetic transformation mediated by a gene gun showed that the number of regenerated seedlings from A3A-nSpG was less than that from AncAD-nSpG (Table 4), which may be related to the cytotoxicity of A3A. Without selection markers, AncAD-nSpG showed a higher editing efficiency (1.96%) compared to A3A-nSpG (Table 4), and all three mutants detected produced base editing in the AABBDD genome of the wheat allohexaploid. Figure 11 This also demonstrates the high efficiency of the new system.

[0095] Table 4. Comparison of the efficiency of gene gun-mediated endogenous target base editing in wheat

[0096]

[0097] Example 3: Compatibility of a New Precise and Efficient Wheat Base Editing System

[0098] Replacing nSpG with nCas9 in the above base editing systems, Anc656-BEmax, A3A-BEmax, AncY16H-BEmax, AncI50A-BEmax, AncG149D-BEmax, AncHA-BEmax, AncHD-BEmax, and AncAD-BEmax were constructed. Wheat protoplast transformation and next-generation sequencing 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 indicate that these variants can improve the efficiency of the base editing system constructed based on nCas9. Figure 12 ).

[0099] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A cytosine deaminase mutant, characterized in that, The cytosine deaminase mutant is AncG149D, AncHD, or AncAD; The cytosine deaminase mutant AncHD is a single-point mutation of Y16H based on the AncG149D sequence, and AncAD is a single-point mutation of I50A based on the AncG149D sequence. The amino acid sequences of AncG149D, AncHD, and AncAD are shown in SEQ ID NO.6, SEQ ID NO.8, and SEQ ID NO.9, respectively.

2. The cytosine deaminase mutant according to claim 1, characterized in that, The cytosine deaminase mutant is AncHD or AncAD, with the amino acid sequence shown in SEQ ID NO.8 or SEQ ID NO.

9.

3. The 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 cytosine base editing system, characterized in that, Includes the cytosine deaminase mutant as described in any one of claims 1-3, the Cas9 nick enzyme, and the uracil-DNA glycosylase repressor UGI; the Cas9 nick enzyme is wild-type nCas9 or an nCas9 variant, 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.

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