A base editor dgHBE4max, base editing method and application thereof

Abstract

The present application relates to a kind of base editor DGhBE4max and base editing method and its application, belong to gene editing technical field.The present application provides a kind of base editor DGhBE4max, its amino acid sequence is as shown in SEQ ID NO.1;The base editor DGhBE4max includes deaminase rAPOBEC1, endotoxin protein dDddA, nCas9 protein, uracil glycosylase inhibitor UGI.The base editor DGhBE4max of the present application can significantly improve the editing efficiency of existing cytosine base editor in plant, can also significantly expand the window of base editing, to provide a kind of stable and efficient technical tool for the research of plant functional gene and the directional evolution of key gene.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, and in particular to a base editor DGhBE4max, a base editing method, and their applications. Background Technology

[0002] Genome editing technology is widely used in various research fields, including biomedicine, animal science, plant science, and microbiology. Base editors are mainly composed of an nCas9 protein fusion deaminase and a uracil glycosylase inhibitor, enabling base substitution without causing double-strand DNA breaks. Commonly used base editors include cytosine base editors (CBE), adenine base editors (ABE), glycosylase base editors (GBE), and deaminase-independent base editors (DAF-CBE). ABE and CBE can achieve A-to-G and C-to-T conversions, while CGBE or GBE, developed based on CBE, can achieve C-to-G and C-to-A transversions.

[0003] Improving the base editing efficiency and altering the base editing range of base editors are the main directions of base editing research. Currently, efforts are being made to enhance the editing activity of base editors by discovering different types of deaminases and performing directed evolution on existing deaminases, or by modifying the deaminase structure of the base editor to provide different editing windows. However, existing modification methods are all aimed at the compositional structure of the base editor, and research on incorporating exogenous modifying proteins to improve base editing efficiency is still lacking.

[0004] Existing technologies have developed only three base editors for plants such as cotton: GhBE3, GhABE7.10n, and GhABE8e. Except for GhABE8e, which exhibits highly efficient A-to-G editing capabilities, the other two cytosine base editors still have relatively low efficiency, and their editing efficiency needs further optimization and improvement. Summary of the Invention

[0005] The purpose of this invention is to provide a base editor DGhBE4max and a base editing method and its application, so as to solve the problems of high difficulty, low efficiency and poor stability in the base editing of plant genome sequences in the prior art.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a base editor DGhBE4max, the amino acid sequence of which is shown in SEQ ID NO.1;

[0008] The base editor DGhBE4max includes the deaminase rAPOBEC1, the endotoxin protein dDddA, the nCas9 protein, and the uracil glycosylation inhibitor UGI.

[0009] The amino acid sequence of the deaminase rAPOBEC1 is shown in SEQ ID NO.2;

[0010] The amino acid sequence of the endotoxin protein dDddA is shown in SEQ ID NO.3;

[0011] The amino acid sequence of the nCas9 protein is shown in SEQ ID NO.4;

[0012] The amino acid sequence of the uracil glycosylation inhibitor UGI is shown in SEQ ID NO.5.

[0013] The present invention also provides a nucleic acid molecule encoding the base editor DGhBE4max, the sequence of which is shown in SEQ ID NO.6.

[0014] This invention also provides a method for preparing the base editor DGhBE4max, wherein the deaminase rAPOBEC1, nCas9 protein, and uracil glycosylation inhibitor UGI are sequentially linked to form the rAPOBEC1-nCas9-UGI-UGI backbone, and the endotoxin protein dDddA is inserted into the rAPOBEC1-nCas9-UGI-UGI backbone to obtain the base editor DGhBE4max.

[0015] The present invention also provides the application of the base editor DGhBE4max in improving the editing efficiency of plant genome sequences and / or expanding the editing window of plant genome sequences, converting base C in the plant genome sequence to base T.

[0016] The present invention also provides the application of the base editor DGhBE4max in the preparation of products that improve the editing efficiency of plant genome sequences and / or products that expand the editing window of plant genome sequences.

[0017] The present invention also provides a recombinant plasmid comprising the base editor DGhBE4max.

[0018] The present invention also provides the application of the recombinant plasmid in improving the editing efficiency of plant genome sequences and / or expanding the editing window of plant genome sequences.

[0019] The present invention also provides a recombinant bacterium, comprising the base editor DGhBE4max.

[0020] The present invention also provides the application of the recombinant bacteria described above in improving the editing efficiency of plant genome sequences and / or expanding the editing window of plant genome sequences.

[0021] This invention also provides a method for improving the editing efficiency of plant genome sequences and / or expanding the editing window of plant genome sequences, by transferring the base editor DGhBE4max, recombinant plasmids, or recombinant bacteria into plant cells for expression;

[0022] The base editor DGhBE4max is the base editor DGhBE4max;

[0023] The recombinant plasmid is the aforementioned recombinant plasmid;

[0024] The recombinant bacteria are the aforementioned recombinant bacteria;

[0025] The plants mentioned include cotton and tobacco;

[0026] The editing of the plant genome sequence involves converting base C in the plant genome sequence to base T.

[0027] The present invention has the following technical effects and advantages:

[0028] The base editor DGhBE4max of this invention incorporates the bacterial endotoxin protein dDddA. Compared with existing cytosine base editors (such as the base editor BE4max), it significantly improves the C-to-T editing efficiency at target sites in plant genome sequences. Simultaneously, it can deaminate and effectively edit PAM proximal sites that are inaccessible to existing cytosine base editors. Furthermore, it expands the editing window of existing cytosine base editors from C4–C8 sites to C1–C13 sites, significantly broadening the base editing window. This provides a stable and efficient gene editing tool for the study of plant functional genes and the directed evolution of key genes. Attached Figure Description

[0029] Figure 1 A schematic diagram of a recombinant plasmid containing the base editor DGhBE4max and sgRNA;

[0030] Figure 2 A schematic diagram of a recombinant plasmid containing the base editor BE4max and sgRNA;

[0031] Figure 3 The editing efficiencies of base editors DGhBE4max and BE4max in cotton are given, where A is the editing efficiency of base editor BE4max in cotton and B is the editing efficiency of base editor DGhBE4max in cotton.

[0032] Figure 4Editing efficiency of base editors DGhBE4max and BE4max in tobacco;

[0033] Figure 5 The images show the editing windows of the base editors DGhBE4max and BE4max in tobacco, where A is the editing window of the base editor BE4max in tobacco and B is the editing window of the base editor DGhBE4max in tobacco. Detailed Implementation

[0034] This invention provides a base editor DGhBE4max, the amino acid sequence of which is shown in SEQ ID NO.1;

[0035] The base editor DGhBE4max includes the deaminase rAPOBEC1, the endotoxin protein dDddA, the nCas9 protein, and the uracil glycosylation inhibitor UGI.

[0036] The amino acid sequence of the deaminase rAPOBEC1 is shown in SEQ ID NO.2;

[0037] The endotoxin protein dDddA is preferably dDddA(E1347A), and the amino acid sequence of dDddA(E1347A) is shown in SEQ ID NO.3;

[0038] The nCas9 protein is preferably nCas9(D10A), and the amino acid sequence of nCas9(D10A) is shown in SEQ ID NO.4;

[0039] The amino acid sequence of the uracil glycosylation inhibitor UGI is shown in SEQ ID NO.5.

[0040] The present invention also provides a nucleic acid molecule encoding the base editor DGhBE4max, the sequence of which is shown in SEQ ID NO.6;

[0041] The nucleotide sequence encoding the deaminase rAPOBEC1 is shown in SEQ ID NO.7;

[0042] The nucleotide sequence encoding the endotoxin protein dDddA is shown in SEQ ID NO. 8;

[0043] The nucleotide sequence encoding the nCas9 protein is shown in SEQ ID NO.9;

[0044] The nucleotide sequence encoding the uracil glycosylase inhibitor UGI is shown in SEQ ID NO.10.

[0045] This invention also provides a method for preparing the base editor DGhBE4max, wherein the deaminase rAPOBEC1, nCas9 protein, and uracil glycosylation inhibitor UGI are sequentially linked to form the rAPOBEC1-nCas9-UGI-UGI backbone, and the endotoxin protein dDddA is inserted into the rAPOBEC1-nCas9-UGI-UGI backbone to obtain the base editor DGhBE4max.

[0046] The present invention also provides the application of the base editor DGhBE4max in improving the editing efficiency of plant genome sequences and / or expanding the editing window of plant genome sequences, converting base C in the plant genome sequence to base T.

[0047] The present invention also provides the application of the base editor DGhBE4max in the preparation of products that improve the editing efficiency of plant genome sequences and / or products that expand the editing window of plant genome sequences.

[0048] The present invention also provides a recombinant plasmid comprising the base editor DGhBE4max.

[0049] The present invention also provides the application of the recombinant plasmid in improving the editing efficiency of plant genome sequences and / or expanding the editing window of plant genome sequences.

[0050] The present invention also provides a recombinant bacterium, comprising the base editor DGhBE4max.

[0051] The present invention also provides the application of the recombinant bacteria described above in improving the editing efficiency of plant genome sequences and / or expanding the editing window of plant genome sequences.

[0052] This invention also provides a method for improving the editing efficiency of plant genome sequences and / or expanding the editing window of plant genome sequences, by transferring the base editor DGhBE4max, recombinant plasmids, or recombinant bacteria into plant cells for expression;

[0053] The base editor DGhBE4max is the base editor DGhBE4max;

[0054] The recombinant plasmid is the aforementioned recombinant plasmid;

[0055] The recombinant bacteria are the aforementioned recombinant bacteria;

[0056] The plants mentioned include cotton and tobacco;

[0057] The editing of the plant genome sequence involves converting base C in the plant genome sequence to base T.

[0058] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0059] In the reagents of this invention, the infusion enzyme (catalog number: C117-02) and Escherichia coli DH5α competent cells (catalog number: C502-02) were purchased from Nanjing Novizan Biotechnology Co., Ltd., and Agrobacterium GV3101 competent cells (catalog number: B528430) were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0060] The components and preparation methods of the co-culture medium, callus induction medium, and rooting medium of the present invention are prepared in accordance with the methods disclosed by Jin Shuangxia in the literature (Jin Shuangxia. Optimization of cotton genetic transformation system and creation of mutants [D]. Huazhong Agricultural University, 2006);

[0061] In the test materials of this invention, the cotton variety is upland cotton Jin668, which was obtained from Huazhong Agricultural University; the tobacco variety is cultivated tobacco K326, which was purchased from Wuhan Tianwen Biotechnology Co., Ltd.

[0062] Example 1: Construction of the base editor DGhBE4max

[0063] The nucleotide sequences encoding rAPOBEC1, nCas9(D10A), and the uracil glycosylation inhibitor UGI were sequentially linked to form the rAPOBEC1-nCas9-UGI-UGI backbone. Based on the amino acid sequence of dDddA(E1347A) obtained from the Addgene website (Addgene ID: 183894), Genscript Biotech Inc. synthesized and optimized the nucleic acid molecule of dDddA(E1347A), which was then inserted into the rAPOBEC1-nCas9-UGI-UGI backbone after the nucleotide sequence encoding rAPOBEC1. At the same time, GGATCT was used as the linker sequence (linker peptide is GS) between the nucleotide sequences encoding rAPOBEC1 and dDddA(E1347A) to obtain the base editor DGhBE4max, whose amino acid sequence is shown in SEQ ID NO.1.

[0064] SEQ ID NO.1:

[0065]

[0066] The amino acid sequence of rAPOBEC1 is shown in SEQ ID NO.2.

[0067] SEQ ID NO.2:

[0068] MSSETGPVAVDPTLRRRIEPPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFI YIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK

[0069] The amino acid sequence of dDddA(E1347A) is shown in SEQ ID NO.3.

[0070] SEQ ID NO.3:

[0071] MGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVAGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0072] The amino acid sequence of nCas9(D10A) is shown in SEQ ID NO.4.

[0073] SEQ ID NO.4:

[0074]

[0075] The amino acid sequence of UGI-UGI is shown in SEQ ID NO.5.

[0076] SEQ ID NO.5:

[0077] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[0078] The sequence of the nucleic acid molecule encoding the base editor DGhBE4max is shown in SEQ ID NO.6.

[0079] SEQ ID NO.6:

[0080]

[0081] The nucleotide sequence encoding rAPOBEC1 is shown in SEQ ID NO.7.

[0082] SEQ ID NO.7:

[0083] ATGAGCAGTGAGACTGGTCCAGTGGCTGTTGATCCTACTTTACGGCGTAGGATTGAACCTCATGAATTTGAGGTTTTCTTTGATCCCCGGGAGCTGAGGAAAGAAACATGCTTGCTTTATGAAATTAATTGGGGAGGAAGGCATTCTATTTGGAGACACACCAGTCAGAACACAAACAAGCATGTAGAAGTCAATTTCATAGAGAAATTTACAACTGAGCGTTACTTCTGCCCAAATACAAGATGTTCTATAACATGGTTTCTTTCATGGAGTCCGTGTGGTGAATGTTCACGCGCAATCACCGAGTTTTTGAGCCGATACCCGCATGTAACCCTTTTCATATACATAGCACGATTATACCATCATGCTGATCCAAGAAACAGACAAGGACTAAGGGACTTGATCTCCTCAGGGGTGACGATTCAGATCATGACTGAACAAGAAAGTGGATATTGTTGGAGGAATTTTGTTAATTATTCGCCTTCCAATGAAGCTCATTGGCCTAGATATCCCCACTTATGGGTGCGTTTATATGTTTTGGAACTGTATTGCATTATTCTTGGCCTACCACCTTGTCTCAACATTCTGAGAAGGAAGCAACCACAGCTCACTTTTTTCACCATCGCCCTTCAATCTTGCCATTATCAAAGATTGCCCCCACACATCCTTTGGGCAACCGGATTGAAA

[0084] The nucleotide sequence encoding dDddA(E1347A) is shown in SEQ ID NO.8.

[0085] SEQ ID NO.8:

[0086] ATGGGGAGCTACGCCCTTGGGCCCTATCAAATCTCAGCCCCCCAGCTCCCTGCGTACAATGGCCAGACAGTGGGAACATTTTATTATGTTAATGATGCTGGCGGTTTGGAGTCGAAGGTCTTCTCCAGTGGTGGGCCAACCCCTTACCCGAATTATGCAAATGCAGGTCATGTTGCTGGTCAATCAGCATTGTTTATGAGGGACAATGGAATATCTGAGGGACTTGTTTTTCACAACAACCCAGAAGGCACTTGTGGTTTTTGTGTTAACATGACGGAAACCCTGTTACCTGAGAACGCTAAAATGACTGTAGTTCCTCCAGAAGGCGCTATTCCAGTGAAGAGAGGTGCAACTGGTGAAACAAAAGTATTCACTGGAAATTCTAATTCACCAAAAAGTCCTACAAAGGGAGGGTGC

[0087] The nucleotide sequence encoding nCas9(D10A) is shown in SEQ ID NO.9.

[0088] SEQ ID NO.9:

[0089]

[0090] The nucleotide sequence encoding UGI-UGI is shown in SEQ ID NO.10.

[0091] SEQ ID NO.10:

[0092] ATGACAAATTTATCTGACATAATTGAAAAAGAAACTGGAAAACAACTTGTTATTCAAGAATCTATTCTTGATGTTACCTGAGGAGGTGGAAGAGGTTATTGGAAACAAACCAGAGAGCGATATACTAGTGCAC ACAGCTTACGATGAGTCCACTGATGAAAATGTTATGCTGTTGACCTCTGATGCTCCTGAATACAAGCCATGGGCACTCGTCATACAAGACTCCAATGGTGAGAACAAGATTAAAATGCTCTCAGGGGGTTCTG GAGGATCGGGCGGGTCAACCAACCTGTCTGATATCATTGAGAAAGAGACGGGCAAGCAGCTTGTCATTCAGGAAAGTATATTAATGCTTCCTGAAGAAGTTGAAGAAGTAATTGGTAACAAGCCGGAATCGGA CATTTTGGTTCATACTGCATATGATGAATCAACTGACGAAAATGTGATGCTTCTTACATCAGATGCACCCGAGTATAAACCTTGGGCTTTGGTAATCCAAGATAGCAATGGAGAGAATAAAATCAAGATGCTA

[0093] Comparative Example 1: Construction of the base editor BE4max

[0094] The rAPOBEC1-nCas9-UGI-UGI skeleton was constructed according to the method described in Example 1, which is the base editor BE4max.

[0095] Experiment 1: Construction of sgRNA vector and transformation of recombinant vector

[0096] Target sgRNAs were designed using the cotton endogenous gene GhALS and the tobacco endogenous gene NtPDS as target genes (as shown in Table 1). A suitable tRNA element for the plant target sequence was ligated to the 5' end of each target sgRNA (as shown in SEQ ID NO. 11), resulting in tRNA-sgRNA02 and tRNA-sgRNA03, respectively. Using infusion enzyme, tRNA-sgRNA02 and tRNA-sgRNA03 were ligated to the BsaI restriction site of the base editor DGhBE4max nucleic acid molecule, and transformed into E. coli DH5α competent cells, resulting in recombinant plasmids DGhBE4max-sgRNA02 and DGhBE4max-sgRNA03, respectively. Figure 1 As shown; simultaneously, tRNA-sgRNA02 and tRNA-sgRNA03 were ligated to the BsaI restriction site of the base editor BE4max nucleic acid molecule using infusion enzyme, and then transformed into E. coli DH5α competent cells, corresponding to the recombinant plasmids BE4max-sgRNA02 and BE4max-sgRNA03, as shown. Figure 2 As shown. Each recombinant plasmid was transformed into Agrobacterium GV3101 competent cells to obtain DGhBE4max-sgRNA02, DGhBE4max-sgRNA03, BE4max-sgRNA02, and BE4max-sgRNA03 bacterial suspensions.

[0097] SEQ ID NO.11:

[0098] AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGG TACAGACCCGGGTTCGATTCCCGGCTGGTGCA

[0099] Table 1 Target sgRNA sequences of cotton and tobacco

[0100] cotton sgRNA02 GCGCCTCCACTGGGGATCAT 12 tobacco sgRNA03 CAATCTTCTGGTCATGGCAC 13

[0101] Experimental Example 2: Determination of Genetic Transformation and Base Editing Results in Cotton

[0102] Under aseptic conditions, hypocotyls of upland cotton Jin668 seedlings were cut into 1 cm segments and placed in DGhBE4max-sgRNA02 and BE4max-sgRNA02 bacterial solutions, respectively. After stirring and standing for 5 minutes, the segments were air-dried and dispersed in co-culture medium lined with filter paper, ensuring that each hypocotyl segment was in contact with the filter paper. The segments were then incubated in the dark at 19°C for 36 hours. After dark culture, each hypocotyl segment was inoculated onto callus induction medium. Every 30 days, hypocotyls with swollen ends and normal growth were selected and subcultured into fresh callus induction medium. After four subcultures, cotton embryogenic callus tissue corresponding to the genetically transformed bacterial solutions was obtained.

[0103] Following the CTAB method disclosed by Wang Pengcheng et al. in the literature (Wang Pengcheng. Functional verification of cotton ubiquitin-binding enzyme gene GhUBC2L and construction of cotton CRISPR / Cas9 system [D]. Huazhong Agricultural University, 2018), genomic DNA was extracted from various cotton embryogenic callus tissues. Based on the nucleotide sequence of the cotton endogenous gene GhALS, primer sequences for detecting sgRNA02 were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., as shown in Table 2.

[0104] PCR amplification of sgRNA02 was performed using genomic DNA from various cotton embryogenic callus tissues as templates. The PCR amplification system was 20 μL, comprising 2 μL genomic DNA from each cotton embryogenic callus tissue, 0.2 μL sgRNA02-F primer, 0.2 μL sgRNA02-R primer, 2 μL 10×EasyTaq Buffer, 0.2 μL EasyTaq, 0.4 μL dNTPs, and the remainder ddH2O. The PCR amplification program was: 95℃ pre-denaturation for 5 min, followed by 95℃ denaturation for 30 s → 56℃ annealing for 30 s → 72℃ extension for 25 s, for a total of 30 cycles. The amplified products were then subjected to first-generation sequencing by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results were analyzed using the EditR analysis website (https: / / moriaritylab.shinyapps.io / editr_v10 / ). The results are as follows: Figure 3 As shown.

[0105] Table 2 Primer sequences for detecting cotton base editing efficiency

[0106] sgRNA02-F AATGTTGGACACTCCTGGAC 14 sgRNA02-R CCACCCTCATTCATTACATACA 15

[0107] The results showed that the base editor BE4max mainly performed base editing at C7 and C8 sites, with editing efficiencies of 39% and 23%, respectively. In contrast, the base editor DGhBE4max achieved editing efficiencies of 88% and 65% at C7 and C8 sites, respectively. This means that compared to BE4max, DGhBE4max improved the editing efficiency at C7 and C8 sites by 1.26 times and 1.74 times, respectively. Furthermore, DGhBE4max expanded the base editing window in cotton, achieving editing efficiencies of 30% and 52% at C5 and C10 sites, respectively, which were previously inaccessible to BE4max.

[0108] Experiment Example 3: Determination of Genetic Transformation and Base Editing Results in Tobacco

[0109] Under aseptic conditions, healthy and tender green leaves of cultivated tobacco K326 were cut into leaf discs and placed separately in DGhBE4max-sgRNA03 and BE4max-sgRNA03 bacterial solutions, stirred well, allowed to stand for 15 minutes, and then air-dried. These discs were then dispersed and inoculated into a co-culture medium lined with filter paper, ensuring that each leaf disc was in contact with the filter paper. The discs were then incubated in the dark at 19°C for 36 hours. After dark incubation, each leaf disc was inoculated onto callus induction medium, and subcultured every 20 days to a fresh callus induction medium until regenerated tobacco seedlings were obtained. Regenerated tobacco seedlings containing two leaflets, exhibiting healthy growth points, and with excess callus tissue removed from the roots were transferred to rooting medium for rooting culture, yielding tobacco seedlings corresponding to the genetically transformed bacterial solutions.

[0110] Following the CTAB method disclosed by Wang Pengcheng et al. in the literature (Wang Pengcheng. Functional verification of cotton ubiquitin-binding enzyme gene GhUBC2L and construction of cotton CRISPR / Cas9 system [D]. Huazhong Agricultural University, 2018), genomic DNA was extracted from the tender leaves of various tobacco seedlings. Based on the nucleotide sequence of the tobacco endogenous gene NtPDS, primer sequences for detecting sgRNA03 were designed. At the same time, a 6bp barcode tag sequence was added to the 5' end of each primer sequence, and the primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., as shown in Table 3.

[0111] Table 3 Primer sequences for detecting tobacco base editing efficiency

[0112] sgRNA03-F AAGAACAACGAAATGCTTACG 16 sgRNA03-R GGCAGGTAGACAATTCTAGCA 17

[0113] sgRNA03 was amplified by PCR using genomic DNA from young tobacco leaves as a template. The PCR amplification system consisted of 20 μL of genomic DNA from each young tobacco leaf, 0.2 μL of sgRNA03-F primer, 0.2 μL of sgRNA03-R primer, 2 μL of 10×EasyTaq Buffer, 0.2 μL of EasyTaq, 0.4 μL of dNTPs, and the remainder ddH2O. The PCR amplification program was set as follows: 95℃ pre-denaturation for 5 min, followed by 95℃ denaturation for 30 s → 56℃ annealing for 30 s → 72℃ extension for 25 s, for a total of 30 cycles. The amplified products were then sent to Beijing Novogene Technology Co., Ltd. for next-generation high-throughput sequencing. A script was written to split the sequencing results based on the barcode tag sequence and primer sequence. Then, CRISPResso2 was used with the parameters set sequentially as follows: "—quantification_window_size10—quantification_window_center-10—base_editor_output—conversion_nuc_fromA—conversion_nuc_to G". The results are as follows: Figures 4-5 As shown.

[0114] The results showed that, compared with the base editor BE4max, the base editor DGhBE4max improved the editing efficiency at C5 and C8 sites by 70% and 97%, respectively. The overall editing efficiency of DGhBE4max reached a maximum of 84.78%, which is 2.4 times higher than the highest editing efficiency of BE4max (25.17%). Furthermore, the base editor DGhBE4max expanded the base editing window in tobacco, achieving editing efficiencies of 6.91% and 6.91% at C1 and C13 sites, respectively, which were previously inaccessible to BE4max.

[0115] As can be seen from the above embodiments, the present invention provides a base editor DGhBE4max, a base editing method, and their applications. The base editor DGhBE4max of the present invention can significantly improve the editing efficiency of existing cytosine base editors (such as the base editor BE4max) in plants, and can also deaminate PAM proximal sites that cannot be edited by existing cytosine base editors, significantly expanding the base editing window.

[0116] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A base editor DGhBE4max, characterized in that, The amino acid sequence of the base editor DGhBE4max is shown in SEQ ID NO.1; The base editor DGhBE4max includes the deaminase rAPOBEC1, the endotoxin protein dDddA, the nCas9 protein, and the uracil glycosylation inhibitor UGI. The amino acid sequence of the deaminase rAPOBEC1 is shown in SEQ ID NO.2; The amino acid sequence of the endotoxin protein dDddA is shown in SEQ ID NO.3; The amino acid sequence of the nCas9 protein is shown in SEQ ID NO.4; The amino acid sequence of the uracil glycosylation inhibitor UGI is shown in SEQ ID NO.5; The method for preparing the base editor DGhBE4max involves sequentially linking the deaminase rAPOBEC1, nCas9 protein, and uracil glycosylation inhibitor UGI to form the rAPOBEC1-nCas9-UGI-UGI backbone, and inserting the endotoxin protein dDddA into the rAPOBEC1-nCas9-UGI-UGI backbone to obtain the base editor DGhBE4max. The base editor DGhBE4max can improve the efficiency of editing plant genome sequences.

2. A nucleic acid molecule encoding the base editor DGhBE4max of claim 1, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID NO.

6.

3. The method for preparing the base editor DGhBE4max according to claim 1, characterized in that, The deaminase rAPOBEC1, nCas9 protein, and uracil glycosylation inhibitor UGI were sequentially linked to form the rAPOBEC1-nCas9-UGI-UGI backbone. The endotoxin protein dDddA was then inserted into the rAPOBEC1-nCas9-UGI-UGI backbone to obtain the base editor DGhBE4max.

4. The application of the base editor DGhBE4max as described in claim 1 in improving the editing efficiency of plant genome sequences and / or expanding the editing window of plant genome sequences, characterized in that, The C base in the plant genome sequence is converted to the T base.

5. The use of the base editor DGhBE4max as described in claim 1 in the preparation of products that improve the editing efficiency of plant genome sequences and / or expand the editing window of plant genome sequences.

6. A recombinant plasmid, characterized in that, Includes the base editor DGhBE4max as described in claim 1.

7. The application of the recombinant plasmid according to claim 6 in improving the editing efficiency of plant genome sequences and / or expanding the editing window of plant genome sequences.

8. A recombinant bacterium, characterized in that, Includes the base editor DGhBE4max as described in claim 1.

9. The application of the recombinant bacteria according to claim 8 in improving the editing efficiency of plant genome sequences and / or expanding the editing window of plant genome sequences.

10. A method for improving the efficiency of plant genome sequence editing and / or expanding the editing window of plant genome sequences, characterized in that, The base editor DGhBE4max, recombinant plasmids, or recombinant bacteria were transferred into plant cells for expression. The base editor DGhBE4max is the base editor DGhBE4max as described in claim 1; The recombinant plasmid is the recombinant plasmid according to claim 6; The recombinant bacteria is the recombinant bacteria according to claim 8; The plants mentioned include cotton and tobacco; The editing of the plant genome sequence involves converting base C in the plant genome sequence to base T.

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