Base editing system GhDA3A-BE4, base editing method and application of base editing system GhDA3A-BE4

Abstract

The invention relates to a base editing system GhDA3A-BE4, a base editing method and application of the base editing system GhDA3A-BE4, and belongs to the technical field of gene editing. The invention provides a base editing system GhDA3A-BE4. The amino acid sequence of the base editing system GhDA3A-BE4 is as shown in SEQ ID NO. 1; the basic group editing system GhDA3A-BE4 comprises a human deaminase APOBEC3A (A3A), an endotoxin protein dDddA, an nCas9 protein and a uracil glycosylase inhibitor UGI (Uracil Glucosylase Inhibitor). According to the base editing system GhDA3A-BE4, the C-to-T editing efficiency at a plant genome sequence target site can be remarkably improved, meanwhile, a base editing window can be remarkably expanded, and more comprehensive gene editing on a coding region and a non-coding region of a plant genome sequence is achieved.

Description

Technical Field

[0001] The present invention relates to the field of gene editing technology, and in particular to a base editing system GhDA3A-BE4 and a base editing method and applications thereof. Background Art

[0002] The base editing system achieves base replacement at the target site by fusing deaminase with Cas proteins (such as dCas9, nCas9, etc.) that have lost their double-stranded cleavage activity and acting on single-stranded DNA (ssDNA). It does not require DNA double-strand breakage and donor DNA provision, and does not produce cytotoxicity. It has a wide range of application potential in base editing of animal and plant genes. Currently, the commonly used base editing systems include cytosine base editor (CBE), adenine base editor (ABE), glycosylase base editor (GBE) and deaminase-independent base editing system (DAF-CBE). ABE and CBE can achieve transitions between A-to-G and C-to-T (transition, purine to purine or pyrimidine to pyrimidine), while CGBE or GBE developed on the basis of CBE can achieve transversions between C-to-G and C-to-A (transversion, pyrimidine to purine). The development of CGBE or GBE benefited from the in-depth study of CBE editing byproducts (including transversions between C-to-G and C-to-A), which expanded the existing technology to study the mechanism of DNA damage repair and the application of base editing systems.

[0003] Deaminase APOBEC1 is the first deaminase to be used in base editing systems, but its editing efficiency is low and there is a certain degree of off-target. Human deaminase APOBEC3A (A3A) has been shown to exhibit certain base editing activity and base editing windows in plants such as rice, corn, and tomatoes, but the current base editing system containing human deaminase APOBEC3A (A3A) still has a small editing range near the PAM and low editing efficiency, which greatly hinders the development of efficient base editing systems for plant gene editing and restricts the research and application of directed evolution to modify plant specific functional genes. Summary of the invention

[0004] The purpose of the present invention is to provide a base editing system GhDA3A-BE4 and a base editing method and application thereof, so as to solve the problems of insufficient base editing activity, low editing efficiency and narrow editing window of the base editing system in the prior art, which make the base editing of plant genome sequences difficult, inefficient and poorly stable.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a base editing system GhDA3A-BE4, the amino acid sequence of the base editing system GhDA3A-BE4 is shown in SEQ ID NO.1;

[0007] The base editing system GhDA3A-BE4 includes human deaminase APOBEC3A (A3A), endotoxin protein dDddA, nCas9 protein, and uracil glycosylase inhibitor UGI;

[0008] The amino acid sequence of the human deaminase APOBEC3A (A3A) is shown in SEQ ID NO.2;

[0009] The amino acid sequence of the endotoxin protein dDddA is shown in SEQ ID NO.3;

[0010] The amino acid sequence of the nCas9 protein is shown in SEQ ID NO.4;

[0011] The amino acid sequence of the uracil glycosylase inhibitor UGI is shown in SEQ ID NO.5.

[0012] The present invention also provides a nucleic acid molecule encoding the base editing system GhDA3A-BE4, and the sequence of the nucleic acid molecule is shown in SEQ ID NO.6.

[0013] The present invention also provides a method for preparing the base editing system GhDA3A-BE4, wherein human deaminase APOBEC3A (A3A), nCas9 protein, and uracil glycosylase inhibitor UGI are sequentially connected to form a vector skeleton A3A-nCas9-UGI-UGI, and the endotoxin protein dDddA is inserted into the vector skeleton A3A-nCas9-UGI-UGI to obtain the base editing system GhDA3A-BE4.

[0014] The present invention also provides the use of the base editing system GhDA3A-BE4 in any one of the following (1) to (4):

[0015] (1) Improve the editing efficiency of plant genome sequences;

[0016] (2) preparing products that improve the editing efficiency of plant genome sequences;

[0017] (3) Expand the editing window of plant genome sequences;

[0018] (4) Prepare products that expand the editing window of plant genome sequences.

[0019] Preferably, the editing of the plant genome sequence comprises converting base C in the plant genome sequence into base T.

[0020] The present invention also provides a recombinant plasmid, comprising the base editing system GhDA3A-BE4.

[0021] The present invention also provides the use of the recombinant plasmid in the following (1) or (2):

[0022] (1) Improve the editing efficiency of plant genome sequences;

[0023] (2) Expand the editing window of plant genome sequences.

[0024] The present invention also provides a recombinant bacterium, comprising the base editing system GhDA3A-BE4.

[0025] The present invention also provides the use of the recombinant bacteria in the following (1) or (2):

[0026] (1) Improve the editing efficiency of plant genome sequences;

[0027] (2) Expand the editing window of plant genome sequences.

[0028] The present invention also provides a method for improving the editing efficiency of a plant genome sequence and / or expanding the editing window of a plant genome sequence, wherein the base editing system GhDA3A-BE4, a recombinant plasmid or a recombinant bacterium is transferred into a plant cell for expression;

[0029] The base editing system GhDA3A-BE4 is the base editing system GhDA3A-BE4;

[0030] The recombinant plasmid is the recombinant plasmid;

[0031] The recombinant bacteria are the recombinant bacteria described above;

[0032] The plants include cotton and tobacco;

[0033] The editing of the plant genome sequence comprises converting base C in the plant genome sequence into base T.

[0034] The present invention has the following technical effects and advantages:

[0035] The base editing system GhDA3A-BE4 of the present invention fuses the bacterial endotoxin protein dDddA at the C-terminus of the human deaminase APOBEC3A (A3A). Compared with the existing base editing system A3A (Y130F) -BE4, the C-to-T editing efficiency at the target site of the plant genome sequence is significantly improved, especially the editing efficiency at the proximal end of PAM is most significantly improved, with a maximum improvement of 11.5 times; at the same time, the base editing system GhDA3A-BE4 of the present invention can expand the editing window of the existing base editing system A3A (Y130F) -BE4 from C6 to C10 sites to C1 to C 13 sites, significantly expanding the C-to-T editing window of the target site of the plant genome sequence, and even producing an editing efficiency of 39.66% at the C1 site that the base editing system A3A(Y130F)-BE4 could not edit, realizing more comprehensive gene editing of the coding and non-coding regions (such as promoters, enhancers, etc.) of the plant genome sequence, and providing a deeper understanding of the distribution and function of transcription factor binding sites, as well as the interaction between sites and the mechanism of their influence on the expression level of plant functional genes, providing a stable and efficient gene editing tool for the study of plant functional genes and the directed evolution of key genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a schematic diagram of the recombinant plasmid containing the base editing system GhDA3A-BE4 and sgRNA;

[0037] Figure 2 This is a schematic diagram of the recombinant plasmid containing the base editing system A3A(Y130F)-BE4 and sgRNA;

[0038] Figure 3 is the editing efficiency of the base editing system GhDA3A-BE4 and A3A(Y130F)-BE4 at the cotton target sgRNA01, wherein A is the editing efficiency of the base editing system A3A(Y130F)-BE4 at the cotton target sgRNA01, and B is the editing efficiency of the base editing system GhDA3A-BE4 at the cotton target sgRNA01;

[0039] Figure 4 is the editing efficiency of the base editing system GhDA3A-BE4 and A3A(Y130F)-BE4 at the cotton target sgRNA02, wherein A is the editing efficiency of the base editing system A3A(Y130F)-BE4 at the cotton target sgRNA02, and B is the editing efficiency of the base editing system GhDA3A-BE4 at the cotton target sgRNA02;

[0040] Figure 5The editing windows of the base editing systems GhDA3A-BE4 and A3A(Y130F)-BE4 in tobacco, where A is the editing window of the base editing system A3A(Y130F)-BE4 in tobacco, and B is the editing window of the base editing system GhDA3A-BE4 in tobacco. DETAILED DESCRIPTION

[0041] The present invention provides a base editing system GhDA3A-BE4, the amino acid sequence of the base editing system GhDA3A-BE4 is shown in SEQ ID NO.1;

[0042] The base editing system GhDA3A-BE4 includes human deaminase APOBEC3A (A3A), endotoxin protein dDddA, nCas9 protein, and uracil glycosylase inhibitor UGI;

[0043] The human deaminase APOBEC3A (A3A) is preferably A3A (Y130F), and the amino acid sequence of A3A (Y130F) is shown in SEQ ID NO. 2;

[0044] The endotoxin protein dDddA is preferably dDddA (E1347A), and the amino acid sequence of the dDddA (E1347A) is shown in SEQ ID NO. 3;

[0045] The nCas9 protein is preferably nCas9(D10A), and the amino acid sequence of nCas9(D10A) is shown in SEQ ID NO.4;

[0046] The amino acid sequence of the uracil glycosylase inhibitor UGI is shown in SEQ ID NO.5.

[0047] The present invention also provides a nucleic acid molecule encoding the base editing system GhDA3A-BE4, the sequence of the nucleic acid molecule is shown in SEQ ID NO.6;

[0048] The nucleotide sequence encoding the human deaminase APOBEC3A (A3A) is shown in SEQ ID NO.7;

[0049] The nucleotide sequence encoding the endotoxin protein dDddA is shown in SEQ ID NO.8;

[0050] The nucleotide sequence encoding the nCas9 protein is shown in SEQ ID NO.9;

[0051] The nucleotide sequence encoding the uracil glycosylase inhibitor UGI is shown in SEQ ID NO.10.

[0052] The present invention also provides a method for preparing the base editing system GhDA3A-BE4, wherein human deaminase APOBEC3A (A3A), nCas9 protein, and uracil glycosylase inhibitor UGI are sequentially connected to form a vector skeleton A3A-nCas9-UGI-UGI, and the endotoxin protein dDddA is inserted into the vector skeleton A3A-nCas9-UGI-UGI to obtain the base editing system GhDA3A-BE4.

[0053] The present invention also provides the use of the base editing system GhDA3A-BE4 in any one of the following (1) to (4):

[0054] (1) Improve the editing efficiency of plant genome sequences;

[0055] (2) preparing products that improve the editing efficiency of plant genome sequences;

[0056] (3) Expand the editing window of plant genome sequences;

[0057] (4) Prepare products that expand the editing window of plant genome sequences.

[0058] In the present invention, the editing of the plant genome sequence includes converting the base C in the plant genome sequence into the base T.

[0059] The present invention also provides a recombinant plasmid, comprising the base editing system GhDA3A-BE4.

[0060] The present invention also provides the use of the recombinant plasmid in the following (1) or (2):

[0061] (1) Improve the editing efficiency of plant genome sequences;

[0062] (2) Expand the editing window of plant genome sequences.

[0063] The present invention also provides a recombinant bacterium, comprising the base editing system GhDA3A-BE4.

[0064] The present invention also provides the use of the recombinant bacteria in the following (1) or (2):

[0065] (1) Improve the editing efficiency of plant genome sequences;

[0066] (2) Expand the editing window of plant genome sequences.

[0067] The present invention also provides a method for improving the editing efficiency of a plant genome sequence and / or expanding the editing window of a plant genome sequence, wherein the base editing system GhDA3A-BE4, a recombinant plasmid or a recombinant bacterium is transferred into a plant cell for expression;

[0068] The base editing system GhDA3A-BE4 is the base editing system GhDA3A-BE4;

[0069] The recombinant plasmid is the recombinant plasmid;

[0070] The recombinant bacteria are the recombinant bacteria described above;

[0071] The plants include cotton and tobacco;

[0072] The editing of the plant genome sequence comprises converting base C in the plant genome sequence into base T.

[0073] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0074] Among the reagents of the present invention, Infusion enzyme (Cat. No.: C117-02) and Escherichia coli DH5α competent cells (Cat. No.: C502-02) were purchased from Nanjing Novozyme Biotechnology Co., Ltd., and Agrobacterium tumefaciens GV3101 competent cells (Cat. No.: B528430) were purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0075] The components and preparation methods of the co-cultivation medium, callus induction medium and rooting medium of the present invention are prepared with reference to the method disclosed by Jin Shuangxia in the literature (Jin Shuangxia. Optimization of cotton genetic transformation system and creation of mutants [D]. Huazhong Agricultural University, 2006);

[0076] In the test materials of the present invention, the cotton variety is upland cotton Jin668, which is from Huazhong Agricultural University; the tobacco variety is cultivated tobacco K326, which is purchased from Wuhan Tianwen Biotechnology Co., Ltd.

[0077] Example 1: Construction of base editing system GhDA3A-BE4

[0078] The nucleotide sequences encoding A3A (Y130F), nCas9 (D10A), and uracil glycosylase inhibitor UGI were sequentially connected to form the vector backbone A3A-nCas9-UGI-UGI. Based on the amino acid sequence of dDddA (E1347A) obtained from the Addgene website (Addgene ID: 183894), GenScript Biotech Co., Ltd. was commissioned to synthesize and optimize the nucleic acid molecule of dDddA (E1347A), which was inserted into the nucleotide sequence encoding A3A (Y130F) in the vector backbone A3A-nCas9-UGI-UGI. GGATCT was used as the connecting sequence (the connecting peptide was GS) between the nucleotide sequences encoding A3A (Y130F) and dDddA (E1347A) to obtain the base editing system GhDA3A-BE4, whose amino acid sequence is shown in SEQ ID NO.1.

[0079] SEQ ID NO.1:

[0080]

[0081] The amino acid sequence of A3A(Y130F) is shown in SEQ ID NO.2.

[0082] SEQ ID NO.2:

[0083] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIFDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN

[0084] The amino acid sequence of dDddA(E1347A) is shown in SEQ ID NO.3.

[0085] SEQ ID NO.3:

[0086] MGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVAGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0087] The amino acid sequence of nCas9(D10A) is shown in SEQ ID NO.4.

[0088] SEQ ID NO.4:

[0089]

[0090] The amino acid sequence of UGI-UGI is shown in SEQ ID NO.5.

[0091] SEQ ID NO.5:

[0092] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[0093] The sequence of the nucleic acid molecule encoding the base editing system GhDA3A-BE4 is shown in SEQ ID NO.6.

[0094] SEQ ID NO.6:

[0095]

[0096] The nucleotide sequence encoding A3A (Y130F) is shown in SEQ ID NO.7.

[0097] SEQ ID NO.7:

[0098] ATGGAAGCATCCCCTGCGTCGGGTCCGAGACATTTAATGGACCCTCATATTTTCACAAGCAATTTCAACAATGGAATTGGAAGACACAAAACTTACTTATGTTATGAAGTTGAGAGGCTCGATAATGGGACTTCAGTGAAGATGGATCAACACAGAGGTTTCCTCCACAACCAGGCAAAAAATTTGCTTTGTGGTTTTTATGGAAGGCATGCTGAGTTGCGTTTCCTTGATCTTGTCCCTTCACTACAGTTGGATCCAGCTCAGATTTATAGAGTCACATGGTTTATTTCATGGTCACCTTGCTTTAGTTGGGGTTGCGCCGGGGAGGTACGCGCTTTTCTTCAAGAAAACACTCATGTTCGGCTGAGGATATTTGCCGCACGAATCTTTGATTATGACCCACTATACAAGGAAGCTTTGCAAATGCTTAGAGATGCTGGAGCACAGGTTAGTATCATGACTTACGATGAGTTCAAGCATTGTTGGGACACGTTTGTTGACCATCAAGGATGCCCATTTCAACCATGGGATGGTTTAGATGAACATTCCCAGGCTCTTAGTGGGCGTTTGCGAGCCATACTGCAAAATCAAGGCAAT

[0099] The nucleotide sequence encoding dDddA (E1347A) is shown in SEQ ID NO.8.

[0100] SEQ ID NO.8:

[0101] ATGGGGAGCTACGCCCTTGGGCCCTATCAAATCTCAGCCCCCCAGCTCCCTGCGTACAATGGCCAGACAGTGGGAACATTTTATTATGTTAATGATGCTGGCGGTTTGGAGTCGAAGGTCTTCTCCAGTGGTGGGCCAACCCCTTACCCGAATTATGCAAATGCAGGTCATGTTGCTGGTCAATCAGCATTGTTTATGAGGGACAATGGAATATCTGAGGGACTTGTTTTTCACAACAACCCAGAAGGCACTTGTGGTTTTTGTGTTAACATGACGGAAACCCTGTTACCTGAGAACGCTAAAATGACTGTAGTTCCTCCAGAAGGCGCTATTCCAGTGAAGAGAGGTGCAACTGGTGAAACAAAAGTATTCACTGGAAATTCTAATTCACCAAAAAGTCCTACAAAGGGAGGGTGC

[0102] The nucleotide sequence encoding nCas9 (D10A) is shown in SEQ ID NO.9.

[0103] SEQ ID NO.9:

[0104]

[0105] The nucleotide sequence encoding UGI-UGI is shown in SEQ ID NO.10.

[0106] SEQ ID NO.10:

[0107] ATGACAAATTTATCTGACATAATTGAAAAAGAAACTGGAAAACAACTTGTTATTCAAGAATCTATTCTTGATGTTACCTGAGGAGGTGGAAGAGGTTATTGGAAACAAACCAGAGAGCGATATACTAGTGCAC ACAGCTTACGATGAGTCCACTGATGAAAATGTTATGCTGTTGACCTCTGATGCTCCTGAATACAAGCCATGGGCACTCGTCATACAAGACTCCAATGGTGAGAACAAGATTAAAATGCTCTCAGGGGGTTCTG GAGGATCGGGCGGGTCAACCAACCTGTCTGATATCATTGAGAAAGAGACGGGCAAGCAGCTTGTCATTCAGGAAAGTATATTAATGCTTCCTGAAGAAGTTGAAGAAGTAATTGGTAACAAGCCGGAATCGGA CATTTTGGTTCATACTGCATATGATGAATCAACTGACGAAAATGTGATGCTTCTTACATCAGATGCACCCGAGTATAAACCTTGGGCTTTGGTAATCCAAGATAGCAATGGAGAGAATAAAATCAAGATGCTA

[0108] Comparative Example 1: Construction of base editing system A3A(Y130F)-BE4

[0109] The vector skeleton A3A-nCas9-UGI-UGI was constructed by referring to the method described in Example 1, namely the base editing system A3A(Y130F)-BE4.

[0110] Experimental Example 1: sgRNA vector construction and recombinant vector transformation

[0111] The target sgRNAs were designed with cotton endogenous gene GhALS and tobacco endogenous gene NtPDS as target genes (as shown in Table 1), and a tRNA cleavable element suitable for plant target sequence (as shown in SEQ ID NO.11) was connected to the 5'-end of each target sgRNA, and tRNA-sgRNA01, tRNA-sgRNA02 and tRNA-sgRNA03 were obtained respectively; tRNA-sgRNA01, tRNA-sgRNA02 and tRNA-sgRNA03 were connected to the BsaⅠ restriction site of the nucleic acid molecule of the base editing system GhDA3A-BE4 using Infusion enzyme, and transformed into Escherichia coli DH5α competent cells, and recombinant plasmids GhDA3A-BE4-sgRNA01, GhDA3A-BE4-sgRNA02 and GhDA3A-BE4-sgRNA03 were obtained respectively, as shown in Table 1. Figure 1 As shown; at the same time, tRNA-sgRNA01, tRNA-sgRNA02 and tRNA-sgRNA03 were connected to the BsaⅠ restriction site of the nucleic acid molecule of the base editing system A3A(Y130F)-BE4 using Infusion enzyme, and transformed into Escherichia coli DH5α competent cells, and the recombinant plasmids A3A(Y130F)-BE4-sgRNA01, A3A(Y130F)-BE4-sgRNA02 and A3A(Y130F)-BE4-sgRNA03 were obtained accordingly, as shown Figure 2 As shown. Each recombinant plasmid was transformed into Agrobacterium GV3101 competent cells, and GhDA3A-BE4-sgRNA01 bacterial solution, GhDA3A-BE4-sgRNA02 bacterial solution, GhDA3A-BE4-sgRNA03 bacterial solution, A3A(Y130F)-BE4-sgRNA01 bacterial solution, A3A(Y130F)-BE4-sgRNA02 bacterial solution, and A3A(Y130F)-BE4-sgRNA03 bacterial solution were obtained.

[0112] SEQ ID NO.11:

[0113] AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGG TACAGACCCGGGTTCGATTCCCGGCTGGTGCA

[0114] Table 1 Target sgRNA sequences of cotton and tobacco

[0115] Species Target sgRNA Sequence (5'~3') SEQ ID NO. cotton sgRNA01 CAAGTCCCTCGTCGGATGAT 12 cotton sgRNA02 GCGCCTCCACTGGGGATCAT 13 tobacco sgRNA03 CAATCTTCTGGTCATGGCAC 14

[0116] Experimental Example 2: Genetic transformation of cotton and determination of base editing results

[0117] Under sterile conditions, the hypocotyls of upland cotton Jin668 seedlings were cut into 1 cm long segments, placed in GhDA3A-BE4-sgRNA01 bacterial solution, A3A(Y130F)-BE4-sgRNA01 bacterial solution, GhDA3A-BE4-sgRNA02 bacterial solution and A3A(Y130F)-BE4-sgRNA02 bacterial solution, stirred, left to stand for 5 minutes and then dried, and scattered inoculated in the co-culture medium with filter paper to ensure that each hypocotyl was in contact with the filter paper, and cultured in the dark at 19°C for 36 hours. Each hypocotyl segment after dark culture was inoculated on the callus induction medium, and the hypocotyls with swollen ends and normal growth were selected every 30 days and subcultured and inoculated into the new callus induction medium. After subculture 4 times, the cotton embryonic callus tissue of each bacterial solution corresponding to the genetic transformation was obtained.

[0118] Referring to 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 of each cotton embryonic callus was extracted, and primer sequences for detecting sgRNA01 and sgRNA02 were designed based on the nucleotide sequence of the cotton endogenous gene GhALS, and commissioned to Sangon Biotech (Shanghai) Co., Ltd. for synthesis, as shown in Table 2.

[0119] The genomic DNA of each cotton embryonic callus was used as a template to perform PCR amplification of sgRNA01 and sgRNA02. The PCR amplification system was 20 μL, including 2 μL of genomic DNA of each cotton embryonic callus, 0.2 μL of each upstream primer (F), 0.2 μL of each downstream primer (R), 2 μL of 10×EasyTaq Buffer, 0.2 μL of EasyTaq, 0.4 μL of dNTP, and the remainder of ddH2O. The PCR amplification program was set as follows: 95°C pre-denaturation for 5 min, followed by 95°C denaturation for 30 s → 56°C annealing for 30 s → 72°C extension for 25 s, for a total of 30 cycles, to obtain each amplification product, and entrusted Sangon Biotech (Shanghai) Co., Ltd. to perform first-generation sequencing. The sequencing results were analyzed using the EditR analysis website (https: / / moriaritylab.shinyapps.io / editr_v10 / ). The results are shown in Figure 2. Figures 3-4 shown.

[0120] Table 2 Primer sequences for detecting cotton base editing efficiency

[0121] name Sequence (5'~3') SEQ ID NO. sgRNA01-F GCTCCTCTGGCATTTCCG 15 sgRNA01-R CATCCACATCAAGAACAAGATAAT 16 sgRNA02-F AATGTTGGACACTCCTGGAC 17 sgRNA02-R CCACCCTCATTCATTACATACA 18

[0122] The detection results of sgRNA01 showed that the editing window of the base editing system A3A(Y130F)-BE4 was mainly C6~C10, and the editing efficiency at the C13 site proximal to the PAM was extremely low, only 8%; the editing efficiency of the base editing system GhDA3A-BE4 was improved at the C6~C13 sites, and the editing efficiency at the proximal end of PAM was most significantly improved, with a maximum increase of 11.5 times, so that the editing efficiency of the C6~C13 sites reached 100%. Compared with the base editing system A3A(Y130F)-BE4, the overall editing efficiency was significantly improved; the detection results of sgRNA02 showed that the base editing system A3A(Y130F)-BE4 had a higher editing efficiency at the C7 and C8 sites, while the base editing system GhDA3A-BE4 was able to expand the editing window, producing editing efficiencies of 18% and 45% at the C4 and C5 sites, respectively. This shows that the base editing system GhDA3A-BE4 of the present invention can improve the editing efficiency near the PAM end and expand the editing window in cotton.

[0123] Experimental Example 3: Genetic transformation of tobacco and determination of base editing results

[0124] Under sterile conditions, the healthy and tender green leaves of K326 tobacco were cut into leaf discs, placed in GhDA3A-BE4-sgRNA03 bacterial solution and A3A(Y130F)-BE4-sgRNA03 bacterial solution, stirred, left to stand for 15 minutes, dried, and dispersed inoculated in the co-cultivation medium with filter paper to ensure that each leaf disc was in contact with the filter paper, and cultured in the dark at 19°C for 36 hours. Each leaf disc after dark culture was inoculated on the callus induction medium, and subcultured to the new callus induction medium every 20 days until regenerated tobacco seedlings were obtained; regenerated tobacco seedlings with 2 leaflets, healthy growth and normal growth points were selected, and after removing the excess callus tissue at the root, they were transferred to the rooting medium for rooting culture, and the corresponding tobacco seedlings of each bacterial solution were obtained.

[0125] Referring to 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 young leaves of each tobacco seedling. Based on the nucleotide sequence of the tobacco endogenous gene NtPDS, primer sequences for detecting sgRNA03 were designed. At the same time, a 6 bp Barcode tag sequence was added to the 5'-end of each primer sequence, and it was commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd., as shown in Table 3.

[0126] Table 3 Primer sequences for detecting tobacco base editing efficiency

[0127] name Sequence (5'~3') SEQ ID NO. sgRNA03-F AAGAACAACGAAATGCTTACG 19 sgRNA03-R GGCAGGTAGACAATTCTAGCA 20

[0128] The genomic DNA of each young tobacco leaf was used as a template to perform PCR amplification of sgRNA03, wherein the PCR amplification system was 20 μL, including 2 μL of genomic DNA of 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 dNTP and the remainder of ddH2O; the PCR amplification program was set as follows: 95°C pre-denaturation for 5 min, followed by denaturation at 95°C for 30 s→renalization at 56°C for 30 s→extension at 72°C for 25 s, for a total of 30 cycles to obtain each amplification product, and Beijing Novogene Technology Co., Ltd. was commissioned to perform second-generation high-throughput sequencing. Write a script to split the sequencing results according to the Barcode tag sequence and primer sequence, and then use CRISPResso2 to set the parameters "—quantification_window_size10—quantification_window_center-10—base_editor_output—conversion_nuc_fromA—conversion_nuc_to G" to analyze the split sequencing results. The results are as follows Figure 5 shown.

[0129] The results showed that the editing efficiency of C-to-T / C-to-A of the base editing system A3A(Y130F)-BE4 at the C1 site was only 4.66%, while the editing efficiency of C-to-T of the base editing system GhDA3A-BE4 at the C1 site could reach 39.66%, indicating that the base editing system GhDA3A-BE4 of the present invention can greatly expand the base editing window and show high editing efficiency.

[0130] From the above embodiments, it can be seen that the present invention provides a base editing system GhDA3A-BE4 and a base editing method and application thereof. The base editing system GhDA3A-BE4 of the present invention can significantly improve the C-to-T editing efficiency at the target site of the plant genome sequence, significantly expand the C-to-T editing window of the target site of the plant genome sequence, and can even perform efficient editing at the C1 site that the base editing system A3A (Y130F) -BE4 cannot edit.

[0131] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A base editing system GhDA3A-BE4, characterized in that: The amino acid sequence of the base editing system GhDA3A-BE4 is shown in SEQ ID NO.1; The base editing system GhDA3A-BE4 includes human deaminase APOBEC3A (A3A), endotoxin protein dDddA, nCas9 protein, and uracil glycosylase inhibitor UGI; The amino acid sequence of the human deaminase APOBEC3A (A3A) 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 glycosylase inhibitor UGI is shown in SEQ ID NO.

5.

2. A nucleic acid molecule encoding the base editing system GhDA3A-BE4 according to claim 1, characterized in that The sequence of the nucleic acid molecule is shown as SEQ ID NO.

6.

3. The method for preparing the base editing system GhDA3A-BE4 according to claim 1, characterized in that: Human deaminase APOBEC3A (A3A), nCas9 protein, and uracil glycosylase inhibitor UGI were connected in sequence to form the vector skeleton A3A-nCas9-UGI-UGI, and the endotoxin protein dDddA was inserted into the vector skeleton A3A-nCas9-UGI-UGI to obtain the base editing system GhDA3A-BE4.

4. Use of the base editing system GhDA3A-BE4 according to claim 1 in any one of the following (1) to (4): (1) Improve the editing efficiency of plant genome sequences; (2) preparing products that improve the editing efficiency of plant genome sequences; (3) Expand the editing window of plant genome sequences; (4) Prepare products that expand the editing window of plant genome sequences.

5. The use according to claim 4, characterized in that: The editing of the plant genome sequence comprises converting base C in the plant genome sequence into base T.

6. A recombinant plasmid, characterized in that: Including the base editing system GhDA3A-BE4 described in claim 1.

7. Use of the recombinant plasmid according to claim 6 in the following (1) or (2): (1) Improve the editing efficiency of plant genome sequences; (2) Expand the editing window of plant genome sequences.

8. A recombinant bacterium, characterized in that: Including the base editing system GhDA3A-BE4 described in claim 1.

9. Use of the recombinant bacterium according to claim 8 in the following (1) or (2): (1) Improve the editing efficiency of plant genome sequences; (2) Expand the editing window of plant genome sequences.

10. A method for improving the editing efficiency of a plant genome sequence and / or expanding the editing window of a plant genome sequence, characterized in that: The base editing system GhDA3A-BE4, recombinant plasmid or recombinant bacteria are transferred into plant cells for expression; The base editing system GhDA3A-BE4 is the base editing system GhDA3A-BE4 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 include cotton and tobacco; The editing of the plant genome sequence comprises converting base C in the plant genome sequence into base T.

Images