Base editor DGhBE4max, base editing method and application thereof

Abstract

The invention relates to a base editor DGhBE4max, a base editing method and application of the base editor DGhBE4max, and belongs to the technical field of gene editing. The invention provides a base editor DGhBE4max. The amino acid sequence of the base editor DGhBE4max is as shown in SEQ ID NO. 1; the base editor DGhBE4max comprises a deaminase rAPOBEC1, an endotoxin protein dDddA, an nCas9 protein, and a uracil glycosylase inhibitor UGI (Uracil Glucosylase Inhibitor). According to the base editor DGhBE4max, the editing efficiency of an existing cytosine base editor in plants can be remarkably improved, meanwhile, a base editing window can be remarkably expanded, and therefore a stable and efficient technical tool is provided for research of plant functional genes and directed evolution of key genes.

Description

Technical Field

[0001] The present invention relates to the field of gene editing technology, and in particular to a base editor DGhBE4max and a base editing method and applications thereof. Background Art

[0002] Genome editing technology is widely used in many research fields such as biomedicine, animals, plants, and microorganisms. Base editors are mainly composed of nCas9 protein fusion deaminase and uracil glycosylase inhibitor, which can replace bases without breaking double-stranded DNA. Commonly used base editors include cytosine base editors (CBE), adenine base editors (ABE), glycosylase base editors (GBE), and deaminase-independent base editors (DAF-CBE), among which ABE and CBE can achieve A-to-G and C-to-T conversion, while CGBE or GBE developed on the basis of CBE can achieve C-to-G and C-to-A transversion.

[0003] Improving the base editing efficiency and changing the base editing range of base editors are the main directions of base editing research. At present, different types of deaminases are explored and directed evolution of existing deaminases is performed to improve their editing activity, or the deaminase structure of base editors is changed to provide different editing windows. However, the existing modification methods are all carried out on the composition structure of base editors, and there is still a lack of research on fusing foreign modified proteins to improve base editing efficiency.

[0004] Existing technologies have only developed three sets of base editors for plants such as cotton, namely GhBE3, GhABE7.10n and GhABE8e. Except for GhABE8e, which exhibits efficient A-to-G editing ability, the efficiency of the other two cytosine base editors is still relatively low, and the editing efficiency also needs to be further optimized and improved. Summary of the invention

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

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

[0008] The base editor DGhBE4max includes deaminase rAPOBEC1, endotoxin protein dDddA, nCas9 protein, and uracil glycosylase 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 glycosylase inhibitor UGI is shown in SEQ ID NO.5.

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

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

[0015] The present invention also provides the use 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 the base C in the plant genome sequence into the base T.

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

[0021] 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 editor DGhBE4max, a recombinant plasmid or a recombinant bacterium is transferred into a plant cell for expression;

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

[0023] The recombinant plasmid is the recombinant plasmid;

[0024] The recombinant bacteria are the recombinant bacteria described above;

[0025] The plants include cotton and tobacco;

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

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

[0028] The base editor DGhBE4max of the present invention fuses the bacterial endotoxin protein dDddA. Compared with the existing cytosine base editors (such as the base editor BE4max), it significantly improves the C-to-T editing efficiency at the target site of the plant genome sequence. At the same time, it can deaminize and effectively edit the PAM proximal sites that cannot be edited by the existing cytosine base editors, and expand the editing window of the existing cytosine base editors from C4 to C8 sites to C1 to C13 sites, significantly expanding the base editing window, thereby 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

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

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

[0031] Figure 3 is the editing efficiency of base editors DGhBE4max and BE4max in cotton, wherein 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 4The editing efficiency of base editors DGhBE4max and BE4max in tobacco;

[0033] Figure 5 These are the editing windows of base editors DGhBE4max and BE4max in tobacco, where A is the editing window of base editor BE4max in tobacco, and B is the editing window of base editor DGhBE4max in tobacco. DETAILED DESCRIPTION

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

[0035] The base editor DGhBE4max includes deaminase rAPOBEC1, endotoxin protein dDddA, nCas9 protein, and uracil glycosylase 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 the 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 glycosylase 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 the nucleic acid molecule 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] The present invention also provides a method for preparing the base editor DGhBE4max, wherein the deaminase rAPOBEC1, nCas9 protein, and uracil glycosylase inhibitor UGI are sequentially connected to form a rAPOBEC1-nCas9-UGI-UGI skeleton, and the endotoxin protein dDddA is inserted into the rAPOBEC1-nCas9-UGI-UGI skeleton to obtain the base editor DGhBE4max.

[0046] The present invention also provides the use 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 the base C in the plant genome sequence into the base T.

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

[0052] 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 editor DGhBE4max, a recombinant plasmid or a recombinant bacterium is transferred into a plant cell for expression;

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

[0054] The recombinant plasmid is the recombinant plasmid;

[0055] The recombinant bacteria are the recombinant bacteria described above;

[0056] The plants include cotton and tobacco;

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

[0058] 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.

[0059] 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.;

[0060] 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);

[0061] 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.

[0062] Example 1: Construction of base editor DGhBE4max

[0063] The nucleotide sequences encoding rAPOBEC1, nCas9 (D10A), and uracil glycosylase inhibitor UGI were sequentially connected 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 Co., Ltd. was commissioned to synthesize and optimize the dDddA (E1347A) nucleic acid molecule, which was inserted after the nucleotide sequence encoding rAPOBEC1 in the rAPOBEC1-nCas9-UGI-UGI backbone. At the same time, GGATCT was used as a connecting sequence (with a connecting peptide of 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 base editor BE4max

[0094] The rAPOBEC1-nCas9-UGI-UGI skeleton was constructed by referring to the method described in Example 1, namely the base editor BE4max.

[0095] Experimental Example 1: sgRNA vector construction and recombinant vector transformation

[0096] The target sgRNAs were designed with the cotton endogenous gene GhALS and the tobacco endogenous gene NtPDS as target genes (as shown in Table 1), and a tRNA element suitable for the plant target sequence (as shown in SEQ ID NO.11) was connected to the 5'-end of each target sgRNA to obtain tRNA-sgRNA02 and tRNA-sgRNA03, respectively; tRNA-sgRNA02 and tRNA-sgRNA03 were connected to the BsaⅠ restriction site of the nucleic acid molecule of the base editor DGhBE4max using Infusion enzyme, and transformed into Escherichia coli DH5α competent cells, and recombinant plasmids DGhBE4max-sgRNA02 and DGhBE4max-sgRNA03 were obtained, as shown in Table 1. Figure 1 As shown; at the same time, tRNA-sgRNA02 and tRNA-sgRNA03 were connected to the BsaⅠ restriction site of the nucleic acid molecule of the base editor BE4max using Infusion enzyme, and transformed into Escherichia coli DH5α competent cells, and the recombinant plasmids BE4max-sgRNA02 and BE4max-sgRNA03 were obtained accordingly, as shown Figure 2 Each recombinant plasmid was transformed into Agrobacterium GV3101 competent cells to obtain DGhBE4max-sgRNA02 bacterial solution, DGhBE4max-sgRNA03 bacterial solution, BE4max-sgRNA02 bacterial solution, and BE4max-sgRNA03 bacterial solution.

[0097] SEQ ID NO.11:

[0098] AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGG TACAGACCCGGGTTCGATTCCCGGCTGGTGCA

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

[0100] Species Target sgRNA Sequence (5'~3') SEQ ID NO. cotton sgRNA02 GCGCCTCCACTGGGGATCAT 12 tobacco sgRNA03 CAATCTTCTGGTCATGGCAC 13

[0101] Experimental Example 2: Genetic transformation of cotton and determination of base editing results

[0102] Under sterile conditions, the hypocotyls of upland cotton Jin668 seedlings were cut into 1 cm long segments, placed in DGhBE4max-sgRNA02 bacterial solution and BE4max-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 segment 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.

[0103] 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 the primer sequence for detecting sgRNA02 was 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.

[0104] The genomic DNA of each cotton embryonic callus was used as a template to perform PCR amplification of sgRNA02, wherein the PCR amplification system was 20 μL, including 2 μL of genomic DNA of each cotton embryonic callus, 0.2 μL of sgRNA02-F primer, 0.2 μL of sgRNA02-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 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, and the sequencing results were analyzed using the EditR analysis website (https: / / moriaritylab.shinyapps.io / editr_v10 / ). The results are as follows: Figure 3 shown.

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

[0106] name Sequence (5'~3') SEQ ID NO. sgRNA02-F AATGTTGGACACTCCTGGAC 14 sgRNA02-R CCACCCTCATTCATTACATACA 15

[0107] The results showed that the base editor BE4max mainly performed base editing at the C7 and C8 sites, with editing efficiencies of 39% and 23%, respectively, while the base editor DGhBE4max had editing efficiencies of 88% and 65% at the C7 and C8 sites, respectively, which means that compared with the base editor BE4max, the base editor DGhBE4max increased the editing efficiency of the C7 and C8 sites by 1.26 times and 1.74 times, respectively. At the same time, the base editor DGhBE4max expanded the window of base editing in cotton, producing 30% and 52% editing efficiencies at the C5 and C10 sites that the base editor BE4max could not edit, respectively.

[0108] Experimental Example 3: Genetic transformation of tobacco and determination of base editing results

[0109] Under sterile conditions, the healthy and tender green leaves of K326 tobacco were cut into leaf discs, placed in DGhBE4max-sgRNA03 bacterial solution and BE4max-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.

[0110] 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.

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

[0112] name Sequence (5'~3') SEQ ID NO. sgRNA03-F AAGAACAACGAAATGCTTACG 16 sgRNA03-R GGCAGGTAGACAATTCTAGCA 17

[0113] 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 Figures 4-5 shown.

[0114] The results showed that compared with the base editor BE4max, the editing efficiency of the base editor DGhBE4max at the C5 and C8 sites increased by 70% and 97% respectively; the overall editing efficiency of the base editor DGhBE4max was as high as 84.78%, which was 2.4 times higher than the highest editing efficiency of the base editor BE4max (25.17%). At the same time, the base editor DGhBE4max expanded the window of base editing in tobacco, producing 6.91% and 6.91% editing efficiencies at the C1 and C13 sites that the base editor BE4max could not edit, respectively.

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

[0116] 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 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 deaminase rAPOBEC1, endotoxin protein dDddA, nCas9 protein, and uracil glycosylase 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 glycosylase inhibitor UGI is shown in SEQ ID NO.

5.

2. A nucleic acid molecule encoding the base editor DGhBE4max 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 editor DGhBE4max according to claim 1, characterized in that: The deaminase rAPOBEC1, nCas9 protein, and uracil glycosylase inhibitor UGI were connected in sequence to form the rAPOBEC1-nCas9-UGI-UGI skeleton, and the endotoxin protein dDddA was inserted into the rAPOBEC1-nCas9-UGI-UGI skeleton to obtain the base editor DGhBE4max.

4. The use of the base editor DGhBE4max according to 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 base C in the plant genome sequence is converted to the base T.

5. Use of the base editor DGhBE4max 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: Including the base editor DGhBE4max described in claim 1.

7. Use 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: Including the base editor DGhBE4max described in claim 1.

9. Use 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 editing efficiency of a plant genome sequence and / or expanding the editing window of a plant genome sequence, characterized in that: The base editor DGhBE4max, recombinant plasmid or recombinant bacteria are transferred into plant cells for expression; The base editor DGhBE4max is the base editor DGhBE4max 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.

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