Adenosine deaminase and related biomaterials and uses thereof

Abstract

The present invention provides adenosine deaminase, related biomaterials, and applications thereof. The name of the protein, whose amino acid sequence is positions 1 to 167 of SEQ ID NO: 2 in the Sequence Listing, is TadA9. Adenine base editing technology mediated by fusion of TadA9 with SpCas9(D10A), SpCas9-NG(D10A), ScCas9(D10A), and SpRY(D10A) exhibits significantly higher editing efficiency than adenine base editing technology mediated by TadA7.10 and the TadA-R mutant, thereby expanding the range of editing activity, improving the efficiency of adenine base editing technology, and broadening its application range.

Description

[Technical field]

[0001] The present invention relates to adenosine deaminase, related biomaterials and uses thereof. [Background technology]

[0002] Genome editing technology is a genetic engineering technique that uses artificial nucleases to modify specific sites in the genome of an organism. This process involves cleaving the target site with a specific nuclease to generate a DNA double-strand break, and then introducing base deletion, insertion, or mismatch phenomena by non-homologous end-joining (NHEJ) repair or homology-directed repair (HDR) to achieve the purpose of mutating the organism. In recent years, artificial nuclease technologies have been successfully developed, mainly ZFN, TALEN, and CRISPR. In particular, CRISPR / Cas technology has attracted the attention of researchers due to its ease of use, high efficiency, and high versatility.

[0003] Single-nucleotide polymorphisms (SNPs) are the genetic basis of crop agronomic traits, and many important agronomic traits in plants are determined by single-nucleotide loci. Currently, SNPs are being discovered one after another and are being used to screen phenotypic traits in plants. With the development of CRISPR / Cas technology, single-base editing techniques (including cytosine base editing systems and adenine base editing systems) that cause single-base changes in the genome based on CRISPR / Cas technology are being used to induce targeted base substitutions in plants. Since their development, base editing technologies have been successfully applied to a wide range of organisms, including animals and plants, because they are efficient, do not depend on double-stranded DNA breaks, and do not require the presence of donor DNA. Cytidine deaminases that have been successfully applied to plant base editing systems include APOBEC1, APOBEC3, and AID, and adenosine deaminases include TadA7.10.

[0004] Adenosine deaminase-based adenine base editing technology mainly uses a fusion protein combining the excision enzyme Cas9n (D10A) and adenosine deaminase to deaminate the target base A within the base editing activity window to hypoxanthine I under the guidance of sgRNA, which gradually replaces it with G after DNA repair and replication, and finally forms a directional substitution from A to G (A>G). However, current plant adenine base editing technology mainly uses adenosine deaminases TadA7.10 (Yan, et al. Molecular Plant, 2018, 11:631-634) and TadA8e (Li, et al. Molecular Plant, 2021, 14: 352-360), but they generally have low editing efficiency and poor compatibility with various Cas proteins, and moreover, target sites present within the base editing activity window still cannot achieve effective base editing events. Summary of the Invention

[0005] The technical problem that the present invention aims to solve is how to improve the efficiency of adenine base editing in plants and / or how to realize expected adenine base editing that is not achievable with current technology.

[0006] In order to solve the above technical problems, the present invention provides an adenosine deaminase.

[0007] The adenosine deaminase provided by the present invention is named TadA9, and is a non-naturally occurring protein that is a highly active adenosine deaminase obtained by artificially mutating TadA7.10, which is a mutant of Escherichia coli tRNA adenosine deaminase TadA. The adenosine deaminase is a protein having an amino acid sequence of positions 1 to 167 of SEQ ID No. 2 in the sequence listing (i.e., a protein encoded by nucleotides (CDS) of positions 7 to 507 of SEQ ID No. 1 in the sequence listing). In order to solve the above technical problems, the present invention also provides a biological material related to the TadA9.

[0008] The TadA9-related biomaterial provided by the present invention is at least one of the following B1) to B7): B1) a nucleic acid molecule encoding said protein; B2) an expression cassette comprising the nucleic acid molecule of B1); B3) A recombinant vector comprising a nucleic acid molecule according to B1) or a recombinant vector comprising an expression cassette according to B2); B4) A recombinant microorganism comprising a nucleic acid molecule according to B1), a recombinant microorganism comprising an expression cassette according to B2), or a recombinant vector according to B3); B5) a transgenic plant cell line comprising a nucleic acid molecule according to B1), a transgenic plant cell line comprising an expression cassette according to B2), or a transgenic plant cell line comprising a recombinant vector according to B3); B6) A transgenic plant tissue comprising a nucleic acid molecule according to B1), a transgenic plant tissue comprising an expression cassette according to B2), or a transgenic plant tissue comprising a recombinant vector according to B3); B7) A transgenic plant comprising a nucleic acid molecule according to B1), a transgenic plant comprising an expression cassette according to B2), or a transgenic plant comprising a recombinant vector according to B3).

[0009] In the above biological material, the nucleic acid molecule described in B1) may be a gene described in B11) or B12) below. B11) The coding sequence (CDS) of the coding strand is a cDNA molecule or a DNA molecule shown in positions 7 to 507 of SEQ ID NO:1; B12) A cDNA or DNA molecule having 80% or more identity with the nucleotide sequence defined in B11) and encoding said protein.

[0010] The present invention also provides adenine base editors.

[0011] In the adenine base editor provided by the present invention, the fusion protein is a fusion protein comprising a Cas protein and an adenosine deaminase, and the adenosine deaminase is TadA9.

[0012] In the adenine base editor, the Cas protein may be SpCas9(D10A), SpCas9-NG(D10A), ScCas9(D10A) or SpRY(D10A).

[0013] In the above adenine base editor, the SpCas9(D10A) is a protein having an amino acid sequence of positions 200 to 1567 of SEQ ID NO: 2 (i.e., a protein encoded by nucleotides (CDS) of positions 604 to 4707 of SEQ ID NO: 1 in the Sequence Listing), and the SpCas9-NG(D10A) is a protein having an amino acid sequence of positions 200 to 1567 of SEQ ID NO: 4 (i.e., a protein encoded by nucleotides (CDS) of positions 604 to 4707 of SEQ ID NO: 3 in the Sequence Listing). ScCas9(D10A) is a protein whose amino acid sequence is 200-1574 of SEQ ID NO:6 (a protein encoded by nucleotides (CDS) of positions 604-4728 of SEQ ID NO:5 in the Sequence Listing), and SpRY(D10A) is a protein whose amino acid sequence is 200-1567 of SEQ ID NO:8 (a protein encoded by nucleotides (CDS) of positions 604-4728 of SEQ ID NO:5 in the Sequence Listing). SpRY(D10A) is a protein whose amino acid sequence is 200-1567 of SEQ ID NO:8 (i.e., a protein encoded by nucleotides 604-4707 (CDS) of SEQ ID NO:7 in the Sequence Listing).

[0014] In the above-mentioned adenine base editor, the adenine base editor may be a protein formed by linking adenosine deaminase, a Cas protein, and a nuclear localization signal (NLS) conjugate.

[0015] In the above-mentioned adenine base editor, the adenine base editor may be specifically TadA9-SpCas9(D10A), TadA9-SpCas9-NG(D10A), TadA9-ScCas9(D10A) or TadA9-SpRY(D10A). The TadA9-SpCas9(D10A) is a protein whose amino acid sequence is SEQ ID NO: 2 (i.e., a protein encoded by nucleotides (CDS) at positions 7 to 4737 of SEQ ID NO: 1 in the sequence listing), and the TadA9-SpCas9-NG(D10A) is a protein whose amino acid sequence is SEQ ID NO: The protein having the amino acid sequence number 4 (i.e., the protein encoded by the nucleotides (CDS) of positions 7 to 4737 of SEQ ID NO: 3 in the Sequence Listing), the TadA9-SCas9(D10A) is a protein having an amino acid sequence of SEQ ID NO: 6 (i.e., the protein encoded by the nucleotides (CDS) of positions 7 to 4758 of SEQ ID NO: 5 in the Sequence Listing), and the TadA9-SpRY(D10A) is a protein having an amino acid sequence of SEQ ID NO: 8 (i.e., the protein encoded by the nucleotides (CDS) of positions 7 to 4737 of SEQ ID NO: 7 in the Sequence Listing).

[0016] Biomaterials related to said adenine base editors also fall within the scope of protection of the present invention.

[0017] The adenine base editor-related biomaterial may in particular be at least one of the following D1) to D7): D1) a nucleic acid molecule encoding the adenine base editor; D2) an expression cassette comprising the nucleic acid molecule of D1); D3) a recombinant vector comprising the nucleic acid molecule according to D1), or the expression cassette according to D2); D4) A recombinant microorganism comprising a nucleic acid molecule according to D1), a recombinant microorganism comprising an expression cassette according to D2), or a recombinant vector according to D3); D5) a transgenic plant cell line comprising a nucleic acid molecule according to D1), a transgenic plant cell line comprising an expression cassette as defined in D2), or a transgenic plant cell line comprising a recombinant vector as defined in D3); D6) A transgenic plant tissue comprising a nucleic acid molecule according to D1), a transgenic plant tissue comprising an expression cassette according to D2), or a transgenic plant tissue comprising a recombinant vector according to D3); D7) A transgenic plant organ comprising a nucleic acid molecule according to D1), a transgenic plant organ comprising an expression cassette according to D2), or a transgenic plant organ comprising a recombinant vector according to D3).

[0018] (a) Among the above biological materials related to adenine base editors, The nucleic acid molecule of D1) may be any of a gene encoding TadA9-SpCas9(D10A), a gene encoding TadA9-SpCas9-NG(D10A), a gene encoding TadA9-SaCAs9(D10A) or a gene encoding TadA9-SpRY(D10A).

[0019] The gene encoding TadA9-SpCas9(D10A) may be any of the following genes: (D131) a cDNA molecule or a DNA molecule whose coding sequence (CDS) of the coding strand is shown in positions 7 to 4737 of SEQ ID NO:1; D132) A cDNA or DNA molecule having 80% or more identity to the base sequence defined in D131) and encoding the TadA9-SpCas9(D10A).

[0020] The gene encoding TadA9-SpCas9-NG(D10A) may be any of the following genes: D141) a cDNA or DNA molecule whose coding sequence (CDS) is set forth in SEQ ID NO:3, 7 to 4737; D142) A cDNA or DNA molecule having 80% or more identity to the nucleotide sequence defined in D141) and encoding the TadA9-SpCas9(D10A). The gene encoding TadA9-ScCas9(D10A) may be any of the following genes: (D111) a cDNA molecule or a DNA molecule whose coding strand coding sequence (CDS) is shown in positions 7 to 4758 of SEQ ID NO:5; D112) A cDNA or DNA molecule encoding the TadA9-ScCas9, having 80% or more identity to the nucleotide sequence defined in D111) (D10A). The gene encoding TadA9-SpRY(D10A) may be any of the genes shown below: D121) a cDNA or DNA molecule whose coding sequence (CDS) of the coding strand is set forth in positions 7 to 4737 of SEQ ID NO:7; D122) A cDNA or DNA molecule having 80% or more identity to the nucleotide sequence defined in D121) and encoding the TadA9-SpRY(D10A).

[0021] In the above, the expression cassette means a DNA capable of expressing the protein in a host cell (e.g., a plant cell, etc.), and the DNA may contain not only a promoter that initiates the transcription of the protein gene, but also a terminator that terminates the transcription of the protein gene. Furthermore, the expression cassette may contain an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue-, organ- and development-specific promoters, and inducible promoters. Examples of promoters include the ubiquitin promoter from maize, the constitutive promoter 35S from the cauliflower mosaic virus, the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiology 120:979-992); the chemically inducible promoter from tobacco, disease history-dependent protein 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxamic acid S-methyl)); the tomato protease inhibitor II promoter (PIN2) or the LAP promoter (both inducible by methyl jasmonate); heat shock promoters (U.S. Patent 5,187,267); tetracycline-inducible promoters (U.S. Patent 5,057,422); the cereal seed-specific promoter pF128 (CN101063139B (China Patent 2007 1 0099169.7)) and seed storage protein specific promoters (e.g., promoters of phaseolin, napin, oleosin and soybean beta-conglycin (Beachy et al. 1985) emboJ. 4:3047-3053)). These may be used alone or in combination with other plant promoters. All references cited herein are hereby incorporated by reference in their entirety.Suitable transcription terminators include the Klebsiella pneumoniae nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline-octopamine synthase terminator (e.g., see Odell et al. (2013)). 985 ) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res. 15:9627), but are not limited to these.

[0022] In one embodiment of the present invention, the expression cassette is composed of a Ubip promoter (nucleotide sequence of SEQ ID NO: 9), a nucleic acid molecule encoding the adenine base editor, and a NOS terminator (nucleotide sequence of SEQ ID NO: 10) linked together.

[0023] Here, the term "identity" refers to sequence similarity between nucleic acid sequences. Identity can be assessed by eye or by computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%) that can be used to assess identity between the sequences. The 80% or higher identity can be 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity.

[0024] In the above, the recombinant microorganism may specifically be a bacterium, a yeast, an alga, or a fungus.

[0025] In addition, any of the following uses are within the scope of protection of the present invention: 1) Use of TadA9 for single-base editing in plants. 2) Use of TadA9-related biological materials for single-base editing in plants. 3) Use of the above adenine base editor in single base editing in plants. 4) Use of biological materials related to the above adenine base editor in single base editing in plants.

[0026] In order to solve the above technical problems, the present invention provides a method for targeted A to G mutation in a plant genome.

[0027] The present invention provides a method for targeted A to G mutation in a plant genome, comprising the steps of: introducing the adenine base editor and a DNA molecule expressing an sgRNA into a recipient plant to obtain a target plant having an A to G targeted mutation; 19-20 PAM-3', 19-20 is 19-20 Ns, the PAM (protospacer adjacent motif) is 3 Ns, and the Ns are A, G, C, or T.

[0028] The DNA molecule expressing the adenine base editor and sgRNA can be introduced into a recipient plant by PEG-mediated transformation, or by using one of the following methods: gene gun or agrobacterial infiltration to introduce the base editing toolbox into rice protoplasts or callus tissue. This can be easily understood by those skilled in the art. As it is well known to those skilled in the art that rice genomic DNA is composed of two strands, the target nucleotide sequence can be located on any complementary strand. This system can be used to obtain a "correction" mutant for the function of a rice gene, for example, when the target nucleotide sequence is located on the sense strand of a functional gene, and one of the mutations can be used to obtain a corresponding functional protein containing a desired amino acid after targeted mutation of A to G at a specific locus of the functional gene, i.e., when the A in the triplet codon can be replaced by G by directly performing base substitution on the sense strand. Alternatively, this system can be used when the target nucleotide sequence is located in the antisense strand of a functional gene and a T to C mutation at a specific site in the functional gene is targeted, where one of the mutations results in a corresponding functional protein containing the desired amino acid, i.e., the amino acid encoded by the triplet codon on the sense strand can be changed by a targeted A to G mutation in the antisense strand, replacing the corresponding complementary T in the sense strand with C, thereby obtaining a "corrective" mutant for the function of the rice gene.

[0029] In the above, the plant may be a dicotyledonous plant or a monocotyledonous plant. The monocotyledonous plant may be rice. Single base editing may replace adenine A with guanine G. [Brief description of the drawings]

[0030] [Figure 1] This is the spectrum of pUbi-rBE. [Diagram 2] Spectrum of pENTR4:sgRNA. [Diagram 3]Spectrum of pUbi-rBE-sgRNA. [Figure 4] Figure 1 shows the effect of adenine base editing mutations in the rice endogenous gene Ostms9 mediated by rBE14, rBE46b, and rBE49b. In the figure, ref indicates the corresponding sequence of the rice reference genome, and the nucleotide sequence is sequence 14. WT indicates the corresponding sequence of the rice japonica cultivar Kitaake, which has not been subjected to base editing, and the nucleotide sequence is sequence 14. T>C indicates the corresponding sequence of the mutant strain, and the nucleotide sequence is sequence 15. [Diagram 5] Figure 1 shows the effect of adenine base editing mutations in the rice endogenous gene OsWRKY45 mediated by rBE14, rBE46b, and rBE49b. In the figure, ref indicates the corresponding sequence of the rice reference genome, and the nucleotide sequence is sequence 16. WT indicates the corresponding sequence of the non-base-edited rice japonica cultivar Kitaake, and the nucleotide sequence is sequence 16. A>G represents the corresponding sequence of the mutant strain, and the nucleotide sequence is sequence 17. [Figure 6] Figure 1 shows the effect of adenine base editing mutations in the endogenous rice gene OsDEP2 mediated by rBE23, rBE50, and rBE53. In the figure, ref indicates the corresponding sequence of the rice reference genome, and the nucleotide sequence is sequence 18. WT indicates the corresponding sequence of the rice japonica cultivar Kitaake, which has not been subjected to base editing, and the nucleotide sequence is sequence 18. 2A>2G represents the corresponding sequence of the mutant strain, and the nucleotide sequence is sequence 19. [Figure 7] Figure 1 shows the effect of adenine base editing mutations in the endogenous rice gene OsWRKY45 mediated by rBE23, rBE50, and rBE53. In the figure, ref indicates the corresponding sequence of the rice reference genome, and the nucleotide sequence is sequence 20. WT indicates the corresponding sequence of the rice japonica cultivar Kitaake, which has not been subjected to base editing, and the nucleotide sequence is sequence 20. T>C represents the corresponding sequence of the mutant strain, and the nucleotide sequence is sequence 21. [Figure 8]Figure 1 shows the effect of mutations on adenine base editing of the rice endogenous gene OsGS1 mediated by rBE26, rBE54, and rBE57. In the figure, ref indicates the corresponding sequence of the rice reference genome, and the nucleotide sequence is sequence 22. WT indicates the corresponding sequence of the rice japonica cultivar Kitaake, which has not been subjected to base editing, and the nucleotide sequence is sequence 22. 3A>3G represents the corresponding sequence of the mutant strain, and the nucleotide sequence is sequence 23. 4A>4G represents the corresponding sequence of the mutant strain, and the nucleotide sequence is sequence 24. [Figure 9] Mutation effect diagram of adenine base editing of rice endogenous gene OsSERK2 mediated by rBE54 and rBE57. In the figure, ref indicates the corresponding sequence of the rice reference genome, and the nucleotide sequence is sequence 25. WT indicates the corresponding sequence of the rice japonica cultivar Kitaake without base editing, and the nucleotide sequence is sequence 25. 2T>2C represents the corresponding sequence of the mutant line, and the nucleotide sequence is sequence 26. [Figure 10] Effect of rBE62- and rBE65-mediated adenine base editing mutations in the rice endogenous gene OsMPK13. In the figure, ref indicates the corresponding sequence of the rice reference genome, and the nucleotide sequence is sequence 27. WT indicates the corresponding sequence of the rice japonica cultivar Kitaake, which has not been subjected to base editing, and the nucleotide sequence is sequence 27. A>G indicates the corresponding sequence of the mutant strain, and the nucleotide sequence is sequence 28. 2A>2G indicates the corresponding sequence of the mutant strain, and the nucleotide sequence is sequence 29. [Figure 11] Mutation effect diagram of adenine base editing of rice endogenous gene OsGSK4 mediated by rBE62 and rBE65. In the figure, ref indicates the corresponding sequence of the rice reference genome, and the nucleotide sequence is sequence 30. WT indicates the corresponding sequence of the rice japonica cultivar Kitaake without base editing, and the nucleotide sequence is sequence 30. T>C represents the corresponding sequence of the mutant strain, and the nucleotide sequence is sequence 31. 2T>2C represents the corresponding sequence of the mutant strain, and the nucleotide sequence is sequence 32. 4T>4C represents the corresponding sequence of the mutant strain, and the nucleotide sequence is sequence 33. [Figure 12] Mutation effect diagram of adenine base editing of rice endogenous gene OsJAR 1 mediated by rBE62 and rBE65. In the figure, ref indicates the corresponding sequence of the rice reference genome, and the nucleotide sequence is sequence 34. WT indicates the corresponding sequence of the rice japonica cultivar Kitaake without base editing, and the nucleotide sequence is sequence 34. 2T>2C represents the corresponding sequence of the mutant strain, and the nucleotide sequence is sequence 35. 3T>3C represents the corresponding sequence of the mutant strain, and the nucleotide sequence is sequence 36. [Figure 13] The target nucleotide sequence information of each target gene. In the figure, the capital letters in the oligonucleotide chain required for double-stranded DNA fragment synthesis correspond to N19 of attB1-sgRNA expression cassette-attB2, the lowercase gtgt corresponds to the BsaI site, and the lowercase tgtt corresponds to the BtgZI site. [Figure 14] Detection primers for each target gene are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present invention will be described in more detail below based on specific examples. The examples provided are provided only to illustrate the present invention, and do not limit the scope of the present invention. The examples provided below do not constitute the limitation of the present invention, and can serve as a guide for further improvement by those skilled in the art.

[0032] The experimental methods in the following examples are commonly used methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the relevant technical field, or according to the product specifications. Materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0033] pUbi-Cas 9 in the following examples is stored and provided by the laboratory to which the inventors belong (H. Zhou, B. Liu, DP Weeks, MH Spalding & B. Yang. Large chromosoma deletions and heritable small genetic changes induced by CRISPR / Cas 9 in rice. Nucleic Acids Res. 2014, 42(17):10903-10914). The biomaterials are publicly available from the inventors' laboratory and will be used only to repeat experiments related to the present invention and will not be used for any other purpose.

[0034] Example 1. Targeted A to G mutation in the rice genome This example provides an artificial mutant of adenosine deaminase TadA7.10 (hereinafter abbreviated as TadA7.10), i.e., adenosine deaminase TadA9 (hereinafter abbreviated as TadA9). TadA7.10 is a protein encoded by base numbers 598 to 1095 of SEQ ID NO: 12. TadA9 has an amino acid sequence of positions 1 to 167 of SEQ ID NO: 2, and has the following mutations in the amino acid sequence compared to TadA7.10: V81S / A108S / T110R / D118N / H121N / Y146D / F148Y / Q153R / T165I / D166N.

[0035] In this embodiment, two control adenosine deaminases for adenine base editing, wtTadA-TadA7.10 (protein encoded by nucleotides 1 to 1095 of SEQ ID NO: 12) and TadA-R (protein encoded by nucleotides 1 to 501 of SEQ ID NO: 13), are provided as TadA9 controls. wtTadA-TadA 7.10 is an existing adenosine deaminase dimer that is commonly used in plants. Compared to TadA 9, TadA-R has two fewer mutations in the amino acid sequence, V 81 S and Q 153 R, and compared to TadA 7.10, the following mutations are present in the amino acid sequence: A 108 S / T 110 R / D 118 N / H 121 N / Y 146 D / F 148 Y / T 165 I / D 166 N。

[0036] This example provides four rice adenine base editor expression vectors pUbi-rBE (FIG. 1) containing TadA 9. The rice adenine base editor expression vectors are named pUbi-rBE 49 b, pUbi-rBE 53, pUbi-rBE 57, and pUbi-rBE 65. The adenine base editor expressed by pUbi-rBE 49 b is a fusion protein named rBE 49 b (also called TadA 9-SpCas 9 (D 10 A)), which is a protein formed by combining an adenosine deaminase named TadA 9, a Cas protein named SpCas 9 (D 10 A), and a nuclear localization signal named NLS. The amino acid sequence of rBE 49 b is SEQ ID NO:2. In sequence 2, positions 1 to 167 are the amino acid sequence of TadA 9, positions 168 to 199 are the amino acid sequence of the binding peptide, positions 200 to 1567 are the amino acid sequence of SpCas 9 (D 10 A), and positions 1568 to 1576 are the amino acid sequence of the NLS. Based on the preference for inecodon usage and the optimization strategy for heterologous protein expression codons, the nucleotide sequence of the chimeric gene rBE 49 b gene was codon-optimized, and then the artificial synthesis of the 4743 bp rBE 49 b gene was requested from Biotechnology (Shanghai) Co., Ltd. The small fragment (Cas 9) between the BamHI recognition site and the BcuI recognition site of pUbi-Cas 9 was replaced with the rBE 49 b gene shown in positions 7 to 4737 of sequence 1, and the other nucleotides of pUbi-Cas 9 were maintained, resulting in the rBE 49 b gene expression vector pUbi-rBE 49 b. In sequence 1, positions 1 to 6 were BamHI recognition sites, positions 7 to 507 were CDSs of TadA 9, positions 508 to 603 were CDSs for binding peptides, positions 604 to 4707 were CDSs of SpCas 9(D 10 A), positions 4708 to 4734 were CDSs of NLS, positions 4735 to 4737 were termination codons TGA, and positions 4738 to 4743 were recognition sites for BcuIno. pUbi-rBE 49 b contained elements attR 1-ccdB-attR 2 for the LR reaction.

[0037] The adenine base editor expressed by pUbi-rBE 53 is a fusion protein named rBE 53 (also called TadA 9-SpCas 9-NG (D 10 A)), which is a protein consisting of an adenosine deaminase named TadA 9, a Cas protein named SpCas 9-NG, and a nuclear localization signal named NLS. The amino acid sequence of rBE 50 is sequence 4 in the sequence listing. In sequence 4, positions 1 to 167 are the amino acid sequence of TadA 9, positions 168 to 199 are the amino acid sequence of the binding peptide, positions 200 to 1567 are the amino acid sequence of SpCas 9-NG (D 10 A), and positions 1568 to 1576 are the amino acid sequence of NLS. We codon-optimized the nucleotide sequence of the chimeric gene rBE 53 gene based on the rice codon usage preference, and then commissioned Biotechnology (Shanghai) Co., Ltd. to artificially synthesize the 4743 bp rBE 53 gene. The small fragment (Cas 9) between the BamHI recognition site and the BcuI recognition site of pUbi-Cas 9 was replaced with the rBE 53 gene shown at positions 7-4737 of sequence 3, while maintaining the other nucleotides of pUbi-Cas 9, to obtain the rBE 53 gene expression vector pUbi-rBE 53. In sequence 3, positions 1 to 6 were BamHI recognition sites, positions 7 to 507 were CDSs of TadA 9, positions 508 to 603 were CDSs for binding peptides, positions 604 to 4707 were CDSs of SpCas 9-NG(D 10 A), positions 4708 to 4734 were CDSs of NLS, positions 4735 to 4737 were termination codons TGA, and positions 4738 to 4743 were BcuI recognition sites. pUbi-rBE 50 contained elements attR 1-ccdB-attR 2 for the LR reaction.

[0038] The adenine base editor expressed by pUbi-rBE 57 is a fusion protein named rBE 57 (also called TadA 9-ScCas 9 (D 10 A)), which is a protein formed by combining an adenosine deaminase named TadA 9, a Cas protein named ScCas 9 (D 10 A), and a nuclear localization signal named NLS. The amino acid sequence of rBE 57 is sequence 6 in the sequence listing. In sequence 6, positions 1 to 167 are the amino acid sequence of TadA 9, positions 168 to 199 are the amino acid sequence of the binding peptide, positions 200 to 1574 are the amino acid sequence of ScCas 9 (D 10 A), and positions 1575 to 1583 are the amino acid sequence of NLS. We codon-optimized the nucleotide sequence of the chimeric gene rBE 57 gene based on the preference of rice codon usage, and then commissioned Biotechnology (Shanghai) Co., Ltd. to artificially synthesize the 4764 bp rBE 57 gene. The small fragment (Cas 9) between the BamHI recognition site and the BcuI recognition site of pUbi-Cas 9 was replaced with the rBE 57 gene shown at positions 7-4758 of sequence 5, and the other nucleotides of pUbi-Cas9 were not changed, resulting in the rBE57 gene expression vector rBE57. In sequence 5, positions 1-6 are BamHI recognition sites, positions 7-507 are the CDS of TadA9, positions 508-603 are the CDS of the connecting peptide, positions 604-4728 are the CDS of ScCas9 (D10A), positions 4729-4755 are the CDS of the NLS, positions 4756-4758 are the terminator codon TGA, and positions 4759-4764 are the recognition sites of BcuI. pUbi-rBE57 contained elements attR1-ccdB-attR2 for the LR reaction of LR.

[0039] The adenine base editor expressed by pUbi-rBE 65 is a fusion protein named rBE 65 (also called TadA 9-SpRY(D 10 A)), which is a protein consisting of an adenosine deaminase named TadA 9, a Cas protein named SpRY(D 10 A), and a nuclear localization signal named NLS. The amino acid sequence of rBE 65 is Sequence 8 in the Sequence Listing. In Sequence 8, positions 1 to 167 are the amino acid sequence of TadA 9, positions 168 to 199 are the amino acid sequence of the binding peptide, positions 200 to 1567 are the amino acid sequence of SpRY(D 10 A), and positions 1568 to 1576 are the amino acid sequence of NLS. We codon-optimized the nucleotide sequence of the chimeric gene rBE 65 gene based on the preference for codon usage, and then commissioned Biotechnology (Shanghai) Co., Ltd. to artificially synthesize the 4743 bp rBE 65 gene. The small fragment (Cas 9) between the BamHI recognition site and the BcuI recognition site of pUbi-Cas 9 was replaced with the rBE 65 gene shown at positions 7-4737 of sequence 7, and the other nucleotides of pUbi-Cas 9 were not changed to obtain the rBE 65 gene expression vector pUbi-rBE 65. In sequence 7, positions 1 to 6 were BamHI recognition sites, positions 7 to 507 were CDSs of TadA 9, positions 508 to 603 were CDSs for linking peptides, positions 604 to 4707 were CDSs of SPRY(D 10 A), positions 4708 to 4734 were CDSs of NLS, positions 4735 to 4737 were termination codons TGA, and positions 4738 to 4743 were BcuI recognition sites. pUbi-rBE 65 contained elements attR 1-ccdB-attR 2 for the LR reaction.

[0040] pUbi-rBE 49 b, pUbi-rBE 53, pUbi-rBE 57, and pUbi-rBE 65 differ only in the coding gene for the adenine base editor rBE. ​​The four adenine base editors, rBE49b, rBE53, rBE57, and rBE65, differ only in the Cas protein.

[0041] The main components of the vectors pUbi-rBE 49 b, pUbi-rBE 53, pUbi-rBE 57, and pUbi-rBE 65 are as follows: RB T-DNA repeat sequence (nucleotide sequence is from position 13973 to position 13997 in genbank accession no. LC 506530.1, dated March 20, 2020), attR 1 (nucleotide sequence is from position 2055 to position 2179 in genbank accession no. KR 233518.1, dated August 8, 2015), ccdB expression cassette (nucleotide sequence is from position 3289 to position 3594 in genbank accession no. KR 233518.1, dated August 8, 2015), attR 2 (nucleotide sequence is from position 3289 to position 3594 in genbank accession no. KR 233518.1, dated August 8, 2015), attR 3 (nucleotide sequence is from position 3289 to position 3594 in genbank accession no. KR 233518.1, dated August 8, 2015), and attR 4 (nucleotide sequence is from position 3289 to position 3594 in genbank accession no. KR 233518.1, dated August 8, 2015). 233518.1, positions 3635 to 3759, dated August 8, 2015), Ubip promoter (nucleotide sequence is shown in SEQ ID NO: 9), rice adenine base editor rBE gene (rBE 49 b gene (nucleotide sequence is shown in SEQ ID NO: 7 to 4737, dated August 8, 2015), rBE 53 gene (nucleotide sequence is shown in SEQ ID NO: 7 to 4737, dated September 1, 2015), rBE 57 gene (nucleotide sequence is shown in SEQ ID NO: 7 to 4758, dated November 2, 2008), or rBE 65 gene (nucleotide sequence is shown in SEQ ID NO: 7 to 4737, dated November 2, 2015), NOS terminator (nucleotide sequence is shown in SEQ ID NO: 10), CaMV 35 S promoter (nucleotide sequence is shown in SEQ ID NO: 10414 to 11092, dated November 26, 2008), and ushiomycin gene (nucleotide sequence is shown in SEQ ID NO: KY 420085.1, positions 511 to 1536, July 10, 2017), CaMV poly(A) terminator (nucleotide sequence is genbank accession no. MK 896900.1, positions 8618 to 8792, September 4, 2019), and LB T-DNA repeat (nucleotide sequence is genbank accession no. LC 506530.1, positions 3635 to 3795, March 20, 2020).

[0042] In this example, seven rice adenine base editor expression vectors were prepared as controls. The names of the control vectors used as controls for pUbi-rBE49b of the present invention are pUbi-rBE14 and pUbi-rBE46b, the names of the control vectors used as controls for pUbi-rBE53 of the present invention are pUbi-rBE23 and pUbi-rBE50, the names of the control vectors for pUbi-rBE57 of the present invention are pUbi-rBE26 and pUbi-rBE54, and the name of the control vector for pUbi-rBE65 of the present invention is pUbi-rBE62.

[0043] The adenine base editor expressed by pUbi-rBE 14 is a fusion protein named rBE 14 (also called wtTadA-TadA 7.10-SpCas 9(D 10 A)-NLS), which is a protein consisting of a wild-type adenosine deaminase named wtTadA, a mutant adenosine deaminase named TadA 7.10, a Cas protein named SpCas 9(D 10 A), and a nuclear localization signal named NLS. The amino acid sequence of rBE14 is the same as that of rBE49b, except that the adenosine deaminase named TadA9 in rBE- 49 b is replaced with a protein wtTadA-TadA7.10 in which a wild-type adenosine deaminase named wtTadA and a mutant adenosine deaminase named TadA7.10 are combined. The rBE14 gene is a DNA molecule obtained by replacing the CDS of TadA 9 (nucleotide sequence: ... In sequence 12, positions 1 to 501 are the CDS of wtTadA, positions 502 to 597 are the CDS of the binding peptide, and positions 598 to 1095 are the CDS of TadA 7.10. pUbi-rBE 14 is an rBE 14 gene expression vector obtained by replacing the rBE 49 b gene of pUbi-rBE 49 b with the rBE 14 gene without changing other nucleotides of pUbi-rBE 49 b.

[0044] The adenine base editor expressed by pUbi-rBE 46 b is a fusion protein named rBE 46 b (also called TadA-R-SpCas 9(D 10 A)-NLS), which is a protein consisting of a mutant adenosine deaminase named TadA-R, a Cas protein named SpCas 9(D 10 A), and a nuclear localization signal named NLS. rBE 46 b has exactly the same amino acid sequence as rBE 49 b, except that the adenosine deaminase named TadA 9 in rBE 49 b is replaced with a mutant adenosine deaminase named TadA-R. The rBE 46 b gene is a DNA molecule obtained by replacing the TadA 9 CDS (nucleotide sequence: positions 7 to 507 of SEQ ID NO:1) of the rBE 49 b gene (nucleotide sequence: positions 7 to 4737 of SEQ ID NO:1) with the TadA-R gene sequence shown in SEQ ID NO:13, without changing any other nucleotides of SEQ ID NO:1. pUbi-rBE 46 b is an rBE 46 b gene expression vector obtained by replacing the rBE 49 b gene of pUbi-rBE 49 b with the rBE 46 b gene, without changing any other nucleotides of pUbi-rBE 49 b.

[0045] The adenine base editor expressed by pUbi-rBE 23 is a fusion protein named rBE 23 (wtTadA-Tada 7.10-SpCas 9-NG(D 10 A)-NLS), which is a protein composed of a wild-type adenine deaminase named wtTadA, a mutant adenine deaminase named TadA 7.10, a Cas protein named SpCas 9-NG(D 10 A), and a nuclear localization signal named NLS. rBE 23 has exactly the same amino acid sequence as rBE 53, except that the adenine deaminase named TadA 9 in rBE 53 is replaced with a protein called wtTadA-TadA 7.10, which is a combination of a wild-type adenine deaminase named wtTadA and a mutant adenine deaminase named TadA 7.10. The rBE 23 gene is a DNA molecule obtained by replacing the CDS of TadA 9 (nucleotide sequence: positions 7 to 507 of sequence 3) in the rBE 53 gene (nucleotide sequence: positions 7 to 4737 of sequence 3) with the wtTadA-TadA 7.10 gene shown in SEQ ID NO: 12, without changing the other nucleotides of sequence 3. pUbi-rBE 23 is an rBE 23 gene expression vector obtained by replacing the rBE 53 gene in pUbi-rBE 53 with the rBE 23 gene, without changing other nucleotides of pUbi-rBE 53.

[0046] The adenine base editor expressed by pUbi-rBE 50 is a fusion protein named rBE 50 (also called wtTadA-TadA 7.10-SpCas 9-NG(D 10 A)-NLS), which is a protein consisting of a mutant adenosine deaminase named TadA-R, a Cas protein named SpCas 9-NG(D 10 A), and a nuclear localization signal named NLS. rBE 50 has exactly the same amino acid sequence as rBE 53, except that the adenosine deaminase named TadA 9 of rBE 53 is replaced with the mutant adenosine deaminase named TadA-R. The rBE 50 gene is a DNA molecule obtained by replacing the TadA 9 CDS (nucleotide sequence: positions 7 to 507 of SEQ ID NO:1) of the rBE 53 gene (nucleotide sequence: positions 7 to 4737 of SEQ ID NO:3) with the TadA-R gene shown in SEQ ID NO:13, without changing any other nucleotides of SEQ ID NO:3. pUbi-rBE 50 is an rBE 50 gene expression vector obtained by replacing the rBE 53 gene in pUbi-rBE 53 with the rBE 50 gene, without changing any other nucleotides of pUbi-rBE 53.

[0047] The adenine base editor expressed by pUbi-rBE 26 is a fusion protein named rBE 26 (wtTadA-TadA 7.10-ScCas 9(D 10 A)-NLS), which is a protein consisting of a wild-type adenine deaminase named wtTadA, a mutant adenine deaminase named TadA 7.10, a Cas protein named ScCas 9(D 10 A), and a nuclear localization signal named NLS. rBE 26 has the same amino acid sequence as rBE 57, except that the adenine deaminase named TadA 9 in rBE 57 is replaced with a protein called wtTadA-TadA 7.10, which is a combination of a wild-type adenine deaminase named wtTadA and a mutant adenine deaminase named TadA 7.10. The rBE 26 gene is a DNA molecule obtained by replacing the CDS (nucleotide sequence: nucleotide sequence 7 to 507 in SEQ ID NO:5) of TadA 9 in the rBE 57 gene (nucleotide sequence: nucleotide sequence 7 to 4758 in SEQ ID NO:5) with the wtTadA-TadA 7.10 gene shown in SEQ ID NO:12, without changing the other nucleotides in SEQ ID NO:5. pUbi-rBE 26 is an rBE 26 gene expression vector obtained by replacing the rBE 57 gene in pUbi-rBE 57 with the rBE 26 gene, without changing any other nucleotides of pUbi-rBE 57.

[0048] The adenine base editor expressed by pUbi-rBE 54 is a fusion protein named rBE 54 (also called TadA-R-ScCas 9(D 10 A)-NLS), which is a protein consisting of a mutant adenosine deaminase named TadA-R, a Cas protein named ScCas 9(D 10 A), and a nuclear localization signal named NLS. rBE 54 has exactly the same amino acid sequence as rBE 57, except that the adenosine deaminase named TadA 9 in rBE 57 is replaced with a mutant adenosine deaminase named TadA-R. The rBE 54 gene is a DNA molecule obtained by replacing the TadA 9 CDS (nucleotide sequence: positions 7 to 507 of SEQ ID NO:5) of the rBE 57 gene (nucleotide sequence: positions 7 to 4758 of SEQ ID NO:5) with the TadA-R gene sequence shown in SEQ ID NO:13, without changing any other nucleotides of SEQ ID NO:5. pUbi-rBE 54 is an rBE 54 gene expression vector obtained by replacing the rBE 57 gene in pUbi-rBE 57 with the rBE 54 gene, without changing any other nucleotides of pUbi-rBE 57.

[0049] The adenine base editor expressed by pUbi-rBE 62 is a fusion protein named rBE 62 (TadA-R-SpRY(D 10 A)-NLS), which is a protein consisting of a mutant adenosine deaminase named TadA-R, a Cas protein named SpRY(D 10 A), and a nuclear localization signal named NLS. rBE 62 has exactly the same amino acid sequence as rBE 65, except that the adenosine deaminase named TadA 9 in rBE 65 is replaced with a mutant adenosine deaminase named TadA-R. The rBE 62 gene is a DNA molecule obtained by replacing the TadA 9 CDS (nucleotide sequence: positions 7 to 507 of SEQ ID NO:7) of the rBE 65 gene (nucleotide sequence: positions 7 to 4737 of SEQ ID NO:7) with the TadA-R gene sequence shown in SEQ ID NO:13, without changing any other nucleotides of SEQ ID NO:7. pUbi-rBE 62 is an rBE 62 gene expression vector obtained by replacing the rBE 65 gene in pUbi-rBE 65 with the rBE 62 gene, without changing any other nucleotides of pUbi-rBE 65.

[0050] 2. Targeted A>G substitution in endogenous rice genes using rice adenine base editor expression vector 1. Construction of the base editing vector pUbi-rBE-sgRNA for the target sequence The genomic DNA sequences of the selected target genes (see Figure 13) were obtained from the rice genome database (https: / / rapdb.dna.affrc.go.jp / ), and the corresponding target sequences and forward and reverse oligonucleotide strands were designed for each base editor according to the requirements for identifying PAM. The synthesis of the forward and reverse oligonucleotide strands (see Figure 13 for specific sequences) of each target sequence (5'-N19PAM-3') in Figure 13 was entrusted to NTT Docomo, Inc. After artificial synthesis, the primers were phosphorylated using T4 polynucleotide kinase and annealed to form double-stranded DNA fragments (5'-N19-3' for target sequences containing sgRNA), and the double-stranded DNA fragments were cloned into the two BtgZI or two BsaI enzyme cleavage sites of the pENTR4:sgRNA (Figure 2, containing attL1-sgRNA expression cassette-attL2) vector, respectively. After sequencing the primer U6p-F1 (5'-AAGAACGAACTAAGCCGGAC-3' (SEQ ID NO: 37) to confirm that the insertion fragment was completely accurate (the insertion fragment was 5'-N19-3' of the target sequence containing sgRNA), the resulting plasmid was linearized by AatII enzyme cutting, and the sgRNA expression cassette (coding DNA containing sgRNA) was cloned into attR1-ccdB-attR2 of the rice adenine base editor expression vector pUbi-rBE (Figure 1) by Gateway LR reaction, respectively, to align the base of each target sequence. The recombinant vector pUbi-rBE-sgRNA was obtained (Figure 3). pUbi-rBE-sgRNA is a recombinant expression vector obtained by replacing the elements attR1-ccdB-attR2 of pUbi-rBE with attB1-sgRNA expression cassette-attB2, without changing other nucleotides of pUbi-rBE. Three base-editing vectors targeting the OsTms9 gene, pUbi-rBE49b-sgRNA-Ostms9, pUbi-rBE14-sgRNA-Ostms9, and pUbi-rBE46b-sgRNA-Ostms9, were obtained, respectively.Three base-editing vectors targeting target sequence 1 (sequence 76, containing NGGPAM) of OsWRKY45 gene were pUbi-rBE49b-sgRNA-OsWRKY45, pUbi-rBE14-sgRNA-OsWRKY45, and pUbi-rBE46b-sgRNA-OsWRKY45, respectively. Three base-editing vectors targeting OsDEP2 gene were pUbi-rBE53-sgRNA-OsDEP2, pUbi-rBE23-sgRNA-OsDEP2, and pUbi-rBE62-sgRNA-OsJAR2, respectively. The three base-editing vectors targeting the target sequence 2 (sequence 79, containing NGT PAM) of the OsWRKY45 gene were pUbi-rBE53-sgRNA-OsWRKY45, pUbi-rBE23-sgRNA-OsWRKY45, and pUbi-rBE50-sgRNA-OsWRKY45, respectively. The three base-editing vectors targeting the OsGS1 gene were pUbi-rBE57-sgRNA-OsGS1, pUbi-rBE54-sgRNA-OsGS1, and pUbi-rBE26-sgRNA-OsGS1, respectively. The two base-editing vectors targeting the OssERK2 gene were pUbi-rBE57-sgRNA-OssERK2 and pUbi-rBE54-sgRNA-OssERK2. The two base-editing vectors targeting the OsMPK13 gene were pUbi-rBE65-sgRNA-OsMPK13 and pUbi-rBE62-sgRNA-OsMPK13. The two base-editing vectors targeting the OsGSK4 gene were pUbi-rBE65-sgRNA-OsGSK4 and pUbi-rBE62-sgRNA-OsGSK4. The two base-editing vectors targeting the OsJAR1 gene were pUbi-rBE65-sgRNA-OsJAR1 and pUbi-rBE62-sgRNA-OsJAR1.

[0051] Here, the construction method of pENTR4:sgRNA is as follows: From the 5' end to the 3' end, the U6 promoter sequence 1, a nucleotide sequence containing two BtgZI enzyme cut sites, an sgRNA Scaffold sequence, a (T)8 terminator sequence, a U6 promoter sequence 2, a nucleotide sequence containing two BsaI enzyme cut sites, an sgRNA Scaffold sequence, and a (T)8 terminator sequence were sequentially bound to form an sgRNA expression cassette. This was artificially synthesized by Seiko Biotechnology (Shanghai) Co., Ltd. Using the gene synthesized by the company as a template, a primer pair (sgRNA-F: 5'-GCAGGCTGTCGACTGGATCCAAGCTTAAGAACGAACTAAGCC-3' (sequence 38) and sgRNA-R: 5'-CAAGAAAGCTGGGTGAATTCGATATCAAGCTTATCGATACCG-3' (sequence 39)) was amplified to obtain a 1 kb sgRNA expression cassette fragment (nucleotide sequence is sequence 11 in the sequence table). Using the pENTR4 (Invitrogen) vector as a template, pENTR4-F1: (5'-CGAATTCACCCAGCTTTCTTGTACAAAGTTGGCATTATAAGA-3' (sequence 40) and pENTR4-R1: (5'-CTTAGTTCGTTCTTAAGCTTGGATCCAGTCGACAGCCTGCTTTTTTGTACAAAGT-3' (sequence 41)) were amplified to obtain a 2.2 kb pENTR4 vector backbone (a DNA fragment in which the fragment of the ccdB gene expression cassette of pENTR4 was removed). The sgRNA expression cassette fragment was infusion-linked to the pENTR 4 vector backbone using the reagent cassette ClonExpress II One Step Cloning Kit (purchased from Nanjing Nuovizan Bioscience and Technology Co., Ltd.), and the vector pENTR 4: sgRNA was obtained (Figure 2). Two BtgZI or two BsaI enzyme cut sites were used in the identification sequence of a specific gene in the clone (5'-N19-3' in the target sequence of sgRNA).In sequence 11, positions 27 to 348 are U6 promoter sequence 1, positions 349 to 389 are a nucleotide fragment containing two BtgZI sites, positions 390 to 465 are an sgRNA scaffold sequence, positions 466 to 473 are a (T)8 terminator sequence, positions 474 to 782 are U6 promoter sequence 2, positions 783 to 806 are a nucleotide fragment containing two BsaI sites, positions 807 to 882 are an sgRNA scaffold sequence, and positions 883 to 890 are a (T)8 terminator sequence.

[0052] 2. Bacillus subtilis mediates stable genetic transformation in rice The obtained editing vectors for each target gene base were transformed into Agrobacterium EHA 105 by electric shock method (EHA 105 electric shock receptor cells were purchased from Beijing Boya Gene Technology Co., Ltd.), and genetic transformation mediated by Agrobacterium was performed with reference to the method disclosed by Hiei et al. (Hiei Y., Ohta S., Komari T., and Kumashiro T. (1994); Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6: 271-282.), to obtain rice materials with edited target genes for each generation in the T0 generation.

[0053] 3. Measurement of base editing efficiency at the target locus in T0 generation transgenic rice The genomic DNA of the T 0 generation transgenic rice materials was extracted using the conventional CTAB method.

[0054] The target sequence information of each target gene and its detection primer are shown in Figure 14. All primers were artificially synthesized by Sanko Biotechnology (Shanghai) Co., Ltd. All PCR reactions involved in the test were performed using HiFi enzyme premix I-5 TMPCR amplification of the target fragment was performed using 2× High-Fidelity Master Mix (purchased from Keloning (Beijing) Biological Science and Technology Co., Ltd.). The PCR product was directly entrusted to Sankyo Biological Engineering (Shanghai) Co., Ltd. for Sanger sequencing.

[0055] The sequencing results are as follows: The targeted base editing efficiency of the NGG PAM target of Ostms 9 by the base editor rBE 14 was 0%, the targeted base editing efficiency of the NGG PAM target of Ostms 9 by the base editor rBE 46 b was 64.58%, and the targeted base editing efficiency of the NGG PAM target of Ostms 9 by the base editor rBE 49 b was 97.92% (Table 1).

[0056] Of the 54 T0 generation trans-pUbi-rBE14-sgRNA-OsTms9 rice lines, 0 lines had adenine A replaced with guanine G by deammonification. Of the 48 T0 generation trans-pUbi-rBE46b-sgRNA-OsTms9 rice lines, 31 lines had adenine A replaced with guanine G by deammonification. In all of them, the 5th A in the 5' to 3' direction of the target sequence (corresponding to T5 in Figure 4) was replaced with G by deammonification. Of the 48 T0 generation trans-pUbi-rBE49b-sgRNA-OsTms9 rice lines tested, 47 lines had adenine A replaced with guanine G by deammonification, and in all of them, the 5th A in the 5' to 3' direction of the target sequence (corresponding to T5 in Figure 4) was replaced with G by deammonification.

[0057] The efficiency of targeted base editing by the base editor rBE 14 on the NGGPAM target of OsWRKY 45 was 0.00%, the efficiency of targeted base editing by the base editor rBE 46 b on the NGG PAM target of OsWRKY 45 was 0.00%, and the efficiency of targeted base editing by the base editor rBE 49 b on the NGG PAM target of OsWRKY 45 was 2.86% (Table 1). Of the 53 T 0 generation trans pUbi-rBE 14-sgRNA-OsWRKY 45 rice plants detected, 0 plants had adenine A replaced with guanine G by deammonification. Of the 48 T 0 generation trans pUbi-rBE 46 b-sgRNA-OsWRKY 45 rice plants detected, 0 plants had adenine A replaced with guanine G by deammonification. Of the 35 T 0 generation trans pUbi-rBE 49 b-sgRNA-OsWRKY 45 rice lines detected, one line had adenine A replaced with guanine G by deammonification, and in each line, the fourth A in the 5' to 3' direction of the target sequence was replaced with G by deammonification (corresponding to A 4 in Figure 5).

[0058] The efficiency of target base editing by the base editor rBE 23 against the NGA PAM target of OsDEP 2 was 0.00%, that by the base editor rBE 50 against the NGA PAM target of OsDEP 2 was 27.08%, and that by the base editor rBE 53 against the NGA PAM target of OsDEP 2 was 78.72% (Table 2). Of the 96 T 0 generation trans pUbi-rBE 23-sgRNA-OsDEP 2 rice plants detected, 0 had adenine A replaced with guanine G by deammonification. Of the 48 T 0 generation trans pUbi-rBE 50-sgRNA-OsDEP 2 rice plants detected, 13 had adenine A replaced with guanine G by deammonification. Among the strains in which A at positions 4 and 6 in the 5' to 3' direction of the target sequence could be replaced with G by deammonification, there were 10 strains in which adenine A at position 4 (corresponding to A4 in Figure 6) was replaced with G by deammonification, and 13 strains in which adenine A at position 6 (corresponding to A6 in Figure 6) was replaced with G by deammonification. Of the 47 T 0 generation trans pUbi-rBE 53-sgRNA-OsDEP 2 rice strains detected, there were 37 strains in which adenine A was replaced with guanine G by deammonification. Among the strains in which A at positions 4 and 6 in the 5' to 3' direction of the target sequence could be replaced with G by deammonification, there were 37 strains in which adenine A at position 4 (corresponding to A4 in Figure 6) was replaced with G by deammonification, and 28 strains in which adenine A at position 6 (corresponding to A6 in Figure 6) was replaced with G by deammonification.

[0059] The target base editing efficiency of the NGPAM target of OsWRKY 45 by the base editor rBE 23 was 0%, the target base editing efficiency of the NGPAM target of OsWRKY 45 by the base editor rBE 50 was 89.36%, and the target base editing efficiency of the NGT PAM target of OsWRKY 45 by the base editor rBE 53 was 93.75% (Table 2). Of the 52 T 0 generation trans pUbi-rBE 23-sgRNA-OsWRKY 45 rice lines detected, 0 lines had adenine A replaced by guanine G by deammonification. Of the 47 T 0 generation trans pUbi-rBE 50-sgRNA-OsWRKY 45 rice strains detected, 42 had adenine A replaced with guanine G by deammonification, and in all cases, the 5th A in the 5' to 3' direction of the target sequence was replaced with G by deammonification (corresponding to T5 in Figure 7). Of the 48 T 0 generation trans pUbi-rBE 53-sgRNA-OsWRKY 45 rice strains detected, 45 had adenine A replaced with guanine G by deammonification, and in all cases, the 5th A in the 5' to 3' direction of the target sequence was replaced with G by deammonification (corresponding to T5 in Figure 7).

[0060] The targeted base editing efficiency of the NAG PAM target of OsGS 1 by the base editor rBE 26 was 0.00%, the targeted base editing efficiency of the NAG PAM target of OsGS 1 by the base editor rBE 54 was 25.00%, and the targeted base editing efficiency of the NAG PAM target of OsGS 1 by the base editor rBE 57 was 54.17% (Table 3). Of the 36 T 0 generation trans pUbi-rBE 26-sgRNA-OsGS 1 rice lines detected, 0 lines had adenine A replaced by guanine G by deammonification. Of the 48 T 0 generation trans pUbi-rBE 54-sgRNA-OsGS 1 rice strains detected, 12 strains had adenine A replaced with guanine G by deammonification, and of the A at positions 3, 6, and 9 in the 5' to 3' direction of the target sequence replaced with G by deammonification, 3 strains had adenine A at position 3 (corresponding to A3 in Figure 8) replaced with G by deammonification, 11 strains had adenine A at position 6 (corresponding to A6 in Figure 8) replaced with G by deammonification, and 12 strains had adenine A at position 9 (corresponding to A9 in Figure 8) replaced with G by deammonification. Of the 48 T 0 generation trans pUbi-rBE 57-sgRNA-OsGS 1 rice strains detected, 23 strains had adenine A replaced with guanine G by deammonification, and of the A at positions 3, 4, 6, 9, and 11 in the 5' to 3' direction of the target sequence replaced with G by deammonification, 26 strains had adenine A at position 3 (corresponding to A3 in Figure 8) replaced with G by deammonification, 13 strains had adenine A at position 4 (corresponding to A4 in Figure 8) replaced with G by deammonification, 26 strains had adenine A at position 6 (corresponding to A6 in Figure 8) replaced with G by deammonification, 26 strains had adenine A at position 9 (corresponding to A9 in Figure 8) replaced with G by deammonification, and 26 strains had adenine A at position 11 (corresponding to A1 in Figure 8) replaced with G by deammonification. 11 In one strain, the α-ammoniamine derivative (corresponding to α-ammoniamine) was replaced by G through deammonification.

[0061] The target base editing efficiency of the NAG PAM target of OssERK2 by the base editor rBE54 was 28.26%, and the target base editing efficiency of the NAG PAM target of OssERK2 by the base editor rBE57 was 72.34% (Table 3). Of the 46 T 0 generation trans pUbi-rBE54-sgRNA-OssERK2 rice strains detected, 13 strains had adenine A replaced with guanine G by deammonification. Of the strains in which A at the 4th and 6th positions in the 5' to 3' direction of the target sequence could be replaced with G by deammonification, 3 strains had adenine A at the 4th position (corresponding to T4 in Figure 9) replaced with G by deammonification, and 13 strains had adenine A at the 6th position (corresponding to T6 in Figure 9) replaced with G by deammonification. Of the 47 T 0 generation trans pUbi-rBE 57-sgRNA-OssERK 2 rice strains detected, 34 strains had adenine A replaced with guanine G by deammonification, and of the strains in which A at positions 4 and 6 in the 5' to 3' direction of the target sequence was replaced with G by deammonification, 34 strains had adenine A at position 4 (corresponding to T4 in Figure 9) replaced with G by deammonification, and 34 strains had adenine A at position 6 (corresponding to T6 in Figure 9) replaced with G by deammonification.

[0062] The efficiency of targeted base editing by the base editor rBE 62 against the NAA PAM target of OsMPK 13 was 29.17%, and the efficiency of targeted base editing by the base editor rBE 65 against the NAA PAM target of OsMPK 13 was 31.91% (Table 4). Of the 48 T 0 generation trans pUbi-rBE 62-sgRNA-OsMPK 13 rice lines detected, 14 lines had adenine A replaced with guanine G by deammonification, and in all cases, A at position 6 in the 5' to 3' direction of the target sequence (corresponding to A6 in Figure 10) was replaced with G by deammonification. Of the 47 T 0 generation trans pUbi-rBE65-sgRNA-OsMK13 rice strains detected, 15 strains had adenine A replaced with guanine G by deammonification, and of the strains in which A at positions 4 and 6 in the 5' to 3' direction of the target sequence was replaced with G by deammonification, 2 strains had adenine A at position 4 (corresponding to A4 in Figure 10) replaced with G by deammonification, and 15 strains had adenine A at position 6 (corresponding to A6 in Figure 10) replaced with G by deammonification.

[0063] The target base editing efficiency of the OsGSK 4 NAC PAM target by the base editor rBE 62 was 51.28%, and the target base editing efficiency of the OsGSK 4 NAC PAM target by the base editor rBE65 was 73.17% (Table 4). Of the 39 T 0 generation trans pUbi-rBE 62-sgRNA-OsGSK 4 rice lines detected, 20 lines had adenine A replaced with guanine G by deammonification, and of the lines in which A at positions 4 and 11 in the 5' to 3' direction of the target sequence could be replaced with G by deammonification, 20 lines had adenine A at position 4 (corresponding to T4 in Figure 11) replaced with G by deammonification, and 10 lines had adenine A at position 11 (corresponding to T1 in Figure 11) replaced with G by deammonification. 11Among the 41 T 0 generation trans pUbi-rBE 65-sgRNA-OsGSK 4 rice strains detected, 30 strains had adenine A replaced with guanine G by deammonification, and among those in which A at positions 4, 8, 9, and 11 in the 5' to 3' direction of the target sequence could be replaced with G by deammonification, 30 strains had adenine A at position 4 (corresponding to T 4 in FIG. 11) replaced with G by deammonification, 1 strain had adenine A at position 8 (corresponding to T 8 in FIG. 11) replaced with G by deammonification, 1 strain had adenine A at position 9 (corresponding to T 9 in FIG. 11) replaced with G by deammonification, and 9 strains had adenine A at position 11 (corresponding to T 11 in FIG. 11) replaced with G by deammonification.

[0064] The efficiency of target base editing by the base editor rBE 62 against the NAT PAM target of OsJAR 1 was 36.17%, and the efficiency of target base editing by the base editor rBE 65 against the NAT PAM target of OsJAR 1 was 45.00% (Table 4). Of the 47 T 0 generation trans pUbi-rBE 62-sgRNA-OsJAR 1 rice strains detected, 17 strains had adenine A replaced with guanine G by deammonification, and of the strains in which A at positions 4 and 6 in the 5' to 3' direction of the target sequence could be replaced with G by deammonification, 17 strains had adenine A at position 4 (corresponding to T4 in Figure 12) replaced with G by deammonification, and 9 strains had adenine A at position 6 (corresponding to T6 in Figure 12) replaced with G by deammonification. Of the 40 T0 generation trans pUbi-rBE65-sgRNA-OsJAR1 rice strains detected, 18 strains had adenine A replaced with guanine G by deammonification, and of the A at positions 2, 4, and 6 in the 5' to 3' direction of the target sequence replaced with G by deammonification, 3 strains had adenine A at position 2 (corresponding to T2 in Figure 12) replaced with G by deammonification, 18 strains had adenine A at position 4 (corresponding to T4 in Figure 12) replaced with G by deammonification, and 18 strains had adenine A at position 6 (corresponding to T6 in Figure 12) replaced with G by deammonification.

[0065] The above results show that the editing efficiency of the adenine base editing technology mediated by the fusion of TadA9 with SpCas9n / ScCas9 / SpCas9-NG / SpRY in the present invention is much higher than that of the adenine base editing technology mediated by TadA-R mutants such as TadA7.10, and its editing activity window can be expanded, thereby improving the efficiency of adenine base editing technology and broadening its scope of application.

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[0070] The present invention has been described in detail above. Those skilled in the art can carry out the present invention in a wide range of equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present invention and without undue experimentation. Although the present invention is given in specific embodiments, it should be understood that further improvements can be made to the present invention. In short, in accordance with the principles of the present invention, the present application includes modifications made using conventional techniques known in the art outside the scope of the disclosure of the present application. It is intended to include any modifications, uses or improvements of the present invention. Applications according to essential features are possible according to the appended claims. [Industrial Applicability]

[0071] The present invention obtains a highly active mutant TadA9 by artificially mutating the TadA protein, and uses it to develop a novel CRISPR / Cas-based editing toolkit. The adenine base editing technology mediated by fusion with SpCas9(D10A), SpCas9-NG(D10A), ScCas9(D10A) and SpRY(D10A) has a significantly higher editing efficiency than the adenine base editing technology mediated by TadA7.10 and TadA-R mutants, and can expand the scope of its editing activity, improve the efficiency of the adenine base editing technology, and widen its range of application. In particular, it can solve the problem of adenine base editing at targets that cannot be edited with base editing vectors mediated by TadA7.10, expand the scope of use for targeted editing of plant genomes, and provide researchers in the fields of plant research and crop gene improvement with an important toolset for gene function research and correction. The present invention can improve the efficiency of adenine base editing and accurately mediate base mutations at target sites, and can be widely applied in rice and even other plant cells.

Claims

1. A protein characterized in that the amino acid sequence of the protein is positions 1 to 167 of SEQ ID NO:2 in the sequence listing.

2. A biological material relating to the protein according to claim 1, characterized in that the biological material is at least one of the following B1) to B7): B1) a nucleic acid molecule encoding the protein; B2) an expression cassette comprising the nucleic acid molecule in B1); B3) a recombinant vector comprising the nucleic acid molecule in B1), or the expression cassette in B2); B4) a recombinant microorganism comprising the nucleic acid molecule in B1), a recombinant microorganism comprising the expression cassette in B2), or a recombinant microorganism comprising the recombinant vector in B3); B5) a transgenic plant cell line comprising the nucleic acid molecule in B1), a transgenic plant cell line comprising the expression cassette in B2), or a transgenic plant cell line comprising the recombinant vector in B3); B6) a transgenic plant tissue comprising the nucleic acid molecule in B1), a transgenic plant tissue comprising the expression cassette in B2), or a transgenic plant tissue comprising the recombinant vector in B3); B7) A transgenic plant organ comprising the nucleic acid molecule in B1), a transgenic plant organ comprising the expression cassette in B2), or a transgenic plant organ comprising the recombinant vector in B3).

3. The biological material according to claim 2, characterized in that the nucleic acid molecule in B1) is a gene shown in B11) or B12) below: B11) a cDNA or DNA molecule whose coding sequence of the coding strand is shown in positions 7 to 507 of SEQ ID NO:1; B12) A cDNA or DNA molecule having 80% or more identity with the nucleotide sequence defined in B11) and encoding said protein.

4. A protein comprising a Cas protein and an adenosine deaminase, A fusion protein, characterized in that the adenosine deaminase is the protein described in claim 1.

5. The fusion protein according to claim 4, wherein the Cas protein is ScCas9(D10A), SpRY(D10A), SpCas9(D10A) or SpCas9-NG(D10A).

6. The fusion protein according to claim 4 or 5, wherein the fusion protein is a protein in which the adenosine deaminase, the Cas protein, and a nuclear localization signal are linked together.

7. the fusion protein is TadA9-ScCas9(D10A), TadA9-SpRY(D10A), TadA9-SpCas9(D10A) or TadA9-SpCas9-NG(D10A); The fusion protein according to claim 6, wherein the TadA9-SpCas9(D10A) is a protein having an amino acid sequence of SEQ ID NO: 2, the TadA9-SpCas9-NG(D10A) is a protein having an amino acid sequence of SEQ ID NO: 4, the TadA9-ScCas9(D10A) is a protein having an amino acid sequence of SEQ ID NO: 6, and the TadA9-SpRY(D10A) is a protein having an amino acid sequence of SEQ ID NO:

8.

8. A biological material relating to the fusion protein according to any one of claims 4 to 7, which is at least one of the following D1) to D7): D1) a nucleic acid molecule encoding the fusion protein; D2) an expression cassette comprising the nucleic acid molecule in D1); D3) a recombinant vector comprising the nucleic acid molecule in D1), or a recombinant vector comprising the expression cassette in D2); D4) a recombinant microorganism comprising the nucleic acid molecule in D1), a recombinant microorganism comprising the expression cassette in D2), or a recombinant microorganism comprising the recombinant vector in D3); D5) a transgenic plant cell line comprising the nucleic acid molecule in D1), a transgenic plant cell line comprising the expression cassette in D2), or a transgenic plant cell line comprising the recombinant vector in D3); D6) a transgenic plant tissue comprising the nucleic acid molecule in D1), a transgenic plant tissue comprising the expression cassette in D2), or a transgenic plant tissue comprising the recombinant vector in D3); D7) A transgenic plant organ comprising the nucleic acid molecule in D1), a transgenic plant organ comprising the expression cassette in D2), or a transgenic plant organ comprising the recombinant vector in D3).

9. The DNA molecule in D1) is a gene encoding TadA9-ScCas9(D10A), a gene encoding TadA9-SpRY(D10A), a gene encoding TadA9-SpCas9(D10A), or a gene encoding TadA9-SpCas9-NG(D10A) according to claim 7; The gene encoding TadA9-SpCas9 (D10A) is D131) a cDNA or DNA molecule whose coding sequence is set forth in positions 7 to 4737 of SEQ ID NO:1; or D132) a cDNA or DNA molecule encoding the TadA9-SpCas9(D10A) having 80% or more identity to the nucleotide sequence defined in D131); The gene encoding TadA9-SpCas9-NG (D10A) is D141) a cDNA or DNA molecule whose coding strand sequence is set forth in positions 7 to 4737 of SEQ ID NO:3; or D142) a cDNA or DNA molecule encoding the TadA9-SpCas9 (D10A) having 80% or more identity with the base sequence defined in D141); The gene encoding TadA9-ScCas9 (D10A) is D111) a cDNA or DNA molecule whose coding sequence is set forth in positions 7 to 4758 of SEQ ID NO:5; or D112) a cDNA or DNA molecule encoding the TadA9-ScCas9(D10A) having 80% or more identity to the nucleotide sequence defined in D111); The gene encoding TadA9-SpRY(D10A) is D121) a cDNA or DNA molecule whose coding sequence is set forth in positions 7 to 4737 of SEQ ID NO:7; or D122) a cDNA molecule or a DNA molecule encoding said TadA9-SpRY(D10A) having 80% or more identity with the nucleotide sequence defined in D121).

10. Use of a protein according to claim 1, a biological material according to claim 2 or 3, a fusion protein according to any one of claims 4 to 7, or a biological material according to claim 8 or 9 for single base editing in plants.

11. The use according to claim 10, wherein the single base editing of the plant is a targeted mutation from A to G in the plant genome.

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