JP2023550932A - Adenosine deaminase and related biomaterials and uses thereof - Google Patents

Abstract

The present invention provides adenosine deaminase and its related biomaterials and applications thereof. The name of the protein whose amino acid sequence of adenosine deaminase is from positions 1 to 167 of SEQ ID NO: 2 in the sequence listing is TadA9. Adenine base editing technology via fusion of TadA9 with SpCas9 (D10A), SpCas9-NG (D10A), ScCas9 (D10A) and SpRY (D10A) can be used to edit adenine bases via TadA7.10 and TadA-R mutants. The editing efficiency is significantly higher than that of editing technology, which can extend the scope of its editing activity, improve the efficiency of adenine base editing technology, and expand its application range. [Selection diagram] None

Description

The present invention relates to adenosine deaminase, biomaterials related thereto and uses thereof.

Genome editing technology is a genetic engineering technology that uses artificial nucleases to modify specific parts of an organism's genome. This process generates DNA double-strand breaks by cleavage of target sites by specific nucleases, followed by non-homologous end-joining (NHEJ) repair or homology-directed repair. HDR) introduces base deletion, insertion, and mismatch phenomena to achieve the purpose of biological mutation. In recent years, we have succeeded in developing artificial nuclease technologies mainly using ZFN, TALEN, and CRISPR. In particular, CRISPR/Cas technology has attracted the attention of researchers due to its ease of use, efficiency, and versatility.

Single-nucleotide polymorphisms (SNPs) are the genetic basis of agricultural traits of crops, and many of the important agricultural traits of plants are determined by single-nucleotide genetic loci. Currently, SNPs are being discovered one after another and are being used to screen for phenotypic traits in plants. With the development of CRISPR/Cas technology, single base editing technology (including cytosine base editing system and adenine base editing system) that causes a single base change on the genome based on this technology will induce targeted base substitutions in plants. It is used for. Since base editing technology is efficient, does not rely on double-strand breaks in DNA, and does not require the presence of donor DNA, it has been successfully applied to a wide range of organisms, including animals and plants. Cytidine deaminases that have been successfully applied to plant base editing systems include APOBEC1, APOBEC3, AID, etc., and adenosine deaminases include TadA7.10.

Adenine base editing technology based on adenosine deaminase mainly uses a fusion protein that combines the excision enzyme Cas9n (D10A) and adenosine deaminase to deaminate target base A within the base editing activity window under the guidance of sgRNA. Hypoxanthine I is gradually substituted 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 deaminase 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, poor compatibility with various Cas proteins, and furthermore, the target site within the base editing activity window is still an effective base. Unable to complete edit event.

The technical problem to be solved by the present invention is how to improve the efficiency of adenine base editing in plants and/or how to realize the expected adenine base editing although it cannot be achieved with current technology. The question is whether to allow it.

In order to solve the above technical problems, the present invention provides adenosine deaminase.

The adenosine deaminase provided by the present invention is named TadA9 and is a non-natural protein that is a highly active adenosine deaminase obtained by artificially mutating TadA7.10, a mutant of Escherichia coli tRNA adenosine deaminase TadA. Adenosine deaminase has the 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) in positions 7 to 507 of SEQ ID No. 1 in the sequence listing). It is a protein with Furthermore, in order to solve the above technical problems, the present invention provides biological materials related to the TadA9.

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 the protein;
B2) an expression cassette comprising the nucleic acid molecule of B1);
B3) A recombinant vector comprising the nucleic acid molecule according to B1) or a recombinant vector comprising the expression cassette according to B2);
B4) A recombinant microorganism comprising the nucleic acid molecule according to B1), a recombinant microorganism comprising the expression cassette according to B2), or a recombinant microorganism comprising the recombinant vector according to B3);
B5) a transgenic plant cell line comprising the nucleic acid molecule according to B1), a transgenic plant cell line comprising the expression cassette according to B2), or a transgenic plant cell line comprising the recombinant vector according to B3);
B6) a transgenic plant tissue comprising the nucleic acid molecule according to B1), a transgenic plant tissue comprising the expression cassette according to B2), or a transgenic plant tissue comprising the recombinant vector according to B3);
B7) A transgenic plant comprising the nucleic acid molecule according to B1), a transgenic plant comprising the expression cassette according to B2), or a transgenic plant comprising the recombinant vector according to B3).

In the above biomaterial, the nucleic acid molecule described in B1) may be a gene shown 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) is a cDNA molecule or DNA molecule that has 80% or more identity with the nucleotide sequence defined in B11) and encodes the protein.

The present invention also provides adenine base editors.

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

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

In the above adenine base editor, the SpCas9 (D10A) is encoded by a protein whose amino acid sequence is from position 200 to position 1567 of SEQ ID NO: 2 (that is, encoded by the nucleotide (CDS) from position 604 to 4707 of SEQ ID NO: 1 in the sequence listing). SpCas9-NG (D10A) is a protein whose amino acid sequence is from positions 200 to 1567 of SEQ ID NO: 4 (i.e., encoded by the nucleotides (CDS) from positions 604 to 4707 of SEQ ID NO: 3 in the sequence listing). ScCas9 (D10A) is a protein whose amino acid sequence is at positions 200-1574 of SEQ ID NO: 6 (a protein encoded by nucleotides (CDS) at positions 604-4728 of SEQ ID NO: 5 in the sequence listing), and SpRY ( D10A) is a protein whose amino acid sequence is at positions 200-1567 of SEQ ID NO: 8 (a protein encoded by the nucleotides (CDS) at positions 604-4728 of SEQ ID NO: 5 in the sequence listing). SpRY (D10A) is a protein whose amino acid sequence is at positions 200-1567 of SEQ ID NO: 8 (ie, a protein encoded by nucleotides 604-4707 (CDS) of SEQ ID NO: 7 in the sequence listing).

In the 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) linkage.

In the above adenine base editor, the adenine base editor may specifically be TadA9-SpCas9 (D10A), TadA9-SpCas9-NG (D10A), TadA9-ScCas9 (D10A) or TadA9-SpRY (D10A). Often, the TadA9-SpCas9 (D10A) is a protein whose amino acid sequence is SEQ ID NO: 2 (i.e., a protein encoded by nucleotides (CDS) from positions 7 to 4737 of SEQ ID NO: 1 in the sequence listing), the TadA9-SpCas9 -NG (D10A) is a protein whose amino acid sequence is SEQ ID NO: 4 (i.e., a protein encoded by nucleotides (CDS) from positions 7 to 4737 of SEQ ID NO: 3 in the sequence listing), and the TadA9-SCas9 (D10A) is a protein whose amino acid sequence is SEQ ID NO: 4. TadA9-SpRY (D10A) is a protein whose amino acid sequence is SEQ ID NO: 6 (that is, a protein encoded by nucleotides (CDS) from positions 7 to 4758 of SEQ ID NO: 5 in the sequence listing), and the TadA9-SpRY (D10A) is a protein whose amino acid sequence is SEQ ID NO: 6. This is the protein number 8 (ie, the protein encoded by nucleotides (CDS) from positions 7 to 4737 of SEQ ID NO: 7 in the sequence listing).

Biological materials related to the adenine base editor also fall within the protection scope of the present invention.

The biomaterial associated with said adenine base editor 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 a recombinant vector comprising the expression cassette according to D2);
D4) A recombinant microorganism comprising the nucleic acid molecule according to D1), a recombinant microorganism comprising the expression cassette according to D2), or a recombinant microorganism comprising the recombinant vector according to D3);
D5) A transgenic plant cell line comprising the nucleic acid molecule according to D1), a transgenic plant cell line comprising the expression cassette as defined in D2), or a transgenic plant cell line comprising the recombinant vector as defined in D3). ;
D6) a transgenic plant tissue comprising the nucleic acid molecule according to D1), a transgenic plant tissue comprising the expression cassette according to D2), or a transgenic plant tissue comprising the 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 recombinant vector according to D3).

(a) Among the biological materials related to the above adenine base editor,
The nucleic acid molecule of D1) contains 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). It may be any of the genes it encodes.

The gene encoding TadA9-SpCas9 (D10A) may be any of the genes shown below:
(D131) A cDNA molecule or DNA molecule in which the coding sequence (CDS) of the coding strand is shown in positions 7 to 4737 of SEQ ID NO: 1;
D132) A cDNA molecule or DNA molecule that has 80% or more identity with the base sequence defined in D131) and encodes the TadA9-SpCas9 (D10A).

The gene encoding TadA9-SpCas9-NG (D10A) may be any of the genes shown below:
D141) A cDNA molecule or DNA molecule whose coding sequence (CDS) of the coding strand is represented by SEQ ID NO: 7 to 4737;
D142) A cDNA molecule or DNA molecule that has 80% or more identity with the nucleotide sequence defined in D141) and encodes the TadA9-SpCas9 (D10A).
The gene encoding TadA9-ScCas9 (D10A) may be any of the genes shown below:
(D111) a cDNA molecule or DNA molecule in which the coding sequence (CDS) of the coding strand is shown in positions 7 to 4758 of SEQ ID NO: 5;
D112) A cDNA molecule or DNA molecule (D10A) that has 80% or more identity with the nucleotide sequence defined in D111) and encodes the TadA9-ScCas9.
The gene encoding TadA9-SpRY (D10A) may be any of the genes shown below:
D121) A cDNA molecule or DNA molecule in which the coding sequence (CDS) of the coding strand is shown in positions 7 to 4737 of SEQ ID NO: 7;
D122) A cDNA molecule or DNA molecule that has 80% or more identity with the nucleotide sequence defined in D121) and encodes the TadA9-SpRY (D10A).

In the above, the expression cassette means a DNA capable of expressing the protein in a host cell (for example, a plant cell, etc.), and the DNA includes not only a promoter that initiates transcription of the protein gene, but also a promoter that initiates transcription of the protein gene. It may contain a terminator that terminates the transcription of. Furthermore, the expression cassette may include 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 cauliflower mosaic virus, the trauma-inducible promoter from tomato, and the leucine aminopeptidase (“LAP”) Chao et al. (1999) Plant Physiology 120:979 -992); chemically inducible promoter from tobacco, history-dependent protein 1 (PR1) (induced by salicylic acid and BTH (S-methyl benzothiadiazole-7-thiohydroxamate)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both inducible by methyl jasmonic acid); heat shock promoter (US Patent 5,187,267); tetracycline inducible promoter (US Patent 5,057,422); cereal seed specific Seed-specific promoters such as the promoter pF128 (CN101063139B (China Patent 2007 1 0099169.7)), seed storage protein specific promoters such as the promoters of phaseolin, napin, oleosin and soybean beta-conglycin (Beachy et al. 1985) emboJ.4:3047-3053)). These can be used alone or in combination with other plant promoters. Note that all documents cited in this specification are cited in their entirety. Suitable transcription terminators include the Bacillus 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., Odell et al. (I 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. l,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.

In one embodiment of the invention, the expression cassette is constructed by linking the Ubip promoter (nucleotide sequence SEQ ID NO: 9), the nucleic acid molecule encoding the adenine base editor, and the NOS terminator (nucleotide sequence SEQ ID NO: 10). It is something that

As used herein, the term "identity" refers to sequence similarity between nucleic acid sequences. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%) that can be used to assess the identity between the sequences. Note that the identity of 80% or more may be 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more.

In the above, the recombinant microorganism may specifically be bacteria, yeast, algae, or fungi.

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

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

The method provided by the present invention for targeted mutation of A to G in a plant genome involves introducing a DNA molecule expressing the adenine base editor and sgRNA into a recipient plant to obtain a target plant in which A is targeted to be mutated to G. the target sequence of the sgRNA is 5'-N _19-20 PAM-3', the N _19-20 is 19-20 N, and the PAM (protospacer adjacent motif) is 5'-N 19-20 PAM-3'; N, and said N is A, G, C or T.

The DNA molecule expressing the adenine base editor and sgRNA may be introduced into the recipient plant using the method of PEG-mediated conversion, or using one of the gene gun method or the agricultural bacillary infection method. A method of introducing the base editing toolbox into rice protoplasts or callus can also be used. This is easily understood by those skilled in the art. Since it is well known to those skilled in the art that rice genomic DNA consists of two strands, the target nucleotide sequence can be located on any complementary strand thereof. This system, for example, involves the target nucleotide sequence being located in the sense strand of a functional gene, and after targeted mutation of A to G at a specific locus of the functional gene, one of the mutations , if it is possible to obtain a corresponding functional protein containing the desired amino acid, i.e., if it is possible to replace A in the triple codon with G by directly performing base substitution on the sense strand, This system can be used to obtain functionally "corrected" mutants of rice genes. Alternatively, the target nucleotide sequence is located in the antisense strand of a functional gene, and one targets a mutation of T to C at a specific site in the functional gene, and one of the mutations creates a corresponding protein containing the desired amino acid. This system can be used even if a functional protein is obtained. That is, according to this system, the amino acid encoded by the triplet codon on the sense strand is replaced with C for the corresponding complementary T on the sense strand by targeted mutation from A to G in the antisense strand. can be modified to obtain "corrected" mutants for the function of the rice gene.

In the above, the plants may be dicots or monocots. The monocot may be paddy rice. Single base editing can replace adenine A with guanine G.

This is a spectrum of pUbi-rBE. This is a spectrum of pENTR4:sgRNA. This is a spectrum of pUbi-rBE-sgRNA. FIG. 4 is a diagram showing the effects 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 without base editing, and the nucleotide sequence is sequence 14. T>C indicates the corresponding sequence of the mutant strain, the nucleotide sequence is sequence 15. FIG. 4 is a diagram showing the effect of adenine base editing mutation in the endogenous rice 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 rice japonica cultivar Kitaake without base editing, and the nucleotide sequence is sequence 16. A>G represents the corresponding sequence of the mutant, and the nucleotide sequence is sequence 17. FIG. 2 is a diagram showing the effect of rBE23, rBE50, and rBE53-mediated editing mutations of the adenine base of the endogenous rice gene OsDEP2. 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 without base editing, and the nucleotide sequence is sequence 18. 2A>2G represents the corresponding sequence of the mutant strain, the nucleotide sequence is sequence 19. FIG. 4 is a diagram showing the effects of rBE23, rBE50, and rBE53-mediated editing mutations of the adenine base of the endogenous rice gene OsWRKY45. 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 without 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. FIG. 3 is a diagram showing the effects of adenine base editing mutations in 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 without base editing, and the nucleotide sequence is sequence 22. 3A>3G represents the corresponding sequence of the mutant strain, the nucleotide sequence is sequence 23. 4A>4G represents the corresponding sequence of the mutant strain, the nucleotide sequence is sequence 24. FIG. 4 is a diagram showing the mutation effect of adenine base editing of the 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 strain, the nucleotide sequence is sequence 26. FIG. 3 is a diagram showing the effects of adenine base editing mutations in the rice endogenous gene OsMPK13 mediated by rBE62 and rBE65. 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 without base editing, and the nucleotide sequence is sequence 27. A>G represents the corresponding sequence of the mutant strain; the nucleotide sequence is sequence 28. 2A>2G represents the corresponding sequence of the mutant strain, the nucleotide sequence is sequence 29. FIG. 4 is a diagram showing the mutation effect of adenine base editing of the endogenous rice gene OsGSK 4 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, the nucleotide sequence is sequence 32. 4T>4C represents the corresponding sequence of the mutant strain, the nucleotide sequence is sequence 33. FIG. 3 is a diagram showing the mutation effect of adenine base editing of the endogenous rice 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, the nucleotide sequence is sequence 35; 3T>3C represents the corresponding sequence of the mutant strain, the nucleotide sequence is sequence 36. This is target nucleotide sequence information for each target gene. In the figure, the uppercase 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 position, and the lowercase tgtt corresponds to the BtgZI position. handle. Detection primers for each target gene are shown.

Hereinafter, the present invention will be explained in more detail based on specific examples. The examples provided are provided only to illustrate the invention rather than to limit its scope. The examples provided below do not constitute a limitation of the invention, but can serve as a guide for further improvements by those skilled in the art.

Experimental methods in the following examples, unless otherwise specified, are commonly used methods and are carried out according to techniques or conditions described in literature in the art or according to product specifications. Materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

pUbi-Cas 9 in the following examples is preserved and provided by the laboratory to which the inventors belong (H. Zhou, B. Liu, D. P. Weeks, M. H. 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 generally available from the inventor's laboratory and shall be used only to repeat experiments related to the present invention and for no other purpose.

Example 1 Targeted mutation from A to G in the rice genome This example provides an artificial mutant of adenosine deaminase TadA7.10 (hereinafter abbreviated as TadA7.10), namely adenosine deaminase TadA9 (hereinafter abbreviated as TadA9). did. TadA7.10 is a protein encoded by base numbers 598 to 1095 of SEQ ID NO: 12. TadA9 has the amino acid sequence at positions 1 to 167 in Sequence Listing 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.

In this example, two proteins, 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), were used as TadA9 controls. provides a control adenosine deaminase for adenine base editing. wtTadA-TadA 7.10 is an existing adenosine deaminase dimer commonly used in plants. TadA-R has two fewer amino acid sequence mutations, V 81 S and Q 153 R, compared to TadA 9, and the following mutations exist on the amino acid sequence compared to TadA 7.10:
A 108 S/T 110 R/D 118 N/H 121 N/Y 146 D/F 148 Y/T 165 I/D 166 N.

This example provides four rice adenine base editor expression vectors pUbi-rBE (Figure 1) containing TadA 9. The names of the rice adenine base editor expression vectors are pUbi-rBE 49 b, pUbi-rBE 53, pUbi-rBE 57, and pUbi-rBE 65, respectively. 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 an adenine base editor named TadA 9, SpCas 9 (D 10 A). It is a protein formed by combining a Cas protein named 10A) and a nuclear localization signal named NLS. The amino acid sequence of rBE 49 b is Sequence 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 NLS. It is an array. After codon optimization of the nucleotide sequence of the rBE49 b gene, which is a chimeric gene, based on Inecodon usage preference and a codon optimization strategy for heterologous protein expression, the 4743 bp rBE was transferred to Biotechnology (Shanghai) Co., Ltd. We requested artificial synthesis of the 49b gene. The small fragment (Cas 9) between the BamHI and BcuI recognition sites of pUbi-Cas 9 was replaced with the rBE 49 b gene shown at positions 7-4737 of sequence 1, maintaining the other nucleotides of pUbi-Cas 9. , the rBE 49 b gene expression vector pUbi-rBE 49 b was obtained. In sequence 1, positions 1 to 6 are the BamHI recognition site, positions 7 to 507 are the CDS of TadA 9, positions 508 to 603 are the CDS that binds the peptide, positions 604 to 4707 are the CDS of SpCas 9 (D 10 A), and 4708 Positions 4734 to 4734 were the CDS of NLS, positions 4735 to 4737 were the termination codon TGA, and positions 4738 to 4743 were the recognition site for BcuIno. pUbi-rBE 49 b contained the elements attR 1-ccdB-attR 2 for the LR reaction.

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)) and an adenosine deaminase named TadA 9, SpCas 9-NG. It is a protein formed by combining a Cas protein named ``NLS'' 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. This is the amino acid sequence of After codon-optimizing the nucleotide sequence of the chimeric gene rBE 53 gene based on the codon usage preference of rice, we 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, maintaining the other nucleotides of pUbi-Cas 9, The rBE 53 gene expression vector pUbi-rBE 53 was obtained. In sequence 3, positions 1 to 6 are the BamHI recognition site, positions 7 to 507 are the CDS of TadA 9, positions 508 to 603 are the CDS that binds the peptide, and positions 604 to 4707 are the CDS of SpCas 9-NG (D 10 A). , positions 4708 to 4734 were the CDS of NLS, positions 4735 to 4737 were the termination codon TGA, and positions 4738 to 4743 were the recognition site for BcuI. pUbi-rBE 50 contained the elements attR 1-ccdB-attR 2 for the LR reaction.

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)) and an adenosine deaminase named TadA 9, ScCas 9 (D 10 A). ) and a nuclear localization signal called 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. It is an array. After codon-optimizing the nucleotide sequence of the chimeric gene rBE 57 gene based on the codon usage preference of rice, we 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, without changing other nucleotides of pUbi-Cas9. The rBE57 gene expression vector rBE57 was obtained. In sequence 5, positions 1 to 6 are the BamHI recognition site, positions 7 to 507 are the CDS of TadA9, positions 508 to 603 are the CDS of the connecting peptide, positions 604 to 4728 are the CDS of ScCas9 (D10A), and positions 604 to 4728 are the CDS of ScCas9 (D10A). The CDS of ScCas9 (D10A), positions 4729-4755 are the CDS of NLS, positions 4756-4758 are the terminator codon TGA, and positions 4759-4764 are the BcuI recognition site. pUbi-rBE57 contained the elements attR1-ccdB-attR2 for the LR reaction of LR.

The adenine base editor expressed by pUbi-rBE 65 is a fusion protein named rBE 65 (also called TadA 9-SpRY (D 10 A)), an adenosine deaminase named TadA 9, and a fusion protein named SpRY (D 10 A). It is a protein formed by combining a Cas protein named Cas 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. It is. After codon-optimizing the nucleotide sequence of the chimeric gene rBE 65 gene based on the preference for use of Inekodon, we 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, without changing other nucleotides of pUbi-Cas 9. , the rBE 65 gene expression vector pUbi-rBE 65 was obtained. In sequence 7, positions 1 to 6 are the BamHI recognition site, positions 7 to 507 are the CDS of TadA 9, positions 508 to 603 are the CDS that connects the peptide, positions 604 to 4707 are the CDS of SPRY (D 10 A), and positions 4708 to Position 4734 was the CDS of NLS, positions 4735 to 4737 were the termination codon TGA, and positions 4738 to 4743 were the BcuI recognition site. pUbi-rBE 65 contained the elements attR 1-ccdB-attR 2 for the LR reaction.

pUbi-rBE 49 b, pUbi-rBE 53, pUbi-rBE 57, and pUbi-rBE 65 differ only in the gene encoding the adenine base editor rBE. The four types of adenine base editors, rBE49b, rBE53, rBE57, and rBE65, differ only in Cas protein.

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 Genbank accession number LC 506530. 1, positions 13973 to 13997, March 20, 2020), attR 1 (nucleotide sequence is Genbank accession number KR 233518.1, positions 2055 to 2179, August 8, 2015), ccdB expression cassette ( The nucleotide sequence is genbank accession number KR 233518.1, positions 3289 to 3594, August 8, 2015), attR 2 (the nucleotide sequence is genbank accession number KR 233518.1, positions 3635 to 3759, August 8, 2015) ), Ubip promoter (nucleotide sequence is sequence 9), rice adenine base editor rBE gene (rBE 49 b gene (nucleotide sequence is position 7 to 4737 of sequence 1), rBE 53 gene (nucleotide sequence is position 7 of sequence 3), -4737 position), rBE 57 gene (nucleotide sequence is position 7-4758 of sequence 5) or rBE 65 gene (nucleotide sequence is position 7-4737 of sequence 7), NOS terminator (nucleotide sequence is sequence 10), CaMV 35 S Promoter (nucleotide sequence is genbank accession number FJ 362600.1, positions 10414 to 11092, November 26, 2008), Ushiomycin gene (nucleotide sequence is genbank accession number KY 420085.1, positions 511 to 1536, July 2017) (September 10, 2019), CaMV poly(A) terminator (nucleotide sequence is genbank accession number MK 896900.1, positions 8618-8792, September 4, 2019), LB T-DNA repeat (nucleotide sequence is genbank accession number LC 506530) .1, 3635-3795, March 20, 2020).

Furthermore, 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, and the names of the control vectors used as controls for pUbi-rBE53 of the present invention are pUbi-rBE23 and pUbi-rBE50, Furthermore, the names of the control vectors of pUbi-rBE57 of the present invention are pUbi-rBE26 and pUbi-rBE54, and the name of the control vector of pUbi-rBE65 of the present invention is pUbi-rBE62.

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) and 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. rBE14 in the amino acid sequence replaces the adenosine deaminase named TadA9 in rBE-49 b with wtTadA-TadA7.10, a protein that combines a wild-type adenosine deaminase named wtTadA and a mutant adenosine deaminase named TadA7.10. The other amino acids are the same as the amino acid sequence of rBE49b, except that The rBE14 gene consists of the wtTadA-TadA 7. CDS of TadA 9 (nucleotide sequence, positions 7 to 507 of sequence 1) of the rBE 49 b gene (nucleotide sequence, positions 7 to 4737 of sequence 1) shown in sequence 12. This is a DNA molecule obtained by replacing 10 genes and leaving the other nucleotides of sequence 1 unchanged. Sequence 12 is the coding gene for the protein wtTadA-TadA 7.10, whose CDS is sequence 12. 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 and leaving other nucleotides of pUbi-rBE 49 b unchanged.

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), and a mutant adenosine base editor named TadA-R. It is a protein formed by combining a Cas protein named deaminase, SpCas 9 (D 10 A), and a nuclear localization signal named NLS. rBE 46 b has 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. be. The rBE 46 b gene is a combination of TadA-R CDS of TadA 9 (nucleotide sequence, positions 7 to 507 of sequence 1) of the rBE 49 b gene (nucleotide sequence, positions 7 to 4737 of sequence 1) shown in sequence 13. This is a DNA molecule obtained by replacing the gene sequence and leaving the other nucleotides of sequence 1 unchanged. 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 and leaving other nucleotides of pUbi-rBE 49 b unchanged. be.

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) and 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 converts the adenine deaminase named TadA 9 in rBE 53 into wtTadA-TadA 7.10, a protein that combines a wild-type adenine deaminase named wtTadA and a mutant adenine deaminase named TadA 7.10. Except for the substitutions, the other amino acids are exactly the same as the amino acid sequence of rBE53. The rBE 23 gene is the wtTadA-TadA 7. CDS of TadA 9 (the nucleotide sequence is from positions 7 to 507 of sequence 3) of the rBE 53 gene (the nucleotide sequence is from positions 7 to 4737 of sequence 3) shown in SEQ ID NO: 12. This is a DNA molecule obtained by replacing 10 genes and leaving the other nucleotides of sequence 3 unchanged. 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 in pUbi-rBE 53.

The adenine base editor expressed in pUbi-rBE 50 is a fusion protein named rBE 50 (also called wtTadA-TadA 7.10-SpCas 9-NG (D 10 A)-NLS) and a fusion protein named TadA-R. It is a protein formed by combining a mutant adenosine deaminase, a Cas protein named SpCas 9-NG (D 10 A), and a nuclear localization signal named NLS. rBE 50 has the same amino acid sequence as rBE 53 except that the adenosine deaminase named TadA 9 of rBE 53 is replaced with a mutant adenosine deaminase named TadA-R. The rBE 50 gene is obtained by replacing the CDS of TadA 9 (nucleotide sequence is from positions 7 to 507 of sequence 1) of the rBE 53 gene (nucleotide sequence is from positions 7 to 4737 of sequence 3) with the TadA-R gene shown in sequence 13. , the DNA molecule obtained without changing the other nucleotides of sequence 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 and leaving other nucleotides of pUbi-rBE 53 unchanged.

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 contains a wild-type adenine deaminase named wtTadA, TadA It is a protein formed by combining a mutant adenine deaminase named 7.10, a Cas protein named ScCas 9 (D 10 A), and a nuclear localization signal named NLS. rBE 26 is a protein wtTadA-TadA 7.10, which combines an adenine deaminase named TadA 9 of rBE 57 with a wild-type adenine deaminase named wtTadA and a mutant adenine deaminase named TadA 7.10. Except for the substitutions, the other amino acids are exactly the same as the amino acid sequence of rBE 57. The rBE 26 gene is wtTadA-TadA 7.10, which has the CDS of TadA 9 (nucleotide sequence, positions 7 to 507 of sequence 5) of the rBE 57 gene (nucleotide sequence, positions 7 to 4758 of sequence 5) shown in sequence 12. This is a DNA molecule obtained by replacing the gene with the other nucleotides of sequence 5 unchanged. 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 and leaving other nucleotides of pUbi-rBE 57 unchanged.

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) and a mutant adenosine deaminase named TadA-R. , is a protein formed by combining a Cas protein named ScCas 9 (D 10 A) and a nuclear localization signal named NLS. rBE 54 has the same amino acid sequence as rBE 57 in other amino acids, 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 consists of the TadA 9 CDS (nucleotide sequence, positions 7 to 507 of sequence 5) of the rBE 57 gene (nucleotide sequence, positions 7 to 4758 of sequence 5) to the TadA-R gene sequence shown in sequence 13. This is the DNA molecule obtained without changing the other nucleotides of sequence 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 other nucleotides in pUbi-rBE 57.

The adenine base editor expressed by pUbi-rBE 62 is a fusion protein named rBE 62 (TadA-R-SpRY (D 10 A)-NLS), and a mutant adenosine deaminase named TadA-R, SpRY ( It is a protein formed by combining a Cas protein named D 10 A) and a nuclear localization signal named NLS. rBE 62 has 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 consists of the TadA 9 CDS (nucleotide sequence, positions 7 to 507 of sequence 7) of the rBE 65 gene (nucleotide sequence, positions 7 to 4737 of sequence 7) to the TadA-R gene sequence shown in sequence 13. This is the DNA molecule obtained by replacing the other nucleotides of sequence 7 without changing them. 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 other nucleotides in pUbi-rBE 65.

2. A>G substitution of the target base of the rice endogenous gene using the rice adenine base editor expression vector 1. Construction of the base editing vector pUbi-rBE-sgRNA for the target sequence Genomic DNA sequence of the selected target gene (see Figure 13) are obtained from the rice genome database (https://rapdb.dna.affrc.go.jp/), and each base editor creates the corresponding target sequence and forward and reverse oligonucleotide strands according to the requirements for identifying PAMs. Designed. The synthesis of forward and reverse oligonucleotide chains (see FIG. 13 for specific sequences) of each target sequence (5'-N19PAM-3') in FIG. 13 was entrusted to NTT DoCoMo, Inc. After artificial synthesis, the primer is phosphorylated using T4 polynucleotide kinase and annealed to form a double-stranded DNA fragment (5'-N19-3' for a target sequence containing sgRNA), and a double-stranded DNA fragment were cloned into the two BtgZI or two BsaI enzyme cleavage sites of the pENTR4:sgRNA (Fig. 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 insert fragment is completely accurate (the insert fragment is 5'-N19 of the target sequence containing the sgRNA) -3'), the obtained plasmid was linearized by AatII enzyme cut, and the sgRNA expression cassette (code DNA containing sgRNA) was transformed into attR1- of the rice adenine base editor expression vector pUbi-rBE (Fig. 1) by Gateway's LR reaction. Each target sequence was cloned into ccdB-attR2 to obtain a base editing vector pUbi-rBE-sgRNA for each target sequence (Figure 3). Recombinant expression vectors obtained by replacing cassette-attB2 and leaving other nucleotides of pUbi-rBE unchanged. pUbi-rBE49b-sgRNA-Ostms9, pUbi-rBE14-sgRNA-Ostms9 targeting OsTms9 gene. , pUbi-rBE46b-sgRNA-Ostms9, and pUbi-rBE46b-sgRNA-Ostms9. rBE49b-sgRNA-OsWRKY45, pUbi-rBE14-sgRNA-OsWRKY45, and pUbi-rBE46b-sgRNA-OsWRKY45.pUbi-rBE53-sgRNA-OsDEP2, pUbi-rBE targeting the OsDEP2 gene. 23-sgRNA-OsDEP2 and pUbi- Three types of base editing vectors, rBE62-sgRNA-OsJAR2, were obtained.Three types of base editing vectors targeting target sequence 2 (sequence 79, including NGT PAM) of the OsWRKY45 gene were obtained, pUbi-rBE53-sgRNA, respectively. The three base editing vectors targeting the OsGS1 gene were pUbi-rBE57-sgRNA-OsGS1 and pUbi-rBE54-, respectively. sgRNA-OsGS1, and pUbi-rBE26-sgRNA-OsGS1. The two base editing vectors targeting the OssERK2 gene were pUbi-rBE57-sgRNA-OssERK2 and pUbi-rBE54-sgRNA-OssERK2. The two types of base editing vectors targeting the OsMPK13 gene were pUbi-rBE65-sgRNA-OsMPK13 and pUbi-rBE62-sgRNA-OsMPK13. The two types of base editing vectors targeting the OsGSK4 gene were pUbi-rBE65-sgRNA-OsGSK4 and pUbi-rBE62-sgRNA-OsGSK4. The two types of base editing vectors targeting the OsJAR1 gene were pUbi-rBE65-sgRNA-OsJAR1 and pUbi-rBE62-sgRNA-OsJAR1.

Here, the construction method of pENTR4:sgRNA is as follows:
In the direction from the 5' end to the 3' end: U6 promoter sequence 1, nucleotide sequence containing two BtgZI enzyme cut sites, sgRNA scaffold sequence, (T)8 terminator sequence, U6 promoter sequence 2, two BsaI enzyme cut sites. The containing nucleotide sequence, sgRNA scaffold sequence, and (T)8 terminator sequence were sequentially combined to form an sgRNA expression cassette. This was outsourced to Seikou Biotechnology (Shanghai) Co., Ltd., and was artificially synthesized. Using the company's synthesized gene as a template, primer pairs (sgRNA-F: 5'-GCAGGCTGTCGACTGGATCCAAGCTTAAGAACGAACTAAGCC-3' (sequence 38) and sgRNA-R: 5'-CAAGAAAGCTGGGTGAATTCGATATCCAAGCTTATCG ATACCG-3' (sequence 39)) was amplified to 1 kb. An sgRNA expression cassette fragment (nucleotide sequence is sequence 11 in the sequence listing) was obtained. Using the pENTR4 (Invitrogen) vector as a template, pENTR4-F1: (5'-CGAATTCACCCAGCTTTCTTGTACAAAGTTGGCATTATAAGA-3' (sequence 40) and pENTR4-R1: (5'-CTTAGTTCGTTCTTAAGCT TGGATCCAGTCGACAGCCTGCTTTTTTGTACAAAGT-3' (sequence 41) was amplified and a 2.2 kb A pENTR4 vector backbone (a DNA fragment from which the ccdB gene expression cassette fragment of pENTR4 was removed) was obtained. Using the reagent cassette ClonExpress II One Step Cloning Kit (purchased from Nanjing Nuoyishan Bioscience and Technology Co., Ltd.), the sgRNA expression cassette was The fragment and the pENTR 4 vector backbone were infusion-ligated to obtain the vector pENTR 4:sgRNA (Fig. 2).Two BtgZI or two BsaI enzyme cut sites were inserted into the identification sequence of the specific gene in the clone (the target of sgRNA). 5'-N19-3') in the sequence.In sequence 11, positions 27-348 are U6 promoter sequence 1, positions 349-389 are a nucleotide fragment containing two BtgZI sites, and positions 390-465. is the sgRNA scaffold sequence, positions 466 to 473 are the (T)8 terminator sequence, positions 474 to 782 are the U6 promoter sequence 2, positions 783 to 806 are the nucleotide fragment containing two BsaI sites, and positions 807 to 882 are the sgRNA scaffold sequence. , 883-890 is a (T)8 terminator sequence.

2. Agricultural bacilli mediate stable genetic transformation of paddy rice The obtained editing vectors for each target gene base were transformed into agricultural bacillus EHA 105 by electric shock method (EHA 105 electric shock receptor cells were purchased from Beijing Boya Gene Technology Co., Ltd. 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.), genetic conversion mediated by rice agricultural bacilli was performed to obtain T0 generation rice materials with base editing for each target gene.

3. Measurement of base editing efficiency of target gene locus in T 0 generation transgenic rice Genomic DNA of the T 0 generation transgenic rice material was extracted using the usual CTAB method.

The target sequence information of each target gene and its detection primers are as shown in FIG. 14, and all the primers were artificially synthesized by Seiko Biotechnology (Shanghai) Co., Ltd. All PCR reactions involved in testing were performed using the high-fidelity enzyme premix I-5 ^TM 2×High-Fidelity Master Mix (purchased from Keluning (Beijing) Biological Science and Technology Co., Ltd.) to perform PCR amplification of the target fragment. Ta. Sanger sequencing of the PCR products was outsourced to Direct Biotechnology (Shanghai) Co., Ltd.

The sequencing results are as follows:
Target base editing efficiency for NGGPAM targets in Ostms 9 by base editor rBE 14 is 0%, target base editing efficiency for NGG PAM targets in Ostms 9 by base editor rBE 46 b is 64.58%, by base editor rBE 49 b , the target base editing efficiency of Ostms 9 for the NGG PAM target was 97.92% (Table 1).

Among the 54 T0 generation trans-pUbi-rBE14-sgRNA-OsTms9 rice lines, there were 0 lines in which adenine A was replaced with guanine G by deammonization. Among the 48 T0 generation trans-pUbi-rBE46b-sgRNA-OsTms9 rice lines, there were 31 lines in which adenine A was replaced with guanine G by deammonia. In all of them, A at position 5 (corresponding to _T5 in FIG. 4) in the 5' to 3' direction of the target sequence could be replaced with G by deammonization. In 47 of the 48 T0 generation trans-pUbi-rBE49b-sgRNA-OsTms9 rice lines tested, adenine A was replaced with guanine G by deammonia, and in all cases, adenine A was replaced with guanine G in the 5' to 3' direction of the target sequence. A at position 5 (corresponding to T5 in FIG. 4) could be replaced with G by deammonization.

Target base editing efficiency of OsWRKY 45 for NGGPAM target by base editor rBE 14 is 0.00%, Target base editing efficiency for OsWRKY 45 for NGG PAM target by base editor rBE 46 b is 0.00%, Base editor rBE 49 The target base editing efficiency of OsWRKY 45 for the NGG PAM target by b was 2.86% (Table 1). Among the 53 T 0 generation trans pUbi-rBE 14-sgRNA-OsWRKY 45 rice plants detected, there were 0 plants in which adenine A was replaced with guanine G by deammonia. Among the 48 T 0 generation trans pUbi-rBE 46 b-sgRNA-OsWRKY 45 rice strains detected, there were 0 strains in which adenine A was replaced with guanine G by deammonia. Among the 35 T 0 generation trans pUbi-rBE 49 b-sgRNA-OsWRKY 45 rice strains detected, there was one strain in which adenine A was replaced with guanine G by deammonization, and in all of them, the 5′ of the target sequence A at the 4th position in the 3' direction could be substituted with G by deammonization (corresponding to A4 in Figure 5).

Target base editing efficiency of OsDEP 2 against NGA PAM target by base editor rBE 23 is 0.00%, target base editing efficiency of OsDEP 2 against NGA PAM target by base editor rBE 50 is 27.08%, base editor rBE 53 The target base editing efficiency of OsDEP 2 against the NGA PAM target was 78.72% (Table 2). Among the 96 T 0 generation trans pUbi-rBE 23-sgRNA-OsDEP 2 rice strains detected, 0 had adenine A replaced with guanine G by deammonia. Among the 48 T 0 generation trans pUbi-rBE 50-sgRNA-OsDEP 2 rice strains detected, there were 13 strains in which adenine A was replaced with guanine G by deammonia. Among those that could replace A at positions 4 and 6 with G in the 5' to 3' direction of the target sequence by deammoniation, adenine A at position 4 (corresponding to A 4 in Figure 6) was replaced with G by deammonia. There were 10 strains with substitutions, and 13 strains with adenine A at position 6 (corresponding to A6 in Figure 6) replaced with G by deammonization. Among 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 deammonia. Among the A in the 4th and 6th positions in the 5' to 3' direction of the target sequence that could be replaced with G by deammoniation, adenine A in the 4th position (corresponding to A 4 in Figure 6) was replaced by G by deammoniation. There were 37 strains in which G was substituted, and 28 strains in which adenine A at position 6 (corresponding to A6 in Figure 6) was substituted with G by deammonia.

Target base editing efficiency of OsWRKY 45 for NGPAM target by base editor rBE 23 is 0%; Target base editing efficiency of OsWRKY 45 for NGPAM target by base editor rBE 50 is 89.36%; OsWRKY 45 by base editor rBE 53 The target base editing efficiency for the NGT PAM target was 93.75% (Table 2). Among the 52 T 0 generation trans pUbi-rBE 23-sgRNA-OsWRKY 45 rice strains detected, there were 0 strains in which adenine A was replaced with guanine G by deammonia. Of the 47 T 0 generation trans pUbi-rBE 50-sgRNA-OsWRKY rice strains detected, there were 42 strains in which adenine A was replaced with guanine G by deammonia, and all of them were found to have a 5′ of the target sequence. A at the 5th position in the 3' direction could be substituted with G by deammoniation (corresponding to T ₅ in FIG. 7). Among the 48 T 0 generation trans pUbi-rBE 53-sgRNA-OsWRKY rice strains detected, there were 45 strains in which adenine A was replaced with guanine G by deammonia, and in all of them, 5' of the target sequence A at the 5-position in the 3' direction could be substituted with G by deammonization (corresponding to _T5 in FIG. 7).

Target base editing efficiency for NAG PAM targets in OsGS 1 by base editor rBE 26 is 0.00%; target base editing efficiency for NAG PAM targets in OsGS 1 by base editor rBE 54 is 25.00%; base editor rBE 57 The target base editing efficiency of OsGS 1 against the NAG PAM target was 54.17% (Table 3). Among the 36 T 0 generation trans pUbi-rBE 26-sgRNA-OsGS 1 rice plants detected, there were 0 plants in which adenine A was replaced with guanine G by deammonia. Among the 48 T 0 generation trans pUbi-rBE 54-sgRNA-OsGS 1 rice strains detected, there were 12 strains in which adenine A was replaced with guanine G by deammonia, and the 5' to 3' of the target sequence Among those in which A at the 3rd, 6th, and 9th positions in the direction of can be replaced with G by deammoniation, adenine A at the 3rd position (corresponding to A ₃ in Figure 8) is replaced by G by deammonia. 3 strains, 11 strains in which adenine A at position 6 (corresponding to A ₆ in Figure 8) was replaced with G by deammonia, and adenine A at position 9 (corresponding to A ₉ in Figure 8) There were 12 strains that were replaced with G by deammonization. Among the 48 T 0 generation trans pUbi-rBE 57-sgRNA-OsGS 1 rice strains detected, there were 23 strains in which adenine A was replaced with guanine G by deammonization, and the 5' to 3' of the target sequence Among the A in the 3rd, 4th, 6th, 9th, and 11th positions in the direction of , which can be replaced with G by deammoniation, adenine A in the 3rd position (corresponding to A ₃ in Figure 8) is replaced by G by deammonia. 26 strains had the substitution, 13 strains had the adenine A at the 4th position (corresponding to _A4 in Figure 8) replaced with G by deammonia, and adenine A at the 6th position (corresponding to A4 in Figure 8). 26 strains had A (corresponding to A ₆ in Figure 8) replaced with G by deammoniation, 26 strains had adenine A at position 9 (corresponding to A 9 in Figure 8) replaced with G by deammonia, and 26 strains had the adenine A at position 9 (corresponding to A ₉ in Figure 8) replaced with G by deammonia, and 26 strains had the 11th position There was one strain in which adenine A (corresponding to _A11 in Figure 8) was replaced with G by deammonia.

The target base editing efficiency for the NAG PAM target of OssERK2 using the base editor rBE54 was 28.26%, and the target base editing efficiency for the NAG PAM target of OssERK2 using the base editor rBE57 was 72.34% (Table 3). Among the 46 T 0 generation trans pUbi-rBE 54-sgRNA-OssERK 2 rice strains detected, there were 13 strains in which adenine A was replaced with guanine G by deammonia. Among the A in the 4th and 6th positions in the 5' to 3' direction of the target sequence that could be replaced with G by deammoniation, the adenine A in the 4th position (corresponding to _T4 in Figure 9) was replaced by G by deammoniation. There were 3 strains in which the substitution was made with G, and 13 strains in which the adenine A at position 6 (corresponding to T ₆ in FIG. 9) was substituted with G by removal of ammonia. Among the 47 T 0 generation trans pUbi-rBE 57-sgRNA-OssERK 2 rice strains detected, there were 34 strains in which adenine A was replaced with guanine G by deammonia, and the 5' to 3' of the target sequence Among those in which A at the 4th and 6th positions in the direction of ₃₄ were replaced with G by deammoniation, 34 There were 34 strains in which adenine A at position 6 (corresponding to T ₆ in FIG. 9) was replaced with G by deammonization.

The target base editing efficiency for the NAA PAM target of OsMPK 13 using the base editor rBE 62 was 29.17%, and the target base editing efficiency for the NAA PAM target of OsMPK 13 using the base editor rBE 65 was 31.91% (Table 4). there were. Among the 48 T 0 generation trans pUbi-rBE 62-sgRNA-OsMPK rice strains detected, there were 14 strains in which adenine A was replaced with guanine G by deammonization, and in all of them, A at position 6 in the 3′ direction (corresponding to A ₆ in FIG. 10) was substituted with G by deammoniation. Among the 47 T 0 generation trans pUbi-rBE 65-sgRNA-OsMK rice strains detected, there were 15 strains in which adenine A was replaced with guanine G by deammonia, and the 5' to 3' of the target sequence Among those in which A at the 4th and 6th positions in the direction of ₂ can be replaced with G by deammoniation, 2 There were 15 strains in which adenine A at position 6 (corresponding to _A6 in FIG. 10) was replaced with G by deammonization.

The target base editing efficiency for the NAC PAM target of OsGSK 4 using the base editor rBE62 was 51.28%, and the target base editing efficiency for the NAC PAM target of OsGSK 4 using the base editor rBE65 was 73.17% (Table 4). Ta. Among the 39 T 0 generation trans pUbi-rBE 62-sgRNA-OsGSK 4 rice strains detected, there were 20 strains in which adenine A was replaced with guanine G by deammonization, and the 5' to 3' of the target sequence Among the strains in which A at the 4th and 11th positions in the direction of A could be replaced with G by deammoniation, 20 strains had adenine A at the 4th position (corresponding to T ₄ in Figure 11) replaced by G by deammonification. There were two strains in which adenine A at position 11 (corresponding to T11 in FIG. ₁₁ ) was replaced with G by deammonization. Of the 41 T0 generation trans pUbi-rBE 65-sgRNA-OsGSK4 rice strains detected, 30 strains had adenine A replaced with guanine G by deammonia, and the 5' to 3' of the target sequence. Among those that were able to replace A at positions 4, 8, 9, and 11 with G by deammoniation, adenine A at the 4th position (corresponding to T 4 in Figure 11) was replaced with G by deammonia. There are 30 strains, one strain has adenine A at position 8 (corresponding to T 8 in Figure 11) replaced with G by deammonia, and adenine A at position 9 (corresponds to T 9 in Figure 11). ) was replaced with G by deammoniation in one strain, and nine strains were found in which adenine A at position 11 (corresponding to T11 in FIG. 11) was replaced by G by deammonization.

The target base editing efficiency for OsJAR 1's NAT PAM target by base editor rBE 62 was 36.17%, and the target base editing efficiency for OsJAR 1's NAT PAM target by base editor rBE 65 was 45.00% (Table 4). there were. Among the 47 T 0 generation trans pUbi-rBE 62-sgRNA-OsJAR 1 rice strains detected, there were 17 strains in which adenine A was replaced with guanine G by deammonization, and the 5' to 3' of the target sequence Among the strains in which A at positions 4 and 6 in the direction of A could be replaced with G by deammoniation, 17 strains had adenine A at position ₄ (corresponding to T4 in Figure 12) replaced by G by deammonia. There were 9 strains in which adenine A at position 6 (corresponding to T ₆ in FIG. 12) was replaced with G by deammonization. Among the 40 T 0 generation trans pUbi-rBE 65-sgRNA-OsJAR 1 rice strains detected, there were 18 strains in which adenine A was replaced with guanine G by deammonization, and the 5' to 3' of the target sequence Of those in which A at the 2nd, 4th, and 6th positions in the direction of can be replaced with G by deammoniation, the adenine A at the 2nd position (corresponding to T 2 in Figure 12) is replaced by G by deammonia. 3 strains, 18 strains in which adenine A at the 4th position (corresponding to T 4 in Figure 12) was replaced with G by deammonia, and adenine A at the 6th position (corresponding to T 6 in Figure 12) There were 18 strains in which G was substituted by deammonia.

From the above results, the editing efficiency of the adenine base editing technology mediated after the fusion of TadA 9 and SpCas 9 n/ScCas 9/SpCas 9-NG/SpRY in the present invention is higher than that of TadA-R mutants such as TadA 7.10. It is much higher than the adenine base editing technology mediated by , and the editing activity window can be extended, increasing the efficiency of the adenine base editing technology and expanding its application range.

The present invention has been described in detail above. Those skilled in the art will be able to practice the invention over a wide range of equivalent parameters, concentrations and conditions without departing from the purpose and scope of the invention and without undue experimentation. Although the invention has been given particular embodiments, it should be understood that further modifications may be made thereto. In summary, in accordance with the principles of the invention, this application includes modifications that may be made using conventional techniques known in the art that are outside the scope of the disclosure of this application. Any adaptations, uses, or improvements of the invention are intended to be covered. Applications of the essential features are possible according to the appended claims.

The present invention obtains a highly active mutant TadA9 by artificially mutating the TadA protein, and uses this to develop a novel CRISPR/Cas-based editing toolkit. Adenine base editing technology via fusion with SpCas9(D10A), SpCas9-NG(D10A), ScCas9(D10A) and SpRY(D10A) is superior to adenine base editing technology via TadA7.10 and TadA-R mutants. It also has extremely high editing efficiency, which can extend the scope of its editing activity, improve the efficiency of adenine base editing technology, and expand its application range. In particular, we solved the problem of adenine base editing at targets that cannot be edited with TadA7.10-mediated base editing vectors, expanded the scope of use for targeted editing of plant genomes, and provided information to researchers in the fields of plant research and crop gene improvement. It can provide an important toolset for functional research and modification. The present invention can improve the efficiency of adenine base editing and accurately mediate base mutations at target sites, and can be widely applied to rice and other plant cells.

Claims (12)

A protein characterized in that the amino acid sequence of the protein is from positions 1 to 167 of SEQ ID NO: 2 in the sequence listing. A biomaterial related to the protein according to claim 1, characterized in that it 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 a recombinant vector comprising 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 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 recombinant vector in B3). The biological material according to claim 2, wherein the nucleic acid molecule in B1) is a gene shown in B11) or B12) below:
B11) A cDNA molecule or DNA molecule in which the coding sequence of the coding strand is represented by positions 7 to 507 of SEQ ID NO: 1;
B12) A cDNA molecule or DNA molecule that has 80% or more identity with the nucleotide sequence defined in B11) and encodes the protein. A protein comprising a Cas protein and adenosine deaminase,
A fusion protein, wherein the adenosine deaminase is the protein according to claim 1. The fusion protein according to claim 4, wherein the Cas protein is ScCas9 (D10A), SpRY (D10A), SpCas9 (D10A) or SpCas9-NG (D10A). 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. The fusion protein is TadA9-ScCas9 (D10A), TadA9-SpRY (D10A), TadA9-SpCas9 (D10A) or TadA9-SpCas9-NG (D10A),
The TadA9-SpCas9 (D10A) is a protein whose amino acid sequence is SEQ ID NO: 2, the TadA9-SpCas9-NG (D10A) is a protein whose amino acid sequence is SEQ ID NO: 4, and the TadA9-ScCas9 (D10A) is a protein whose amino acid sequence is SEQ ID NO: 4. 7. The fusion protein according to claim 6, wherein the protein number 6, TadA9-SPRY (D10A), is a protein whose amino acid sequence is SEQ ID NO: 8. A biomaterial related 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 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 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 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 recombinant vector in D3). The DNA molecule in D1) is the gene encoding TadA9-ScCas9 (D10A), the gene encoding TadA9-SpRY (D10A), the gene encoding TadA9-SpCas9 (D10A), or TadA9-SpCas9 according to claim 4. - A gene encoding NG (D10A);
The gene encoding TadA9-SpCas9 (D10A) is
D131) A cDNA molecule or a DNA molecule in which the coding sequence of the coding strand is represented by positions 7 to 4737 of SEQ ID NO: 1; or
D132) A cDNA molecule or DNA molecule having 80% or more identity with the nucleotide sequence defined in D131) and encoding the TadA9-SpCas9 (D10A);
The gene encoding TadA9-SpCas9-NG (D10A) is
D141) A cDNA molecule or DNA molecule whose coding strand sequence is represented by SEQ ID NOs: 3, 7 to 4737; or
D142) A cDNA molecule or DNA molecule that has 80% or more identity with the base sequence defined in D141) and encodes the TadA9-SpCas9 (D10A);
The gene encoding TadA9-ScCas9 (D10A) is
(D111) A cDNA molecule or DNA molecule whose coding sequence of the coding strand is represented by positions 7 to 4758 of SEQ ID NO: 5; or
D112) A cDNA molecule or a DNA molecule that has 80% or more identity with the nucleotide sequence defined in D111) and encodes the TadA9-ScCas9 (D10A);
The gene encoding TadA9-SpRY (D10A) is
(D121) a cDNA molecule or DNA molecule in which the coding sequence of the coding strand is represented by positions 7 to 4737 of SEQ ID NO: 7; or
D122) A cDNA molecule or a DNA molecule having 80% or more identity with the nucleotide sequence defined in D121) and encoding the TadA9-SpRY (D10A); 8. The biomaterial according to 8. The protein according to claim 1, the biomaterial according to claim 2 or 3, the fusion protein according to any one of claims 4 to 7, or the biomaterial according to claim 8 or 9 in plants. Use for single base editing. Use according to claim 10, characterized in that the plant single base editing is a targeted mutation from A to G in the plant genome. introducing a DNA molecule expressing an adenine base editor and sgRNA into a recipient plant to obtain a target plant with a targeted mutation of A to G;
The target sequence of the sgRNA is 5'-N _19-20 PAM-3', where the N _19-20 is 19-20 N, the PAM is 3 N, and the N is A, A method for targeted mutation of A to G in a plant genome, characterized in that it is G, C or T.

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