Corn genome editing vector and method and application thereof

Abstract

The invention discloses a corn gene editing vector, a gene editing method of the corn gene editing vector and application of the corn gene editing vector, and particularly provides a nucleotide sequence for encoding CasWM protein and aiming at corn genome codon optimization, and a nucleic acid construct and a kit for gene editing. High gene editing efficiency can be obtained, and the method can be used for improving corn characters and cultivating new corn varieties.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, to a maize genome editing vector, its method and application. Background Art

[0002] In many bacteria and most archaea, there is a defense system for resisting foreign DNA (such as phages), called the CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated Cas endonuclease) system (Bhaya et al., 2011). The CRISPR / Cas system contains two basic components: one is the endonuclease Cas protein, and the other is the guide RNA (gRNA). Under specific conditions, the gRNA can form a complex with the Cas protein and guide the Cas protein to the target DNA through base complementary pairing, so that the Cas protein binds to and cleaves the target double-stranded DNA, resulting in a double-strand break. Subsequently, the DNA repair mechanism of the cell will repair the damaged double-stranded DNA, including non-homologous end joining (NHEJ) and homologous recombination repair (HDR) (Hsu et al., 2014).

[0003] The plant gene editing technology developed using the CRISPR / Cas system is a key technology in crop breeding. Researchers have carried out a large number of downstream technology development and application studies based on classic gene editing nucleases such as Cas9 and Cpf1, and have achieved a series of important results in the field of plant gene editing.

[0004] According to the number and composition of Cas proteins involved in endonuclease cleavage of the genome, the CRISPR / Cas system is divided into class I (types I, III, and IV) and class II (types II, V, and VI) systems. In class II systems, the type II endonuclease Cas9 contains an HNH nuclease domain inserted into the RuvC nuclease domain; these two domains work together to cleave double-stranded DNA 3 bases upstream of the PAM (5’-NGG-3’) site, generating blunt ends. By introducing point mutations in the nuclease domain, nickase-Cas9 (nCas9) or dead-Cas9 (dCas9) can be generated for the development of base editors and gene expression regulators. Different from Cas9, type V Cas12a has a single RuvC nuclease domain, which cleaves double-stranded DNA by recognizing a T-rich PAM sequence (5’-TTTN-3’), generating sticky ends, thus expanding the available target range.

[0005] In the past few years, Cas9 (type II) and Cas12a have been widely used in genome editing of various organisms, including microorganisms, animals, and plants. Due to the diversity of type V CRISPR / Cas systems, many newly reported Cas proteins are members of the large family of type V endonucleases. Among them, CRISPR / Cas12 shows potential as a good genome editing tool because of its relatively small size, simple crRNA, "TTN" PAM requirement, and pre-crRNA processing ability.

[0006] Cas12 is a single crRNA-guided effector that mainly cleaves the non-spacer complementary strand of the target DNA and cleaves the spacer complementary strand with a lower efficiency, generating double-stranded DNA breaks. Some Cas12 family members, including Cas12i1, Cas12i2, Cas12i3, Cas12i7, Cas12i10, Cas12i11, and Cas12i12, have demonstrated their gene editing activity in mammalian cells. In 2023, researchers identified Cas12i3, a member of the endonuclease family V-I, to determine its application in plant genome editing. This protein can recognize the PAM of TTN, and its average editing efficiency at four rice endogenous loci (OsYSA, OsNAL, OsMIR396e, OsPYL6) reached 36.31%. The engineered Cas12i3 (Cas-SF01) has an extended PAM range, can effectively recognize the PAMs of NTTN as well as non-canonical NATN and TTVN, and the editing efficiency is further improved, up to 80% in rice.

[0007] Currently, the number of domestically self-patented Cas12i endonucleases is small, which to a certain extent hinders the process of gene editing crop breeding in China. In addition, most researchers focus on optimizing the amino acid sequence of nuclease proteins, resulting in a relatively single protein efficiency optimization strategy. Currently, there are very few reports on the efficient realization of Cas12 protein-mediated gene editing in maize, and the gene editing efficiency is also low. Summary of the Invention

[0008] Aiming at at least one defect or improvement requirement of the prior art, the inventors of the present invention have greatly improved the editing efficiency of the CasWM protein by combining a variety of editing vector optimization strategies, including optimizing the nucleotide sequence of the CasWM protein and the crRNA expression structure, and finally screened out a new gene editing vector with high editing efficiency for the maize genome. This new editing tool further expands the plant Cas12 family gene editing tools and can be used to cultivate new maize varieties.

[0009] To achieve the above object, according to one aspect of the present invention, there is provided a codon-optimized nucleotide sequence encoding CasWM protein, and the nucleotide sequence is SEQ ID NO.1 or SEQ ID NO.2, preferably SEQ ID NO.2.

[0010] In another embodiment, the present invention provides a nucleic acid construct for editing the maize genome, and the nucleic acid construct includes:

[0011] A first expression cassette for expressing CasWM protein;

[0012] A second expression cassette for expressing sgRNA;

[0013] And a third expression cassette for expressing a maize transformation selection marker;

[0014] The first expression cassette includes the nucleotide sequence described in claim 1 and a first promoter; the second expression cassette includes sgRNA and a second promoter.

[0015] Preferably, the second expression cassette includes an element of formula I:

[0016] P1-X1-L1-H1:

[0017] Wherein P1 is a U6 composite promoter sequence; X1 is a tRNA sequence; L1 is a crRNA sequence; H1 is an HDV sequence; each "-" is independently a bond or a nucleotide linking sequence.

[0018] Preferably, the U6 composite promoter includes an element of formula II:

[0019] P2-X2-L2;

[0020] Wherein P2 is a 35S enhancer sequence; X2 is a CMYLCV sequence; L2 is a U6 promoter; the nucleotide sequence of the U6 promoter is any one of SEQ ID NO.3 to SEQ ID NO.5, preferably the nucleotide sequence shown in SEQ ID NO.5.

[0021] In another embodiment, the first promoter initiates the expression of CasWM. In a preferred example, the first expression cassette uses the maize ubiquitin gene promoter (ZmUBI) to initiate the expression of CasWM, adds NLS (nuclear localization signal peptide) to the C-terminus and N-terminus of CasWM, and uses the NOS terminator;

[0022] In another embodiment, the third expression cassette includes P3-X3-L3(II)

[0023] Among them, P3 is the third promoter sequence; including but not limited to the 35S promoter, the CMV promoter, the CAG promoter, and the PGK promoter;

[0024] X3 is a maize transformation selection marker, preferably the Bar selection marker;

[0025] L3 is the polyA sequence;

[0026] Furthermore, an expression vector is also provided, and the expression vector contains the nucleic acid construct described in any one of the above.

[0027] Furthermore, a host cell is also provided, and the cell contains the above expression vector, or its genome integrates the nucleic acid construct described in any one of the above.

[0028] Furthermore, a kit for maize genome editing is also provided, including: the above nucleic acid construct, or an expression vector containing the nucleic acid construct.

[0029] Furthermore, a maize breeding method is also provided, including the following steps:

[0030] (i) Provide the plant or plant cell to be edited;

[0031] (ii) Introduce the expression vector containing the nucleic acid construct described in claim 3 into the plant or plant cell to be edited, so as to achieve the editing of the target gene of the plant or plant cell.

[0032] Generally speaking, compared with the prior art through the above technical solutions conceived by the present invention, the following beneficial effects can be achieved:

[0033] (1) For a maize genome editing vector, its method and application provided by the present invention, in order to improve the editing efficiency of the Cas12 family in maize, the inventors optimized the structure of the editing vector from multiple aspects. First, the codons of the CasWM nucleotide sequence were optimized and T5 exonuclease was added to improve the editing efficiency of the CasWM protein in maize; secondly, the expression structure of crRNA was optimized. By using the optimized U6 composite promoter to express crRNA, and adding tRNA and HDV sequences to the upstream and downstream of crRNA respectively to stabilize the structure of crRNA, while enhancing the expression level of sgRNA and stabilizing its structure, the editing efficiency of the CasWM protein was further greatly improved, and finally a gene editing vector with extremely high editing efficiency in maize was obtained.

[0034] (2) The maize genome editing vector provided by the present invention, its method and application. The maize genome editing vector uses the finally optimized vector with the best editing effect to edit endogenous genes in maize. After stable genetic transformation of maize, efficient editing has been achieved at multiple endogenous target sites in maize, and the highest editing efficiency has reached more than 97%. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a schematic structural diagram of the gene editing vector provided by an embodiment of the present invention;

[0036] Figure 2 is a schematic structural diagram of three U6 composite promoters provided by an embodiment of the present invention;

[0037] Figure 3 is a gene editing result diagram of three U6 composite promoters provided by an embodiment of the present invention;

[0038] Figure 4 is a first-generation sequencing peak diagram of the editing target sites of three endogenous genes (ZmLAC1, ZmBGLU11, and ZmNL4) in maize by the gene editing vector provided by an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0039] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0040] It should be noted that the details not elaborated in the present invention are well known to those skilled in the art. The experimental methods without specific conditions in the following embodiments are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer, and the experimental steps not described in detail.

[0041] As used herein, the term "plant promoter" refers to a nucleic acid sequence capable of initiating nucleic acid transcription in plant cells. The plant promoter can be derived from plants, microorganisms (such as bacteria, viruses) or animals, etc., or can be an artificially synthesized or modified promoter.

[0042] As used herein, the term "CasWM protein" refers to a nuclease. In the present invention, the CasWM protein belongs to the Cas12 protein, and its amino acid sequence is as shown in SEQ ID NO.3:

[0043] The coding sequence of the CasWM protein obtained by codon optimization of the present invention can significantly increase the expression level of the CasWM protein and also significantly improve the editing efficiency of the CasWM protein in plants.

[0044] As used herein, the term "plant" includes whole plants, plant organs (such as leaves, stems, roots, etc.), seeds, and plant cells and their progeny. There is no particular limitation on the types of plants that can be used in the methods of the present invention, and generally includes any type of higher plant that can be subjected to transformation techniques, including monocotyledonous, dicotyledonous plants, and gymnosperms.

[0045] As used herein, the term "expression cassette" refers to a polynucleotide sequence of a sequence component containing a gene to be expressed and elements required for expression. The components required for expression include a promoter and a polyadenylation signal sequence. In addition, the expression cassette of the present invention may or may not contain other sequences, including (but not limited to): enhancers, secretion signal peptide sequences, etc.

[0046] As used herein, the term "sgRNA", also referred to as "guide RNA", is a single-stranded RNA with a sequence complementary to the target DNA at the 5' end. The gRNA targets Cas9 to a specific genomic locus through this complementary sequence. Usually, in a gene editing system, there is a PAM sequence behind (or in front of) the gRNA.

[0047] As used herein, "selectable marker gene" refers to a gene used to screen transgenic cells or transgenic animals during the transgenic process. There is no particular limitation on the selectable marker genes that can be used in the present application, including various selectable marker genes commonly used in the field of transgenics. Representative examples include (but not limited to): luciferase protein, or luciferase (such as firefly luciferase, Renilla luciferase), green fluorescent protein, yellow fluorescent protein, red fluorescent protein, or combinations thereof.

[0048] As used herein, "HDV sequence" refers to the ribonucleic acid (RNA) sequence of hepatitis delta virus (HDV) present in a plasmid. In this article, the nucleotide sequence of the "HDV sequence" is: GGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCT TCGGCATGGCGAATGGGAC.

[0049] As used herein, "CMYLCV sequence" refers to the following sequence:

[0050] GGCAGACATACTGTCCCACAAATGAAGATGGAATCTGTAAAAGAAAACG

[0051] CGTGAAATAATGCGTCTGACAAAGGTTAGGTCGGCTGCCTTTAATCAATA

[0052] CCAAAGTGGTCCCTACCACGATGGAAAAACTGTGCAGTCGGTTTGGCTT

[0053] TTTCTGACGAACAAATAAGATTCGTGGCCGACAGGTGGGGGTCCACCAT

[0054] GTGAAGGCATCTTCAGACTCCAATAATGGAGCAATGACGTAAGGGCTTA

[0055] CGAAATAAGTAAGGGTAGTTTGGGAAATGTCCACTCACCCGTCAGTCTA

[0056] TAAATACTTAGCCCCTCCCTCATTGTTAAGGGAGCAAAATCTCAGAGAG

[0057] ATAGTCCTAGAGAGAGAAAGAGAGCAAGTAGCCTAGAAGTAGTCAAGG

[0058] CGGCGAAGTATTCAGGCACGTGGCCAGGAAGAAGAAAAGCCAAGACG

[0059] ACGAAAACAGGTAAGAGCTAAGC

[0060] Figure 1 is a schematic diagram of the gene editing vector structure provided by the present invention. As Figure 1 shown, the gene editing vector provided by the present invention includes:

[0061] a first expression cassette for expressing CasWM protein;

[0062] a second expression cassette for expressing sgRNA;

[0063] and a third expression cassette for expressing a maize transformation selection marker;

[0064] The first expression cassette includes the nucleotide sequence described in claim 1 and a first promoter; the second expression cassette includes sgRNA and a second promoter.

[0065] In a preferred embodiment, the first promoter in the first expression cassette is the maize ubiquitin gene promoter (ZmUBI); NLS (nuclear localization signal peptide) sequences are added to the C-terminus and N-terminus of CasWM, and the NOS terminator is used.

[0066] The second expression cassette includes elements of the structure of formula I:

[0067] P1-X1-L1-H1:

[0068] Wherein, P1 is a U6 composite promoter sequence; X1 is a tRNA sequence; L1 is a crRNA sequence; H1 is an HDV sequence; each "-" is independently a bond or a nucleotide linking sequence.

[0069] The U6 composite promoter comprises an element of formula II:

[0070] P2-X2-L2;

[0071] Wherein, P2 is a 35S enhancer sequence; X2 is a CMYLCV sequence; L2 is a U6 promoter; the nucleotide sequence of the U6 promoter is any one of SEQ ID NO.3 to SEQ ID NO.5, preferably the nucleotide sequence shown in SEQ ID NO.5.

[0072] Optimized design of the nucleotide sequence of CasWM protein in Example 1

[0073] Since the efficiency of the Cas12 family in plant gene editing has always been poor, in order to improve the editing efficiency of CasWM protein in maize, the inventors considered optimizing it from multiple aspects. First, we performed codon optimization on the nucleotide sequence according to the CasWM amino acid sequence. Since the GC content of most gene expression frames in maize is relatively high, based on this characteristic, we designed multiple pairs of CasWM nucleotide sequences with different GC contents for comparison of gene editing efficiency. The inventors found that there were significant differences in the gene editing efficiency of CasWM nucleotide sequences with different GC contents. Finally, a high-GC version of the CasWM nucleotide sequence, named Caswm01, was screened out from a large number of experiments, and its nucleotide sequence is shown in SEQ ID NO.1; secondly, the inventors tried to add T5 exonuclease to the CasWM protein nucleotide sequence to further improve the editing efficiency, named Caswm02, and its nucleotide sequence is shown in SEQ ID NO.2; in order to compare the gene editing effects of Caswm01 and Caswm02, the inventors designed sgRNA, used Caswm01 and Caswm02 to edit the ZmLAC1 gene of maize, and performed transient transformation experiments of maize protoplasts to verify the editing efficiency. We used the inbred maize variety KN5585 selected by Weimi Biotechnology (Jiangsu) Co., Ltd. to prepare protoplasts. The methods for isolating and transforming maize protoplasts can be found in the reference [1] . The results are shown in Table 1.

[0074] As can be seen from the results in Table 1, after codon optimization, Caswm01 and Caswm02 have a certain editing efficiency for maize genes. For Caswm02 with the addition of T5 exonuclease, the gene editing efficiency is greatly improved. Compared with Caswm01, the editing efficiency of Caswm02 is doubled to about 77.8%, indicating that the optimization strategy for the CasWM nucleotide sequence in this example effectively improves the editing efficiency.

[0075] Table 1 Results of Caswm01 and Caswm02 editing maize ZmLAC1 gene

[0076] Editor Gene sgRNA Efficiency Caswm01 zmLAC1 TTCTTCCTTCCCTTCATCCTGCTGGC 8 / 21 38.1％ Caswm02 zmLAC1 TTCTTCCTTCCCTTCATCCTGCTGGC 14 / 18，77.8％

[0077] Example 2 Optimization of the crRNA expression structure of CasWM protein

[0078] Furthermore, on the basis of the optimization results in Example 1, the inventors optimized the crRNA expression structure of CasWM protein.

[0079] On the one hand, the inventors optimized the promoter and used the U6 composite promoter to express sgRNA. The U6 composite promoter includes the U6 promoter, the upstream 35s enhancer and the CNYLCV element. In order to test which U6 composite promoter is the optimal choice in maize, the inventors carried out a large number of screenings and optimization designs on the U6 promoter, and finally selected three U6 promoters, ATU6, ZmU61 and ZmU62, to form multiple versions of the U6 composite promoter, which were named U6 composite v0, U6 composite v1 and U6 composite v2 respectively, and their nucleotide sequences are shown in SEQ ID NO.3 to SEQ ID NO.5; on the other hand, the inventors added tRNA and HDV structures to the upstream and downstream of crRNA respectively to stabilize the expression of crRNA. The schematic diagram of the structure is shown in Figure 2 。

[0080] The inventors designed sgRNA according to the maize endogenous gene ZmLAC1 and constructed it into the pCas-WM01 vector, and used the above three U6 composite promoters to initiate expression respectively to obtain three gene editing vectors, pCasWM+U6composite v0, pCasWM+U6composite v1 and pCasWM+U6 composite v2. The three constructed gene editing vectors were transiently transformed into maize cells by protoplast transformation. After culturing for 24 hours, cell DNA was extracted and genotyping detection was carried out. We used the inbred maize variety KN5585 selected by Weimi Biotechnology (Jiangsu) Co., Ltd. to prepare protoplasts. The methods for maize protoplast isolation and transformation can be found in the references[1] 。

[0081] The detection results are as Figure 3 shown. All three gene editing vectors have certain gene editing effects. Among them, the U6composite v2 promoter shows the best editing efficiency, with an editing efficiency exceeding 90%.

[0082] Example 3: Testing the editing effects of the optimized gene editing vectors on different endogenous genes in maize

[0083] Furthermore, according to the optimized experimental results of the crRNA expression structure in Example 2, we used the pCasWM+U6composite v2 gene editing vector to test the editing effects on three endogenous targets (ZmLAC1, ZmBGLU11, and ZmNL4) in maize.

[0084] First, sgRNAs were designed for ZmLAC1, ZmBGLU11, and ZmNL4 respectively and cloned into the pCasWM+U6 composite v2 gene editing vector. The vector was used for stable genetic transformation of maize, and 56 regenerated plants were obtained. First-generation sequencing genotyping was performed on each T0 plant. The results are shown in Table 2. pCas-WM01 achieved high-efficiency editing in all three maize genes, with efficiencies ranging from 93.7% to 97.5%, and the highest reaching 97.5%. This indicates that the optimized pCas-WM01 has good editing ability. Figure 4 Figure 1 is the first-generation sequencing peak map of the editing target sites of the three gene editing vectors in this example for three endogenous genes (ZmLAC1, ZmBGLU11, and ZmNL4) in maize. It can be seen from the Figure 4 first-generation sequencing peak map of the target sites that at the ZmLAC1 gene locus, the pCasWM+U6 composite v2 editing vector achieved the effect of homozygous deletion fragment ( Figure 4 A), and at the ZmBGLU11 and ZmNL4 loci ( Figure 4 B, Figure 4 C), the pCasWM+U6 composite v2 obtained the effect of heterozygous large fragment deletion. Generally speaking, using the pCasWM+U6 composite v2 gene editing vector can achieve efficient editing in maize, and most of the editing cases are base fragment deletions.

[0085] Table 2 Editing results of the pCasWM+U6 composite v2 gene editing vector

[0086]

[0087] In summary, the present invention first optimizes the amino acid codons of CasWM for high GC content and adds T5 exonuclease. Secondly, it optimizes the expression structure of crRNA, uses the optimized U6 composite promoter to express crRNA, and adds tRNA and HDV structures upstream and downstream of crRNA to stabilize the expression of crRNA. The last part of the sequence of the U6 composite promoter is AtU6, and experiments have shown that it is not the best choice in maize. The inventors innovatively discovered two U6 promoters, ZmU61 and ZmU62. In transient experiments, it was shown that the ZmU62 version of the U6 composite promoter has the best editing effect. This shows that our optimization strategy is feasible and effective. Moreover, in maize stably genetically transformed plants, the optimized editing vector also achieved efficient editing at three maize endogenous targets, with the highest efficiency reaching 97.5%, and its editing characteristic was the occurrence of fragment deletion. Our new editing tool further expands the plant Cas12 family gene editing tools and helps to cultivate new maize varieties.

[0088] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

[0089] It is easy for those skilled in the art to understand that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0090] References: [1] Tu, X., et al., Reconstructing the maize leaf regulatory network using ChIP-seq data of 104 transcription factors. Nat Commun, 2020. 11(1): p. 5089. SEQ ID NO.1

[0091] atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcGCCACCAAGAC

[0092] CATCGTGCGCCCGTACACGTCCAACCTCTCCCCCAACGCCGCCAAGAAG

[0093] GCCATGCTCGACGAGTCCTTCAAGTTCTTCGACCACGCCTACACCGTGT

[0094] TCTTCTCCGTGTTCATCAAGCTCTGGGGCGGCGTGAAGCCGACCCAAGT

[0095] GGCCCTCGTGGAGAACGACACCAACAAGATCGACGCCATCTGCTCCATC

[0096] CTCTGGTTCCGCCTGCAGACCAAGACCGACTCCACCAACATCACCCTGC

[0097] AGTCCGCCGAGGAGCGCATCCGCCGCTTCAAGGAGTACGCGCAGCACG

[0098] ACCCGTCCCCGCTCGCCCTCTCCTACCTCACCGGCAACCTCGACCCGGA

[0099] GAAGCACGAGTGGGTGGACTGCCGCGAACTCTATCAGAACTGGTGCGC

[0100] CGAACTCAAGTGCGACCTCGCCACCGACATCGAaACCATGATCAACCAC

[0101] AATCTCCTCCCGATCTCCGCCAAGCAAGAGTACAACTGCTACTCCTCCTT

[0102] CTCCAACCTCTTCGGCGAGGCCGAGAAGGAGAACAAGGCGGAGAAGG

[0103] TGGAGTGGTATCGCTCCGTGGTGGCCGCCCTCAAGTCCAAGTCCGCCTG

[0104] GAACTGGGACGAGTACCGCAAGATGCTCTTCGAGAAAATCGGCTGTACC

[0105] GCGCCGAAGGGGGGCGGCCGCCTCTCCCTCCTCTTCCTCGACATGCGCT

[0106] CCGACACCACCGGCCCGATCTCCTCCCAAGAGATCACCAAGCGCATCAC

[0107] GTCCTTCGAGAAGGTGGCCGAGGAGAAGTCCAAGACCTACGAACTCCC

[0108] GCACCGCCTCACCGTGAAGCAGTACATCTGCAATCAGCTCGCCCACCCG

[0109] TTTCAGCTCACCATCTGGTCCGCCGTCGCCAACTACGCCATCTCCTCCAT

[0110] CCGCTCCAAGAACTCCATCAACATGCTCTACTCCGACGAGAAGCTCAAC

[0111] CGCGAAACCATCATCGCCAAGATCAAGTCCCAAAACCCGAACATCGAGC

[0112] AAGTGCAAGAGATCCTCTCCCAATTCCGCGCCGGCGAGAACAACGCCTT

[0113] CGTGATCGAGCCGCGCCACCTCAGCAACCTCAAGTCCCTCTTCCGCTTC

[0114] TGGTCCAACTCCTCCATGGACGAGGGCATCACCACCCTCCTGCAGATCC

[0115] CGCAGTCCGAGTACGAGCGCTCCCCGATCGTGGATCTCCTCCGCCACAT

[0116] CCACCCGCACCGCAACAAGTTCACCGCCGAGCAGTTCATCGTGGCGGC

[0117] CGAACTCAACGGCATCGAGTTCGACAACGCCATGAAGAAGATCCACCC

[0118] GACCGTGTACGGCAAGACCACCGTGGACTACGGCCCGAAGTCCACCAT

[0119] CTACGGCTCCATCACCCCGCCGTCCAAGCTCATTCGCGGCGTGCACGCC

[0120] GGCTCCCACGGCATGATGTGGGTGACCATGACCCTCCTCGACAACGGCA

[0121] AGTGGATCAAGCACCACATCCCGTTCCACAACTCCCGCTACTACGAGGA

[0122] GATGTATGCGTATCGCGAGGGGCTGCCGGTGGGCGAGAAGCCGCGCCGC

[0123] CCGGTGCTCGGCAACCGCGTGGGCAACCACATCGACAACACCGGCCTC

[0124] ATCAACAAAAAGTGCCGCAAGGCCTCCAAGGCCTACCTCCGCACCATTC

[0125] AGAACATGAACCACAACGTGTGGTTCGACCCGGACACCCACTTCCTCGT

[0126] GCAGCGCAATGGGGACAACTTCGCCATTCAGATCTCCTCCCGCATCAAG

[0127] GAGTCCAAGCCGAAGTCCAACGTGGAGGTGGGCGACCGCATCCTCGGC

[0128] ATGGACCAAAACCAAACCGCCTCCAACACCTACTCCGTGGTGGAGGTG

[0129] GTGCGCGAGGGCACCGAGAACTCCCACCCGTACAACAACTACTTCGTG

[0130] AAGGTGATCGAGGACGGCAACGTCACGTCCTGCACCACCAAGAACGAT

[0131] CGCGGCGAGTTCGATCAGCTCTCCTACGAGGGCCTCGCCTACTCCGAGT

[0132] TCGAGATCTGGCGCCAAGCCCGCATCGCCTTCCTCTCCGCCCACGACCC

[0133] GGAACTCGCGCAGAAGATGATCGACAAGACCAACGAGTCCCTCTACAA

[0134] GTGGAACAATAACTACGCCTACTACCTCAAGGTGTGCATGCGCAACAAG

[0135] ATGAACGGCGACAACCACGCCCTCTTCCGCAACGAGATCAAGGAGTTC

[0136] ATCGAGGGCATGCCGGTGGCCGGCAAGAACGAGAAGCACAAGTTCTTC

[0137] GGCTCCATTCGCGGCTCCCTCTCCCTGGAGTCCCTGGAGGGCCTCTCCA

[0138] AGTCCCGCTCCCTCATCTCCTGCTACTTCTATCTCCTCGAGAAGAAGGAG

[0139] ATCGAGCAGCAGAAGGAGTTCGACTCCGACCTCTTCAAGCTCGGCGAG

[0140] TGCCTCTCCGAGAAGCGCGTGAACAAGCGCGAGGAGCGCGCCAACCGC

[0141] ATCGTGTCCTCCGTGCTGCAGATCTGCTCCCGCCTCAACGTGTCCCGCAT

[0142] CGTGATCGAGAACAAGCTCCCGACCGCCAACCACGAGAACAAGTCCTC

[0143] CGCCAACCGCCGCGCCACCGACTGGTGCCCGCGCAAGGTGCAGCAGAA

[0144] GCTCCTCGACGCCGTGAAGATGGTGGGCATCAAGGTGCTCGCCGTGAA

[0145] GCCGTACAACACCTCCCACATCGACCCGTTCGTGAACGGCGAGTCCAAC

[0146] CGCCAAGCCCTGGAGGCCCGCTTCATGGACGTGGACGTGAAGGACATC

[0147] ACCGACCGCAACATCAAGCAGTTCAAGAAGATGCACCGCAACCAAGTG

[0148] GGCGTGCTCAACTCCATCTACCACAACGCCCTCCGCTCCTTCGCCGCCA

[0149] ACTACGGCCTCAACTGGAACGAACTCCCGAACATGAATCTCGAGCAGAT

[0150] CAAGAACGCCCTCAAGGACCACGTGCGCGTGATGTTCCCGCAGTGGGG

[0151] CGGCCGCTCCTTCATGTCCACCCACAACGTGACCCGCAACTCCGTGCGC

[0152] GTGTCCTACAACAACCGCACCCGCTGGCTCAACTTCTCCGACGTGATCG

[0153] CCGCCCTCAACATCGCCCTGCGCGGCTCCGGCAACTATGAGCCGAAGGG

[0154] CGACTCCCAAAACACCGCCCCGTCCCGCAATtcctcaggagggtccAAGCGGAC

[0155] TGCGGATGGGTCTGAGTTCGAGTCACCAAAGAAGAAGAGGAAGGTGgga

[0156] tccGGCTCCAAACGCCCGGCCGCCACCAAAAAAGCCGGCCAAGCCAAGA

[0157] AAAAGAAAACGTCCTCCGGCGGGTCCGCCTCCCCCAAGCGCCCGCGCG

[0158] ATAGGCACGACGGCGAACTCGGCGGCCGCAAGCGCGCGCGCGGTTAASEQ ID NO.2

[0159] atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcGCCACCAAG

[0160] ACCATCGTGCGCCCGTACACGTCCAACCTCTCCCCCAACGCCGCCAA

[0161] GAAGGCCATGCTCGACGAGTCCTTCAAGTTCTTCGACCACGCCTACA

[0162] CCGTGTTCTTCTCCGTGTTCATCAAGCTCTGGGGCGGCGTGAAGCCGA

[0163] CCCAAGTGGCCCTCGTGGAGAACGACACCAACAAGATCGACGCCATC

[0164] TGCTCCATCCTCTGGTTCCGCCTGCAGACCAAGACCGACTCCACCAA

[0165] CATCACCCTGCAGTCCGCCGAGGAGCGCATCCGCCGCTTCAAGGAGT

[0166] ACGCGCAGCACGACCCGTCCCCGCTCGCCCTCTCCTACCTCACCGGC

[0167] AACCTCGACCCGGAGAAGCACGAGTGGGTGGACTGCCGCGAACTCT

[0168] ATCAGAACTGGTGCGCCGAACTCAAGTGCGACCTCGCCACCGACATC

[0169] GAaACCATGATCAACCACAATCTCCTCCCGATCTCCGCCAAGCAAGAG

[0170] TACAACTGCTACTCCTCCTTCTCCAACCTCTTCGGCGAGGCCGAGAAG

[0171] GAGAACAAGGCGGAGAAGGTGGAGTGGTATCGCTCCGTGGTGGCCG

[0172] CCCTCAAGTCCAAGTCCGCCTGGAACTGGGACGAGTACCGCAAGATG

[0173] CTCTTCGAGAAAATCGGCTGTACCGCGCCGAAGGGGGGCGGCCGCCT

[0174] CTCCCTCCTCTTCCTCGACATGCGCTCCGACACCACCGGCCCGATCTC

[0175] CTCCCAAGAGATCACCAAGCGCATCACGTCCTTCGAGAAGGTGGCCG

[0176] AGGAGAAGTCCAAGACCTACGAACTCCCGCACCGCCTCACCGTGAAG

[0177] CAGTACATCTGCAATCAGCTCGCCCACCCGTTTCAGCTCACCATCTGG

[0178] TCCGCCGTCGCCAACTACGCCATCTCCTCCATCCGCTCCAAGAACTCC

[0179] ATCAACATGCTCTACTCCGACGAGAAGCTCAACCGCGAAACCATCATC

[0180] GCCAAGATCAAGTCCCAAAACCCGAACATCGAGCAAGTGCAAGAGAT

[0181] CCTCTCCCAATTCCGCGCCGGCGAGAACAACGCCTTCGTGATCGAGC

[0182] CGCGCCACCTCAGCAACCTCAAGTCCCTCTTCCGCTTCTGGTCCAACT

[0183] CCTCCATGGACGAGGGCATCACCACCCTCCTGCAGATCCCGCAGTCC

[0184] GAGTACGAGCGCTCCCCGATCGTGGATCTCCTCCGCCACATCCACCCG

[0185] CACCGCAACAAGTTCACCGCCGAGCAGTTCATCGTGGCGGCCGAACT

[0186] CAACGGCATCGAGTTCGACAACGCCATGAAGAAGATCCACCCGACCG

[0187] TGTACGGCAAGACCACCGTGGACTACGGCCCGAAGTCCACCATCTAC

[0188] GGCTCCATCACCCCGCCGTCCAAGCTCATTCGCGGCGTGCACGCCGG

[0189] CTCCCACGGCATGATGTGGGTGACCATGACCCTCCTCGACAACGGCA

[0190] AGTGGATCAAGCACCACATCCCGTTCCACAACTCCCGCTACTACGAG

[0191] GAGATGTATGCGTATCGCGAGGGGCTGCCGGTGGGCGAGAAGCCGCG

[0192] CCGCCCGGTGCTCGGCAACCGCGTGGGCAACCACATCGACAACACCG

[0193] GCCTCATCAACAAAAAGTGCCGCAAGGCCTCCAAGGCCTACCTCCGC

[0194] ACCATTCAGAACATGAACCACAACGTGTGGTTCGACCCGGACACCCA

[0195] CTTCCTCGTGCAGCGCAATGGGGACAACTTCGCCATTCAGATCTCCTC

[0196] CCGCATCAAGGAGTCCAAGCCGAAGTCCAACGTGGAGGTGGGCGAC

[0197] CGCATCCTCGGCATGGACCAAAACCAAACCGCCTCCAACACCTACTC

[0198] CGTGGTGGAGGTGGTGCGCGAGGGCACCGAGAACTCCCACCCGTAC

[0199] AACAACTACTTCGTGAAGGTGATCGAGGACGGCAACGTCACGTCCTG

[0200] CACCACCAAGAACGATCGCGGCGAGTTCGATCAGCTCTCCTACGAGG

[0201] GCCTCGCCTACTCCGAGTTCGAGATCTGGCGCCAAGCCCGCATCGCCT

[0202] TCCTCTCCGCCCACGACCCGGAACTCGCGCAGAAGATGATCGACAAG

[0203] ACCAACGAGTCCCTCTACAAGTGGAACAATAACTACGCCTACTACCTC

[0204] AAGGTGTGCATGCGCAACAAGATGAACGGCGACAACCACGCCCTCTT

[0205] CCGCAACGAGATCAAGGAGTTCATCGAGGGCATGCCGGTGGCCGGCA

[0206] AGAACGAGAAGCACAAGTTCTTCGGCTCCATTCGCGGCTCCCTCTCC

[0207] CTGGAGTCCCTGGAGGGCCTCTCCAAGTCCCGCTCCCTCATCTCCTGC

[0208] TACTTCTATCTCCTCGAGAAGAAGGAGATCGAGCAGCAGAAGGAGTT

[0209] CGACTCCGACCTCTTCAAGCTCGGCGAGTGCCTCTCCGAGAAGCGCG

[0210] TGAACAAGCGCGAGGAGCGCGCCAACCGCATCGTGTCCTCCGTGCTG

[0211] CAGATCTGCTCCCGCCTCAACGTGTCCCGCATCGTGATCGAGAACAA

[0212] GCTCCCGACCGCCAACCACGAGAACAAGTCCTCCGCCAACCGCCGCG

[0213] CCACCGACTGGTGCCCGCGCAAGGTGCAGCAGAAGCTCCTCGACGCC

[0214] GTGAAGATGGTGGGCATCAAGGTGCTCGCCGTGAAGCCGTACAACAC

[0215] CTCCCACATCGACCCGTTCGTGAACGGCGAGTCCAACCGCCAAGCCC

[0216] TGGAGGCCCGCTTCATGGACGTGGACGTGAAGGACATCACCGACCGC

[0217] AACATCAAGCAGTTCAAGAAGATGCACCGCAACCAAGTGGGCGTGCT

[0218] CAACTCCATCTACCACAACGCCCTCCGCTCCTTCGCCGCCAACTACGG

[0219] CCTCAACTGGAACGAACTCCCGAACATGAATCTCGAGCAGATCAAGA

[0220] ACGCCCTCAAGGACCACGTGCGCGTGATGTTCCCGCAGTGGGGCGGC

[0221] CGCTCCTTCATGTCCACCCACAACGTGACCCGCAACTCCGTGCGCGT

[0222] GTCCTACAACAACCGCACCCGCTGGCTCAACTTCTCCGACGTGATCGC

[0223] CGCCCTCAACATCGCCCTGCGCGGCTCCGGCAACTATGAGCCGAAGG

[0224] GCGACTCCCAAAACACCGCCCCGTCCCGCAATtccTCTGGGGGAAGTA

[0225] GCGGTGGAAGCAGTGGCAGCGAAACCCCTGGAACAAGCGAAAGCGC

[0226] TACCCCCGAATCTAGCGGCGGTTCATCAGGTGGATCTTCAAAGAGTTG

[0227] GGGCAAGTTCATTGAGGAGGAGGAAGCCGAAATGGCCAGTCGGCGC

[0228] AATCTCATGATTGTTGACGGCACCAATCTCGGCTTCAGATTCAAGCAC

[0229] AACAACTCCAAGAAACCCTTTGCTTCCAGTTATGTGAGCACGATTCAA

[0230] AGCCTGGCCAAGTCATATTCTGCTCGCACTACGATTGTGCTGGGGGAC

[0231] AAAGGGAAAAGCGTGTTCAGACTTGAGCACCTGCCAGAGTACAAAG

[0232] GAAATCGAGATGAGAAGTACGCCCAAAGAACTGAAGAGGAGAAGGC

[0233] CCTGGATGAGCAGTTTTTCGAGTACCTGAAAGATGCTTTCGAGCTTTG

[0234] CAAAACGACCTTCCCAACATTCACAATTCGCGGGGTTGAGGCCGATG

[0235] ACATGGCAGCCTACATTGTCAAGTTGATCGGACACCTGTATGACCATG

[0236] TGTGGCTTATCAGCACTGATGGAGACTGGGATACTCTTCTGACAGACA

[0237] AAGTATCTCGGTTCAGCTTCACCACGAGGCGAGAATATCACCTGAGG

[0238] GATATGTACGAGCACCATAATGTGGACGATGTTGAGCAGTTCATTTCTC

[0239] TGAAGGCGATTATGGGCGACCTGGGGGACAATATAAGAGGAGTTGAG

[0240] GGAATCGGGGCCAAGAGAGGGTACAACATCATACGAGAGTTTGGGAA

[0241] CGTACTTGATATCATCGACCAGCTGCCATTGCCTGGGAAGCAAAAATA

[0242] TATACAGAACCTGAACGCTTCCGAAGAGTTGCTGTTTAGGAACCTGAT

[0243] CCTGGTGGATTTGCCCACCTATTGCGTCGACGCAATAGCAGCAGTAGG

[0244] TCAGGACGTGCTCGATAAATTTACCAAGGATATACTTGAGATCGCTGA

[0245] GCAAtcaggagggtccAAGCGGACTGCGGATGGGTCTGAGTTCGAGTCACC

[0246] AAAGAAGAAGAGGAAGGTGggatccGGCTCCAAACGCCCGGCCGCCAC

[0247] CAAAAAAGCCGGCCAAGCCAAGAAAAAGAAAACGTCCTCCGGCGGG

[0248] TCCGCCTCCCCCAAGCGCCCGCGCGATAGGCACGACGGCGAACTCGG

[0249] CGGCCGCAAGCGCGCGCGCGGT

[0250] SEQ ID NO.3

[0251] ATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAG

[0252] ACTGGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGA

[0253] AAATCTTCGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAA

[0254] TATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAA

[0255] CAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTG

[0256] TCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGC

[0257] CATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACA

[0258] GTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAG

[0259] AAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATTGGCA

[0260] GACATACTGTCCCACAAATGAAGATGGAATCTGTAAAAGAAAACGCGTG

[0261] AAATAATGCGTCTGACAAAGGTTAGGTCGGCTGCCTTTAATCAATACCAA

[0262] AGTGGTCCCTACCACGATGGAAAAACTGTGCAGTCGGTTTGGCTTTTTC

[0263] TGACGAACAAATAAGATTCGTGGCCGACAGGTGGGGGTCCACCATGTG

[0264] AAGGCATCTTCAGACTCCAATAATGGAGCAATGACGTAAGGGCTTACGA

[0265] AATAAGTAAGGGTAGTTTGGGAAATGTCCACTCACCCGTCAGTCTATAA

[0266] ATACTTAGCCCCTCCCTCATTGTTAAGGGAGCAAAATCTCAGAGAGATAG

[0267] TCCTAGAGAGAGAAAGAGAGCAAGTAGCCTAGAAGTAGTCAAGGCGGC

[0268] GAAGTATTCAGGCACGTGGCCAGGAAGAAGAAAAGCCAAGACGACGA

[0269] AAACAGGTAAGAGCTAAGCATCTAGAAAGTTGAAAACAATCTTCAAAA

[0270] GTCCCACATCGCTTAGATAAGAAAACGAAGCTGAGTTTATATACAGCTAG

[0271] AGTCGAAGTAGTGATT

[0272] SEQ ID NO.4

[0273] ATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAG

[0274] ACTGGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGA

[0275] AAATCTTCGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAA

[0276] TATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAA

[0277] CAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTG

[0278] TCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGC

[0279] CATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACA

[0280] GTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAG

[0281] AAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATTGGCA

[0282] GACATACTGTCCCACAAATGAAGATGGAATCTGTAAAAGAAAACGCGTG

[0283] AAATAATGCGTCTGACAAAGGTTAGGTCGGCTGCCTTTAATCAATACCAA

[0284] AGTGGTCCCTACCACGATGGAAAAACTGTGCAGTCGGTTTGGCTTTTTC

[0285] TGACGAACAAATAAGATTCGTGGCCGACAGGTGGGGGTCCACCATGTG

[0286] AAGGCATCTTCAGACTCCAATAATGGAGCAATGACGTAAGGGCTTACGA

[0287] AATAAGTAAGGGTAGTTTGGGAAATGTCCACTCACCCGTCAGTCTATAA

[0288] ATACTTAGCCCCTCCCTCATTGTTAAGGGAGCAAAATCTCAGAGAGATAG

[0289] TCCTAGAGAGAGAAAGAGAGCAAGTAGCCTAGAAGTAGTCAAGGCGGC

[0290] GAAGTATTCAGGCACGTGGCCAGGAAGAAGAAAAGCCAAGACGACGA

[0291] AAACAGGTAAGAGCTAAGCATCTAGAgctgtttttgttagccccatcgaatccttgacataatga

[0292] tcccgcttaaataagcaacctcgcttgtatagttccttgtgctctaacacacgatgatgataagtcgtaaaatagtggtgtc

[0293] caaagaatttccaggcccagttgtaaaagctaaaatgctattcgaatttctactagcagtaagtcgtgtttagaaattattttt

[0294] ttatataccttttttccttctatgtacagtaggacacagtgtcagcgccgcgttgacggagaatatttgcaaaaaagtaaaa

[0295] gagaaagtcatagcggcgtatgtgccaaaaacttcgtcacagagagggccataagaaacatggcccacggcccaat

[0296] acgaagcaccgcgacgaagcccaaacagcagtccgtaggtggagcaaagcgctgggtaatacgcaaacgttttgtc

[0297] ccaccttgactaatcacaagagtggagcgtaccttataaaccgagccgcaagcaccgaattGSEQ IDNO.5

[0298] ATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAG

[0299] ACTGGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGA

[0300] AAATCTTCGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAA

[0301] TATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAA

[0302] CAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTG

[0303] TCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGC

[0304] CATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACA

[0305] GTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAG

[0306] AAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATTGGCA

[0307] GACATACTGTCCCACAAATGAAGATGGAATCTGTAAAAGAAAACGCGTG

[0308] AAATAATGCGTCTGACAAAGGTTAGGTCGGCTGCCTTTAATCAATACCAA

[0309] AGTGGTCCCTACCACGATGGAAAAACTGTGCAGTCGGTTTGGCTTTTTC

[0310] TGACGAACAAATAAGATTCGTGGCCGACAGGTGGGGGTCCACCATGTG

[0311] AAGGCATCTTCAGACTCCAATAATGGAGCAATGACGTAAGGGCTTACGA

[0312] AATAAGTAAGGGTAGTTTGGGAAATGTCCACTCACCCGTCAGTCTATAA

[0313] ATACTTAGCCCCTCCCTCATTGTTAAGGGAGCAAAATCTCAGAGAGATAG

[0314] TCCTAGAGAGAGAAAGAGAGCAAGTAGCCTAGAAGTAGTCAAGGCGGC

[0315] GAAGTATTCAGGCACGTGGCCAGGAAGAAGAAAAGCCAAGACGACGA

[0316] AAACAGGTAAGAGCTAAGCATCTAGACTAATTGGCCCTTACAAAATAGC

[0317] TAGACGTGCAGGTGGCTGGATGTGCGCTCCCTGAATATCAACTTGTGTCT

[0318] CCTCCGATTCAGTCCGCAGATGAAACTTGGTAATAACTGCAGCTGATCCG

[0319] TCGTCATTCATGCTATGCAGGGGATTCGATCTTCAGCATGTGCAGTGCAG

[0320] GCAACAACAATCTACGTTGTCTGGGCTTGCGATAGGTACACGACCACGA

[0321] GGGAAGGCAACGCGTGATGTATGGGCCGCGCCTAAGCATCCAGCCCACG

[0322] CGGGCGTGCGCGTCGTCGCTACGGCTTGCGGGGGAAGGGATCAAGGGA

[0323] CGAACCGAGAACTAGTACCAGACCGGCCAGCGAGCATTGCAGACACCG

[0324] GCTTATAAGTTCAGCTGCGACCACCGCTCCG

Claims

1. A codon-optimized nucleotide sequence encoding the CasWM protein, characterized in that, The nucleotide sequence is SEQ ID NO.1 or SEQ ID NO.2, preferably SEQ ID NO.

2.

2. A nucleic acid construct for editing the maize genome, characterized in that, The nucleic acid construct comprises: a first expression cassette for expressing CasWM protein; a second expression cassette for expressing sgRNA; and a third expression cassette for expressing a maize transformation selection marker; The first expression cassette comprises the nucleotide sequence as claimed in claim 1 and a first promoter; the second expression cassette comprises sgRNA and a second promoter.

3. The nucleic acid construct for editing the maize genome according to claim 2, wherein The second expression cassette comprises an element of the structure of formula I: P1-X1-L1-H1: wherein P1 is a U6 composite promoter sequence; X1 is a tRNA sequence; L1 is a crRNA sequence; H1 is an HDV sequence; each "-" is independently a bond or a nucleotide linker sequence.

4. The nucleic acid construct for editing the maize genome according to claim 3, characterized in that, The U6 composite promoter comprises an element of the structure of formula II: P2-X2-L2; wherein P2 is a 35S enhancer sequence; X2 is a CMYLCV sequence; L2 is a U6 promoter; the nucleotide sequence of the U6 promoter is any one of SEQ ID NO.3 to SEQ ID NO.5, preferably the nucleotide sequence shown in SEQ ID NO.

5.

5. An expression vector, characterized in that, The expression vector contains the nucleic acid construct as claimed in any one of claims 2 to 4.

6. A host cell, characterized in that, The cell contains the expression vector expressing the nucleic acid construct as claimed in claim 5, or its genome is integrated with the nucleic acid construct as claimed in any one of claims 2 to 4.

7. A kit for maize genome editing, characterized in that, Comprises: the nucleic acid construct as claimed in claim 2, or an expression vector containing the nucleic acid construct.

8. A maize breeding method, characterized in that, Comprises the following steps: (i) providing a plant or plant cell to be edited; (ii) introducing an expression vector containing the nucleic acid construct as claimed in claim 3 into the plant or plant cell to be edited, so as to achieve editing of the target gene of the plant or plant cell.

9. Use of the nucleotide sequence as claimed in claim 1, the nucleic acid construct as claimed in any one of claims 2 to 4, the expression vector as claimed in claim 5, the host cell as claimed in claim 6, the kit as claimed in claim 7, and the breeding method as claimed in claim 8 in plant breeding, wherein the plant is maize.

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