Corn gene editor based on glycosidase and application thereof

Abstract

The invention discloses a novel corn gene editor based on glycosidase and application of the novel corn gene editor, the gene editor realizes accurate base editing of corn genes, G-to-T base replacement, and / or G-to-A base replacement, and / or G-to-C base replacement, and the gene editor has high editing efficiency in corn protoplast and transgenic corn plants. The invention also provides a corn gene fixed-point editing method. The corn gene editor DNA based on glycosidase can realize directional base replacement in corn, is high in accuracy, provides an efficient tool for corn genome editing, fills the blank of corn glycosidase base editor, and can be widely applied to corn molecular breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of gene editing, and more particularly relates to a maize gene editor based on glycosidase and application thereof. BACKGROUND

[0002] Base editing technology (BEs) refers to a gene editing technology capable of causing single base change on a genome, and is a revolutionary tool for realizing precise genome modification. The principle thereof is to fuse cytosine deaminase (APOBEC) or adenosine deaminase with Cas9n (D10A) to form, and to depend on CRISPR principle to cause modification of a single base away from the 4-7 positions of a PAM end.

[0003] At present, some engineered glycosidase base editors have been successfully applied to mammalian cells to realize G-to-W (G-to-A / T) and T-to-S (T-to-C / G) base editing [1-3] However, in plants, base editing tools capable of directly modifying thymine (T) and guanine (G) in plants have not been developed to a great extent, and still have problems of low efficiency, many by-products and the like. At present, only a small amount of research reports base editing based on glycosidase in rice, but a glycosidase-based G / T base editing tool has not been developed in maize. Therefore, it is urgent to develop a single base editing tool capable of realizing base substitution in maize. SUMMARY

[0004] In view of at least one defect or improvement demand of the prior art, the present application provides a maize gene editor based on glycosidase, which aims to solve the problem of single base editing in a maize genome.

[0005] To achieve the above-mentioned purpose, according to one aspect of the present application, a maize gene editor based on glycosidase is provided, characterized in that the base editor has the following structural formula:

[0006] NLS-Z-NLS-L-M-NLS

[0007] wherein,

[0008] NLS is a nuclear localization signal element; L is an optional connecting sequence; Z is an nCas9 (D10A) nucleotide sequence; M is a methyl purine DNA glycosidase; and each "-" is independently a bond or a nucleotide connecting sequence.

[0009] In another preferred embodiment, the gene editor achieves G to T base substitution, and / or G to A base substitution, and / or G to C base substitution.

[0010] In another preferred embodiment, the nucleotide sequence of the methylpurine DNA glycosylase is as shown in SEQ ID NO: 1.

[0011] In another preferred embodiment, the nucleotide sequence of the base editor is as shown in SEQ ID NO: 7.

[0012] According to a second aspect of the present application, there is also provided a nucleic acid construct for evaluating the editing efficiency of a maize gene editor, the nucleic acid construct comprising a first expression cassette expressing a reference protein; and a second expression cassette expressing a reporter gene;

[0013] In another preferred embodiment, the reference protein is luciferase; the luciferase comprises Firefly luciferase (Rluc).

[0014] The reporter gene is mNluc, the nucleotide sequence of which is as shown in SEQ ID NO: 3.

[0015] The nucleic acid construct is used for evaluating the editing efficiency of the gene editor according to any one of the above.

[0016] According to a third aspect of the present application, there is also provided an expression cassette comprising the maize gene editor according to any one of the above.

[0017] According to a fourth aspect of the present application, there is also provided an expression vector comprising the maize gene editor or the expression cassette according to any one of the above.

[0018] According to a fifth aspect of the present application, there is also provided a host cell comprising the vector according to the above, or having integrated into its genome the base editor gene sequence according to any one of the above.

[0019] According to a sixth aspect of the present application, there is also provided a method for maize genome site-directed editing, comprising the following steps:

[0020] (i) providing a host cell and a first vector and a second vector, wherein the first vector comprises the expression cassette of the gene editor according to any one of the above, and the second vector comprises an expression cassette expressing sgRNA;

[0021] (ii) infecting the host cell with the first vector and the second vector, thereby performing base site-directed editing in the cell, wherein the cell is a maize cell.

[0022] According to a seventh aspect of the present application, there is further provided the use of the above-mentioned corn gene editor, the above-mentioned nucleic acid sequence, the above-mentioned expression cassette, the above-mentioned expression vector, the above-mentioned host cell, and the above-mentioned method for corn genome site-directed editing in corn breeding.

[0023] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described hereinafter (such as the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they will not be listed one by one here.

[0024] The corn gene editor based on glycosidase and the application thereof provided by the present application have the beneficial effects that:

[0025] 1. The present application provides a corn gene editor based on glycosidase. The inventors select two human-derived glycosidases for sequence optimization aiming at corn codons, and design to combine the glycosidases and their mutants into different positions of nCas9 to obtain multiple base editors. Through corn protoplast transient editing efficiency test, a new type of base editor ZmgGBE based on glycosidase is finally screened, which can realize directional base substitution in corn and has high editing efficiency. The ZmgGBE editor realizes G-to-T, G-to-C and G-to-A base substitution in corn protoplast, and the editing efficiency is 0.54% to 4.72%, and the editing efficiency in transgenic corn plants can be as high as 66.7%. The corn editor based on glycosidase of the present application greatly expands the mutation types that can be generated by corn base editing, fills the gap of corn glycosidase base editor, and has important significance for expanding crop genetic diversity.

[0026] 2. The present application provides a corn gene editor based on glycosidase. A luciferase reporter gene detection system is developed. The system is based on the Nluc reporter system and is improved by introducing a stop codon, so as to quickly detect the editing efficiency of the base editor. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a schematic diagram of screening a glycosidase editor in corn protoplast provided by an embodiment of the present application, wherein (A) indicates a schematic diagram of inducing base mutation using the ZmgGBE editor, and (B) indicates a pDual-LucM reporter system schematic diagram for screening a glycosidase base editor; Figure 2 is a transient editing experiment result graph of a base editor in corn protoplast provided by an embodiment of the present application; Figure 3is a base editing efficiency diagram of three endogenous target points of the ZmgGBE editor provided by the embodiment of the present application in a corn protoplast; Figure 4 is a base editing result diagram of the ZmgGBE provided by the embodiment of the present application in a transgenic corn plant; wherein A and B are target site sequences and insertion / deletion frequencies (%) of the edited ZmGA20ox3 (A) and ZmEPSPS (B), compared with the wild type (WT), the edited sequence shows G→T (sgRNA2-#01) and G→A (sgRNA2-#08) substitution; C is ZmGA20ox3 a comparison diagram of wild type (WT) and ZmGBE edited type sequences; D is ZmPDS a comparison diagram of wild type (WT) and ZmGBE edited type sequences; E is a phenotype comparison diagram of wild type (WT) plants and edited type sgRNA5-T0#03 plants showing a white phenotype, the left is the wild type (WT) plant, and the right is the white phenotype plant. DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application 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 application and should not be used to limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0031] As used herein, the term "base mutation" refers to substitution, insertion and / or deletion of a base at a certain position of a nucleotide sequence.

[0032] As used herein, the term "base substitution" refers to a base mutation of a base at a certain position of a nucleotide sequence to another different base, such as mutation of C to T.

[0033] As used herein, the term "nCas9 protein", Cas9 protein is a class 2 effector protein in the large class of Cas proteins. Cas9 protein usually contains two domains with cleavage activity: HNH domain and RuvC domain, where HNH domain cleaves the DNA strand complementary to crRNA, and RuvC domain cleaves the non-complementary strand. Mutations are made to Cas9 to make one of the two catalytic domains, RuvC and HNH, lose nuclease activity, forming nCas9, which only produces a single-stranded cut when nCas9 interacts with DNA. Typically, an aspartate-to-alanine conversion in the RuvC domain will produce an nCas9 protein, or a histidine-to-alanine conversion (e.g., H840A or H839A) in the HNH domain will also produce an nCas9 protein.

[0034] Uracil DNA Glycosylase (UDG or UNG for short) is an important DNA repair enzyme, belonging to the glycosylase family. Its main function is to recognize and remove uracil in DNA, prevent mutation accumulation and maintain the stability of the genome, thereby initiating the DNA repair process. In the following text of this patent, uracil glycosylase is referred to as "UNG".

[0035] Methylpurine DNA Glycosylase (MPG or AAG for short) is an important DNA repair enzyme, which is one of the key enzymes in the Base Excision Repair (BER) pathway. Its main function is to recognize and remove damaged bases in DNA, especially methylated or alkylated purine bases, thereby initiating the DNA repair process. In the following text of this patent, uracil glycosylase is referred to as "UNG".

[0036] As used herein, the term "sgRNA", also known as "guide RNA", is a single-stranded RNA with a 5' end complementary to the target DNA sequence, and the gRNA targets Cas9 to a specific genomic site through the complementary sequence, usually followed (or preceded) by a PAM sequence in the gene editing system.

[0037] As used herein, the term "nuclear localization signal (NLS)", also known as "nuclear localization sequence (Nuclear localization sequence)", is usually a short amino acid sequence that can interact with nuclear importers to allow proteins to be transported into the nucleus, consisting of at least 4-8 basic amino acids such as Pro, Lys and Arg.

[0038] As used herein, "screening marker gene" refers to a gene used to screen transgenic cells or transgenic animals during the transgenic process. The screening marker gene used in the present application is not particularly limited and includes various screening marker genes commonly used in the transgenic field. Representative examples include, but are not limited to, luciferin protein or luciferase (e.g., firefly luciferase, sea pansy luciferase), green fluorescent protein, yellow fluorescent protein, red fluorescent protein, or a combination thereof.

[0039] As used herein, the term "expression cassette" refers to a polynucleotide sequence containing a gene to be expressed and components required for expression. The components required for expression include a promoter and a polyadenylation signal sequence. In addition, the expression cassette of the present application can or can not contain other sequences, including, but not limited to, enhancers, secretory signal peptide sequences, etc.

[0040] As used herein, the term "reporter gene" is a gene encoding a protein or enzyme that is easily detected in the background of an endogenous protein. The reporter gene is linked to a gene of interest or a regulatory sequence, and the transcriptional activity or expression level of the gene in the cell is intuitively reflected by indirectly or directly detecting signals of the reporter gene coding product, such as reporter protein, mRNA, enzyme, etc. The general characteristics of a reporter gene are non-toxic, non-immunogenic, easy to detect, and specific. Among them, "Nluc" refers to nanoluciferase, which is a small and high-brightness luciferase commonly used in reporter gene experiments; "mNluc" is a modified version of Nluc, which is usually optimized by mutation to improve stability, brightness, or specificity.

[0041] As used herein, the term "internal reference gene" (also referred to as reference gene) is a gene used to correct experimental errors. In a dual luciferase experiment, a stably expressed gene is often selected as an internal reference gene for normalizing the expression level of the target gene. The expression of the internal reference gene should remain relatively stable under different experimental conditions and should not be affected by the factors to be tested. By using an internal reference gene, the effects of sample processing and detection errors in the experiment can be eliminated, making the results more accurate and reliable. Commonly used internal reference genes include GAPDH (glycolytic enzyme) and B-actin (B-actin), but the selection of an internal reference gene should be based on the characteristics of the specific experimental design and research object. In a dual luciferase experiment, Fluc, Rluc, etc. are often used as internal reference genes.

[0042] The application first optimizes two human-derived glycosidases, methylpurine DNA glycosylase (MPG) and uracil DNA glycosylase (UNG), for maize codons, including but not limited to, codon usage bias, elimination of secondary structures (such as hairpin structures) that are not conducive to expression, alteration of GC content, CpG dinucleotide content, mRNA secondary structure, cryptic splice sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA instability regions, repetitive sequences (direct repeats, inverted repeats, etc.), and restriction sites that can affect cloning, etc. The application optimizes a large number of nucleotide sequences based on the two glycosidases, and not all optimized nucleotide sequences can be used for maize gene editing.

[0043] The application tests a plurality of codon-optimized nucleotide sequences, and after a large number of screenings, it is found that only the methylpurine DNA glycosylase (MPG) and the uracil DNA glycosylase (UNG) with the nucleotide sequences of SEQ ID NO. 1 and SEQ ID NO. 2, respectively, have relatively high availability. Therefore, these two sequences are selected for subsequent development of maize base editors. The methylpurine DNA glycosylase (MPG) after codon preference optimization provided by the application and shown in SEQ ID NO. 1 is named ZmMPG; and the uracil DNA glycosylase (UNG) after codon preference optimization and shown in SEQ ID NO. 2 is named ZmUPG.

[0044] In order to further obtain a glycosidase base editor with the best gene editing effect, the application optimizes the glycosidase sequence, further designs mutations of different amino acid sites of the glycosidase, and combines the optimized glycosidase and its mutants into different positions of nCas9 to obtain a plurality of base editors, which are respectively named ZmGBE, ZmTDG, ZmTSBE3, ZmDAF-TBE, and ZmgTBEv5 in the following, and the construction process of these base editors is specifically introduced below.

[0045] Example 1 Efficiency screening of different glycosidase and mutant base editors

[0046] (1) Construction of pDual-LucM, ZmGBE, ZmTDG, ZmTSBE3, ZmDAF-TBE, and ZmTDGv5 vectors

[0047] In order to quickly screen the editing efficiency of different glycosidase and its mutants in corn protoplast in the embodiment, the inventors developed a tool that can efficiently and sensitively monitor the transient expression efficiency of base editor in corn protoplast, named pDual-LucM. The pDual-LucM is a dual luciferase reporter gene detection system, which comprises a first expression cassette for expressing a reference protein; and a second expression cassette for expressing a reporter gene; the reference protein is luciferase; the reporter gene is mNluc, and the nucleotide sequence is shown as SEQ ID NO: 3. The tool is based on the Nluc reporter system and is improved to obtain mNluc. The third and fourth amino acids of nano luciferase (Nluc) are replaced with stop codon (TAG) on the dual luciferase vector, which causes the inactivation of nano luciferase, and the stop codon is introduced, and the vector structure is as shown in Figure 1 In the pDual-LucM, the inventors designed a universal guide sequence to guide the base editor in the application to anchor to the pDual-LucM. The base editor restores the Nluc activity by correcting the stop codon in the pDual-LucM, so that the gene restores normal expression and emits fluorescence.

[0048] In order to screen the base editor with the highest editing efficiency, the inventors combined ZmMPG, ZmUNG and its multiple different mutants with different positions of nCas9 to obtain multiple base editors. Specifically, ZmMPG was fused to the C-terminal of nCas9 (D10A) to create ZmGBE editor; ZmUNG was fused to the N-terminal of nCas9 (D10A) to create ZmTDG editor; the first mutant of ZmUNG was fused between the 1047th and 1064th amino acids of nCas9 (D10A) to obtain ZmgTBEv5 editor, the first mutant of UNG is that four amino acids in UNG are replaced and the first 88 amino acids are deleted, and the replaced amino acids include Y156A, A214T, Q259A, Y284D; the second mutant of UNG was fused between the 1046th and 1063rd amino acids of nCas9 (D10A) to obtain ZmTSBE3 editor, and the second mutant of UNG is that three amino acids in UNG are replaced, including G107E, Y147A, R260K; the third mutant of UNG was fused to the N-terminal of nCas9 (D10A) to obtain ZmDAF-TBE editor, and the third mutant of UNG is that 11 amino acids in UNG are mutated and the first 84 amino acids are deleted, and the mutated amino acids include F85L, G107E, L142V, Y147A, K175E, S237L, A255V, K259E, T266A, V274A, V275S;

[0049] The above glycosidase and its mutant nucleotide sequences are artificially synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd., and the nucleotide sequences of mNluc, the first mutant of UNG, the second mutant of UNG and the third mutant of UNG are shown in SEQ ID NO: 3-SEQ ID NO: 6, respectively.

[0050] (2) Protoplast transformation and fluorescence value determination

[0051] We use the corn inbred line KN5585 variety (obtained from the University of Missouri) to prepare protoplasts, and the corn protoplast isolation and transformation method can refer to the literature [4] . The vector pDual-LucM carrying inactivated nanoluciferase is transformed into protoplasts as a control group, and the base editor of the application is co-transfected with the corresponding pDual-LucM vector into protoplasts as an experimental group. The experimental steps of protoplast transformation and dual-luciferase reporter gene detection are briefly described as follows: co-transfect pDual-LucM vector and corresponding base editor into 100 μl corn protoplasts, incubate for 14 hours, then add 100 ul 1x Passive Lysis Buffer (Promega) to the protoplasts; after incubation for 15 minutes, add an equal volume of ONE-Glo EX reagent to the protoplasts. After 3 minutes of incubation, an equal volume of NanoDLR Stop&Glo reagent was added to evaluate luciferase activity. In order to detect the editing efficiency in protoplasts, the transformed protoplasts were collected for genomic DNA extraction. The editing efficiency was analyzed by deep amplicon sequencing.

[0052] The results of transient editing experiments in corn protoplasts are shown in Figure 2 , it can be seen from Figure 2 that ZmgGBE has the highest editing efficiency. Based on the above results, the inventors selected ZmgGBE base editor as a candidate base editor for further development, and the nucleotide sequence of ZmgGBE base editor is shown in SEQ ID NO: 7, and the schematic diagram of inducing base editing is shown in Figure 1 A.

[0053] Example Two: Editing efficiency of ZmgGBE base editor in corn protoplasts

[0054] Based on the results in Example One, in this example, the inventors further evaluated the editing efficiency of the base editor in maize protoplasts, and designed three sgRNAs, sgRNA2, sgRNA3 and sgRNA4, targeting three genes of maize, ZmGA20ox3, ZmEPSPS and ZmALS2, respectively. The nucleotide sequences are shown in Table 1; deep amplicon sequencing (Hi-TOM sequencing) was performed for the first round of PCR amplification primers of sgRNA, as shown in Table 2. The test results are shown in Figure 3 Table 3. For the three target genes ZmGA20ox3, ZmEPSPS and ZmALS2, the ZmgGBE editor successfully induced G-to-T, G-to-C and G-to-A base substitutions from G4 to G10 positions, with an editing efficiency of 0.54% to 4.72%.

[0055] Table 1 sgRNA sequence

[0056] sgRNA Nucleotide sequence sgRNA2 GCAGCTCTTGTACCGCCCGTTGG sgRNA3 ACTCAAGTACTGACTGCTGATGG sgRNA4 AGTGTATGCAAATTATGCAGTGG

[0057] Table 2 sgRNA sequence Hi-TOM sequencing first round PCR amplification primer

[0058] sgRNA2-HF ggagtgagtacggtgtgcGTACTAACTCCCCCCACCCC sgRNA2-HR gagttggatgctggatggGCGCGGGCACAGGAAGAA sgRNA3-HF ggagtgagtacggtgtgcGGGGCATTGCCTGTTTTCTAG sgRNA3-HR gagttggatgctggatggCAATGATTTCAATCTCCACATCCCCA sgRNA4-HF ggagtgagtacggtgtgcTTATGGGCCTTGGCAACTTCC sgRNA4-HR gagttggatgctggatggCCTCAATTTTCCCTGTCACACGATC

[0059] Example Three Editing efficiency of ZmgGBE base editor in transgenic maize plants

[0060] Further, in view of the performance of the ZmgGBE editor in maize protoplasts, in this example, the inventors further evaluated its editing efficiency in transgenic maize plants using the same target sites (Table 1).

[0061] (1) Agrobacterium-mediated maize transformation

[0062] The ZmgGBE vector was transformed into maize according to the Agrobacterium transformation method, and the maize inbred line KN5585 of the company was selected for transformation. The glufosinate resistance gene was used as a screening marker, and positive resistant calli were obtained after conventional tissue culture screening. Further differentiation obtained stable transformed plants, and a total of 54 T0 generation transgenic maize plants were obtained.

[0063] (2) Genotyping of T0 plants

[0064] Genomic DNA was extracted from the leaves of transgenic maize plants using the CTAB method, and about 200bp DNA fragments covering the target region were amplified using specific primers. These amplification products were analyzed by Sanger sequencing, and then adapter primers were added and a sequencing library was constructed. Genotyping was performed by Hi-Tom sequencing [5] .

[0065] The same targets as in Example 2: sgRNA2, sgRNA3, sgRNA4 were used for editing efficiency test, and the results are shown in Table 3. ZmgGBE editors, which achieved G-to-A base conversion at sgRNA2 and sgRNA3 targets, with efficiencies of 5.3% (1 / 19) and 16.7% (3 / 18), respectively; achieved G-to-T base conversion at sgRNA2 and sgRNA4 targets, with efficiencies of 31.6% (6 / 19) and 29.4% (5 / 17), respectively.

[0066] To further verify the editing effect of ZmgGBE in maize, we additionally designed four sgRNAs—sgRNA5, sgRNA6, sgRNA7 and sgRNA8, which target two genes ZmPDS and ZmLG1, respectively. The editing results are shown in Table 3. In addition to G-to-T base conversion, ZmgGBE at the four sgRNA sites had an editing efficiency of up to 66.7%, with an average efficiency of 31.4%, and some events produced G-to-C base conversion.

[0067] Table 3 Editing efficiency of ZmgGBE editor in T0 transgenic maize plants

[0068]

[0069] We selected representative plants for one-generation sequencing analysis, and the sequencing peak chart results showed that sgRNA2 produced a heterozygous G-to-T base transition at G10 position, successfully modifying tyrosine (Y) into a stop codon (Figure 4C). Similarly, sgRNA5 produced a homozygous G-to-T base transition at G7 position, modifying glycine (G) into a stop codon (Figure 4D), causing ZmPDS gene mutation, and corn produced a white phenotype at the seedling stage (Figure 4E).

[0070] Since the base editor editing process produces AP sites that can be cleaved by AP lyase, from Figure 4 It can be seen from Table 3 that some T0 transgenic maize plants (0%-85.7%) carried insertion and deletion (InDel) mutations, and double alleles or heterozygous editing was detected in some plants, indicating that the gene editing induced by the base editor ZmgGBE of the application is heritable.

[0071] References:

[0072] [1] Tong, H., Liu, N., Wei, Y., Zhou, Y., Li, Y., Wu, D., Jin, M., Cui, S., Li, H., Li, G., et al. (2023). Programmable deaminase-free base editor for G-to-Y conversion via engineered glycosidases. Natl Sci Rev. 10: nwad143.

[0073] [2] Tong, H., Wang, H., Wang, X., Liu, N., Li, G., Wu, D., Li, Y., Jin, M., Li, H., Wei, Y., et al. (2024). Deaminase-free T-to-S and C-to-G base editors developed via engineered human uracil DNA glycosylase. Nat Commun. 15: 4897.

[0074] [3] Ye, L., Zhao, D., Li, J., Wang, Y., Li, B., Yang, Y., Hou, X., Wang, H., Wei, Z., Liu, X., et al. (2024). Glycosidase-based base editors enable efficient T-to-G and C-to-G editing in mammalian cells. Nat Biotechnol. 42: 1538-47.

[0075] [4] 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.

[0076] [5] Liu, Q., Wang, C., Jiao, X., Zhang, H., Song, L., Li, Y., Gao, C., and Wang, K. (2019). Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR / Cas systems. Sci China Life Sci. 62: 1-7.

[0077] It is readily apparent to those skilled in the art that the above-described preferred embodiments of the application are only illustrative and not restrictive, and that any modifications, equivalent replacements and improvements made within the spirit and principle of the application shall be included in the scope of protection of the application.

[0078] SEQ ID NO: 1

[0079] GTCACCCCGGCGCTGCAGATGAAGAAACCAAAGCAGTTCTGCCGCCGCATGGGCCA

[0080] GAAGAAGCAAAGGCCTGCGCGCGCGGGGCAGCCCCACTCCTCGAGCGACGCCGCGC

[0081] AGGCGCCGGCCGAGCAGCCACACTCGTCAAGTGATGCTGCACAAGCACCCTGCCCG

[0082] CGGGAGAGGTGTTTGGGACCACCGACCACGCCGGGGCCCTACCGCTCCATCTACTTC

[0083] TCCAGCCCCAAAGGCCATCTGACACGGTTGGGCCTCGAGTTCTTCGACCAGCCCGCT

[0084] GTCCCTCTCGCTCGTGCCTTCCTCGGGCAAGTCTTAGTAAGGCGTCTCCCTAATGGA

[0085] ACTGAACTTCGGGGCAGAATAGTTGAAACAGAGGCCTACCTGGGTCCCGAGGACGA

[0086] GGCTGCTCATTCAAGAGGTGGGCGTCAAACACCAAGGAACAGGGGGATGTTCATGA

[0087] AGCCTGGTACTCTCTACGTGTACATCATTTATAGGATGTACTTCTGCATGGGCATTTC

[0088] ATCACAAGGAAGAGGTGCTAATGTTCTTCTCAGAGCTTTGGAGCCGCTGGAGGGCTT

[0089] GGAAACCATGAGGCAGCTCCGGGCCACGCTGCGCGCCGCGACTGCAGCGCGGGTGC

[0090] TGGCTGATAGGGAGCTGTGTTCGGGGCCGTCCAAACTTTGCCAGGCGCTGGCCATCA

[0091] ACAAGAGCTTTGATCAGAGGGACCTCGCGCAGGACGAAGCCGTGTGGCTGGAGAGA

[0092] GGTCCTCTTGAACCATCTGAACCTGCTGTCGTCGCGGCCGCCAGAGTTGGGGTGGGC

[0093] CACGCGGGCGAGTGGGCCAGGAAGCCCTTGCGCTTCTATGTGCGCGGTAGCCCATG

[0094] GGTGTCTGTTGTGGACCGCGTCGCCGAG AGGGATACACAAGCA

[0095] SEQ ID NO:2

[0096] ATGATCGGCCAGAAGACGCTCTACTCCTTTTTCTCCCCCAGCCCCGCCAGGAAGCGA

[0097] CACGCCCCCAGCCCCGAGCCGGCCGTCCAGGGGACCGGCGT

[0098] GGCTGGGGTGCCTGAGGAAAGCGGAGATGCGGCGGCCATCCCAGCCAAGAAGGCC

[0099] CCGGCTGGGCAGGAGGAGCCTGGGACGCCGCCCTCCTCGCCGCTGAGTGCCGAGCA

[0100] GTTGGACCGGATCCAGAGGAACAAGGCCGCGGCCCTGCTCAGACTCGCGGCCCGCA

[0101] ACGTGCCCGTGGGCTTTGGAGAGAGCTGGAAGAAGCACCTCAGCGGGGAGTTCGGG

[0102] AAACCGTATTTTATCAAGCTAATGGGATTTGTTGCAGAAGAAAGAAAGCATTACACT

[0103] GTTTATCCACCCCCACACCAAGTCTTCACCTGGACCCAGATGTGTGACATAAAAGAT

[0104] GTGAAGGTTGTCATCCTGGGACAGGACCCATATCATGGACCTAATCAAGCTCACGG

[0105] GCTCTGCTTTAGTGTTCAAAGGCCTGTTCCGCCTCCGCCCAGTTTGGAGAACATTTAT

[0106] AAAGAGTTGTCTACAGACATAGAGGATTTTGTTCATCCTGG

[0107] CCATGGAGATTTATCTGGGTGGGCCAAGCAAGGTGTTCTCCTTCTCAACGCTGTCCT

[0108] CACGGTTCGTGCCCATCAAGCCAACTCTCATAAGGAGCGAGGCTGGGAGCAGTTCA

[0109] CTGATGCAGTTGTGTCCTGGCTAAATCAGAACTCGAATGGCCTTGTTTTCTTGCTCTG

[0110] GGGCTCTTATGCTCAGAAGAAGGGCAGTGCCATTGATAGGAAGCGGCACCATGTAC

[0111] TACAGACGGCTCATCCCTCCCCTTTGTCAGTGTATAGAGGGTTCTTTGGATGTAGAC

[0112] ACTTTTCAAAGACCAATGAGCTGCTGCAGAAGTCTGGCAAGAAGCCCATTGACTGG

[0113] AAGGAGCTG

[0114] SEQ ID NO:3

[0115] ATGCACCACTGACACCACCATTCAGGGGGTCTTCACACTCGAAGATTTCGTTGGGGA

[0116] CTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGT

[0117] CCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACT

[0118] CCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGT

[0119] CATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTT

[0120] TTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCA

[0121] CACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATG

[0122] AAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAAC

[0123] GGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGA

[0124] GTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAASEQ ID NO:4

[0125] TTTTTCTCCCCCAGCCCCGCCAGGAAGCGACACGCCCCCAGCCCCGAGAACGTGCCC

[0126] GTGGGCTTTGGAGAGAGCTGGAAGAAGCACCTCAGCGGGGAGTTCGGGAAACCGTA

[0127] TTTTATCAAGCTAATGGGATTTGTTGCAGAAGAAAGAAAGCATTACACTGTTTATCC

[0128] ACCCCCACACCAAGTCTTCACCTGGACCCAGATGTGTGACATAAAAGATGTGAAGG

[0129] TTGTCATCCTGGGACAGGACCCAGCTCATGGACCTAATCAAGCTCACGGGCTCTGCT

[0130] TTAGTGTTCAAAGGCCTGTTCCGCCTCCGCCCAGTTTGGAGAACATTTATAAAGAGT

[0131] TGTCTACAGACATAGAGGATTTTGTTCATCCTGGCCATGGAGATTTATCTGGGTGGG

[0132] CCAAGCAAGGTGTTCTCCTTCTCAACACGGTCCTCACGGTTCGTGCCCATCAAGCCA

[0133] ACTCTCATAAGGAGCGAGGCTGGGAGCAGTTCACTGATGCA

[0134] GTTGTGTCCTGGCTAAATCAGAACTCGAATGGCCTTGTTTTCTTGCTCTGGGGCTCTT

[0135] ATGCTGCTAAGAAGGGCAGTGCCATTGATAGGAAGCGGCACCATGTACTACAGACG

[0136] GCTCATCCCTCCCCTTTGTCAGTGGATAGAGGGTTCTTTGGATGTAGACACTTTTCAA

[0137] AGACCAATGAGCTGCTGCAGAAGTCTGGCAAGAAGCCCATTGACTGGAAGGAGCTG

[0138] ACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATC

[0139] SEQ ID NO:5

[0140] ATGTTTTTCTCCCCCAGCCCCGCCAGGAAGCGACACGCCCCCAGCCCCGAGCCGGCC

[0141] GTCCAGGGGACCGGCGTGGCTGGGGTGCCTGAGGAAAGCGGAGATGCGGCGGCCAT

[0142] CCCAGCCAAGAAGGCCCCGGCTGGGCAGGAGG

[0143] AGCCTGGGACGCCGCCCTCCTCGCCGCTGAGTGCCGAGCAGTTGGACCGGATCCAG

[0144] AGGAACAAGGCCGCGGCCCTGCTCAGACTCGCGGCCCGCAACGTGCCCGTGGGCTT

[0145] TGGAGAGAGCTGGAAGAAGCACCTCAGCGGGGAGTTCGGGAAACCGTATTTTATCA

[0146] AGCTAATGGAGTTTGTTGCAGAAGAAAGAAAGCATTACACTGTTTATCCACCCCCAC

[0147] ACCAAGTCTTCACCTGGACCCAGATGTGTGACATAAAAGATGTGAAGGTTGTCATCC

[0148] TGGGACAGGATCCAGCTCATGGACCTAATCAAGCTCACGGGCTCTGCTTTAGTGTTC

[0149] AAAGGCCTGTTCCGCCTCCGCCCAGTTTGGAGAACATTTATAAAGAGTTGTCTACAG

[0150] ACATAGAGGATTTTGTTCATCCTGGCCATGGAGATTTATCTGGGTGGGCCAAGCAAG

[0151] GTGTTCTCCTTCTCAACGCTGTCCTCACGGTTCGTGCCCATCAAGCCAACTCTCATAA

[0152] GGAGCGAGGCTGGGAGCAGTTCACTGATGCAGTTGTGTCCTGGCTAAATCAGAACT

[0153] CGAATGGCCTTGTTTTCTTGCTCTGGGGCTCTTATGCTCAGAAGAAGGGCAGTGCCA

[0154] TTGATAGGAAGAAGCACCATGTACTACAGACGGCTCATCCCTCCCCTTTGTCAGTGT

[0155] ATAGAGGGTTCTTTGGATGTAGACACTTTTCAAAGACCAATGAGCTGCTGCAGAAGT

[0156] CTGGCAAGAAGCCCATTGACTGGAAGGAGCTGGGCAGTAGCGGTAGT

[0157] SEQ ID NO:6

[0158] GGAGAGAGCTGGAAGAAGCACCTCAGCGGGGAGTTCGGGAAACCGTATTTTATCAA

[0159] GCTAATGGAATTTGTTGCAGAAGAAAGAAAGCATTACACTGTTTATCCACCCCCACA

[0160] CCAAGTCTTCACCTGGACCCAGATGTGTGACATAAAAGATGTGAAGGTTGTCATCGT

[0161] GGGACAGGACCCAGCTCATGGACCTAATCAAGCTCACGGGCTCTGCTTTAGTGTTCA

[0162] AAGGCCTGTTCCGCCTCCGCCCAGTTTGGAGAACATTTATGAAGAGTTGTCTACAGA

[0163] CATAGAGGATTTTGTTCATCCTGGCCATGGAGATTTATCTGGGTGGGCCAAGCAAGG

[0164] TGTTCTCCTTCTCAACGCTGTCCTCACGGTTCGTGCCCATCAAGCCAACTCTCATAAG

[0165] GAGCGAGGCTGGGAGCAGTTCACTGATGCAGTTGTGTCCTGGCTAAATCAGAACTT

[0166] GAATGGCCTTGTTTTCTTGCTCTGGGGCTCTTATGCTCAGAAGAAGGGCAGTGTCAT

[0167] TGATAGGGAGCGGCACCATGTACTACAGGC

[0168] GGCTCATCCCTCCCCTTTGTCAGCGTCTAGAGGGTTCTTTGGATGTAGACACTTTTCA

[0169] AAGACCAATGAGCTGCTGCAGAAGTCTGGCAAGAAGCCCATTGACTGGAAGGAGCT

[0170] G

[0171] SEQ ID NO:7

[0172] ATGGTACCAAAGAAGAAGCGGAAGGTCCCTAAGAAGAAGAGAAAAGTTGGATCCA

[0173] TGAAGAGGACAGCCGACGGCTCTGAGTTCGAGTCCCCGAAGAAGAAGCGCAAGGTT

[0174] TCCGGCGGATCCGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGT

[0175] GGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGC

[0176] TGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTC

[0177] GACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGAT

[0178] ACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATG

[0179] GCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGA

[0180] GGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCT

[0181] ACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACC

[0182] GACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGG

[0183] GGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCT

[0184] GTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACG

[0185] CCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGG

[0186] CTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAA

[0187] CCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGC

[0188] CGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACC

[0189] TGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGT

[0190] CCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCC

[0191] CCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCT

[0192] GCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCG

[0193] ACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGA

[0194] GTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGC

[0195] TCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGC

[0196] AGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGA

[0197] AGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCT

[0198] TCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGA

[0199] TGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGAC

[0200] AAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCT

[0201] GCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGT

[0202] ATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTC

[0203] CTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAA

[0204] AGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACT

[0205] CCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCAC

[0206] GATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGA

[0207] CATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCG

[0208] AGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTG

[0209] AAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCA

[0210] TCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTC

[0211] GCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGA

[0212] CATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCA

[0213] ATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTG

[0214] GACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAAT

[0215] GGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATG

[0216] AAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACC

[0217] CCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAAT

[0218] GGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGA

[0219] TGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGT

[0220] GCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAG

[0221] GTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTAC

[0222] CCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTG

[0223] GATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCA

[0224] CGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGC

[0225] TGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGG

[0226] AAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGA

[0227] CGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGG

[0228] AAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCC

[0229] AAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACAT

[0230] CATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGC

[0231] CTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGAT

[0232] TTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGAC

[0233] CGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCG

[0234] ATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGAC

[0235] AGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTC

[0236] CAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGC

[0237] AGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAA

[0238] AAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCC

[0239] GGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTG

[0240] CCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGC

[0241] TCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCT

[0242] GGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACG

[0243] CTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGA

[0244] GAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCC

[0245] GCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGA

[0246] GGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGA

[0247] TCGACCTGTCTCAGCTGGGAGGCGACTCTGGCGGCTCTAAGCGGACTGCGGATGGG

[0248] TCTGAGTTCGAGTCACCAAAGAAGAAGAGGAAGGTGCTCGGCGGCGACAGCGGGG

[0249] GATCTGGCGGGAGTGGTGGGTCCGTCACCCCGGCGCTGCAGATGAAGAAACCAAAG

[0250] CAGTTCTGCCGCCGCATGGGCCAGAAGAAGCAAAGGCCTGCGCGCGCGGGGCAGCC

[0251] CCACTCCTCG AGCGACGCCG CCGCAGGCGC CGGCCGAGCA GCCACACTCG TCAAGTG

[0252] ATGCTGCACA AGCACCCTGC CCGCGGGAGA GGTGTTTGGG ACCACCGACC ACGCCG

[0253] GGGCCCTACC GCTCCATCTA CTTCTCCAGC CC AAAGGCCAT CTGACACGGT TGGGC

[0254] CTCGAGTTCT TCGACCAGCC CGCTGTCCCT CTCGCTCGTG CCTTCCTCGG GCAAGTCT

[0255] TAGTAAGGCG TCTCCCTAAT GGAACTGAACTTCGGGGC AGAATAGTTG AAACAGAG

[0256] GCCTACCTGG GTCCCGAGGA CGAGGCTGCT CATTCAAGAG GTGGGCGTCA AACACC

[0257] AAGGAACAGG GGGATGTTCAT GAAGCCTGGT ACTCTCTACG TGTACATCAT TTATAG

[0258] GATGTACTTC TGCATGGGCA TTTCATCACA AGGAAGAGGT GCTAATGTTCT TCTCAG

[0259] AGCTTTGGAG CCGCTGGAGG GCTTGGAAAC CATGAGGCAG CTCCGGGCCA CGCTGC

[0260] GCGCCGCGAC TGCAGCGCGG GTGCTGGCTG ATAGGGAGCT GTGTTCGGGG CCGTCC

[0261] AAACTTTGCC AGGCGCTGGC CATCAACAAG AGCTTTGATC AGAGGGACCT CGCGCA

[0262] GGACGAAGCC GTGTGGCTGG AGAGAGGTCC TCTTGAACCA TCTGAACCTG CTGTCGT

[0263] CGCGGCCGCCAGAGTTGGGGTGGGCCACGCGGGCGAGTGGGCCAGGAAGCCCTTGC

[0264] GCTTCTATGTGCGCGGTAGCCCATGGGTGTCTGTTGTGGACCGCGTCGCCGAGAGGG

[0265] ATACACAAGCATCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGAGC

[0266] CCCAAGAAGAAGAGGAAAGTCAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGG

[0267] CAAAAAAGAAAAAGAAGAGGCCGGCGGCAACCAAGAAGGCTGGCCAGGCCAAGAA

[0268] GAAAAAA

Claims

1. A glycosidase-based maize gene editor, characterized in that, The base editor has the following structural formula: NLS-Z-NLS-L-M-NLS wherein, NLS is a nuclear localization signal element; L is an optional connecting sequence; Z is a nCas9(D10A) nucleotide sequence; M is a methyl-purine DNA glycosylase; each “-” is independently a bond or a nucleotide connecting sequence.

2. The glycosidase-based maize gene editor of claim 1, wherein, The gene editor achieves G to T base substitution, and / or G to A base substitution, and / or G to C base substitution.

3. The glycosidase-based maize gene editor of claim 1, wherein, The nucleotide sequence of the methyl-purine DNA glycosylase is shown in SEQ ID NO:

1.

4. The glycosidase-based maize gene editor of claim 1, wherein, The nucleotide sequence of the base editor is shown in SEQ ID NO:

7.

5. A nucleic acid construct for assessing editing efficiency of a maize gene editor, comprising, The nucleic acid construct comprises a first expression cassette expressing a reference protein; and a second expression cassette expressing a reporter gene; the reference protein is luciferase; the reporter gene is mNluc, and the nucleotide sequence thereof is shown in SEQ ID NO:

3.

6. An expression cassette comprising, The expression cassette comprises the corn gene editor of any one of claims 1-5.

7. An expression vector, characterized by, The expression vector comprises the corn gene editor of any one of claims 1-5 or the expression cassette of claim 7.

8. A host cell, characterized in that, The host cell contains the vector of claim 8, or the genome of which is integrated with the gene editor nucleic acid sequence of any one of claims 1-5.

9. A method of site-directed editing of a maize genome, characterized in that, The method comprises the following steps: (i) providing a host cell and a first vector and a second vector, wherein the first vector contains an expression cassette of the gene editor of any one of claims 1-4, and the second vector contains an expression cassette expressing sgRNA; (ii) infecting the host cell with the first vector and the second vector, thereby performing base site-directed editing in the cell, wherein the cell is a corn cell.

10. Use of the corn gene editor of claims 1-4, the nucleic acid sequence of claim 5, the expression cassette of claim 6, the expression vector of claim 7, the host cell of claim 8, the method of corn genome site-directed editing of claim 9 in corn breeding.

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