Application of a compact CRISPR / SwCas9 system in maize gene editing

Abstract

The application discloses application of a compact CRISPR / SwCas9 system in corn gene editing. Zmzb7 and ZmF3H The editing efficiency of T0 plants targeting the genes is 60% and 75% respectively. The application provides a novel tool with independent intellectual property right, compact size and high efficiency for corn gene knockout and multi-target point editing.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a compact CRISPR / SwCas9 system in plant gene editing. Background Technology

[0002] The CRISPR / Cas system can guide RNA-mediated nucleases to create DNA double-strand breaks at specific sites in the genome, thereby enabling gene knockout, insertion, or replacement via intracellular repair mechanisms. It has become an important technology for plant functional gene research and crop genetic improvement. Maize is an important food and feed crop, but its genetic transformation cycle is long and significantly limited by genotype, placing high demands on the delivery efficiency, expression stability, and vector capacity of editing elements (Svitashev et al., 2016).

[0003] Currently, maize gene editing mainly relies on SpCas9 derived from Streptococcus pyogenes. While this system is well-established, the large size and long coding sequence of the SpCas9 protein mean it occupies significant space in Agrobacterium-mediated transformation, gene gun delivery, viral vector delivery, and multi-sgRNA module assembly, potentially reducing the ease of vector construction and delivery. Furthermore, the editing efficiency varies considerably between different targets, making it difficult to meet the needs of multi-gene, multi-site, and high-throughput breeding applications (Jing et al., 2021; Shi et al., 2024).

[0004] To reduce vector burden, studies have attempted to use small or different-sourced Cas nucleases such as SaCas9 and St1Cas9 for plant gene editing. However, some systems are still limited by PAM recognition range, plant cell expression adaptability, target selectivity, or editing efficiency stability. Especially in maize, there is still a lack of compact Cas9 tools that can balance molecular size, editing efficiency, PAM applicability, and multi-target expansion capabilities.

[0005] Furthermore, a significant portion of the core nucleases in existing plant gene-editing tools are derived from publicly available systems, resulting in a relatively concentrated intellectual property portfolio. For maize molecular breeding and industrial applications, developing a Cas9 nuclease and its accompanying sgRNA system with independent intellectual property rights would not only enrich the gene-editing toolkit but also help reduce intellectual property risks in subsequent technology transfer and application promotion.

[0006] Therefore, there is an urgent need in the field for a compact gene editing system based on the proprietary Cas9, suitable for maize cell expression and delivery, capable of recognizing effective PAMs and stably inducing mutations at target sites. The SwCas9 involved in this invention is derived from Staphylococcus warwick (…). Warneri StaphylococcusThe SwCas9 protein, with a length of 1054 amino acids, is more compact than the commonly used SpCas9. Currently, there are no reports on the application of this SwCas9 in plant cells or plant gene editing. Its expression in maize cells, sgRNA adaptation, PAM recognition characteristics, and editing activity all need to be established and validated. This invention constructs a SwCas9 nuclease, matches it with an sgRNA backbone and a plant expression vector, and validates its editing activity in stable transformed maize plants and protoplasts. This solves the problems of heavy vector burden, insufficient selection of compact Cas9 nucleases, and lack of plant application validation in existing maize gene editing tools, providing a new technical solution for maize gene knockout, multi-target editing, and molecular breeding. Summary of the Invention

[0007] The technical problem to be solved by this invention is that the large size of the SpCas9 protein molecule commonly used in existing maize gene editing technologies limits the construction, delivery, and assembly of plant expression vectors and multi-target editing modules. At the same time, the editing activity, PAM recognition characteristics, or expression stability of some existing small Cas systems in plant cells cannot fully meet the needs of maize gene editing. This invention provides a compact CRISPR / SwCas9 system suitable for maize gene editing and its application.

[0008] To address the aforementioned technical problems, this invention provides an application of a compact CRISPR / SwCas9 system in maize gene editing, wherein the CRISPR / SwCas9 system comprises: (1) A compact SwCas9 nuclease protein, the amino acid sequence of which is shown in SEQ ID NO.1, and the CDS sequence of which is shown in SEQ ID NO.2, wherein the SwCas9 nuclease protein is derived from Staphylococcus warwickii ( Staphylococcus warneri The SwCas9 nuclease has an amino acid length of 1054 amino acids and is a compact type. Its molecular size is smaller than that of the SpCas9 protein derived from Streptococcus pyogenes, which has 1368 amino acids. (2) A single-stranded guide RNA (sgRNA) that matches the SwCas9 nuclease protein, the nucleotide sequence of the sgRNA backbone being shown in SEQ ID NO.3, wherein the sgRNA includes a backbone sequence capable of forming a complex with the SwCas9 nuclease protein, and a targeting sequence capable of complementary pairing with a target sequence of the maize genome. (3) A plant expression vector for expressing the SwCas9 nuclease protein and the sgRNA in plant cells, wherein the expression of the SwCas9 nuclease protein is regulated by a plant ubiquitin promoter or a promoter with equivalent function, preferably by the maize ZmUbi promoter; and the expression of the sgRNA is regulated by a plant RNA polymerase III promoter, preferably by the OsU6 promoter.

[0009] The SwCas9 nuclease protein shown in SEQ ID NO.1, guided by the sgRNA, can recognize the NNGG type PAM sequence adjacent to the target sequence and generate double-strand DNA breaks at the target site in the maize genome. After the double-strand DNA breaks are repaired by the endogenous DNA repair pathway in maize cells, site-directed mutation, gene knockout, gene insertion, or gene replacement in the maize genome can be achieved, with gene knockout being the preferred method.

[0010] In a preferred embodiment, the sgRNA backbone is capable of forming a stable ribonucleoprotein complex (RNP) with the SwCas9 nuclease protein shown in SEQ ID NO.1, thereby mediating specific cleavage of target DNA with an adjacent NNGG type PAM sequence.

[0011] In a preferred embodiment, the target of the maize gene editing is an endogenous maize gene, including but not limited to... Zmzb7 , ZmF3H , ZmKRN2-2 , ZmKRN2-1 or ZmCLE Gene.

[0012] The present invention also provides a method for maize gene editing using the compact CRISPR / SwCas9 system, comprising: designing a target sequence based on the maize target gene sequence; ligating the target sequence into an sgRNA expression module containing the sgRNA backbone shown in SEQ ID NO.3; constructing the sgRNA expression module and the SwCas9 nuclease protein expression module shown in SEQ ID NO.1 into a plant expression vector; and introducing the resulting recombinant vector into maize cells, tissues, or recipient materials, so that the SwCas9 nuclease protein and sgRNA are expressed in maize cells and form a complex, thereby achieving targeted editing of the maize target gene.

[0013] The beneficial effects of this invention are as follows: (1) The SwCas9 nuclease protein provided by the present invention is a compact nuclease with 1054 amino acids. Compared with the commonly used SpCas9, it has a smaller molecular size, which can reduce the Cas9 coding sequence's occupation of plant expression vector capacity, which is beneficial to simplify vector construction, improve the convenience of application in Agrobacterium-mediated transformation, gene gun delivery and other plant cell delivery systems, and reserve more vector space for the assembly of multiple sgRNA expression cassettes or multiple gene editing modules. (2) The SwCas9 nuclease protein described in this invention can form an effective complex with a specific sgRNA backbone, recognize the target sequence of NNGG type PAM, and generate double-strand DNA breaks at the target site of the maize genome, thus expanding the selection of compact Cas9 tools suitable for maize gene editing; (3) The system described in this invention has been verified through stable transformation in maize and can transform maize endogenous genes. Zmzb7 and ZmF3H The site produces insertion or deletion mutations, among which the targeted Zmzb7 The T0 generation of plants showed an editing efficiency of 60% for the gene, targeting... ZmF3H The editing efficiency of the T0 generation plants was 75%, indicating that the system has high editing activity at the maize plant level. (4) The system described in this invention can also generate expected insertion or deletion mutations at multiple endogenous target sites in maize protoplasts, indicating that it has detectable and reproducible editing capabilities in different maize cell systems. (5) This invention can effectively supplement the existing maize gene editing tool system, and provide a compact, efficient and easy vector construction and delivery technical solution for maize functional gene research, target trait improvement and molecular breeding. It has high application value and promotion prospects. (6) The SwCas9 nuclease and its matching sgRNA system developed in this invention have independent intellectual property rights, which helps to reduce the intellectual property risks in subsequent technology transformation and application promotion. Attached Figure Description

[0014] Figure 1 Schematic diagram of the compact SwCas9 protein structure The SwCas9 protein is 1054 amino acids in size: amino acids 1 to 41, 438 to 483, and 654 to 775 are RuvC domains; amino acids 42 to 74 are Bridge helix (BH) domains; amino acids 75 to 428 are REC domains; amino acids 524 to 631 are HNH domains; amino acids 790 to 909 are WED domains; and amino acids 910 to 1054 are PI domains.

[0015] Figure 2Schematic diagram of the structure of a compact CRISPR / SwCas9 plant expression vector Targeted corn Zmzb7 and ZmF3H Diagram of gene recombination vectors. sgRNA is driven by the OsU6 promoter; compact SwCas9 is driven by the ZmUbi promoter.

[0016] Figure 3 Compact CRISPR / SwCas9 Targeted Corn Zmzb7 Schematic diagram of gene editing results a. Phenotypic images of regenerated maize plants #1 and #5, where plant #1 exhibits complete albinoization; b. Genotyping results of the five regenerated maize plants: #1 has a homozygous +1 bp (+A) mutation at the target site, #2 has a homozygous +1 bp (+T) mutation at the target site, #3 has a homozygous +1 bp (+C) mutation at the target site, and #4 and #5 are wild-type.

[0017] Figure 4 Compact CRISPR / SwCas9 Targeted Corn ZmF3H Schematic diagram of gene editing results Genotyping results of 8 regenerated maize plants: #1, #5 and #8 were heterozygous mutations at the target site; #2 was homozygous mutation at the target site; #4 and #6 were biallelic mutations at the target site; #3 and #7 were wild-type at the target site.

[0018] Figure 5 Results of editing efficiency of compact CRISPR / SwCas9 in maize protoplasts The compact CRISPR / SwCas9 targets three different endogenous gene sites in maize. ZmKRN2-2 , ZmKRN2-1 and ZmCLE Editing efficiency results. Amplicon deep sequencing analysis results show that, compared with the reference sequence, compact CRISPR / SwCas9 produces small insertions or deletions at all three target sites. The red boxes represent inserted sequences, the horizontal lines represent deleted bases, and the bolded bases represent substituted bases. Detailed Implementation

[0019] Example 1: Construction of a compact CRISPR / SwCas9 system plant expression vector This embodiment illustrates the construction process of the plant expression vector for the compact CRISPR / SwCas9 system described in this invention. The vector constructed in this embodiment can express the compact SwCas9 nuclease protein shown in SEQ ID NO.2 and its matching sgRNA in maize cells, and can be used for subsequent gene editing targeting maize endogenous genes. Unless otherwise specified, restriction endonucleases, ligases, and Golden-Gate assembly reagents were used in accordance with their respective product instructions, and all positive clones were confirmed by colony PCR, restriction enzyme digestion, and Sanger sequencing.

[0020] (1) Preparation of compact SwCas9 coding sequence and expression cassette.

[0021] The DNA sequence shown in SEQ ID NO.2 is used as the coding sequence for the compact SwCas9 nuclease, which encodes the 1054-amino acid SwCas9 protein shown in SEQ ID NO.1. This SwCas9 protein contains functional domains including RuvC, Bridgehelix (BH), REC, HNH, WED, and PI. The arrangement of each domain in the protein and their corresponding amino acid ranges are shown below. Figure 1 As shown, this demonstrates that it maintains the core cleavage structure of SwCas9 while possessing a relatively compact molecular size. The SwCas9 coding sequence was placed downstream of the maize ubiquitin promoter ZmUbi and upstream of the Nos terminator to form the ZmUbi-SwCas9-Nos expression cassette; this expression cassette was then assembled into the pICH47742 vector using the Golden-Gate method to obtain the SwCas9 expression module vector pICH47742-ZmUbi-SwCas9-Nos.

[0022] (2) Preparation of sgRNA backbone expression cassette.

[0023] A DNA fragment containing the rice OsU6 promoter, a target sequence insertion region, and the sgRNA backbone sequence shown in SEQ ID NO.3 was artificially synthesized, with BbsI restriction endonuclease recognition sites flanking the target sequence insertion region. This DNA fragment was ligated into the pBM16K plasmid to obtain the pBM16K-OsU6-sgRNA backbone vector. After extracting the backbone vector plasmid, the vector was completely digested with BbsI and purified to linearize it for insertion into the core target sequences of different maize target genes.

[0024] (3) Design and annealing of target sequence oligonucleotides.

[0025] This embodiment uses targeted corn Zmzb7 Genes and ZmF3H Recombinant vectors were constructed using genes as an example.

[0026] Targeted Zmzb7 The core target sequence of the gene is Zmzb7-T1 ( SEQ ID NO.4): 5'-CAGCGACAACTACAACCGCA-3'; The core target sequence targeting the ZmF3H gene is ZmF3H-T1 (SEQ ID NO.5): 5'-CCGCTGCCGCCCGGGCCGA-3'.

[0027] For each core target sequence, a pair of complementary oligonucleotides were synthesized: the forward oligonucleotide consisted of a linker sequence complementary to the sticky 5' end of the BbsI digest and the core target sequence, while the reverse oligonucleotide consisted of a linker sequence complementary to the sticky 3' end of the BbsI digest and the reverse complementary sequence of the core target sequence. The forward and reverse oligonucleotides were annealed to form a double-stranded target sequence insert with BbsI-compatible sticky ends.

[0028] (4) Construction of OsU6-sgRNA target expression cassette. The double-stranded target sequence insert obtained in step (3) was ligated to the ppBM16K-OsU6-sgRNA backbone vector digested with BbsI obtained in step (2), and transformed into competent E. coli cells. After resistance selection, positive clones were selected, and sequencing confirmed that the target sequence insertion direction and sequence were correct, and pBM16K-OsU6-sgRNA-Zmzb7 and pBM16K-OsU6-sgRNA-ZmF3H were obtained respectively.

[0029] (5) The sgRNA expression module was transferred into the plant module vector. The OsU6-sgRNA-Zmzb7 expression cassette and the OsU6-sgRNA-ZmF3H expression cassette obtained in step (4) were assembled into the pICH47732 vector using the Golden-Gate method to obtain the sgRNA expression module vectors pICH47732-OsU6-sgRNA-Zmzb7 and pICH47732-OsU6-sgRNA-ZmF3H.

[0030] (6) Construction of the final plant expression vector. The SwCas9 expression module vector obtained in step (1), the sgRNA expression module vector obtained in step (5), and the selection marker module containing the 35S promoter, bar selection marker gene, and 35S terminator were assembled into the pAGM8031 vector using the Golden-Gate method to obtain compact CRISPR / SwCas9 plant expression vectors for maize genetic transformation, named pCas9-U6-sgRNA (Zmzb7) and pCas9-U6-sgRNA (ZmF3H), respectively. The arrangement of OsU6-sgRNA, ZmUbi-SwCas9-Nos, and the 35S-bar selection marker module in the two recombinant vectors is shown in the figure. Figure 2 This figure illustrates that when targeting different maize genes, only the sgRNA target sequence needs to be replaced, while the SwCas9 expression cassette and the selection marker expression cassette can remain consistent.

[0031] (7) Recombinant vector verification. The recombinant vector obtained in step (6) was identified by restriction enzyme digestion and verified by Sanger sequencing to confirm that the OsU6 promoter, target sequence, sgRNA backbone, ZmUbi promoter, SwCas9 coding sequence shown in SEQ ID NO.2, Nos terminator, and bar selection marker gene were all connected in the predetermined direction and order. The verified recombinant vector was used for subsequent maize genetic transformation or protoplast transformation experiments.

[0032] Among them, pICH47732 (addgene ID: #48000), pICH47742 (addgene ID: #48001), and pAGM8031 (addgene ID: #48037) are derived from Addgene. If a compact CRISPR / SwCas9 vector targeting other maize genes is constructed, only the core target sequence in step (3) needs to be replaced with the target sequence of the adjacent NNGG type PAM sequence in the target gene, while the remaining SwCas9 expression module, sgRNA backbone sequence, and vector assembly method remain unchanged.

[0033] Example 2: Targeted Stable Transformation of Maize Zmzb7 ( Zm00001d033896 Gene editing efficiency analysis This embodiment illustrates that the compact CRISPR / SwCas9 system constructed in Example 1 can perform site-specific editing of the endogenous gene Zmzb7 in stable maize transformant plants.

[0034] (1) Target selection and vector preparation. Targeting maize Zmzb7 Gene( Zm00001d033896The target sequence selected in this embodiment is SEQ ID NO.4 Zmzb7-T1. Following the method described in Example 1, this core target sequence is inserted into the OsU6-sgRNA backbone expression module and assembled with the ZmUbi-SwCas9-Nos expression module and the 35S-bar selection marker module to obtain the target. Zmzb7 The plant expression vector pCas9-U6-sgRNA ( Zmzb7 ).

[0035] (2) Agrobacterium-mediated transformation and maize genetic transformation. The pCas9-U6-sgRNA, verified by sequencing, was transformed... Zmzb7 The plasmid was introduced into Agrobacterium EHA105 via heat shock. After selecting positive Agrobacterium clones for expansion culture, Agrobacterium-mediated transformation was performed using freshly peeled immature embryos of maize inbred line B104 (approximately 1.5 mm in diameter) as recipient material. Resistance selection and regeneration culture were then carried out using the bar selection marker in the vector to obtain T0 generation maize regenerated plants.

[0036] (3) Phenotypic observation. Zmzb7 Homozygous frameshift mutations in the gene can lead to albino phenotypes in maize regenerated plants. Phenotypic observation of the obtained T0 generation regenerated plants revealed that plants with homozygous frameshift mutations at the target site exhibited complete albinoness. The relevant phenotypes are detailed below. Figure 3 a.

[0037] (4) Target site genotyping. Genomic DNA was extracted from leaves of T0 generation plants using the CTAB method. Zmzb7 PCR primers were designed based on the upstream and downstream sequences of the target site to amplify the genomic fragment containing the target site. The primer sequences are as follows: Zmzb7-F1 (SEQ ID NO.6): 5'-GGGAAACACAAACACCAGGC-3'; Zmzb7-R1 (SEQ ID NO. 7): 5'-CAGCCATTCGCCGTCC-3'.

[0038] The PCR products were purified and then subjected to Sanger sequencing. The sequencing results were compared with the wild-type reference sequence to determine whether there were insertion, deletion, or substitution mutations near the target site. The editing efficiency was calculated as "number of T0 generation plants with target site editing / total number of T0 generation plants tested × 100%".

[0039] (5) Editing Results. Five T0 generation regenerated plants were analyzed, and the results showed that three of them underwent editing at the target site, with an editing efficiency of 60%. Specifically, plant #1 had a homozygous +1 bp (+A) mutation at the target site and exhibited an albino phenotype; plant #2 had a homozygous +1 bp (+T) mutation at the target site; plant #3 had a homozygous +1 bp (+C) mutation at the target site; and plants #4 and #5 were wild-type. These results demonstrate that the compact CRISPR / SwCas9 system of this invention can effectively mediate editing in a stable maize transformation system. Zmzb7 Gene knockout, related sequencing results can be found in Figure 3 b.

[0040] Example 3: Targeted Corn ZmF3H ( Zm00001eb351260 Gene editing This embodiment illustrates the effect of the compact CRISPR / SwCas9 system described in this invention on another maize endogenous gene. ZmF3H The system's editing capabilities will be further validated to determine its applicability to different maize target genes.

[0041] (1) Target selection and vector preparation. Targeting maize ZmF3H The gene (Zm00001eb351260) was used as a target, and the core target sequence selected in this embodiment was SEQ ID NO.5 ZmF3H-T1. Following the method described in Example 1, this core target sequence was inserted into the OsU6-sgRNA backbone expression module, and then assembled with the ZmUbi-SwCas9-Nos expression module and the 35S-bar selection marker module to obtain the target gene. ZmF3H The plant expression vector pCas9-U6-sgRNA ( ZmF3H ).

[0042] (2) Maize genetic transformation. The pCas9-U6-sgRNA, verified by sequencing, was transformed... ZmF3H The plasmid was introduced into Agrobacterium EHA105 by heat shock, and the same Agrobacterium-mediated transformation method for maize immature embryos as in Example 2 was used. The immature embryos of maize inbred line B104 with an diameter of about 1.5 mm were used as recipient materials. T0 generation maize plants were obtained through resistance screening and regeneration culture.

[0043] (3) Target site genotyping. Genomic DNA was extracted from leaves of T0 generation plants using the CTAB method. ZmF3H PCR primers were designed based on the upstream and downstream sequences of the target site to amplify the genomic fragment containing the target site. The primer sequences are as follows: ZmF3H-F1 (SEQ ID NO.8): 5'-TGGAGCTCTTCGTCACTACA-3'; ZmF3H-R1 (SEQ ID NO. 9): 5'-CACGTTGATGGCCTTCCAA-3'.

[0044] The PCR products were purified and subjected to Sanger sequencing. The sequencing results were compared with the wild-type reference sequence to determine the mutation type of the target site in the T0 generation plants. The editing efficiency was calculated as "number of T0 generation plants with target site editing / total number of T0 generation plants tested × 100%".

[0045] (4) Editing Results. Eight T0 generation regenerated plants were tested, and the results showed that gene editing occurred at the target site in six of them, with an editing efficiency of 75%. Specifically, plants #1, #5, and #8 had heterozygous mutations at the target site; plant #2 had a homozygous mutation; plants #4 and #6 had biallelic mutations; and plants #3 and #7 were wild-type. These results demonstrate that the compact CRISPR / SwCas9 system described in this invention can not only edit the Zmzb7 gene but also efficiently edit it. ZmF3H The sequencing results indicate that this system has reproducible editing activity at different endogenous gene loci in maize. (See [link to sequencing data]). Figure 4 .

[0046] Example 4: Detection of editing efficiency of compact CRISPR / SwCas9 in maize protoplasts This embodiment illustrates the editing activity of the compact CRISPR / SwCas9 system described in this invention in a transient expression system in maize cells. Through transient protoplast transformation and amplicon deep sequencing, the system's ability to induce insertion or deletion mutations at multiple endogenous target sites in maize can be rapidly evaluated.

[0047] (1) Target selection and vector construction. Based on the maize B104 inbred line, three maize endogenous gene target sites were selected for detection. The sequences of each target site are listed below in the 5' to 3' direction: ZmKRN2-2 Target site ZmKRN2-2-T (SEQ ID NO.10): 5'-AGGGGAGCCGGGCAGCACTCTAGG-3'; ZmKRN2-1 Target site ZmKRN2-1-T (SEQ ID NO.11): 5'-GGCCCTGCATTGCCGTGGTCGGG-3'; ZmCLE Target site ZmCLE-T (SEQ ID NO.12): 5'-CGGCCTTCCTTTGCCTCCTGGCGG-3'.

[0048] Based on the target sequence adjacent to the NNGG type PAM in the above target site, sgRNA guide sequences were designed, and corresponding compact CRISPR / SwCas9 plant expression vectors were constructed according to the method described in Example 1.

[0049] (2) Isolation and transformation of maize protoplasts. Maize protoplasts were prepared using the maize B104 inbred line. For each target site, 20 μg of the corresponding gene-editing plasmid was transformed into maize protoplasts via PEG-mediated transformation. The transformed protoplasts were cultured at room temperature for 48 hours to allow the compact SwCas9 protein and the corresponding sgRNA to be expressed in the cells and act on the target site.

[0050] (3) Target site amplification and sequencing analysis. After culture, protoplast cells were collected, and plant genomic DNA was extracted using the CTAB method. PCR primers were designed based on the upstream and downstream sequences of each target site to amplify DNA fragments covering the target sites. The PCR products were purified and subjected to amplicon deep sequencing. The sequencing reads were compared with wild-type reference sequences using the CRISPResso2 online analysis tool to statistically predict small insertion or deletion mutations near the cleavage sites.

[0051] (4) Editing results. Amplicon deep sequencing results show that, ZmKRN2-2 At the target site, 0.39% of reads (3601 reads) were deletions of 1 bp / -A mutations, 0.28% of reads (2619 reads) were insertions of 1 bp / +T mutations, and 0.21% of reads (1936 reads) were insertions of 1 bp / +A mutations; at the ZmKRN2-1 target site, 0.34% of reads (4067 reads) were insertions of 1 bp / +T mutations; ZmCLE At the target site, 1.20% of the reads (14420 reads) showed a 1 bp deletion / -C mutation, and 0.45% of the reads (5434 reads) showed a 1 bp insertion / +A mutation. These mutations were all small fragment insertions or deletions near the target site, consistent with the characteristics of mutations arising from cellular repair following double-strand DNA breaks mediated by Cas9 nuclease. Related results are shown in [link to relevant results]. Figure 5 .

[0052] (5) Conclusion. Examples 2 and 3 demonstrate that the compact CRISPR / SwCas9 system described in this invention can achieve gene knockout in stable maize transformants; this example further demonstrates that the system can also generate detectable insertion or deletion mutations at multiple maize endogenous gene target sites in a transient expression system of maize protoplasts.

[0053] The above results collectively demonstrate that the compact CRISPR / SwCas9 system described in this invention is suitable for maize gene editing, exhibiting good stability and reproducibility. This invention can effectively supplement the existing maize gene editing tool system, providing a compact, efficient, and easy-to-construct and deliver vector technical solution for maize functional gene research, target trait improvement, and molecular breeding. Furthermore, the SwCas9 nuclease and its matching sgRNA system developed in this invention have independent intellectual property rights and possess high application value and promising prospects for promotion.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0055] References: Svitashev, S., Schwartz, C., Lenderts, B., Young, JK & Cigan, AM Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nature Communications 7, 13274 (2016). Jing, C., Wei, M., Fang, P., Song, R. & Qi, W. Pollen-SpecificCRISPR / Cas9 system to increase heritable gene mutations in maize. Agriculture 11, 751 (2021). Shi, Y., Wang, J., Yu, T., Song, R. & Qi, W. Callus-specific CRISPR / Cas9 system to increase heritable gene mutations inmaize. Planta 260, 16 (2024).

Claims

1. A compact CRISPR / SwCas9 system for maize gene editing, characterized in that, The compact CRISPR / SwCas9 system includes: (1) SwCas9 nuclease protein, wherein the SwCas9 nuclease protein is derived from Staphylococcus warwick (… Staphylococcus warneri The amino acid sequence of the SwCas9 nuclease protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the SwCas9 nuclease protein is shown in SEQ ID NO.

2. (2) A single-stranded guide RNA matching the SwCas9 nuclease protein described in (1), namely sgRNA, wherein the sgRNA comprises an sgRNA backbone sequence and a target sequence, the sgRNA backbone sequence being shown in SEQ ID NO.3; (3) Plant expression vectors, including SwCas9 expression cassettes and sgRNA expression cassettes.

2. The application of the compact CRISPR / SwCas9 system as described in claim 1 in maize gene editing, characterized in that, The SwCas9 nuclease protein described in claim 1 is able to recognize NNGG-type PAM sequences adjacent to the target sequence in the maize genome under the guidance of the sgRNA described in claim 1, and generate double-strand DNA breaks at the target site in the maize genome.

3. A plant expression vector for maize gene editing, characterized in that, The plant expression vector comprises: (1) a SwCas9 expression cassette, the SwCas9 expression cassette comprising a nucleotide sequence encoding the SwCas9 nuclease protein shown in SEQ ID NO.1; and (2) an sgRNA expression cassette, the sgRNA expression cassette comprising the sgRNA backbone sequence shown in SEQ ID NO.3 and a targeting sequence that is complementary to the maize genome target sequence; the maize genome target sequence having a neighboring NNGG type PAM sequence.

4. A method for gene editing in maize, characterized in that, The procedure includes the following steps: selecting a target sequence adjacent to an NNGG-type PAM sequence based on the maize target gene sequence; ligating the target sequence into an sgRNA expression module containing the sgRNA backbone sequence shown in SEQ ID NO.3; constructing the sgRNA expression module and a SwCas9 expression module encoding the SwCas9 nuclease protein shown in SEQ ID NO.1 into a plant expression vector; and introducing the resulting plant expression vector into maize cells, tissues, or recipient materials, so that the SwCas9 nuclease protein and the sgRNA are expressed in maize cells and form a complex, thereby achieving targeted editing of the maize target gene.

5. The method according to claim 4, characterized in that, The corn cells, tissues, or receptor materials are corn embryos or corn protoplasts; the introduction method is Agrobacterium-mediated transformation or PEG-mediated transformation.

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