Cas12i3 protein mutants and uses thereof

Abstract

This invention discloses Cas12i3 protein mutants and their applications. Based on the Cas12i3_S7R protein, this invention utilizes structure-guided design and a combination of individual and combined mutations at key amino acid sites to target and optimize the Cas12i3 protein. Experiments demonstrate that, compared to the Cas12i3_S7R protein, the Cas12i3 mutant combinations provided by this invention significantly improve gene editing efficiency at multiple target sites in maize, offering a new pathway for Cas protein modification. This has significant application value and can be widely used in biological genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology, specifically relating to Cas12i3 protein mutants and their applications. Background Technology

[0002] CRISPR-Cas system-mediated gene editing technology is currently the most widely used and versatile technical approach in crop genetic improvement and gene function research. As my country's largest grain crop, the precise genetic improvement of yield and quality-related traits in maize is of great significance to ensuring national food security; and efficient and precise gene editing tools are the core foundation for maize molecular breeding and gene function analysis. Cas12i3 is a novel Cas nuclease, belonging to type VI of the class II CRISPR-Cas system. Naturally derived from prokaryotes, its natural function is to defend against exogenous nucleic acid invasion, and it is a core component of the prokaryotic acquired immune system. Unlike Cas9, Cas12i3 has independent intellectual property rights in my country and is a monomeric protein. Smaller than Cas9, it is easier to deliver in practical applications. This nuclease has the potential for plant gene editing applications; however, when wild-type Cas12i3 is directly applied to maize gene editing scenarios, there is insufficient adaptability to the editing needs of the plant host, resulting in low target editing efficiency and failing to meet the practical application requirements of maize molecular breeding.

[0003] To enhance the editing activity of Cas12i3, existing technologies have obtained the Cas12i3 amino acid mutant Cas12i3_S7R through amino acid site mutation screening. This mutant exhibits significantly enhanced nuclease activity compared to wild-type Cas12i3, enabling gene editing at certain specific targets. However, in practical applications, the enhanced activity of Cas12i3_S7R shows a clear target-dependent effect. At target sites of several key functional genes in maize, the editing efficiency still falls short of the practical application standards for maize gene editing. Therefore, further development of Cas12i3 protein mutants with higher editing efficiency is needed in the field of maize gene editing to provide efficient and precise gene editing tools for maize molecular breeding and gene function analysis. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the purpose of this invention is to provide a Cas12i3 protein mutant and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a Cas12i3 protein mutant, which, compared with the amino acid sequence of the wild-type Cas12i3 protein (SEQ ID NO:1), exhibits the following mutations: a) S7R; and b) Amino acid substitutions at any one or more of the following amino acid sites: E5, Y124, D166, E321, V460, Y468, S582, N586, E601.

[0006] Furthermore, the amino acid substitution refers to the substitution with arginine.

[0007] Furthermore, the E321 can also be replaced with glycine.

[0008] Furthermore, the Cas12i3 protein mutant is selected from any one of the following: Cas_S7R_V460R, Cas_S7R_Y468R, Cas_S7R_S582R, Cas_S7R_N586R, Cas_S7R_S811R, Cas_S7R_E321R_V460R, Cas_S7R_E5R_V460R, Cas_S7R_E321G_V460R, Cas_S7R_Y124R_D166R, Cas_S7R_V460R_Y468R, Cas_S7R_Y124R_S582R , Cas_S7R_V460_E601R, Cas_S7R_Y124R_V460R_Y468R, Cas_E5R_S7R_Y124R_V460R, Cas_S7R_Y124R_D166R_V460R, Cas_S7R_Y12 4R_V460R_Y468R, Cas_S7R_Y124R_D166R_V460R_Y468R, Cas_E5R_S7R_Y124R_V460R_N586R, Cas_E5R_S7R_Y124R_V460R_S811R.

[0009] A second aspect of the present invention provides a fusion protein comprising the Cas12i3 protein mutant described in the first aspect of the present invention and one or more functional domains.

[0010] A third aspect of the present invention provides a polynucleotide that encodes the Cas12i3 protein mutant described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention.

[0011] A fourth aspect of the present invention provides a carrier comprising the polynucleotide described in the third aspect of the present invention.

[0012] The fifth aspect of the present invention provides any of the following products: 1) A CRISPR-Cas system, the system comprising: a) The Cas12i3 protein mutant described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, or the vector described in the fourth aspect of the present invention; and b) Guide RNA, polynucleotide encoding the guide RNA, or vector carrying the guide RNA.

[0013] 2) An engineered host cell comprising the Cas12i3 protein mutant of the first aspect of the present invention, the fusion protein of the second aspect of the present invention, the polynucleotide of the third aspect of the present invention, the vector of the fourth aspect of the present invention, or the CRISPR-Cas system.

[0014] 3) A kit comprising the Cas12i3 protein mutant of the first aspect of the present invention, the fusion protein of the second aspect of the present invention, the polynucleotide of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the CRISPR-Cas system or the host cell.

[0015] Furthermore, a) and b) can exist independently or combine to form a complex.

[0016] Furthermore, the complex may also include the target sequence of the guide RNA described in b).

[0017] Furthermore, the host cells include prokaryotic cells and eukaryotic cells.

[0018] The sixth aspect of the present invention provides any of the following methods: 1) A method for preparing the host cell according to the fifth aspect of the present invention, the method comprising: introducing the polynucleotide according to the third aspect of the present invention or the vector according to the fourth aspect of the present invention into the host cell.

[0019] 2) A method for preparing the Cas12i3 protein mutant according to the first aspect of the present invention, the method comprising: culturing the host cell according to the fifth aspect of the present invention, inducing host cell expression, separating and purifying the expression product, thereby obtaining the Cas12i3 protein mutant.

[0020] 3) A gene editing method, the method comprising contacting a target sequence with the CRISPR-Cas system described in the fifth aspect of the present invention.

[0021] 4) A method for improving plant traits, the method comprising editing at least one target gene of a plant sample using the gene editing method, regenerating plants based on the edited sample, and improving plant traits through screening or breeding.

[0022] Furthermore, the target sequence can be an ex vivo sequence or a sequence present in a biological sample.

[0023] Furthermore, the samples include animal-derived samples and plant-derived samples.

[0024] Furthermore, the plant-derived samples include corn, rice, chili peppers, wheat, soybeans, sorghum, and millet.

[0025] Furthermore, the plant in question is corn.

[0026] The seventh aspect of the present invention provides an improved plant tissue or cell, said improved plant tissue or cell being obtained by the method for improving plant traits described in the sixth aspect of the present invention.

[0027] Furthermore, the plants include corn, rice, chili peppers, wheat, soybeans, sorghum, and millet.

[0028] Furthermore, the plant in question is corn.

[0029] The eighth aspect of the present invention provides any of the following applications: 1) The application of the Cas12i3 protein mutant described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, or the product described in the fifth aspect of the present invention in gene editing.

[0030] 2) The use of the Cas12i3 protein mutant described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, or the product described in the fifth aspect of the present invention in the preparation of reagents or kits for gene editing.

[0031] 3) The application of the Cas12i3 protein mutant described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, or the product described in the fifth aspect of the present invention in the construction of biological models.

[0032] 4) The application of the Cas12i3 protein mutant described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, or the product described in the fifth aspect of the present invention in improving plant traits.

[0033] 5) The use of the Cas12i3 protein mutant described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, or the product described in the fifth aspect of the present invention in the preparation of a medicament for treating gene-related diseases.

[0034] Furthermore, the biological models include cell models, animal models, and plant models.

[0035] Advantages and beneficial effects of the present invention: This invention utilizes a structure-guided design, based on the Cas12i3_S7R protein, and employs a combination of individual and combined mutations at key amino acid sites to target and optimize the Cas12i3 protein. Experiments have demonstrated that, compared to the Cas12i3_S7R protein, the Cas12i3 mutant combination significantly improves editing efficiency at multiple target sites in maize, providing a new pathway for Cas protein modification. This approach has significant application value and can be widely used in biological genetic improvement. Attached Figure Description

[0036] Figure 1 This diagram illustrates the structure of the Cas12i3-CrRNA-target DNA complex and the screening of mutation sites. A is a three-dimensional structural diagram of the wild-type Cas12i3-CrRNA-target DNA complex, with the gray portion representing the Cas12i3 protein backbone, the blue portion representing CrRNA, and the red portion representing target DNA. B is a diagram illustrating the labeling of key amino acid sites in the DNA unwinding region, with yellow markings indicating candidate amino acid sites within the DNA unwinding region. C is a flipped view of B. D is a diagram illustrating the labeling of key amino acid sites in the protein-heteroduplex binding region, with green markings indicating candidate amino acid sites within the binding region.

[0037] Figure 2 This is a schematic diagram of the vector structure of the Cas12i3 protein mutant.

[0038] Figure 3 The editing efficiency of the maize sbe4 gene by the Cas12i3 protein double mutant.

[0039] Figure 4 The editing efficiency of the maize sbe4 gene by the Cas12i3 protein multisite combination mutant.

[0040] Figure 5 The editing efficiency of the maize rel2_1 gene by the Cas12i3 protein multisite combination mutant.

[0041] Figure 6 The editing efficiency of the maize rel2_3 gene by the Cas12i3 protein multisite combination mutant. Detailed Implementation

[0042] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0043] The reagents, raw materials, and experimental consumables used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are typically performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0044] In some embodiments, the amino acid sequence of the parental Cas protein of the Cas12i3 protein mutant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with SEQ ID No:1.

[0045] In some embodiments, conserved amino acid substitutions may be performed at one or more predicted non-essential amino acid residues. “Non-essential” amino acid residues are those that can be altered (deleted, substituted, or replaced) without changing biological activity, while “essential” amino acid residues are required for biological activity. A “conserved amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Amino acid substitutions may be performed in the non-conserved regions of the aforementioned Cas mutant protein. Generally, such substitutions are not performed on conserved amino acid residues, or on amino acid residues located within conserved motifs, where such residues are required for protein activity. However, those skilled in the art will understand that functional variants may have fewer conserved or non-conserved alterations in conserved regions.

[0046] In some implementations, the term "AxxB" indicates that amino acid A at position xx is changed to amino acid B. For example, S7R indicates that S at position 7 is mutated to R. When multiple amino acid sites are mutated simultaneously, it can be expressed in a form similar to A1xxB1_A2xxB2. For example, S7R_V460R represents that S at position 7 is mutated to R and V at position 460 is mutated to R.

[0047] In this invention, amino acid residues can be represented by a single letter or a three-letter symbol, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamic acid (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), and arginine (Arg, R).

[0048] In some embodiments, the functional domains are selected from: nuclear localization signals, nuclear output signals, base editing domains, base excision domains, uracil glycosylation enzyme inhibitors or their catalytic domains, uracil glycosylation enzymes or their catalytic domains, methylpurine glycosylation enzymes or their catalytic domains, methyltransferases or their catalytic domains, demethylases or their catalytic domains, transcriptional activation domains, transcriptional repression domains, reverse transcriptases or their catalytic domains, exonucleases or their catalytic domains, destabilization domains, histone residue modification domains, nuclease catalytic domains, transcriptional modification factors, and light-gated factors. Chemically inducible factors, chromatin visualization factors, targeting peptides that provide binding to cell surface portions on target cells or target cell types, reporter peptides or detection tags, localization signals, peptide targeting moieties, DNA-binding domains, epitope tags, transcription release factors, HDACs, moieties with ssRNA cleaving activity, moieties with dsRNA cleaving activity, moieties with ssDNA cleaving activity, moieties with dsDNA cleaving activity, DNA or RNA ligases, functional domains exhibiting activity of modifying target DNA and their catalytic domains and functional fragments thereof, and any combination thereof.

[0049] In some embodiments, the activity of the modified target DNA is selected from: methyltransferase activity, DNA repair activity, DNA damage activity, superoxide dismutase activity, alkylation activity, dealkylation activity, depurination activity, oxidation activity, deoxygenation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylation activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristylation activity, demyristylation activity, glycosylation activity (e.g., from O-GlcNAc transferase), and deglycosylation activity.

[0050] In some embodiments, the polynucleotide is isolated or purified. The sequence of the polynucleotide can be obtained using conventional techniques. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. Alternatively, the relevant sequence can be synthesized artificially, especially when the fragment length is short. Generally, longer fragments can be obtained by first synthesizing multiple small fragments and then ligating them.

[0051] In this invention, the term "encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes. These polymers and macromolecules have defined nucleotide sequences (e.g., rRNA, tRNA, and mRNA) or defined amino acid sequences and the biological properties derived therefrom. Therefore, if the transcription and translation of mRNA corresponding to the gene produces a protein in a cell or other biological system, then the gene, cDNA, or RNA encodes a protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically provided in the sequence listing, and the non-coding strand, which serves as a template for transcribing the gene or cDNA, can be referred to as the protein or other product encoding that gene or cDNA.

[0052] In some embodiments, various vectors known in the art can be used, such as commercially available vectors, and then the polynucleotide encoding the above-mentioned Cas12i3 protein mutant can be operatively linked to the expression regulatory sequence to form an expression vector. In some embodiments, the vector includes, but is not limited to, plasmids, phage particles, granules, artificial chromosomes, and virus-derived vectors.

[0053] In some embodiments, the vector may also contain one or more marker genes that allow selection of host cells for transformation or transfection. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.); and prototrophic complementation in auxotrophic hosts. Suitable marker genes for the expression vector of this invention include neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, and puromycin resistance genes.

[0054] In some embodiments, the guide RNA is any polynucleotide sequence that is sufficiently complementary to the target sequence to hybridize with the target sequence and guide the CRISPR / Cas complex to specifically bind to the target sequence. In one embodiment, the complementarity between the guide RNA and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% when optimal alignment is achieved. Determining optimal alignment is within the capabilities of a person skilled in the art. For example, publicly available and commercially available alignment algorithms and programs exist, such as, but not limited to, ClustalW, the Smith-Waterman algorithm in MATLAB, Bowtie, Geneious, Biopython, and SeqMan.

[0055] In some implementations, the "target sequence" refers to a polynucleotide targeted by a guide sequence in the guide RNA, such as a sequence complementary to the guide sequence, wherein hybridization between the target sequence and the guide sequence will promote the formation of a CRISPR / Cas complex (including the Cas protein and the guide RNA). Perfect complementarity is not required, as long as sufficient complementarity exists to induce hybridization and promote the formation of a CRISPR / Cas complex.

[0056] In some embodiments, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsiae. The bacteria include *Escherichia coli*, *Bacillus subtilis*, *Salmonella typhimurium*, *Pseudomonas*, *Streptomyces*, and *Staphylococcus*. The eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0057] In some embodiments, the components of the kit may be packaged in an aqueous medium or in lyophilized form. The container of the kit typically includes at least one vial, test tube, flask, bottle, syringe, or other container in which the components can be placed, and preferably appropriately aliquoted. In cases where the kit contains more than one component, the kit typically also includes a second, third, or other additional container in which the additional components can be placed individually. However, various combinations of components may be contained in the vial. The kit may also include tools for containing buffers and / or other diluents. Optionally, instructions for use of such a kit are provided.

[0058] In some implementations, the methods for introduction include, but are not limited to, physical, chemical, and biological methods. Physical methods include, but are not limited to, microinjection, electroporation, calcium phosphate precipitation, lipid transfection, and particle bombardment; chemical methods include, but are not limited to, colloidal dispersion systems and lipid-based systems; colloidal dispersion systems include, but are not limited to, macromolecular complexes, nanocapsules, microspheres, and beads; lipid-based systems include, but are not limited to, oil-in-water emulsions, micelles, mixed micelles, and liposomes; and biological methods include, but are not limited to, DNA vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, and adeno-associated virus vectors.

[0059] In some implementations, the gene editing includes modifying genes, knocking out genes, altering the expression of gene products, repairing mutations, and / or inserting polynucleotides, or gene mutations.

[0060] In some implementations, the term "treatment" refers to treating or curing a disease, delaying the onset of symptoms, and / or slowing the progression of the disease.

[0061] Example 1: Obtaining the Cas12i3 protein mutant I. Experimental Methods 1. Identification of mutation sites: This invention first uses Alphafold3 software to predict the three-dimensional structure of the complex formed by Cas12i3 protein, CrRNA (CRISPR RNA), and target DNA. Then, PyMOL software is used to perform a visual and rational analysis of the predicted three-dimensional structure: nucleic acid chains (CrRNA and target DNA) in the complex are selected, and amino acid sites of Cas12i3 protein that interact with the nucleic acid chains within a 5 angstrom range are screened out. During the analysis, the focus is on the DNA unwinding region, as well as the negatively charged amino acid sites and nonpolar amino acid sites in the region where Cas12i3 protein binds to the heteroduplex (the heteroduplex formed by CrRNA and target DNA).

[0062] Wild-type Cas12i3 protein sequence: 2. Mutation Induction: Reverse PCR amplification was performed using NEB Q5® High-Fidelity DNA Polymerase. Target mutations were introduced using primers designed and applied according to standard techniques in the field. The 50 μL reaction system was prepared as shown in Table 1. Table 1 Reaction System

[0063] The PCR reaction program was set as follows: Pre-denaturation: 98℃, 30 s; Amplification cycles (18 cycles): 98℃ denaturation for 10 s, annealing temperature adjusted according to primer Tm value (60–72℃), annealing for 30 s, extension at 72℃ (extension time calculated based on full vector length, 1kb / 30 s); Final extension: 72℃, 5 min; Incubation: 4℃, infinity.

[0064] After PCR amplification, 1 μL of DpnI restriction endonuclease (10 U / μL) was added directly to the reaction system, gently mixed, and incubated in a 37℃ water bath for 1–2 h to specifically digest the methylated wild-type template plasmid from E. coli, retaining only the PCR product of the unmethylated mutant plasmid to remove the interference of the wild-type template on subsequent experiments.

[0065] Using a single mutant plasmid that has been verified by sequencing as a template, the above experimental steps were repeated to introduce a second, third, and more target mutation sites in sequence to construct a multi-mutation combination of Cas12i3 protein mutants.

[0066] 3. Transformation and Validation of the Mutant Plasmid: 5 μL of the PCR product digested with DpnI was slowly added to 50 μL of *E. coli* DH5α chemocompetent cells and incubated on ice for 30 min; then, heat-shocked at 42℃ for 45 s, and rapidly transferred to ice for 2 min to complete the transformation. 450 μL of antibiotic-free LB broth was added to the transformation system, and the cells were incubated at 37℃ with shaking at 220 rpm for 1 h to restore cell viability and expression of the resistance gene.

[0067] Take 200 μL of the revived bacterial culture and spread it evenly on LB agar plates containing kanamycin (50 μg / mL). Incubate at 37°C upside down for 12–16 h to obtain single colonies. Randomly select 3–5 single colonies for each mutation site and inoculate them into 5 mL of LB liquid medium containing kanamycin. Incubate at 37°C with shaking at 220 rpm for 12 h. After extracting plasmid DNA using a plasmid extraction kit, send the samples to a sequencing company for Sanger full-length sequencing verification.

[0068] Sequencing result verification criteria: The target mutation site is correctly introduced (e.g., in the E321R mutant, the 321st codon is successfully mutated from GAG to AGG), there are no additional random mutations, the sequencing peaks are clear and free of impurities, and the plasmid that meets the requirements is the correct mutant plasmid, which should be stored at -20℃ for later use.

[0069] II. Experimental Results The results are as follows Figure 1 As shown, through visual rational analysis, 24 candidate amino acid sites were finally screened in the DNA unwinding region and 24 candidate amino acid sites were screened in the protein-heteroduplex interaction region. Site-directed single mutations or combination mutations were performed on the above sites to obtain the Cas12i3 protein mutant.

[0070] Example 2: Validation of gene editing efficiency of Cas12i3 protein mutant I. Experimental Methods 1. Preparation of etiolated maize seedlings: Select plump, uniform, disease-free, and mold-free B73 maize inbred line seeds. Disinfect the seeds with 75% (v / v) ethanol for 30 seconds, and rinse three times with sterile, enzyme-free water to thoroughly remove residual ethanol. Spread the disinfected seeds evenly in a sterile petri dish lined with moist sterile filter paper and place it in a constant temperature incubator at 25°C in complete darkness for static cultivation. When the seedlings grow to the longest leaf length of 13-17 cm, select etiolated seedlings with uniform growth, no mechanical damage, and no abnormal yellowing as donor materials for protoplast preparation. Strict aseptic operation procedures are followed throughout the process.

[0071] 2. Preparation of maize protoplasts: Take the upper part of the leaves of the etiolated seedlings that are full of cells and have no damaged veins. Remove the main vein with a sterile scalpel and quickly cut them into uniform filaments with a width of 0.5-1 mm, avoiding water loss and mechanical damage to the leaves throughout the process. Immerse the leaf filaments completely in the pre-prepared enzymatic hydrolysis solution at a material-to-liquid ratio of 1 g:10 mL and place them in a constant temperature shaker at 25℃ for 2.8-3.8 h in the dark with shaking at 40 r / min. After enzymatic hydrolysis, filter the enzymatic hydrolysis mixture through a 400-mesh sterile cell sieve to remove undigested leaf tissue residue. Collect the filtrate in a sterile round-bottom centrifuge tube and centrifuge at 100×g for 2 min with the centrifuge speed set to level 1. After centrifugation, gently discard the supernatant to avoid disturbing the protoplast precipitate. Resuspend the protoplast precipitate in W5 washing solution pre-cooled at 4℃. Repeat the above centrifugation and washing steps twice. Finally, resuspend the precipitate in MMG solution pre-cooled at 4℃.

[0072] 3. Construction and transfection of recombinant vectors: A Cas12i3 protein mutant vector was constructed using the ampicillin resistance T vector (Beijing Bomaide Gene Technology Co., Ltd., PMD18-T) as the backbone. The vector structure is shown below. Figure 2As shown. The constructed Cas12i3 protein mutant vector and the corresponding guide RNA (sequence shown in Table 2) targeting expression recombinant plasmid were transformed into *E. coli* Mach-T1 chemocompetent cells (Beijing Bomed Gene Technology Co., Ltd., BC102-02). The cells were plated on LB solid medium containing 100 μg / mL ampicillin and incubated at 37°C with inverted incubation for 12–16 h. Single colonies were picked and inoculated into 50 mL of LB liquid medium containing 100 μg / mL ampicillin and incubated at 37°C with shaking at 220 r / min for 12–16 h until the bacterial culture reached OD. 600 The plasmid concentration was 0.6–0.8. An endotoxin-free plasmid extraction kit (Tiangen Biotech (Beijing) Co., Ltd., DP117) was used, and plasmid extraction and purification were performed strictly according to the kit instructions. Plasmid integrity was verified by 1% (w / v) agarose gel electrophoresis, requiring a single band, no degradation, and no impurities. The purity and concentration of the plasmid were detected using a Thermo Nanodrop 2000 nucleic acid quantification instrument, requiring an OD value of 0.6–0.8. 260 / OD 280 =1.8~2.0, adjust the final concentration of all plasmids to 1000 ng / μL, and store at -20℃ protected from light for later use.

[0073] Table 2 Guide RNA Sequences

[0074] Take 100 μL of the above concentration, which is 1×10 6 Add 10 μg of Cas12i3 protein mutant vector and 10 μg of the corresponding target CrRNA expression plasmid to a sterile centrifuge tube. Gently pipette to mix, avoiding air bubbles that could damage the protoplasts. Immediately add 100 μL of freshly prepared PEG4000 transfection buffer, gently invert to mix, and incubate at room temperature in the dark for 25 min. After incubation, add 400 μL of W5 wash buffer pre-chilled at 4℃ and gently invert to mix, terminating the transfection reaction. Centrifuge at 100×g for 2 min (Eppendorf 5810R centrifuge, speed setting 1), and discard the supernatant. Resuspend the protoplast pellet in WI medium and incubate at 25℃ in the dark for 48 h, maintaining a stable temperature throughout the incubation process and avoiding light and vibration.

[0075] 4. Construction of Hi-TOM high-throughput sequencing libraries using a two-round PCR method: 1) First round of PCR: Using protoplast genomic DNA as a template, specific primers designed for upstream and downstream of the editing target sites sbe4, rel2_1, rel2_2, and rel2_3 were used (see Table 3) to prepare 50 μL PCR reaction systems: 25 μL of 2×PhantaMax high-fidelity PCR premix, 2 μL each of upstream and downstream primers (10 μmol / L), 2 μL of genomic DNA template (50 ng / μL), and 19 μL of sterile enzyme-free water.

[0076] PCR amplification program settings: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ final extension for 5 min, and incubation at 4℃.

[0077] After amplification, 5 μL of PCR product was subjected to 1% agarose gel electrophoresis to verify successful amplification of the target band (single band, no obvious impurities). No gel extraction or purification was performed, and the remaining product was stored at 4°C for later use.

[0078] Table 3 Primer sequences for the first round of PCR

[0079] 2) Second round PCR: Take 0.5 μL of the first round PCR product as a template and use primers containing both Illumina universal sequencing adapters and Hi-TOM sample-specific paired-end splitting index adapters (see Table 4) for the second round of amplification. Prepare a 50 μL PCR reaction system: 25 μL of 2×PhantaMax high-fidelity PCR premix, 2 μL each of upstream and downstream adapter primers (10 μmol / L), 0.5 μL of the first round PCR product template, and 20.5 μL of sterile enzyme-free water.

[0080] PCR amplification program settings: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ final extension for 5 min, and incubation at 4℃.

[0081] After amplification, 5 μL of PCR product was taken for 1% agarose gel electrophoresis to verify the integrity of the bands. Then, the target band was excised and purified using a DNA gel extraction kit to obtain a next-generation sequencing library that could be directly sequenced. After quantification with Qubit, the library was stored at -20°C in the dark for later use.

[0082] Table 4 Primer sequences for the second round of PCR

[0083] 5. High-throughput sequencing and gene editing efficiency analysis: All constructed sample sequencing libraries were subjected to 150 bp paired-end high-throughput sequencing using the Illumina NovaSeq 6000 platform to obtain raw sequencing data. Using the official splitting tool provided with the Hi-TOM platform, samples were split according to the corresponding Hi-TOM paired-end index adapter sequences to remove adapter contamination sequences, low-quality reads (base quality value Q<20), and invalid sequences shorter than 50 bp, obtaining clean sequencing data. The clean sequencing data was uploaded to the Hi-TOM 2.0 online analysis platform. The target editing site sequence, primer sequences, and the corresponding sequence of the maize B73 reference genome were input. The platform automatically completed sequence alignment, mutation site identification, and mutation type statistics, ultimately accurately calculating the gene editing efficiency of the target sites in each experimental group and control group. Simultaneously, the Hi-TOM platform was used to predict the top 10 potential off-target sites for each target and detect editing efficiency, assess the editing specificity of different Cas12i3 protein mutants, export a complete analysis report, and retain all raw sequencing data.

[0084] II. Experimental Results The results showed that, after unit point mutations were performed on the Cas12i3_S7R mutant, some mutants (Cas_S7R_V460R, Cas_S7R_Y468R, Cas_S7R_S582R, Cas_S7R_N586R, Cas_S7R_S811R) exhibited significantly better editing efficiency. Figure 3 We then performed combinatorial mutations on the Cas12i3 protein, identifying 14 combinatorial mutants with superior editing efficiency compared to the Cas12i3_S7R mutant (Cas_S7R_E321R_V460R, Cas_S7R_E5R_V460R, Cas_S7R_E321G_V460R, Cas_S7R_Y124R_D166R, Cas_S7R_V460R_Y468R, Cas_S7R_Y124R_S582R, Cas_S7R_V460_E601R). , Cas_S7R_Y124R_V460R_Y468R, Cas_E5R_S7R_Y124R_V460R, Cas_S7R_Y124R_D166R_V460R, Cas_S7R_Y124R_V460R_Y 468R, Cas_S7R_Y124R_D166R_V460R_Y468R, Cas_E5R_S7R_Y124R_V460R_N586R, Cas_E5R_S7R_Y124R_V460R_S811R) ( Figure 4-6 ).

[0085] This invention systematically screened and validated the editing performance of various Cas12i3 combinatorial variants through multi-target, multi-repetition maize protoplast gene editing experiments, ultimately obtaining 19 combinatorial variants with superior editing efficiency: Cas_S7R_V460R, Cas_S7R_Y468R, Cas_S7R_S582R, Cas_S7R_N586R, Cas_S7R_S811R, Cas_S7R_E321R_V460R, Cas_S7R_E5R_V460R, Cas_S7R_E321G_V460R, Cas_S7R_Y124R_D166R, and Cas_S7R_V460R_Y. 468R, Cas_S7R_Y124R_S582R, Cas_S7R_V460_E601R, Cas_S7R_Y124R_V460R_Y468R, Cas_E5R_S7R_Y124R_V460R, Cas_S7R_Y124R_D166R_V460R , Cas_S7R_Y124R_V460R_Y468R, Cas_S7R_Y124R_D166R_V460R_Y468R, Cas_E5R_S7R_Y124R_V460R_N586R, Cas_E5R_S7R_Y124R_V460R_S811R. This invention provides an efficient and precise gene editing tool for the field of gene editing, especially for maize gene editing, and has broad application value.

[0086] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A mutant of a Casl2i3 protein, characterized in that, The Cas12i3 protein mutant, compared with the wild-type Cas12i3 protein (SEQ ID NO:1), exhibits the following mutations: a) S7R; and b) Amino acid substitutions at any one or more of the following amino acid sites: E5, Y124, D166, E321, V460, Y468, S582, N586, E601.

2. The Casl2i3 protein mutant of claim 1, wherein, The amino acid substitution refers to the substitution with arginine; Preferably, E321 can also be replaced with glycine.

3. The Casl2i3 protein mutant of claim 1, wherein The Cas12i3 protein mutant is selected from any one of the following: Cas_S7R_V460R, Cas_S7R_Y468R, Cas_S7R_S582R, Cas_S7R_N586R, Cas_S7R_S811R, Cas_S7R_E321R_V460R, Cas_S7R_E5R_V460R, Cas_S7R_E321G_V460R, Cas_S7R_Y124R_D166R, Cas_S7R_V460R_Y468R, Cas_S7R_Y124R_S582R, C as_S7R_V460_E601R, Cas_S7R_Y124R_V460R_Y468R, Cas_E5R_S7R_Y124R_V460R, Cas_S7R_Y124R_D166R_V460R, Cas_S7R_Y124 R_V460R_Y468R, Cas_S7R_Y124R_D166R_V460R_Y468R, Cas_E5R_S7R_Y124R_V460R_N586R, Cas_E5R_S7R_Y124R_V460R_S811R.

4. A fusion protein, characterized in that, The fusion protein comprises the Cas12i3 protein mutant as described in any one of claims 1-3 and one or more functional domains.

5. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-4. The polynucleotide encodes the Cas12i3 protein mutant of any one of claims 1-3 or the fusion protein of claim 4.

6. A vector, characterized in that, The vector comprises the polynucleotide of claim 5.

7. Any of the following products: 1) A CRISPR-Cas system, the system comprising: a) the Cas12i3 protein mutant according to any one of claims 1-3, the fusion protein according to claim 4, the polynucleotide according to claim 5, or the vector according to claim 6; and b) Guide RNA, polynucleotide encoding the guide RNA, or vector carrying the guide RNA; 2) An engineered host cell, said host cell comprising the Cas12i3 protein mutant of any one of claims 1-3, the fusion protein of claim 4, the polynucleotide of claim 5, the vector of claim 6, or the CRISPR-Cas system; 3) A kit comprising the Cas12i3 protein mutant of any one of claims 1-3, the fusion protein of claim 4, the polynucleotide of claim 5, the vector of claim 6, the CRISPR-Cas system, or the host cell; Preferably, a) and b) can exist independently or combine to form a complex; Preferably, the complex may further include the target sequence of the guide RNA described in b); Preferably, the host cell includes prokaryotic cells and eukaryotic cells.

8. Any of the following methods: 1) A method of making the host cell of claim 7, the method comprising: The polynucleotide of claim 5 or the vector of claim 6 is introduced into host cells. ； 2) A method for preparing the Cas12i3 protein mutant according to any one of claims 1-3, the method comprising: culturing the host cell according to claim 7, inducing host cell expression, separating and purifying the expression product, thereby obtaining the Cas12i3 protein mutant; 3) A method for gene editing, the method comprising contacting a target sequence with the CRISPR-Cas system of claim 7; 4) A method for improving plant traits, the method comprising editing at least one target gene of a plant sample using the gene editing method, regenerating plants based on the edited sample, and improving plant traits through screening or breeding; Preferably, the target sequence can be an in vitro sequence or a sequence present in a biological sample; Preferably, the samples include animal-derived samples and plant-derived samples; Preferably, the plant-derived samples include corn, rice, chili peppers, wheat, soybeans, sorghum, and millet; Preferably, the plant is corn.

9. An improved plant tissue or cell, characterized in that, The improved plant tissue or cells are obtained by the method for improving plant traits as described in claim 8; Preferably, the plants include corn, rice, chili peppers, wheat, soybeans, sorghum, and millet; Preferably, the plant is corn.

10. Any of the following applications: 1) The use of the Cas12i3 protein mutant as described in any one of claims 1-3, the fusion protein as described in claim 4, the polynucleotide as described in claim 5, the vector as described in claim 6, or the product as described in claim 7 in gene editing; 2) The use of the Cas12i3 protein mutant of any one of claims 1-3, the fusion protein of claim 4, the polynucleotide of claim 5, the vector of claim 6, or the product of claim 7 in the preparation of reagents or kits for gene editing; 3) The use of the Cas12i3 protein mutant as described in any one of claims 1-3, the fusion protein as described in claim 4, the polynucleotide as described in claim 5, the vector as described in claim 6, or the product as described in claim 7 in the construction of biological models; 4) The use of the Cas12i3 protein mutant as described in any one of claims 1-3, the fusion protein as described in claim 4, the polynucleotide as described in claim 5, the vector as described in claim 6, or the product as described in claim 7 in improving plant traits; 5) The use of the Cas12i3 protein mutant of any one of claims 1-3, the fusion protein of claim 4, the polynucleotide of claim 5, the vector of claim 6, or the product of claim 7 in the preparation of a medicament for treating gene-related diseases; Preferably, the biological model includes a cell model, an animal model, or a plant model.

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