SgRNA composition for targeted editing of CCM1-CCM4, expression vector composition, and application and method of sgRNA composition and expression vector composition in plant growth and development

Abstract

The invention provides a sgRNA composition for targeted editing of CCM1-CCM4, an expression vector composition and application and a method in plant growth and development, and belongs to the technical field of plant genetic engineering. The invention provides an sgRNA composition. The sgRNA composition comprises a sgRNA of a targeted CCM1 gene, a sgRNA of a targeted CCM2 gene, a sgRNA of a targeted CCM3 gene and a sgRNA of a targeted CCM4 gene, wherein the nucleotide sequences of the sgRNA are sequentially shown as SEQ ID NO: 1-SEQ ID NO: 4. In the embodiment of the invention, the sgRNA composition can realize accurate targeting on multiple regeneration key genes in the cell Acidinium paracellum, effectively promote the growth and development of plants, improve the regeneration rate of protoplast cells, shorten the cultivation time of protofilament regeneration propagation, greatly shorten the cultivation time of Acidinium paracellum protofilament propagation, and improve the survival rate of Acidinium paracellum. And the cultivation efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to an sgRNA composition for targeted editing of CCM1-CCM4, an expression vector composition, and its application and method in plant growth and development. Background Technology

[0002] A crucial aspect of plant growth and development is plant regeneration. The fundamental mechanisms of plant regeneration include plant hormones, cell division, epigenetic remodeling, and transcription factors, all of which play key roles in somatic cell regeneration. The pluripotency of plant cells depends on the activity of stem cells, which determines their ability to self-renew and differentiate into specific tissues or organs.

[0003] Physcomitrium patens, as a model organism for non-seed plant research, can form various stem cells. Its simple plant structure and high regenerative capacity provide a suitable system for studying plant regeneration and stem cell induction mechanisms at the cellular level. Physcomitrium patens, with its efficient homologous recombination ability and evolutionary uniqueness, has become a "living fossil" for research on key gene regulation in plant regeneration. However, the regeneration system of Physcomitrium patens is still limited by existing technological shortcomings, such as low transformation efficiency, complexity of multi-gene regulation, and limited regulatory targets. Conserved regulatory genes in the plant carbon concentrating mechanism (CCM) are involved in plant photosynthesis, biotic or abiotic stress responses, amino acid or lipid biosynthesis, growth and development, and cell differentiation. Currently, there are few reports on designing sgRNAs targeting multiple genes in the plant carbon concentrating mechanism. Summary of the Invention

[0004] In view of this, the present invention provides an sgRNA composition that can precisely target multiple genes, namely the key genes for cell regeneration, CCM1, CCM2, CCM3 and CCM4, effectively promoting plant growth and development, increasing the regeneration rate of protoplasts, and shortening the cultivation time for protoplast regeneration and reproduction.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The present invention provides an sgRNA composition comprising CCM1 sgRNA, CCM2 sgRNA, CCM3 sgRNA and CCM4 sgRNA as shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 4.

[0007] The present invention provides an sgRNA expression vector containing the sgRNA composition.

[0008] The present invention provides an expression vector composition comprising the sgRNA expression vector and the Cas protein expression vector.

[0009] The present invention provides the application of the sgRNA composition, the sgRNA expression vector, or the expression vector composition in promoting plant growth and development.

[0010] In this invention, the plant includes non-seed plants.

[0011] In this invention, the growth and development includes cell growth and / or protonema growth.

[0012] This invention provides a method for promoting plant growth and development by inhibiting the biological functions of CCM1, CCM2, CCM3 and CCM4 proteins in plants or by inhibiting the expression of CCM1, CCM2, CCM3 and CCM4 genes in plants to promote plant growth and development.

[0013] The amino acid sequences of the CCM1, CCM2, CCM3, and CCM4 proteins are shown in SEQ ID NO: 5-8, respectively.

[0014] The nucleotide sequences of the CCM1, CCM2, CCM3 and CCM4 genes are shown in SEQ ID NO: 9-12, respectively.

[0015] In this invention, the method for inhibiting the biological function of CCM1, CCM2, CCM3, and CCM4 proteins in plants or inhibiting the expression of CCM1, CCM2, CCM3, and CCM4 genes in plants includes transferring the sgRNA composition, the sgRNA expression vector, or the expression vector composition into plants to perform targeted gene editing of the CCM1, CCM2, CCM3, and CCM4 genes.

[0016] In this invention, the reagent for CCM1 gene detection includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:13 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:14;

[0017] The reagent for CCM2 gene detection includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:15 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:16;

[0018] The reagent for CCM3 gene detection includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:17 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:18;

[0019] The reagents for CCM4 gene detection include a forward primer with a nucleotide sequence as shown in SEQ ID NO:19 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:20.

[0020] In this invention, the plant includes non-seed plants.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] This invention provides an sgRNA composition comprising CCM1 sgRNA, CCM2 sgRNA, CCM3 sgRNA, and CCM4 sgRNA, with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 4. In embodiments of this invention, the sgRNA composition is capable of precise multi-gene targeting of the key regeneration genes CCM1, CCM2, CCM3, and CCM4 in *Pseudomonas aeruginosa*.

[0023] The present invention provides an sgRNA expression vector containing the sgRNA composition.

[0024] The present invention provides an expression vector composition comprising the sgRNA expression vector and the Cas protein expression vector.

[0025] This invention provides the application of the sgRNA composition, the sgRNA expression vector, or the expression vector composition in promoting plant growth and development. In embodiments of this invention, expression vectors containing one (CCM1 sgRNA), two (CCM1-CCM2 sgRNA), three (CCM1-CCM3 sgRNA), and four (CCM1-CCM4 sgRNA) from the sgRNA composition were genetically transformed into *Moss simonii* to obtain a CCM1 single mutant (ccm1), a CCM1-CCM2 double mutant (ccmd), a CCM1-CCM3 triple mutant (ccmt), and a CCM1-CCM4 quadruple mutant (ccmq). Cell regeneration culture of protoplasts and protonema growth rates were compared between the mutant materials and the wild type. The results showed that the CCM1-CCM4 quadruple mutant exhibited a significantly higher number of newly formed cells, a significantly higher area of ​​newly formed protonema, and a significantly higher growth rate compared to the wild type, while the other mutant materials showed no significant differences compared to the wild type. The sgRNA composition, the sgRNA expression vector, or the expression vector composition can be used to efficiently edit multiple regulatory gene target sites, which greatly shortens the reproduction time of protonema cell regeneration in *Styrax styrax* and improves the protonema culture efficiency, which is of great significance to the healthy development of the moss industry. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 A is a schematic diagram of the CCM1 sgRNA expression vector and the sequencing maps of the target sites in the wild-type (WT) and gene-edited genetic line (ccm1#1); B is a schematic diagram of the CCM1 target expression vector; CCM1 sgRNA site sequencing maps in WT and ccm1#1.

[0028] Figure 2 A is a schematic diagram of the CCM1-CCM2 sgRNA expression vector and the sequencing maps of the target sites in WT and the gene-editing genetic line (ccmd#1); B is a schematic diagram of the CCM1 and CCM2 tandem target expression vector; C is a sequencing map of the CCM1 sgRNA site in WT and ccmd#1; D is a sequencing map of the CCM2 sgRNA site in WT and ccmd#1.

[0029] Figure 3A is a schematic diagram of the CM1-CCM3 sgRNA expression vector and the sequencing maps of the target sites in WT and the gene-edited genetic line (ccmt#1); B is a schematic diagram of the CCM1-CCM3 tandem target expression vector; C is a sequencing map of the CCM1 sgRNA site in WT and ccmt#1; D is a sequencing map of the CCM2 sgRNA site in WT and ccmt#1.

[0030] Figure 4 A is a schematic diagram of the CCM1-CCM4 sgRNA expression vector and the sequencing maps of the target sites in WT and the gene-editing genetic line (ccmq#1); B is a schematic diagram of the CCM1-CCM4 tandem target expression vector; C is a sequencing map of the CCM1 sgRNA site in WT and ccmq#1; D is a sequencing map of the CCM3 sgRNA site in WT and ccmq#1; E is a sequencing map of the CCM4 sgRNA site in WT and ccmq#1.

[0031] Figure 5 The graph shows the comparison of protoplast cell regeneration capacity among WT, ccm1#1, ccmd#1, ccmt#1, and ccmq#1. A is a schematic diagram of protoplasts after 7 days of growth; B is a statistical graph of the number of regenerated cells after 7 days of growth. *** corresponds to p<0.001, and ns indicates no significant difference.

[0032] Figure 6 Figure A shows the comparison of protonema regeneration capacity among WT, ccm1#1, ccmd#1, ccmt#1, and ccmq#1 ​​protonema; Figure B shows the cell regeneration after 7 days of growth of equal amounts of protonema; Figure C shows the protonema area; Figure C shows the protonema growth rate; *** corresponds to p<0.001, and ns indicates no significant difference. Detailed Implementation

[0033] The present invention provides an sgRNA composition comprising CCM1 sgRNA, CCM2 sgRNA, CCM3 sgRNA and CCM4 sgRNA as shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 4.

[0034] In this invention, the CCM1 sgRNA targets the CCM1 gene, the CCM2 sgRNA targets the CCM2 gene, the CCM3 sgRNA targets the CCM3 gene, and the CCM4 sgRNA targets the CCM4 gene. The sgRNA composition can precisely target multiple genes, namely the key genes for cell regeneration, CCM1-CCM4.

[0035] The present invention provides an sgRNA expression vector containing the sgRNA composition.

[0036] In this invention, the sgRNA expression vector is preferably a vector containing CCM1 sgRNA, CCM2 sgRNA, CCM3 sgRNA, and CCM4 sgRNA simultaneously. The sgRNA expression vector can simultaneously express CCM1 sgRNA, CCM2 sgRNA, CCM3 sgRNA, and CCM4 sgRNA targeting the CCM1-CCM4 genes in cells. The preferred method for constructing the sgRNA expression vector is to insert a CCM1 sgRNA-CCM4 sgRNA expression cassette into a backbone vector to obtain the CCM1 sgRNA-CCM4 sgRNA expression vector. The backbone vector preferably includes pU6-sgRNA, which is disclosed in the prior art (Pu, X., Liu, L., Li, P., Huo, H., Dong, X., Xie, K., Yang, H., & Liu, L. (2019). A CRISPR / LbCas12a-based method for highly efficient multiplex gene editing in Physcomitrella patens. The Plant Journal: for cell and molecular biology, 100(4), 863-872. https: / / doi.org / 10.1111 / tpj.14478), with cloning sites of NcoI and XbaI. This invention does not specifically limit the method of insertion into the backbone vector; conventional methods are acceptable. The CCM1sgRNA-CCM4sgRNA expression cassette is obtained by sequentially tandemly connecting crRNA1, crRNA2, crRNA3, and crRNA4. The crRNA1, crRNA2, crRNA3, and crRNA4 were obtained by adding DR sequences to the 5' end of the CCM1 sgRNA, CCM2 sgRNA, CCM3 sgRNA, and CCM4 sgRNA, respectively.

[0037] The present invention provides an expression vector composition comprising the sgRNA expression vector and the Cas protein expression vector.

[0038] In this invention, the expression vector composition includes an sgRNA expression vector that expresses the target sgRNA composition. The Cas protein expression vector expresses the Cas protein. The expression vector composition is used for gene editing of multiple genes in plants, specifically, the sgRNA composition can target a target gene, guiding the Cas protein to cleave the target DNA sequence. The expression vector composition preferably also includes a resistance gene expression vector. The resistance gene expression vector is used to express antibiotics in cells, and positive transformants can be screened using the expressed antibiotics. In this invention, the expression vector composition is preferably obtained by mixing the sgRNA expression vector, the Cas protein expression vector, and the resistance gene expression vector in a mass ratio of 1:1:1. The independent concentrations of the sgRNA expression vector, the Cas protein expression vector, and the resistance gene expression vector are preferably 0.9–1.2 μg / μL, more preferably 1 μg / μL. In this embodiment of the invention, the Cas protein expression vector is a Cas12a expression vector, full name pActLbCas12a. The resistance gene expression vector is a G418 resistance gene expression vector, named pBNRF. pActLbCas12a and pBNRF are widely used in existing technologies (Pu, X., Liu, L., Li, P., Huo, H., Dong, molecularbiology, 100(4), 863-872. https: / / doi.org / 10.1111 / tpj.14478).

[0039] The present invention provides the application of the sgRNA composition, the sgRNA expression vector, or the expression vector composition in promoting plant growth and development.

[0040] In this invention, the plants preferably include non-seed plants, more preferably plants of the family Cucurbitaceae, more preferably plants of the genus *Physcomitrium*, and most preferably *Physcomitrium patens*. Since *Physcomitrium patens* is a model organism for non-seed plant research, the embodiments of this invention verify that the sgRNA composition, the sgRNA expression vector, or the expression vector composition can promote the growth and development of *Physcomitrium patens*, demonstrating the same function in non-seed plants. In the embodiments of this invention, *Physcomitrium patens* material with good growth status and free from impurities and diseases was selected.

[0041] In this invention, the growth and development preferably includes cell growth and / or protonema growth. Cell growth preferably includes cell proliferation and / or regeneration. Plant cell regeneration refers to the ability of plant cells or tissues to recover into an intact plant or specific tissue (cell totipotency or pluripotency) after being damaged or under in vitro culture conditions. This ability is fundamental to plant adaptation to the environment, asexual reproduction, and tissue culture techniques, and has wide applications in agriculture and biotechnology. Its regulatory mechanism is complex, involving multi-level signaling networks, including hormonal signals (such as auxin and cytokinin), transcription factors, epigenetic regulation, cell cycle, and energy states (such as ATP and carbon metabolism levels). Plant cell regeneration capacity is closely related to intracellular gene expression regulation; therefore, identifying key regulatory factors and precisely controlling the regeneration process is crucial for optimizing regeneration frequency and shortening the regeneration cycle.

[0042] In this embodiment of the invention, expression vectors containing one (CCM1 sgRNA), two (CCM1-CCM2 sgRNA), three (CCM1-CCM3 sgRNA), and four (CCM1-CCM4 sgRNA) from the sgRNA composition were genetically transformed into *Moss simonii* to obtain a CCM1 single mutant (ccm1), a CCM1-CCM2 double mutant (ccmd), a CCM1-CCM3 triple mutant (ccmt), and a CCM1-CCM4 quadruple mutant (ccmq). Cell regeneration culture of protoplasts and protonema growth rates were compared between the four mutant materials and the wild type. The results showed that the CCM1-CCM4 quadruple mutant exhibited a significantly higher number of newly formed cells, a significantly higher area of ​​newly formed protonema, and a significantly higher growth rate compared to the wild type, while the other mutant materials showed no significant differences compared to the wild type. Therefore, the sgRNA composition, the sgRNA expression vector, or the expression vector composition can be used to promote plant growth and development.

[0043] This invention provides a method for promoting plant growth and development by inhibiting the biological functions of CCM1, CCM2, CCM3 and CCM4 proteins in plants or by inhibiting the expression levels of CCM1, CCM2, CCM3 and CCM4 genes in plants to promote plant growth and development.

[0044] The amino acid sequences of the CCM1, CCM2, CCM3, and CCM4 proteins are shown in SEQ ID NO: 5-8, respectively.

[0045] The nucleotide sequences of the CCM1, CCM2, CCM3 and CCM4 genes are shown in SEQ ID NO: 9-12, respectively.

[0046] In the present invention, the amino acid sequence of the CCM1 protein is as shown in SEQ ID NO: 5 (MEAVQVGSSPTAGFASDGFLARKSAHVAPKIGPCTAPCSAWRPQSSEPRGFAWRPVADNGIAKVVKFSRHVTTRVALMRGLGGMLKEGYVLRKHEILDEHTPVQSTGDVIEKLKNGFKNFKKNEYNQKPDLYAKLAEGQEPKVMMITCADSRVCPTMLHGLEAGEAFIVRNVANLVPPCEGSGEHHGTSAAIEFAVTVLQVERIVVMGHSNCGGIRALMTRDIYSGDFVGSWVRIGLPAKEKALSLMAGKSFDEQCGFCEQEAVNVSLVNLLTFPFIEERVKAGKLRIFGMHYDFVQGHLTSWEIEREDDFVHA); the amino acid sequence of the CCM2 protein is as shown in SEQ ID NO: 6 (MFIFSFFCFWVGHGLCLMPRRLRLVSSTRGGICLLLLNLKSLLVVVIVAVRVVFSGFSAVVSVVTGNASRCIDDSEVHVQGLELSLSRRVVLIIGLGSVCSSRLGLCCVAMEALERGLGLVGRSASTPGFSVEDSSRVRVTLRKGLVKVVSAARPLRSMEGVEFVGRRRIAESERVQERVDARSVTAHAAMMGGLGDSLKSGYVLKYLVVVDEHTEVDSTGDVIEKLKNGFRNFKVTQYNQKPDLYARLAEGQQPKVMMITCADSRVCPTMLHGLEAGEAFIVRNVANLVPPCEESGEHHGTSAAIEFAVTVLGVERIVVMGHSNCGGIRALMTRDAFSGDFVGSWIRIGLPAKKKALSVMKGKPLQEQCRFCEQEAVNVSLANLLTFPFIEERVKSGKLRIHGMHYNFIDGQLTSWEIEPEEAPVYS).The amino acid sequence of the CCM3 protein is shown in SEQ ID NO: 7 (MAASLNQKVDALVKSNPEAAKKLEETIAALEKTYLSGESAEGPIGRLGRGF QTFKSNVYDKDSALFDKLKTGQWPKYMIIACSDSRVDPATIFGLNPGEAFMVRNVANMVPAWEPKGGYPSVSSALEYAVKHLKVEHIIVIGHRLCGGIKALVTTEEGQGSHDFIENWLEIGKPARAATKAVSGSDEVDEQCKFCEKESVNVSLTNLLSYPWVKEKVVGKKLSIHGGFYDFVEGSFQVWDFELNVSHSQKF); the amino acid sequence of the CCM4 protein is shown in SEQ ID NO: 8 (MDQLSTPKAGRPHGLGEILNDGYALTDHVVVDENHTFDGNNDVIEKLITGF). The gene numbers of the CCM1-CCM4 genes can be found in the Phytozome genome database, namely: Pp6c24_4570 (CCM1), Pp6c20_10150 (CCM2), Pp6c1_9650 (CCM3), and Pp6c2_8540 (CCM4). The nucleotide sequence of the CCM1 gene is shown in SEQ ID NO: 9; the nucleotide sequence of the CCM2 gene is shown in SEQ ID NO: 10; the nucleotide sequence of the CCM3 gene is shown in SEQ ID NO: 11; and the nucleotide sequence of the CCM4 gene is shown in SEQ ID NO: 12.

[0047] In this invention, the method for inhibiting the biological function of CCM1, CCM2, CCM3, and CCM4 proteins in plants or for inhibiting the expression of CCM1, CCM2, CCM3, and CCM4 genes in plants includes transferring the sgRNA composition, the sgRNA expression vector, or the expression vector composition into plants to perform targeted gene editing of the CCM1, CCM2, CCM3, and CCM4 genes.

[0048] In this invention, the introduced plant preferably includes the introduction of the expression vector composition into a plant via PEG-mediated protoplast transformation. This invention does not specifically limit the PEG-mediated protoplast transformation method; conventional protoplast transformation methods in the art are acceptable. The plant is preferably the same as that used in the above applications and will not be described again here. After introduction into the plant, positive transformants are screened. The screening method is preferably G418 resistance screening. The screening medium is preferably BCDAT medium. After screening, positive transformants are preferably identified by comparing the differences in the CCM1-CCM4sgRNA site sequences between the positive transformants and wild-type CCM1-CCM4 to determine whether the CCM1, CCM2, CCM3, and CCM4 genes have been edited.

[0049] In this invention, the reagent for CCM1 gene detection preferably includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:13 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:14;

[0050] The reagent for CCM2 gene detection includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:15 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:16;

[0051] The reagent for CCM3 gene detection includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:17 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:18;

[0052] The reagents for CCM4 gene detection include a forward primer with a nucleotide sequence as shown in SEQ ID NO:19 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:20.

[0053] Cell regeneration is crucial for plant development and morphogenesis. The method in this invention enables rapid and precise simultaneous genetic manipulation of multiple key regeneration genes (CCM1-CCM4) to promote the development of *Sphaerocera dorsalis* protoplast cells and shorten the cell proliferation time. In this embodiment, the expression vector composition was transferred into plants to obtain a CCM1-CCM4 quad mutant (ccmq) with all four genes (CCM1-CCM4) edited. Sequencing results showed that, compared to wild-type material, the CCM1-CCM4 quad mutant (ccmq) had a deletion of 8-23 bp in the CCM1 gene, 11 bp in the CCM2 gene, 11-30 bp in the CCM3 gene, and 5-23 bp in the CCM4 gene. Further phenotypic analysis revealed that, compared to wild-type material, the CCM1-CCM4 quad mutant (ccmq) exhibited significantly improved protoplast cell regeneration capacity and protoplast growth rate, achieving more precise and efficient artificial control of the regeneration process and realizing genetic improvement of the *Sphaerocera dorsalis* regeneration system.

[0054] To ensure consistent cell growth during phenotypic observation, this invention preferably uses *Sphaerocarpus spp.* protoplasts with plump chloroplasts and uniform diameter, ideally 25–35 μm in diameter. To ensure uniformity of the initial protoplasts, this invention preferably uses materials with uniform area obtained after 7 days of polishing and culturing for regeneration culture; the initial protoplast area is preferably 0.11–0.12 mm². 2 The protoplasts used for the transformation of *Sphaerocera spp.* protoplasts are preferably plant materials that have grown uniformly and are in good condition after being pounded in a mortar. The culture time after pounding is 6–10 days, more preferably 7 days. The cell wall degrading enzyme used for the preparation of *Sphaerocera spp.* protoplasts is a composition containing cellulase, hemicellulase, and pectinase (in a mass ratio of 1:1:1).

[0055] In this embodiment of the invention, the culture of *Sphaerothora spp.* is preferably carried out in a constant temperature incubator at a temperature of 23–25°C, more preferably 25°C; the growth time after protoplast transformation can be 14 days, the resistance screening time can be 21 days, the time for materials used in protoplast preparation can be 7 days, the protoplast regeneration experiment time can be 7 days, and the protonema treatment experiment time can be 7 days. The light conditions during culture preferably include alternating light and dark culture. Each alternating light and dark culture cycle is 24 hours. The light culture time in each alternating light and dark culture is preferably 15–17 hours, more preferably 16 hours. The dark culture time in each alternating light and dark culture is preferably 7–9 hours, more preferably 8 hours. The light intensity of the light culture is preferably 60–80 lx, more preferably 80 lx. The humidity of the incubator is preferably 40–50%, more preferably 45%. The selection of culture temperature, light intensity, and light-dark culture cycle in this invention is aimed at promoting the growth and development of *Sphaerothora spp.* protoplasts or protonema.

[0056] The present invention does not have any special restrictions on the source of the reagents and consumables; conventional products can be used. All of them need to be sterilized by high temperature and high pressure or by filtration before use.

[0057] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0058] 1. Materials and Methods

[0059] Methods for preparing protoplast suspension:

[0060] A. Place the 7-day-old young protonema material of *Moss simonii* in the prepared cell wall degrading enzyme solution and mix thoroughly.

[0061] B. Place in an incubator at 23-25℃ and keep in the dark, gently rotating to mix every 5 minutes;

[0062] C. After pyrolysis for 60 min, the material slurry was filtered through an 80 μm sterile nylon filter.

[0063] D. Centrifuge the obtained filtrate at 800 rpm for 8 min at room temperature;

[0064] E. Gently aspirate the supernatant, add 40 mL of 8% mannitol, mix gently, wash, and then centrifuge at 800 rpm for 8 min at room temperature;

[0065] F. Repeat step E twice, and add 0.5 mL of protoplast buffer on the last time to obtain the protoplast suspension.

[0066] The steps for preparing the cell wall degrading enzyme composition solution for the protoplasts of *Sclerotium spp.* are as follows:

[0067] Add 0.5g of a mixture containing cellulase, hemicellulase, and pectinase (in a mass ratio of 1:1:1) to a 50mL centrifuge tube; add 25mL of 8% mannitol (final concentration 2%); mix thoroughly on a shaker or vortex mixer at 23–25℃ for 30min; centrifuge the mixture at 4000rpm for 5min; collect the supernatant and filter it through a 0.45μm filter for sterilization.

[0068] The preparation method of 40% PEG solution is as follows: Add 100 μL of 1M Tris-HCl (pH 8.0), 1 mL of 1M Ca(NO3)2 and 9 mL of 8% mannitol to a 15 mL centrifuge tube, mix well and filter through a 0.22 μm filter to prepare PEG buffer for later use; take 5 mL of PEG buffer to dissolve 2 g of PEG reagent that has been sterilized by high temperature and autoclave to obtain 40% PEG solution.

[0069] The specific preparation method of BCDAT medium is as follows: 1000 mL contains 10 mL of 100 mM MgSO4, 10 mL of 184 mM KH2PO4, 10 mL of 1 M KNO3, 10 mL of 4.5 mM FeSO4, 1 mL of 0.22 mM CuSO4, 1 mL of 10 mM H3BO3, 1 mL of 0.23 mM CoCl2, 1 mL of 0.1 mM Na2MoO4, 1 mL of 2 mM MnCl2, 1 mL of 0.19 mM ZnSO4, 1 mL of 0.17 mM KI, 10 mL of 500 mM ammonium tartrate, 20 mL of 500 mM CaCl2, and 8 g of agar. After making up to volume with distilled water, autoclave at 121°C for 20 min. Dispense into 9-12 cm Petri dishes and store at 4°C.

[0070] The preparation method of protoplast buffer is as follows: Weigh 0.91g mannitol into a 15mL centrifuge tube; add 0.15mL of 1M MgCl2, 1mL of 1% MES (pH 5.6) and 8.85mL of sterile water; after the mannitol and each liquid are mixed evenly, filter the mixture through a 0.22μm filter to remove bacteria, and the protoplast buffer is prepared.

[0071] Example 1

[0072] sgRNA design and expression vector construction

[0073] 1. Design of sgRNA and crRNA

[0074] sgRNAs targeting the CCM1, CCM2, CCM3, and CCM4 genes were designed. DR is a direct repeat sequence, sgRNA is a small guide RNA, and crRNA is a CRISPR RNA sequence composed of DR and sgRNA.

[0075] The nucleotide sequence of CCM1 sgRNA targeting the CCM1 gene is: GCCAGUGAUGGGUUCUUGGCCAGA (SEQ ID NO: 1). A DR sequence (UAAUUUCUACUAAGUGUAGAU, SEQ ID NO: 21) is added to the 5' end of the CCM1 sgRNA to obtain CCM1 crRNA.

[0076] The nucleotide sequence of CCM2 sgRNA targeting the CCM2 gene is: CGUCCAGAGUUCGAGUGACGCUGA (SEQ ID NO: 2). By adding a DR sequence to the 5' end of CCM2 sgRNA, CCM2 crRNA is obtained.

[0077] The nucleotide sequence of CCM3 sgRNA targeting the CCM3 gene is: ACUCAACCCUGGAGAAGCCUUU (SEQ ID NO: 3). By adding a DR sequence to the 5' end of the CCM3 sgRNA, CCM3 crRNA is obtained.

[0078] The nucleotide sequence of CCM4 sgRNA targeting the CCM4 gene is: GACGGAACAACGAUGUAAUAGAG (SEQ ID NO: 4). By adding a DR sequence to the 5' end of CCM4 sgRNA, CCM4 crRNA is obtained.

[0079] 2. Construction method of sgRNA expression vector

[0080] (1) The CCM1 gene editing target site expression vector is referred to as the CCM1 sgRNA expression vector. It is obtained by inserting the DNA sequence of CCM1 crRNA into the NcoI and XbaI sites of the pU6-sgRNA vector through enzyme digestion and ligation. See the schematic diagram below. Figure 1 The A. sgRNA expression vector uses U6 and PolyT as the promoter and termination signal, respectively;

[0081] (2) The expression vectors for the CCM1 and CCM2 gene editing target sites are designated as CCM1-CCM2 sgRNA expression vectors. These vectors are obtained by inserting the DNA sequence of CCM1-CCM2 crRNA into the NcoI and XbaI sites of the pU6-sgRNA vector via enzyme digestion and ligation. A schematic diagram is shown below. Figure 2 China A;

[0082] CCM1-CCM2 crRNA is derived from crRNA1 and crRNA2 tandemly;

[0083] (3) The expression vectors for the CCM1, CCM2, and CCM3 gene editing target sites are designated as CCM1-CCM3sgRNA expression vectors. These vectors are obtained by inserting the DNA sequence of CCM1-CCM3 crRNA into the NcoI and XbaI sites of the pU6-sgRNA vector via enzyme digestion and ligation. A schematic diagram is shown below. Figure 3 China A;

[0084] CCM1-CCM3 crRNA is derived from crRNA1, crRNA2 and crRNA3 in tandem;

[0085] (4) The gene editing target site expression vectors for CCM1, CCM2, CCM3, and CCM4 are designated as CCM1-CCM4 sgRNA expression vectors. These vectors are obtained by inserting the DNA sequence of CCM1-CCM4 crRNA into the NcoI and XbaI sites of the pU6-sgRNA vector via enzyme digestion and ligation. A schematic diagram is shown below. Figure 4 China A;

[0086] CCM1-CCM4 crRNA is derived from crRNA1, crRNA2, crRNA3 and crRNA4 in tandem.

[0087] Example 2

[0088] Methods for constructing mutants

[0089] 1. Construction method of CCM1 single mutant (ccm1)

[0090] (1) Methods of genetic transformation

[0091] A. Preparation of protoplast suspensions of wild-type protofilament materials;

[0092] B. Mix the CCM1 sgRNA expression vector, Cas12a expression vector, and G418 (genetic mycotoxin) resistance gene expression vector at a mass ratio of 10 μg:10 μg:10 μg (total volume of 30 μL) to obtain a vector fragment mixture;

[0093] C. Gently mix 30 μL of the carrier fragment mixture, 300 μL of protoplast suspension, and 300 μL of 40% PEG (polyethylene glycol) solution into a 10 mL centrifuge tube;

[0094] D. After heat shock in a 45℃ water bath for 5 min, transfer to a 20℃ water bath for 10 min; add 300 μL, 600 μL, 1 mL, and 2 mL of 8% mannitol solution to the conversion mixture in sequence, with an interval of 2-3 min each time;

[0095] E. Centrifuge the above mixture at 800 rpm for 8 min at room temperature; discard the supernatant, gently mix the lower protoplast precipitate, pour it into BCDAT medium, and incubate in an incubator.

[0096] F. After 14 days of culture, G418 antibiotic was screened on BCDAT medium at a concentration of 20 mg / mL. Transformed seedlings were obtained after 21 days. The incubator was maintained at 45% humidity, 25°C, 80 lx light intensity, and a 16-hour light-8-hour dark cycle.

[0097] (2) Detection of CCM1 single mutant (ccm1)

[0098] After the transformed seedlings were screened on resistant culture medium, genomic DNA was extracted from positive transformants and wild-type seedlings, and PCR amplification and sequencing were performed on the target sequences of CCM1, CCM2, CCM3 and CCM4.

[0099] The primer sequences used for CCM1 target sequence detection are as follows:

[0100] CCM1-F: TGGTATCCCAAT CGTCAC (SEQ ID NO: 13);

[0101] CCM1-R: CATGTTTCCTCAACACATAGC (SEQ ID NO: 14);

[0102] The primer sequences used for CCM2 target sequence detection are as follows:

[0103] CCM2-F: GGTCTGTGTTTAATGCCTC (SEQ ID NO: 15);

[0104] CCM2-R:ACCCTTCCTGCACTCTCTC (SEQ ID NO: 16);

[0105] The primer sequences used for CCM3 target sequence detection are as follows:

[0106] CCM3-F: GAACAGCAAGGACAGCG (SEQ ID NO: 17);

[0107] CCM3-R: GCCGAGAAAACCTTGGA (SEQ ID NO: 18);

[0108] The primer sequences used for CCM4 target sequence detection are as follows:

[0109] CCM4-F: AGCACTTCCCTTCCAAA (SEQ ID NO: 19);

[0110] CCM4-R: CTTACAACGTTGATATGCAGC (SEQ ID NO: 20).

[0111] The PCR amplification system consisted of: 2 μL of forward primer, 2 μL of reverse primer, 25 μL of DNA polymerase, 5 μL of DNA template, and 16 μL of ddH2O.

[0112] PCR amplification was performed using a standard DNA polymerase (purchased from Vazyme, catalog number P515).

[0113] The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 50℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min; storage at 4℃.

[0114] PCR products were used for agarose gel electrophoresis and sequencing identification. By comparing the sgRNA site sequence of positive transformants with the wild-type sequence, materials with changes such as base reduction or substitution were identified as CCM1 single mutants (ccm1).

[0115] See sequencing analysis results Figure 1 See Table B and Table 1.

[0116] Table 1 Sequencing results of CCM1 sgRNA sites in positive transformants

[0117]

[0118] Note: — indicates that the sequence is the same as the wild type.

[0119] According to Table 1 and Figure 1 As shown in Figure B, the DNA base sequence of the ccm1 positive transformant is 8–23 bp missing at the CCM1sgRNA site compared to that of the wild-type CCM1 in Comparative Example 1.

[0120] 2. Construction method of CCM1-CCM2 double mutant (ccmd)

[0121] The construction method is the same as that of the CCM1 single mutant (ccm1), except that the CCM1 sgRNA expression vector is replaced with the CCM1-CCM2 sgRNA expression vector.

[0122] The detection method for the CCM1-CCM2 double mutant (ccmd) is the same as that for the CCM1 single mutant (ccm1), the difference being that the target sequences of CCM1, CCM2, CCM3 and CCM4 are amplified by PCR and sequenced.

[0123] Sequencing analysis results of positive transformant sgRNA site sequence and wild-type sequence are shown below. Figure 2 The sequencing results of the sgRNA sites of the B-positive transformants are shown in Table 2.

[0124] Table 2 Sequencing results of CCM1 and CCM2 sgRNA sites in positive transformants

[0125]

[0126]

[0127] Note: — indicates that the sequence is the same as the wild type.

[0128] According to Table 2 and Figure 2 As shown in Figure B, the DNA base sequences of the ccmd positive transformants at the wild-type CCM1 and CCM2 sgRNA sites were edited compared to those in Comparative Example 1, with CCM1 having a deletion of 15–23 bp and CCM2 having a deletion of 11 bp.

[0129] 3. Construction method of CCM1-CCM3 triple mutant (ccmt)

[0130] The construction method is the same as that of the CCM1-CCM2 double mutant (ccmd), except that the CCM1-CCM2 sgRNA expression vector is replaced with the CCM1-CCM3 sgRNA expression vector.

[0131] The detection method for the CCM1-CCM3 triple mutant (ccmd) is the same as that for the CCM1-CCM2 double mutant, the difference being that the target sequences of CCM1, CCM2, CCM3 and CCM4 are amplified by PCR and sequenced.

[0132] Sequencing analysis results of positive transformant sgRNA site sequence and wild-type sequence are shown below. Figure 3 The sequencing results of the sgRNA sites of the B-positive transformants are shown in Table 3.

[0133] Table 3 Sequencing results of CCM1-CCM3 sgRNA sites in positive transformants

[0134]

[0135] Note: — indicates that the sequence is the same as the wild type.

[0136] According to Table 3 and Figure 3As shown in Figure B, the DNA base sequences of the CCM1-CCM3sgRNA sites in the CCMt positive transformants were all edited compared to those in Comparative Example 1, with CCM1 having a deletion of 8-23 bp, CCM2 having a deletion of 11 bp, and CCM3 having a deletion of 11-30 bp.

[0137] 4. Construction method of CCM1-CCM4 quad mutant ccmq

[0138] The construction method is the same as that of the CCM1-CCM3 triple mutant (ccmt), except that the CCM1-CCM3 sgRNA expression vector is replaced with the CCM1-CCM4 sgRNA expression vector.

[0139] The detection method for the CCM1-CCM4 quad mutant (ccmt) is the same as that for the CCM1-CCM3 triple mutant, the difference being that the target sequences of CCM1, CCM2, CCM3 and CCM4 are amplified by PCR and sequenced.

[0140] Sequencing analysis results of positive transformant sgRNA site sequence and wild-type sequence are shown below. Figure 4 The sequencing results of the sgRNA sites of the B-positive transformants are shown in Table 3.

[0141] Table 4 Sequencing results of CCM1-CCM4 sgRNA sites in positive transformants

[0142]

[0143] According to Table 4 and Figure 4 As shown in Figure B, in Example 4, the DNA base sequences of the ccmq positive transformants at the CCM1-CCM4 sgRNA sites were all edited compared to those in Comparative Example 1, with CCM1 having a deletion of 8-23 bp, CCM2 having a deletion of 11 bp, CCM3 having a deletion of 11-30 bp, and CCM4 having a deletion of 5-23 bp.

[0144] Example 3

[0145] Phenotypic detection

[0146] The protoplast and protonema phenotypes of wild-type and CCM1 single mutant ccm1#1, CCM1 and CCM2 double mutant ccmd#1, CCM1-CCM3 triple mutant ccmt#1, and CCM1-CCM4 quadruple mutant ccmq#1 ​​were tested respectively.

[0147] Protoplast phenotypic detection methods:

[0148] One to two dishes of protoplast material were collected after 7 days of mortar grinding, including wild-type, CCM1 single mutant ccm1#1, CCM1 and CCM2 double mutant ccmd#1, CCM1-CCM3 triple mutant ccmt#1, and CCM1-CCM4 quadruple mutant ccmq#1. Protoplast suspensions were prepared using cell wall degrading enzyme buffer. The protoplast suspensions were poured into BCDAT medium and cultured in an incubator (incubator conditions as in Example 2) for 7 days, and the phenotypes were observed.

[0149] Protoplast phenotypic detection methods:

[0150] Take 1-2 dishes of protonemata, including wild-type, CCM1 single mutant ccm1#1, CCM1 and CCM2 double mutant ccmd#1, CCM1-CCM3 triple mutant ccmt#1, and CCM1-CCM4 quadruple mutant ccmq#1, after 7 days of grinding in a mortar and pestle. Use tweezers to grasp pieces with an area of ​​approximately 0.11-0.12 mm². 2 The protonemae were used as homogenized samples and grown in BCDAT medium for 7 days to observe the phenotype (incubator conditions and parameters were the same as in Example 2).

[0151] A schematic diagram of protoplast regeneration and the results of the analysis of the number of regenerated cells are shown below. Figure 5 See Table 5. Schematic diagram of protonema homogenization culture; protonema area and growth rate are shown in Table 5. Figure 6 Tables 6 and 7.

[0152] The number of newly formed protoplast cells, protoplast area, and growth rate were statistically analyzed using GraphPadprism 8.0 software and expressed as Means ± SEM values. The Student's-test was used to analyze the differences in the data. Significance was indicated by an asterisk, *** corresponds to p < 0.001, and ns indicates no significant difference.

[0153] The calculation method for the growth rate of protofilament area is given in Formula I.

[0154] Protoplast area growth rate (%) = (Area after 7 days of culture - Initial area at 0 days) ÷ Initial area at 0 days

[0155] ×100% Formula I

[0156] Table 5. Number of newly formed cells after 7 days of protoplast culture.

[0157]

[0158] Table 6. Protoneal area (mm²) after 7 days of homogenization culture. 2 )

[0159]

[0160] Table 7. Growth rate of protonema homogenization culture on day 7.

[0161]

[0162] According to Table 5 and Figure 5 It can be seen that after 7 days of protoplast culture, the number of newly formed cells from the CCM1-CCM4 quadrupole strain was significantly increased compared to the wild-type strain. (Based on Tables 6 and 7) Figure 6 It can be seen that after 7 days of homogenization culture of protonema, the area and growth rate of newly formed protonema of the CCM1-CCM4 quad mutant were significantly increased compared with those of the wild type.

[0163] In summary, the multi-gene precise targeting of CCM1-CCM4 proposed in this invention using CRISPR / Cas12a effectively promotes the regeneration of *Sclerotium spp.* cells and improves the culture efficiency of protonema.

[0164] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An sgRNA composition, characterized in that, This includes CCM1 sgRNA, CCM2 sgRNA, CCM3 sgRNA and CCM4 sgRNA, whose nucleotide sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 4, respectively.

2. An sgRNA expression vector, characterized in that, The sgRNA expression vector contains the sgRNA composition of claim 1.

3. An expression vector composition, characterized in that, The expression vector composition comprises the sgRNA expression vector and the Cas protein expression vector as described in claim 2.

4. The use of the sgRNA composition of claim 1, the sgRNA expression vector of claim 2, or the expression vector composition of claim 3 in promoting plant growth and development.

5. The application according to claim 4, characterized in that, The plants mentioned include non-seed plants.

6. The application according to claim 4 or 5, characterized in that, The growth and development includes cell growth and / or protonema growth.

7. A method for promoting plant growth and development, characterized in that, Inhibiting the biological functions of CCM1, CCM2, CCM3, and CCM4 proteins in plants or inhibiting the expression of CCM1, CCM2, CCM3, and CCM4 genes in plants can promote plant growth and development. The amino acid sequences of the CCM1, CCM2, CCM3, and CCM4 proteins are shown in SEQ ID NO: 5-8, respectively. The nucleotide sequences of the CCM1, CCM2, CCM3 and CCM4 genes are shown in SEQ ID NO: 9-12, respectively.

8. The method according to claim 7, characterized in that, A method for inhibiting the biological function of CCM1, CCM2, CCM3, and CCM4 proteins in plants or for inhibiting the expression of CCM1, CCM2, CCM3, and CCM4 genes in plants includes transferring the sgRNA composition of claim 1, the sgRNA expression vector of claim 2, or the expression vector composition of claim 3 into plants to perform targeted gene editing of the CCM1, CCM2, CCM3, and CCM4 genes.

9. The method according to claim 8, characterized in that, The reagent for CCM1 gene detection includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:13 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:14; The reagent for CCM2 gene detection includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:15 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:16; The reagent for CCM3 gene detection includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:17 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:18; The reagents for CCM4 gene detection include a forward primer with a nucleotide sequence as shown in SEQ ID NO:19 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:

20.

10. The method according to claim 9, characterized in that, The plants mentioned include non-seed plants.

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