Application of CRISPR / SaCas9 system in soybean gene editing

Abstract

The application discloses application of a CRISPR / SaCas9 system in soybean gene editing. Experiments prove that SgRNA is designed according to genes GmFT2a or GmFT5a in a soybean genome, and then a coding gene of the SgRNA and a gene coding a SaCas9 protein are introduced into the soybean to knock out the soybean genes GmFT2a and GmFT5a; a PAM sequence recognized by the SaCas9 protein is 5'-NNGRRT-3', N is A, T, G or C, and R is A or G. Therefore, the CRISPR / SaCas9 system can be used for soybean gene editing, and the application has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the CRISPR / SaCas9 system in soybean gene editing. Background Technology

[0002] The emergence of CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-Associated 9) genome editing technology represents a massive and radical technological revolution in the life sciences. Its diverse characteristics and constantly evolving new functions provide new tools for achieving targeted gene knockout, long-fragment deletion, single-base editing, targeted knock-in, target fragment replacement, transcriptional activation, and repression in plants. This technology also provides a powerful tool for in-depth research into plant gene function and the creation of specific mutant materials.

[0003] The CRISPR / Cas9 system targets and cleaves specific DNA sites adjacent to the protospacer-jacent motif (PAM). Soybean (Glycine max) is rich in protein and oil, making it an important dual-purpose crop for both food and oil production. Currently, the most frequently used Cas9 enzyme in soybean genome editing is the SpCas9 protein from *Streptococcus pyogenes*. The PAM sequence of the SpCas9 protein's target site is 5'-NGG-3' (where N represents any base), which limits the range of selectable targets in the genome and thus restricts the application of various techniques in soybean, such as deletion of target fragments, single-base editing, knock-in, and substitution. In contrast, the SaCas9 protein from *Staphylococcus aureus* is smaller, consisting of 1053 amino acids, making it easier to deliver in vivo. Summary of the Invention

[0004] The purpose of this invention is to perform soybean gene editing.

[0005] This invention first protects a method for targeted editing of soybean target genes, which may include the following steps:

[0006] (1) Design SgRNA based on the target genes in the soybean genome that are expected to be edited in a targeted manner;

[0007] (2) The gene encoding SgRNA and the gene encoding SaCas9 protein were introduced into soybeans to achieve targeted editing of target genes in soybeans;

[0008] The PAM sequence for the SaCas9 protein's target recognition site is 5'-NNGRRT-3', where N is A, T, G, or C, and R is A or G.

[0009] In the above method, the target gene can be the GmFT2a gene.

[0010] In the above method, when the target gene is the GmFT2a gene, the target site recognized by SgRNA can be A1), A2), or A3):

[0011] A1) The DNA molecule shown in SEQ ID NO: 1 from position 191 to 217 starting from the 5' end;

[0012] A2) The DNA molecule shown in SEQ ID NO: 1 from position 81 to 107 starting from the 5' end;

[0013] A3) The DNA molecule shown in SEQ ID NO: 1 from position 100 to 126 starting from the 5' end.

[0014] In the above method, the target gene can be the GmFT5a gene.

[0015] In the above method, when the target gene is the GmFT5a gene, the target site recognized by SgRNA can be B1) or B2):

[0016] B1) The DNA molecule shown in SEQ ID NO: 4, positions 177-203 from the 5' end;

[0017] B2) The DNA molecule shown in SEQ ID NO: 4, positions 67-93 from the 5' end.

[0018] In the above method, the introduction of the SgRNA encoding gene and the SaCas9 protein encoding gene into soybean can be specifically achieved by introducing a vector for targeted editing of soybean target genes into soybean.

[0019] The vector for targeted editing of soybean target genes may include genes encoding SaCas9 protein and genes encoding SgRNA.

[0020] In the vectors used for targeted editing of soybean target genes, when the target gene is the GmFT2a gene, the target site recognized by the SgRNA can be A1), A2), or A3):

[0021] A1) The DNA molecule shown in SEQ ID NO: 1 from position 191 to 217 starting from the 5' end;

[0022] A2) The DNA molecule shown in SEQ ID NO: 1 from position 81 to 107 starting from the 5' end;

[0023] A3) The DNA molecule shown in SEQ ID NO: 1 from position 100 to 126 starting from the 5' end.

[0024] In the vectors used for targeted editing of soybean target genes, when the target gene is the GmFT5a gene, the target site recognized by the SgRNA can be either B1) or B2):

[0025] B1) The DNA molecule shown in SEQ ID NO: 4, positions 177-203 from the 5' end;

[0026] B2) The DNA molecule shown in SEQ ID NO: 4, positions 67-93 from the 5' end.

[0027] This invention also protects a method for targeted editing of the soybean genome, which can be used to perform genome editing on soybeans to be edited using the CRISPR / SaCas9 system; wherein the PAM sequence of the SaCas9 protein recognition target in the CRISPR / SaCas9 system is 5'-NNGRRT-3', where N is A, T, G or C, and R is A or G.

[0028] This invention also protects the application of the CRISPR / SaCas9 system in soybean gene editing; the PAM sequence of the SaCas9 protein recognition target in the CRISPR / SaCas9 system is 5'-NNGRRT-3', where N is A, T, G or C, and R is A or G.

[0029] The present invention also protects a vector for targeted editing of soybean target genes, which may include a gene encoding SaCas9 protein and a gene encoding SgRNA; the target gene may be a GmFT2a gene or a GmFT5a gene.

[0030] In the above vectors, when the target gene is the GmFT2a gene, the target site recognized by SgRNA can be A1), A2), or A3):

[0031] A1) The DNA molecule shown in SEQ ID NO: 1 from position 191 to 217 starting from the 5' end;

[0032] A2) The DNA molecule shown in SEQ ID NO: 1 from position 81 to 107 starting from the 5' end;

[0033] A3) The DNA molecule shown in SEQ ID NO: 1 from position 100 to 126 starting from the 5' end.

[0034] In the above vectors, when the target gene is the GmFT5a gene, the target site recognized by SgRNA can be either B1) or B2):

[0035] B1) The DNA molecule shown in SEQ ID NO: 4, positions 177-203 from the 5' end;

[0036] B2) The DNA molecule shown in SEQ ID NO: 4, positions 67-93 from the 5' end.

[0037] The vector for targeted editing of soybean target genes described above can specifically be recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT2a SP1, recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT2a SP2, recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT2a SP3, recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT5a SP1, or recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT5a SP2.

[0038] The method for constructing the recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT2a SP1 is as follows:

[0039] (a1) The vector pUC57-SgRNA-SaCas9 was digested with restriction endonucleases NHeI and BbsI, and the vector backbone of about 3201 bp was recovered.

[0040] The nucleotide sequence (circular) of the vector pUC57-SgRNA-SaCas9 is shown in SEQ ID NO: 2.

[0041] (a2) Prepare the reaction system, and then anneal the reaction system to form oligo dimers.

[0042] The reaction system consisted of 25 μL, comprising 5 μL of the aqueous solution of GmFT2a-SaCas9-SP1-F mentioned in the example (concentration of 100 μM), 5 μL of the aqueous solution of GmFT2a-SaCas9-SP1-R mentioned in the example (concentration of 100 μM), and 15 μL of ddH2O.

[0043] The annealing procedure is as follows: 95℃ for 3 min, anneal at 0.1℃ / s to 16℃, and hold at 16℃ for 10 min.

[0044] (a3) Connect the oligo dimer and the carrier backbone recovered in step (a1) to obtain an intermediate carrier.

[0045] (a4) The intermediate vector was digested with restriction endonucleases PacI and PmeI to recover a DNA fragment of approximately 586 bp.

[0046] (a5) The PTF101-SaCas9-GFP-bar plasmid was digested with restriction endonucleases PacI and PmeI, and the vector backbone of about 14.7 kb was recovered.

[0047] The nucleotide sequence (circular) of the PTF101-SaCas9-GFP-bar plasmid is shown in SEQ ID NO: 3.

[0048] (a6) The DNA fragment recovered in step (a4) and the vector backbone recovered in step (a5) are ligated to obtain the recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT2a SP1.

[0049] Following the steps described above, replace GmFT2a-SaCas9-SP1-F with GmFT2a-SaCas9-SP2-F, and GmFT2a-SaCas9-SP1-R with GmFT2a-SaCas9-SP2-R, while keeping all other steps unchanged, to obtain the recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT2a SP2.

[0050] Following the steps described above, replace GmFT2a-SaCas9-SP1-F with GmFT2a-SaCas9-SP3-F, and GmFT2a-SaCas9-SP1-R with GmFT2a-SaCas9-SP3-R, while keeping all other steps unchanged, to obtain the recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT2a SP3.

[0051] The construction method of the recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT5a SP1 is as follows:

[0052] (b1) The vector pUC57-SgRNA-SaCas9 was digested with restriction endonucleases NHeI and BbsI, and the vector backbone of about 3201 bp was recovered.

[0053] (b2) Prepare the reaction system, and then anneal the reaction system to form oligo dimers.

[0054] The reaction system consisted of 25 μL of 5 μL of the aqueous solution of GmFT5a-SaCas9-SP1-F mentioned in the example (concentration of 100 μM), 5 μL of the aqueous solution of GmFT5a-SaCas9-SP1-R mentioned in the example (concentration of 100 μM), and 15 μL of ddH2O.

[0055] The annealing procedure is as follows: 95℃ for 3 min, anneal at 0.1℃ / s to 16℃, and hold at 16℃ for 10 min.

[0056] (b3) Connect the oligo dimer and the carrier backbone recovered in step (b1) to obtain an intermediate carrier.

[0057] (b4) The intermediate vector was digested with restriction endonucleases PacI and PmeI to recover a DNA fragment of approximately 586 bp.

[0058] (b5) The PTF101-SaCas9-GFP-bar plasmid was digested with restriction endonucleases PacI and PmeI, and the vector backbone of about 14.7 kb was recovered.

[0059] (b6) The DNA fragment recovered in step (b4) and the vector backbone recovered in step (b5) are ligated to obtain the recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT5a SP1.

[0060] Following the steps 3-8 above, replace GmFT5a-SaCas9-SP1-F with GmFT5a-SaCas9-SP2-F and GmFT5a-SaCas9-SP1-R with GmFT5a-SaCas9-SP2-R, while keeping all other steps unchanged, to obtain the recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT5a SP2.

[0061] Experiments have shown that CRISPR / SaCas9 can knock out the soybean genes GmFT2a and GmFT5a. This demonstrates that the CRISPR / SaCas9 system can perform soybean gene editing. This invention has significant application value. Attached Figure Description

[0062] Figure 1 This shows the structure of the GmFT2a gene and the CRISPR / SaCas9 target sequence. Blue bands represent exons; black lines represent introns; gray bands represent untranslated regions; blue letters represent target sequences; and red letters represent PAM sequences, i.e., prototypical spacer adjacent motifs.

[0063] Figure 2 This is a CRISPR / SaCas9-mediated site-directed mutation at the GmFT2a target site. A, B, and C are sequencing peak diagrams of the wild-type and mutant sequences at the three target sites: GmFT2a-SaCas9-SP1, GmFT2a-SaCas9-SP2, and GmFT2a-SaCas9-SP3, respectively. Underlines represent base insertions; short dashes represent base deletions; and red arrows indicate the location of the mutation.

[0064] Figure 3 This shows the structure of the GmFT5a gene and the CRISPR / SaCas9 target sequence. Green bands represent exons; black lines represent introns; gray bands represent untranslated regions; blue letters represent target sequences; and red letters represent PAM sequences, i.e., prototypical spacer adjacent motifs.

[0065] Figure 4 This is a CRISPR / SaCas9-mediated site-directed mutation at the GmFT5a target site. A and B are sequencing peak diagrams of the wild-type and mutant sequences at the GmFT5a-SaCas9-SP1 and GmFT5a-SaCas9-SP2 target sites, respectively. Underlines represent base insertions; short dashes represent base deletions; letters with green backgrounds indicate that the base A is changed to T at this position; red arrows indicate the location of the mutation. Detailed Implementation

[0066] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0068] The soybean cultivar Jack is described in the following literature: Chen, L., Cai, Y., Liu, X., Yao, W., Guo, C., Sun, S., Wu, C., Jiang, B., Han, T. and Hou, W. (2018) Improvement of soybean Agrobacterium-mediated transformation efficiency by adding glutamine and asparagine into the culture media. International Journal of Molecular Sciences 19, 3039. It is available to the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences (i.e., the applicant).

[0069] Agrobacterium rhizogenes K599 is described in the following literature: Cai, Y., Chen, L., Liu, X., Sun, S., Wu, C., Jiang, B., Han, T. and Hou, W. (2015) CRISPR / Cas9-mediated genome editing in soybean hairy roots. PLoS ONE 10, e0136064. It is available to the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences (i.e., the applicant).

[0070] MS salt and B5 salt are both products of Phytotech, with product numbers M524 and G768 respectively.

[0071] Example 1: Site-specific knockout of soybean gene GmFT2a using CRISPR / SaCas9

[0072] I. SgRNA Target Sequence Selection

[0073] The soybean GmFT2a gene sequence (Glyma.16G150700) shown in SEQ ID NO: 1 was obtained from the Phytozome database. Subsequently, three target sites, GmFT2a-SaCas9-SP1, GmFT2a-SaCas9-SP2, and GmFT2a-SaCas9-SP3, were selected on the first exon of the GmFT2a gene.

[0074] The nucleotide sequence of GmFT2a-SaCas9-SP1 is 5'-CTCACAAGTTGTCAACCAACCAAGGGT-3' (SEQ ID NO: 1, positions 191-217 from the 5' end), and its PAM sequence is AAGGGT.

[0075] The nucleotide sequence of GmFT2a-SaCas9-SP2 is 5'-CTCGTTGTTGGGGGAGTAATTGGGGAT-3' (SEQ ID NO: 1, positions 81-107 from the 5' end), and its PAM sequence is GGGGAT.

[0076] The nucleotide sequence of GmFT2a-SaCas9-SP3 is 5'-TTGGGGATGTATTGGATCCTTTTGAAT-3' (SEQ ID NO: 1, positions 100-126 from the 5' end), and its PAM sequence is TTGAAT.

[0077] II. Construction of CRISPR / SaCas9 vectors

[0078] 1. The vector pUC57-SgRNA-SaCas9 was digested with restriction endonucleases NHeI and BbsI, and the vector backbone of approximately 3201 bp was recovered.

[0079] The nucleotide sequence (circular) of the vector pUC57-SgRNA-SaCas9 is shown in SEQ ID NO: 2.

[0080] 2. Based on the nucleotide sequence of GmFT2a-SaCas9-SP1 from step one, synthesize primers GmFT2a-SaCas9-SP1-F and GmFT2a-SaCas9-SP1-R. Based on the nucleotide sequence of GmFT2a-SaCas9-SP2 from step one, synthesize primers GmFT2a-SaCas9-SP2-F and GmFT2a-SaCas9-SP2-R. Based on the nucleotide sequence of GmFT2a-SaCas9-SP3 from step one, synthesize primers GmFT2a-SaCas9-SP3-F and GmFT2a-SaCas9-SP3-R. The nucleotide sequences of each primer are shown in Table 1.

[0081] Table 1

[0082] Primer name nucleotide sequence GmFT2a-SaCas9-SP1-F 5′-TCGAAGTAGTGATTGCTCACAAGTTGTCAACCAACCGTTTTAGTACTCTGG-3′ GmFT2a-SaCas9-SP1-R 5′-CCAGAGTACTAAAACGGTTGGTTGACAACTTGTGAGCAATCACTACTTCGA-3′ GmFT2a-SaCas9-SP2-F 5′-TCGAAGTAGTGATTGCTCGTTGTTGGGGGAGTAATTGTTTTAGTACTCTGG-3′ GmFT2a-SaCas9-SP2-R 5′-CCAGAGTACTAAAACAATTACTCCCCCAACAACGAGCAATCACTACTTCGA-3′ GmFT2a-SaCas9-SP3-F 5′-TCGAAGTAGTGATTGTTGGGGATGTATTGGATCCTTGTTTTAGTACTCTGG-3′ GmFT2a-SaCas9-SP3-R 5′-CCAGAGTACTAAAACAAGGATCCAATACATCCCCAACAATCACTACTTCGA-3′

[0083] 3. First, prepare the reaction system, and then anneal the reaction system to form oligo dimers.

[0084] The reaction system consisted of 25 μL of 5 μL of GmFT2a-SaCas9-SP1-F aqueous solution (concentration 100 μM), 5 μL of GmFT2a-SaCas9-SP1-R aqueous solution (concentration 100 μM), and 15 μL of ddH2O.

[0085] The annealing procedure is as follows: 95℃ for 3 min, anneal at 0.1℃ / s to 16℃, and hold at 16℃ for 10 min.

[0086] 4. Connect the oligo dimer with the carrier backbone recovered in step 1 to obtain an intermediate carrier.

[0087] 5. The intermediate vector was digested with restriction endonucleases PacI and PmeI to recover a DNA fragment of approximately 586 bp.

[0088] 6. Digest the PTF101-SaCas9-GFP-bar plasmid with restriction endonucleases PacI and PmeI, and recover the vector backbone of approximately 14.7 kb.

[0089] The nucleotide sequence (circular) of the PTF101-SaCas9-GFP-bar plasmid is shown in SEQ ID NO: 3.

[0090] 7. Ligate the DNA fragment recovered in step 5 and the vector backbone recovered in step 6 to obtain the recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT2a SP1.

[0091] 8. The recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT2a SP1 was transformed into Agrobacterium rhizogenes K599 by electroporation to obtain recombinant Agrobacterium K599 / GmFT2a SP1.

[0092] Following the steps 3-8 above, replace GmFT2a-SaCas9-SP1-F with GmFT2a-SaCas9-SP2-F and GmFT2a-SaCas9-SP1-R with GmFT2a-SaCas9-SP2-R, while keeping all other steps unchanged, to obtain recombinant Agrobacterium K599 / GmFT2a SP2.

[0093] Following the steps 3-8 above, replace GmFT2a-SaCas9-SP1-F with GmFT2a-SaCas9-SP3-F and GmFT2a-SaCas9-SP1-R with GmFT2a-SaCas9-SP3-R, while keeping all other steps unchanged, to obtain recombinant Agrobacterium K599 / GmFT2a SP3.

[0094] III. Genetic Transformation of Soybean Hairy Roots

[0095] 1. Sterilization

[0096] Soybean cultivar Jack (hereinafter referred to as soybean) seeds were surface sterilized with chlorine gas. Then, plump soybean seeds with smooth skin and no wrinkles or damage were selected and spread evenly in a glass dish. The glass dish was then placed in a desiccator, and a 100mL beaker was placed in it. 90mL of sodium hypochlorite solution (with an active chlorine content of about 5%) was added to the beaker. 4mL of concentrated hydrochloric acid was slowly added along the wall of the beaker. The desiccator was then quickly covered with a lid, which was sealed with petroleum jelly. The sterilization time was about 16-20 hours.

[0097] 2. Seed germination

[0098] After completing step 1, open the sterilized glass dish in a laminar flow hood to release any residual chlorine. Then, use sterile forceps to pick up soybean seeds and inoculate them onto germination medium (solute and concentration of 3.21 g / L B5 salt, 20 g / L sucrose and 8 g / L agar, solvent is water, pH 5.8), and incubate at 25°C with alternating light and dark conditions (18 h light and 6 h dark) for 4-5 days.

[0099] 3. Preparation of bacterial culture

[0100] 50 μL of recombinant Agrobacterium (recombinant Agrobacterium K599 / GmFT2a SP1, recombinant Agrobacterium K599 / GmFT2a SP2, or recombinant Agrobacterium K599 / GmFT2a SP3) was inoculated into 50 mL of LB liquid medium and shaken overnight at 28 °C (for activation purposes) to obtain culture solution 1.

[0101] Add 50 mL of bacterial culture 1 to LB liquid medium at a ratio of 1:1000 and incubate at 28°C with shaking (for activation purposes) to obtain OD. 600nm 2. Culture medium of approximately 0.6-0.8.

[0102] 4. Infection and co-culture

[0103] (1) Take the germinated soybean seeds obtained in step 2, retain about 0.5cm of the hypocotyl, peel off the seed coat, cut the cotyledons and hypocotyl longitudinally, remove the buds, and make several cuts at the junction of the cotyledons and hypocotyl with a scalpel to obtain the explant.

[0104] (2) Soak the explants in culture medium 2 for 30 min (for infection), then discard the liquid phase; spread a layer of dry sterilized filter paper on the co-culture medium, place the infected explants with the cut side down on the filter paper, cover the dish with the lid, seal with sealing film, and culture at 22℃ with alternating light and dark (16 h light and 8 h dark) for 5 days to obtain soybean cotyledons.

[0105] The co-culture medium consisted of 0.43 g / L MS salt, 30 g / L sucrose, 8 g / L agar, 3.9 g / L MES, 150 mg / L DTT, and 200 mM As, with water as the solvent and a pH of 5.4.

[0106] (3) Wash soybean cotyledons several times with root induction liquid medium (until the liquid is clear), then soak soybean cotyledons in root induction liquid medium for 30 minutes and discard the medium; then insert soybean cotyledons at a 45-degree angle into root induction solid medium with the scratched side facing up, cover the dish and seal it, culture at 25℃ with alternating light and dark (18h light and 6h darkness) for about 12 days to obtain hairy roots.

[0107] The root induction liquid culture medium contained 2.17 g / L MS salt, 30 g / L sucrose, 0.6 g / L MES, 250 mg / L Cef, and 250 mg / L Cb in water, with a pH of 5.8.

[0108] The root induction solid medium consisted of 2.17 g / L MS salt, 30 g / L sucrose, 8 g / L agar, 0.6 g / L MES, 250 mg / L Cef, and 250 mg / L Cb, with water as the solvent and a pH of 5.8.

[0109] (4) Observe the hair roots obtained in step (3) using a stereomicroscope (Nikon SMZ1500) with a fluorescence excitation module. If the hair roots emit green fluorescence under excitation light (the transformed hair roots have GFP selection markers), it indicates that the hair roots are positive hair roots.

[0110] IV. Detection of mutation types and efficiency of CRISPR / SaCas9 at the GmFT2a target site

[0111] 1. Genomic DNA was extracted from positive hairy roots using the CTAB method and used as a template. PCR amplification was performed using primer pairs consisting of GmFT2a-SaCas9-F: 5′-AAGCAAACGAGTATATAAGAAAGCA-3′ and GmFT2a-SaCas9-R: 5′-TGGATGGTCAAAAACAATAACGTC-3′, yielding a PCR amplification product of approximately 585 bp.

[0112] The reaction system is 50 μL, from Max Buffer 25 μL, dNTP Mix (10 mM each) 1 μL, Genomic DNA from positive hairy roots (200 ng / μL) 2 μL, GmFT2a-SaCas9-F aqueous solution (10 pmol / μL) 2 μL, GmFT2a-SaCas9-R aqueous solution (10 pmol / μL) 2 μL The mixture consisted of 1 μL of Max Super-Fidelity DNA Polymerase and 17 μL of ddH2O.

[0113] Max Super-Fidelity DNA Polymerase is a product of Nanjing Novizan Biotechnology Co., Ltd., with the product code P505-d3. Max Buffer is Components in Max Super-Fidelity DNA Polymerase.

[0114] The reaction program was as follows: 95℃ for 3 min; 95℃ for 15 sec, 56℃ for 15 sec, 72℃ for 45 sec, 35 cycles; 72℃ for 5 min.

[0115] 2. After completing step 1, sequence the approximately 585bp PCR amplification product. Then, calculate the mutation efficiency of CRISPR / SaCas9 at the GmFT2a target site using the following formula: Mutation efficiency = Number of hair roots mutated at the target site / Number of hair roots detected × 100%

[0116] Sequencing results showed that the mutation efficiencies of CRISPR / SaCas9 at the target sites GmFT2a-SaCas9-SP1, GmFT2a-SaCas9-SP2 and GmFT2a-SaCas9-SP3 were 73.3% (22 / 30), 34.8% (8 / 23) and 34.5% (10 / 29), respectively.

[0117] 3. To detect the type of mutation produced, subcloning sequencing was performed.

[0118] Some test results can be found Figure 2 .

[0119] The results showed that five mutation types were generated at the GmFT2a-SaCas9-SP1 target site (see...). Figure 2 The mutations (A) in the GmFT2a-SaCas9-SP2 target site were 4-bp deletion (SEQ ID NO: 1, positions 206-209), 5-bp deletion (SEQ ID NO: 1, positions 206-210), 1-bp deletion (SEQ ID NO: 1, position 208), 1-bp A insertion (SEQ ID NO: 1, position 209), and 1-bp G insertion (SEQ ID NO: 1, position 209). Three mutation types were generated at the GmFT2a-SaCas9-SP2 target site (see [link to relevant documentation]). Figure 2 The mutations (B) represent 2-bp base deletions (SEQ ID NO: 1, positions 96-97), 1-bp base deletions (SEQ ID NO: 1, position 98), and 1-bp A base insertions (SEQ ID NO: 1, position 100); five mutation types were generated at the GmFT2a-SaCas9-SP3 target site (see [link to relevant documentation]). Figure 2 The C in the SEQ ID NO: 1 contains 12-bp base deletions (SEQ ID NO: 1, positions 107-118), 9-bp base deletions (SEQ ID NO: 1, positions 111-119), 8-bp base deletions (SEQ ID NO: 1, positions 111-118), 8-bp base deletions (SEQ ID NO: 1, positions 110-117), and 1-bp base deletions (SEQ ID NO: 1, position 117).

[0120] Example 2: Site-specific knockout of soybean gene GmFT5a using CRISPR / SaCas9

[0121] I. SgRNA Target Sequence Selection

[0122] The soybean GmFT5a gene (Glyma.16G044100) shown in SEQ ID NO:4 was obtained from the Phytozome database. Two target sites, GmFT5a-SaCas9-SP1 and GmFT5a-SaCas9-SP2, were then selected on the first exon of the GmFT5a gene.

[0123] The nucleotide sequence of GmFT5a-SaCas9-SP1 is 5'-CTCTCAAGTTGTTAATCGCCCTAGGGT-3' (SEQ ID NO: 4, positions 177-203 from the 5' end), and its PAM sequence is TAGGGT.

[0124] The nucleotide sequence of GmFT5a-SaCas9-SP2 is 5'-CTTGTTATTGGTGGTGTGATTGGGGAT-3' (SEQ ID NO: 4, positions 67-93 from the 5' end), and its PAM sequence is GGGGAT.

[0125] II. Construction of CRISPR / SaCas9 vectors

[0126] 1. The vector pUC57-SgRNA-SaCas9 was digested with restriction endonucleases NHeI and BbsI, and the vector backbone of approximately 3201 bp was recovered.

[0127] 2. Based on the nucleotide sequence of GmFT5a-SaCas9-SP1 from step one, synthesize primers GmFT5a-SaCas9-SP1-F and GmFT5a-SaCas9-SP1-R. Based on the nucleotide sequence of GmFT5a-SaCas9-SP2 from step one, synthesize primers GmFT5a-SaCas9-SP2-F and GmFT5a-SaCas9-SP2-R. The nucleotide sequences of each primer are shown in Table 2.

[0128] Table 2

[0129] Primer name nucleotide sequence GmFT5a-SaCas9-SP1-F 5′-TCGAAGTAGTGATTGCTCTCAAGTTGTTAATCGCCCGTTTTAGTACTCTGG-3′ GmFT5a-SaCas9-SP1-R 5′-CCAGAGTACTAAAACGGGCGATTAACAACTTGAGAGCAATCACTACTTCGA-3′ GmFT5a-SaCas9-SP2-F 5′-TCGAAGTAGTGATTGCTTGTTATTGGTGGTGTGATTGTTTTAGTACTCTGG-3′ GmFT5a-SaCas9-SP2-R 5′-CCAGAGTACTAAAACAATCACACCACCAATAACAAGCAATCACTACTTCGA-3′

[0130] 3. First, prepare the reaction system, and then anneal the reaction system to form oligo dimers.

[0131] The reaction system consisted of 25 μL of 5 μL FT5a-SaCas9-SP1-F aqueous solution (concentration 100 μM), 5 μL FT5a-SaCas9-SP1-R aqueous solution (concentration 100 μM), and 15 μL ddH2O.

[0132] The annealing procedure is as follows: 95℃ for 3 min, anneal at 0.1℃ / s to 16℃, and hold at 16℃ for 10 min.

[0133] 4. Connect the oligo dimer with the carrier backbone recovered in step 1 to obtain an intermediate carrier.

[0134] 5. The intermediate vector was digested with restriction endonucleases PacI and PmeI to recover a DNA fragment of approximately 586 bp.

[0135] 6. Digest the PTF101-SaCas9-GFP-bar plasmid with restriction endonucleases PacI and PmeI, and recover the vector backbone of approximately 14.7 kb.

[0136] 7. Ligate the DNA fragment recovered in step 5 and the vector backbone recovered in step 6 to obtain the recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT5a SP1.

[0137] 8. The recombinant plasmid PTF101-SaCas9-GFP-bar-GmFT5a SP1 was transformed into Agrobacterium rhizogenes K599 by electroporation to obtain recombinant Agrobacterium K599 / GmFT5a SP1.

[0138] Following the steps 3-8 above, replace GmFT5a-SaCas9-SP1-F with GmFT5a-SaCas9-SP2-F and GmFT5a-SaCas9-SP1-R with GmFT5a-SaCas9-SP2-R, while keeping all other steps unchanged, to obtain recombinant Agrobacterium K599 / GmFT5a SP2.

[0139] III. Genetic Transformation of Soybean Hairy Roots

[0140] 1. Sterilization

[0141] Soybean cultivar Jack (hereinafter referred to as soybean) seeds were surface sterilized with chlorine gas. Then, plump soybean seeds with smooth skin and no wrinkles or damage were selected and spread evenly in a glass dish. The glass dish was then placed in a desiccator, and a 100mL beaker was placed in it. 90mL of sodium hypochlorite solution (with an active chlorine content of about 5%) was added to the beaker. 4mL of concentrated hydrochloric acid was slowly added along the wall of the beaker. The desiccator was then quickly covered with a lid, which was sealed with petroleum jelly. The sterilization time was about 16-20 hours.

[0142] 2. Seed germination

[0143] After completing step 1, open the sterilized glass dish in a laminar flow hood to release any residual chlorine. Then, use sterile forceps to pick up soybean seeds and inoculate them onto the germination medium. Incubate at 25°C with alternating light and dark conditions (18 hours of light and 6 hours of darkness) for 4-5 days.

[0144] 3. Preparation of bacterial culture

[0145] 50 μL of recombinant Agrobacterium (recombinant Agrobacterium K599 / GmFT5a SP1 or recombinant Agrobacterium K599 / GmFT5a SP2) was inoculated into 50 mL of LB liquid medium and shaken overnight at 28 °C (for activation) to obtain culture solution 1.

[0146] Add 50 mL of bacterial culture 1 to LB liquid medium at a ratio of 1:1000 and incubate at 28°C with shaking (for activation purposes) to obtain OD. 600nm 2. Culture medium of approximately 0.6-0.8.

[0147] 4. Infection and co-culture

[0148] (1) Take the germinated soybean seeds obtained in step 2, retain about 0.5cm of the hypocotyl, peel off the seed coat, cut the cotyledons and hypocotyl longitudinally, remove the buds, and make several cuts at the junction of the cotyledons and hypocotyl with a scalpel to obtain the explant.

[0149] (2) Soak the explants in culture medium 2 for 30 min (for infection), then discard the liquid phase; spread a layer of dry sterilized filter paper on the co-culture medium, place the infected explants with the cut side down on the filter paper, cover the dish with the lid, seal with sealing film, and culture at 22℃ with alternating light and dark (16 h light and 8 h dark) for 5 days to obtain soybean cotyledons.

[0150] (3) Wash soybean cotyledons several times with root induction liquid medium (until the liquid is clear), then soak soybean cotyledons in root induction liquid medium for 30 minutes and discard the medium; then insert soybean cotyledons at a 45-degree angle into root induction solid medium with the scratched side facing up, cover the dish and seal it, culture at 25℃ with alternating light and dark (18h light and 6h darkness) for about 12 days to obtain hairy roots.

[0151] (4) Observe the hair roots obtained in step (3) using a stereomicroscope (Nikon SMZ1500) with a fluorescence excitation module. If the hair roots emit green fluorescence under excitation light (the transformed hair roots have GFP selection markers), it indicates that the hair roots are positive hair roots.

[0152] IV. Detection of mutation types and efficiency of CRISPR / SaCas9 at the GmFT5a target site

[0153] 1. Genomic DNA was extracted from positive hairy roots using the CTAB method and used as a template. PCR amplification was performed using primer pairs consisting of GmFT5a-SaCas9-F: 5′-GCAGATGCTAAGGTGGAAAAATA-3′ and GmFT5a-SaCas9-R: 5′-TGCATCCACCATAACCTGAGAT-3′, yielding a PCR amplification product of approximately 462 bp.

[0154] The reaction system is 50 μL, from Max Buffer 25 μL, dNTP Mix (10 mM each) 1 μL, Genomic DNA from positive hairy roots (200 ng / μL) 2 μL, GmFT5a-SaCas9-F aqueous solution (10 pmol / μL) 2 μL, GmFT5a-SaCas9-R aqueous solution (10 pmol / μL) 2 μL It consists of 1 μL of Max Super-Fidelity DNA Polymerase and 17 μL of ddH2O.

[0155] The reaction program was as follows: 95℃ for 3 min; 95℃ for 15 sec, 56℃ for 15 sec, 72℃ for 45 sec, 35 cycles; 72℃ for 5 min.

[0156] 2. After completing step 1, sequence the approximately 462 bp PCR amplification product. Then, calculate the mutation efficiency of CRISPR / SaCas9 at the GmFT5a target site using the following formula: Mutation efficiency = Number of hair roots mutated at the target site / Number of hair roots detected × 100%

[0157] Sequencing results showed that the mutation efficiencies of CRISPR / SaCas9 at the target sites GmFT5a-SaCas9-SP1 and GmFT5a-SaCas9-SP2 were 42.9% (12 / 28) and 57.1% (16 / 28), respectively.

[0158] 3. To detect the type of mutation produced, subcloning sequencing was performed.

[0159] Some test results can be found Figure 4 .

[0160] The results showed that four mutation types were generated at the GmFT5a-SaCas9-SP1 target site (see...). Figure 4The mutations (A) represent 14-bp deletions (SEQ ID NO: 4, positions 194-207), 15-bp deletions (SEQ ID NO: 4, positions 189-203), 1-bp deletions (SEQ ID NO: 4, position 195), and 2-bp T-base insertions (SEQ ID NO: 4, positions 195-196); four mutation types were generated at the GmFT5a-SaCas9-SP2 target site (see [link to SEQ ID NO: 4]). Figure 4 The B-bases are 2-bp deletion (SEQ ID NO: 4, positions 85-86), 1-bp A base insertion (SEQ ID NO: 4, position 85), 1-bp T base insertion (SEQ ID NO: 4, position 84), and 1-bp SNP (SEQ ID NO: 4, position 85, where A is replaced by T).

[0161] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for targeted editing of soybean target genes, comprising the following steps: (1) Design SgRNA based on the target genes in the soybean genome that are expected to be edited in a targeted manner; (2) The gene encoding SgRNA and the gene encoding SaCas9 protein were introduced into soybeans to achieve targeted editing of target genes in soybeans; The PAM sequence for the SaCas9 protein target recognition site is 5'-NNGRRT-3', where N is A, T, G, or C, and R is A or G; The target gene is GmFT2a Gene; The GmFT2a The target sites recognized by the SgRNA of the gene are A1), A2), or A3). A1) The DNA molecule shown in SEQ ID NO: 1 from position 191 to 217 starting from the 5' end; A2) The DNA molecule shown in SEQ ID NO: 1 from position 81 to 107 starting from the 5' end; A3) The DNA molecule shown in SEQ ID NO: 1 from position 100 to 126 starting from the 5' end.

2. A method for targeted editing of the soybean genome GmFT2a Genetic methods are used to develop soybeans for editing using the CRISPR / SaCas9 system. GmFT2a Genes are edited; in the CRISPR / SaCas9 system, the PAM sequence of the SaCas9 protein recognition target is 5'-NNGRRT-3', where N is A, T, G or C, and R is A or G; The GmFT2a The target sites recognized by the SgRNA of the gene are A1), A2), or A3). A1) The DNA molecule shown in SEQ ID NO: 1 from position 191 to 217 starting from the 5' end; A2) The DNA molecule shown in SEQ ID NO: 1 from position 81 to 107 starting from the 5' end; A3) The DNA molecule shown in SEQ ID NO: 1 from position 100 to 126 starting from the 5' end.

3. Application of CRISPR / SaCas9 system in soybean gene editing; the PAM sequence of the SaCas9 protein recognition target in the CRISPR / SaCas9 system is 5'-NNGRRT-3', where N is A, T, G or C, and R is A or G; The target gene of the gene editing is GmFT2a Gene; The GmFT2a The target sites recognized by the SgRNA of the gene are A1), A2), or A3). A1) The DNA molecule shown in SEQ ID NO: 1 from position 191 to 217 starting from the 5' end; A2) The DNA molecule shown in SEQ ID NO: 1 from position 81 to 107 starting from the 5' end; A3) The DNA molecule shown in SEQ ID NO: 1 from position 100 to 126 starting from the 5' end.

4. A vector for targeted editing of soybean target genes, comprising a gene encoding SaCas9 protein and a gene encoding SgRNA; The target gene is GmFT2a Gene; The GmFT2a The target sites recognized by the SgRNA of the gene are A1), A2), or A3). A1) The DNA molecule shown in SEQ ID NO: 1 from position 191 to 217 starting from the 5' end; A2) The DNA molecule shown in SEQ ID NO: 1 from position 81 to 107 starting from the 5' end; A3) The DNA molecule shown in SEQ ID NO: 1 from position 100 to 126 starting from the 5' end.

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