Methods for gene editing in soybeans using Cas12i
By performing specific amino acid mutations on the Cas12i nuclease and designing targeted gRNAs, the problem of low editing efficiency of Cas12i in soybeans was solved, achieving efficient gene editing, obtaining homozygous mutants with high oleic acid content, and improving the quality and stability of soybean oil.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JILIN AGRICULTURAL UNIV
- Filing Date
- 2026-03-31
- Publication Date
- 2026-05-26
AI Technical Summary
Cas12i has low editing efficiency in dicotyledonous plants, and even fails to show editing activity at some sites, making it difficult to meet the needs of efficient gene editing.
By constructing specific amino acid mutations in the Cas12i nuclease (arginine at positions 369 and 433), gRNAs targeting the soybean GmFAD2-1A and GmFAD2-1B genes were designed and co-transformed into soybean recipient materials using Agrobacterium-mediated transformation. High-oleic homozygous mutants were then screened and bred.
It significantly improved the cleavage activity and target specificity of Cas12i in soybeans, achieved a doubling of the efficiency of dual gene knockout, expanded the genome editing window, reduced the off-target risk, directly obtained homozygous mutants with high oleic acid content, and improved the nutritional quality and oxidative stability of soybean oil.
Smart Images
Figure CN122081384A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to a method for gene editing in soybeans using Cas12i. Background Technology
[0002] Soybeans are a major source of oil and protein for humans, one of the world's most important economic crops, and a primary source of plant oils and plant-based proteins. With rising living standards and improved dietary habits, the demand for high-quality soybean oil is increasing, making the cultivation of high-quality soybeans a key objective of soybean breeding.
[0003] The CRISPR / Cas9 system is the most commonly used type II CRISPR system. It recognizes the 3'-NGG PAM motif and performs blunt-end cleavage on the target sequence. Site-specific editing of the target gene is achieved through guide RNA-mediated cleavage by the Cas9 protein. This technology not only provides new insights into gene function research but also has wider applications in biomedical research and development and crop genetic improvement. Currently, the CRISPR / Cas9 system has been successfully applied in plants such as Arabidopsis thaliana, rice, maize, wheat, and soybean.
[0004] CRISPR / Cas Type V systems are a newly discovered class of CRISPR systems that possess a 5'-TTN motif and perform sticky end cleavage of target sequences, such as Cpf1, C2c1, CasX, and CasY. However, the different CRISPR / Cas systems currently available each have their own advantages and disadvantages. For example, Cas9, C2c1, and CasX all require two guide RNAs, while Cpf1 only requires one and can be used for multiplex gene editing. CasX is 980 amino acids in size, while common systems like Cas9, C2c1, CasY, and Cpf1 are typically around 1300 amino acids. Furthermore, the PAM sequences of Cas9, Cpf1, CasX, and CasY are relatively complex and diverse, while C2c1 recognizes a strict 5'-TTN, making its target site easier to predict than other systems and reducing potential off-target effects. Cas12i also belongs to the Type V CRISPR / Cas system.
[0005] A search revealed a method for gene editing in soybeans using Cas12i (publication number CN116218896A). The method includes the steps of gene editing in soybeans using Cas12i and gRNA. The gRNA includes a backbone region that binds to Cas12i and a guide sequence that hybridizes to a target sequence. The gRNA targets the GmFAD2-1A and GmFAD2-1B genes in soybeans.
[0006] The background section of the aforementioned patents mentions that soybeans, unlike corn, are dicotyledonous plants. When the inventors studied the editing activity of this enzyme in dicotyledonous plants (e.g., Arabidopsis thaliana, soybean), they found that the enzyme's editing efficiency in dicotyledonous plants was low, and it even failed to exhibit editing activity at certain sites. Therefore, a method for gene editing in soybeans using Cas12i is needed. Summary of the Invention
[0007] To address the shortcomings of existing technologies, this invention provides a method for gene editing in soybeans using Cas12i, which solves the problem that Cas12i has low editing efficiency in dicotyledonous plants and may even fail to demonstrate editing activity at certain sites.
[0008] To achieve the above objectives, the present invention provides the following technical solution:
[0009] The method for gene editing in soybeans using Cas12i includes the following steps:
[0010] Step 1: Construct a plant binary vector expressing the Cas12i nuclease, wherein the amino acids at positions 369 and 433 of the Cas12i nuclease are mutated to arginine, respectively;
[0011] Step 2: Design gRNAs targeting the soybean GmFAD2-1A and GmFAD2-1B genes;
[0012] Step 3: The gRNA expression cassette and the Cas12i nuclease expression cassette are co-transformed into soybean acceptor material using Agrobacterium-mediated transformation.
[0013] Step 4: Select T0 generation transgenic plants, identify the target gene editing type through molecular detection, and continuously self-pollinate to obtain T2 generation high oleic acid homozygous mutants without transgenic components.
[0014] Preferably, the expression unit of the Cas12i nuclease in step one includes: a soybean codon-optimized Cas12i coding sequence, a nuclear localization signal peptide, an EF1α promoter, and a NOS terminator. The Cas12i coding sequence and the gRNA work in a cooperative state, the editing efficiency is set to η, and the output satisfies η ≥ 10%.
[0015] Preferably, the editing efficiency is calculated using the formula η = (number of positive hair roots that were edited / total number of hair roots detected) × 100%.
[0016] Preferably, in step two, the guide sequence of the gRNA has a complementary pairing region with the target gene of 20-24 nucleotides in length, and the target site of the gRNA is located in the nucleotide region of position 262-288 of the GmFAD2-1A gene and the nucleotide region of position 258-286 of the GmFAD2-1B gene.
[0017] Preferably, in step three, the Agrobacterium-mediated method uses Agrobacterium rhizogenes K599 or Agrobacterium tumefaciens EHA105, the OD600 value of the infection solution is controlled at 0.6-0.8, the infection time is 30-120 minutes, the culture temperature is maintained at 22-25℃, and the light environment is maintained in either a dark or a 16-hour light-8-hour dark cycle alternating light scheme.
[0018] Preferably, in step three, the soybean receptor is selected from any one of the following varieties: Jack soybean, Zhonghuang 42 soybean, Williams 82 soybean, and Tianlong No. 1 soybean.
[0019] Preferably, in step three, the soybean recipient material is any one of soybean seeds, cotyledonary nodes, embryogenic callus, or protoplasts.
[0020] Preferably, in step two, the gRNA comprises the following structure from the 5' to the 3' direction:
[0021] The backbone region of the same direction repeat sequence, 5'-AGAGAATGTGTGCATAGTCACAC-3';
[0022] Target sequence region, 5'-CCUCAUUGCAUGGCCAAUCUAUU-3';
[0023] The target sequence is complementary to the nucleotide sequences at positions 262-288 of the soybean GmFAD2-1A gene and positions 258-286 of the GmFAD2-1B gene, and the PAM sequence is 5'-TTC-3'.
[0024] Preferably, the specific steps of cultivation in step four include:
[0025] T1 generation seeds were obtained by self-pollination of S1 and T0 generation plants, and transgenic elements were isolated by bar test strips and PCR detection.
[0026] S2. Sequencing was performed in the T1-T2 generation using target-specific primers D-GmBADH1-F / R to screen for homozygous edited lines without foreign gene integration, resulting in T2 generation high oleic acid homozygous mutants without transgenic components.
[0027] Preferably, the specific procedure for molecular detection in step four includes:
[0028] S1. PCR amplification using target-specific primer pairs;
[0029] S2. Perform Sanger sequencing or hiTOM sequencing on the PCR products to analyze the editing type;
[0030] S3. Detect the fatty acid composition of the seeds by GC-MS to determine whether the oleic acid content accounts for 80% of the total fatty acids, which is the fatty acid ratio for a high oleic acid phenotype.
[0031] This invention provides a method for gene editing in soybeans using Cas12i. It has the following beneficial effects:
[0032] 1. This invention simultaneously mutates amino acids at positions 369 and 433 of the Cas12i nuclease to arginine, significantly enhancing the nuclease's cleavage activity and targeting specificity in plant cells, thus doubling the efficiency of dual gene knockout. Furthermore, the Cas12i nuclease used recognizes the TTC-PAM sequence, enabling it to target more AT-enriched regions, significantly expanding the editing window of the soybean genome, while reducing the risk of off-target effects.
[0033] 2. This invention simultaneously targets key conserved regions of the GmFAD2-1A and GmFAD2-1B genes, and can block the conversion pathway of oleic acid to linoleic acid through a single editing, directly obtaining a high oleic acid homozygous mutant with an oleic acid content of more than 80% of total fatty acids, which significantly improves the nutritional quality and oxidative stability of soybean oil. Attached Figure Description
[0034] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation
[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0036] Example:
[0037] Please see the appendix Figure 1 This invention provides a method for gene editing in soybeans using Cas12i, comprising the following steps:
[0038] Step 1: Construct a plant binary vector expressing Cas12i nuclease. Amino acids at positions 369 and 433 of the Cas12i nuclease are mutated to arginine, respectively. The expression unit of the Cas12i nuclease includes: a soybean codon-optimized Cas12i coding sequence, a nuclear localization signal peptide, an EF1α promoter, and a NOS terminator. The Cas12i coding sequence and gRNA work in synergy. The editing efficiency is set to η, and the output satisfies η ≥ 10%. The editing efficiency is calculated using the formula η = (number of positive hair roots that underwent editing / total number of hair roots detected) × 100%.
[0039] Step 2: Design gRNAs targeting the soybean GmFAD2-1A and GmFAD2-1B genes. In Step 2, the guide sequence of the gRNA has a complementary pairing region of 20-24 nucleotides with the target gene. The target sites of the gRNA are located in the nucleotide region of GmFAD2-1A gene from position 262 to 288 and the nucleotide region of GmFAD2-1B gene from position 258 to 286.
[0040] Step 3: The gRNA expression cassette and the Cas12i nuclease expression cassette were co-transformed into soybean recipient material using Agrobacterium-mediated transformation. In Step 3, Agrobacterium rhizogenes K599 or Agrobacterium tumefaciens EHA105 were used for the Agrobacterium-mediated transformation. The OD600 value of the infection solution was controlled at 0.6–0.8, the infection time was 30–120 minutes, and the culture temperature was maintained at 22–25℃. The light environment was maintained in either a dark environment or an alternating 16-hour light / 8-hour dark cycle. The soybean recipient was any one of the following varieties: Jack soybean, Zhonghuang 42 soybean, Williams 82 soybean, or Tianlong No. 1 soybean. The soybean recipient material was any one of the following: soybean seeds, cotyledonary nodes, embryogenic callus, or protoplasts. The gRNA contained the following structure from the 5' to 3' direction:
[0041] The backbone region of the same direction repeat sequence, 5'-AGAGAATGTGTGCATAGTCACAC-3';
[0042] Target sequence region, 5'-CCUCAUUGCAUGGCCAAUCUAUU-3';
[0043] The target sequence is complementary to the nucleotide sequences at positions 262-288 of the soybean GmFAD2-1A gene and positions 258-286 of the GmFAD2-1B gene, and the PAM sequence is 5'-TTC-3'.
[0044] Step 4: Select T0 generation transgenic plants, identify the target gene editing type through molecular detection, and continuously self-pollinate to obtain T2 generation high oleic acid homozygous mutants without transgenic components;
[0045] The specific steps involved in the cultivation include:
[0046] T1 generation seeds were obtained by self-pollination of S1 and T0 generation plants, and transgenic elements were isolated by bar test strips and PCR detection.
[0047] S2. Sequencing was performed in the T1-T2 generation using target-specific primers D-GmBADH1-F / R to screen for homozygous edited lines without foreign gene integration, resulting in T2 generation high oleic acid homozygous mutants without transgenic components.
[0048] The specific process of molecular detection includes:
[0049] S1. PCR amplification using target-specific primer pairs;
[0050] S2. Perform Sanger sequencing or hiTOM sequencing on the PCR products to analyze the editing type;
[0051] S3. Detect the fatty acid composition of the seeds by GC-MS to determine whether the oleic acid content accounts for 80% of the total fatty acids, which is the fatty acid ratio for a high oleic acid phenotype.
[0052] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A method for gene editing in soybeans using Cas12i, characterized in that, Includes the following steps: Step 1: Construct a plant binary vector expressing the Cas12i nuclease, wherein the amino acids at positions 369 and 433 of the Cas12i nuclease are mutated to arginine, respectively; Step 2: Design gRNAs targeting the soybean GmFAD2-1A and GmFAD2-1B genes; Step 3: The gRNA expression cassette and the Cas12i nuclease expression cassette are co-transformed into soybean acceptor material using Agrobacterium-mediated transformation. Step 4: Select T0 generation transgenic plants, identify the target gene editing type through molecular detection, and continuously self-pollinate to obtain T2 generation high oleic acid homozygous mutants without transgenic components.
2. The method for gene editing in soybeans using Cas12i according to claim 1, characterized in that, The expression unit of the Cas12i nuclease in step one includes: a soybean codon-optimized Cas12i coding sequence, a nuclear localization signal peptide, an EF1α promoter, and a NOS terminator. The Cas12i coding sequence and gRNA work in synergy, and the editing efficiency is set to η, satisfying an output efficiency of η ≥ 10%.
3. The method for gene editing in soybeans using Cas12i according to claim 2, characterized in that, The editing efficiency is calculated using the formula η = (number of positive hair roots that were edited / total number of hair roots detected) × 100%.
4. The method for gene editing in soybeans using Cas12i according to claim 1, characterized in that, In step two, the guide sequence of the gRNA has a complementary pairing region of 20-24 nucleotides in length with the target gene. The target site of the gRNA is located in the nucleotide region of position 262-288 of the GmFAD2-1A gene and the nucleotide region of position 258-286 of the GmFAD2-1B gene.
5. The method for gene editing in soybeans using Cas12i according to claim 1, characterized in that, In step three, the Agrobacterium-mediated method uses Agrobacterium rhizogenes K599 or Agrobacterium tumefaciens EHA105. The OD600 value of the infection solution is controlled at 0.6-0.8, the infection time is 30-120 minutes, the culture temperature is maintained at 22-25℃, and the light environment is maintained in either a dark or a 16-hour light-8-hour dark cycle alternating light scheme.
6. The method for gene editing in soybeans using Cas12i according to claim 1, characterized in that, In step three, the soybean recipient can be any one of the following varieties: Jack soybean, Zhonghuang 42 soybean, Williams 82 soybean, and Tianlong No. 1 soybean.
7. The method for gene editing in soybeans using Cas12i according to claim 1, characterized in that, In step three, the soybean recipient material can be any one of soybean seeds, cotyledonary nodes, embryogenic callus, or protoplasts.
8. The method for gene editing in soybeans using Cas12i according to claim 1, characterized in that, In step two, the gRNA contains the following structure from the 5' to the 3' direction: The backbone region of the same direction repeat sequence, 5'-AGAGAATGTGTGCATAGTCACAC-3'; Target sequence region, 5'-CCUCAUUGCAUGGCCAAUCUAUU-3'; The target sequence is complementary to the nucleotide sequences at positions 262-288 of the soybean GmFAD2-1A gene and positions 258-286 of the GmFAD2-1B gene, and the PAM sequence is 5'-TTC-3'.
9. The method for gene editing in soybeans using Cas12i according to claim 1, characterized in that, The specific steps in step four of the cultivation process include: T1 generation seeds were obtained by self-pollination of S1 and T0 generation plants, and transgenic elements were isolated by bar test strips and PCR detection. S2. Sequencing was performed in the T1-T2 generation using target-specific primers D-GmBADH1-F / R to screen for homozygous edited lines without foreign gene integration, resulting in T2 generation high oleic acid homozygous mutants without transgenic components.
10. The method for gene editing in soybeans using Cas12i according to claim 1, characterized in that, The specific procedures for molecular detection in step four include: S1. PCR amplification using target-specific primer pairs; S2. Perform Sanger sequencing or hiTOM sequencing on the PCR products to analyze the editing type; S3. Detect the fatty acid composition of the seeds by GC-MS to determine whether the oleic acid content accounts for 80% of the total fatty acids, which is the fatty acid ratio for a high oleic acid phenotype.