Application of CRISPR / Cas12a in cotton multi-gene editing

Abstract

The invention discloses an application of CRISPR (clustered regularly interspaced short palindromic repeats) / Cas12a (CRISPR / Cas12a) in The method comprises the following steps: selecting AP1, FAD2, TCP18 and AFP3 as target genes, and respectively designing one sgRNA on exons of the genes; the method comprises the following steps: synthesizing a serial sgRNA segment, subcloning the serial sgRNA segment to a pKSE401-CRISPR / Cas12a vector to obtain a cotton knockout vector named as pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3, obtaining a CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 gene knockout plant through an agrobacterium tumefaciens-mediated cotton genetic transformation method, extracting genome DNA (deoxyribonucleic acid) of a plant leaf, and carrying out gene knockout on the plant leaf to obtain the gene knockout plant. Through verification, it is confirmed that at most three sites in an event of pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 are successfully edited at the same time, the editing efficiency of the site AP1 is 62%, the editing efficiency of the site FAD2 is 54%, the editing efficiency of the site TCP18 is 8%, the efficiency of simultaneous editing of the two sites is 46%, and the efficiency of simultaneous editing of the three sites is 8%.

Description

Technical Field

[0001] The present invention relates to the application of CRISPR / Cas12a gene editing system in cotton gene editing. Specifically, the present invention utilizes CRISPR / Cas12a gene editing system to simultaneously target cotton AP1, FAD2, TCP18 and other genes, screens out mutant materials with multiple genes edited simultaneously, provides new germplasm for creating excellent cotton varieties, and belongs to the field of gene editing technology. Background Art

[0002] Cotton is an important crop that can provide fiber, edible oil and feed, and has high economic value in industrial and agricultural production. In cotton breeding, traditional breeding has a long cycle and is highly blind; physical and chemical mutagenesis breeding has random mutation sites and is difficult to control; transgenic breeding has a long safety evaluation cycle and complex supervision; and CRISPR / Cas system-mediated gene editing technology has the advantages of low operating threshold, high editing efficiency, and fast results transformation. It is an ideal way to create excellent mutant materials and is widely used in cotton gene editing breeding.

[0003] In addition to the representative CRISPR / Cas9, CRISPR / Cas12a is another new member with gene editing capabilities, which greatly expands the editable range of gene editing systems. Cas12a, also known as cpf1, belongs to the 2nd type V CRISPR effector protein. Its protein molecular weight is smaller than that of Cas9. Cas12a has both DNA nuclease and RNA nuclease functions. It does not require the assistance of tracrRNA for editing the target gene, but only requires the guidance of crRNA. This greatly promotes the application of Cas12a in multi-gene editing; Cas12a can recognize thymine T-rich 5'-TTTV-3' and form sticky ends after cutting, so it can edit AT-rich regions in the genome with extremely low off-target efficiency.

[0004] Plant type, early maturity, yield and stress resistance are the problems that need to be solved in cotton production. Excellent cotton plant type is the key to mechanized harvesting, and early maturity, yield and stress resistance are the most concerned traits in the breeding process of new cotton varieties. Therefore, creating cotton suitable for mechanized harvesting, early maturity and strong stress resistance is the foundation for the healthy and stable development of the cotton industry. Therefore, the present invention selects some negative regulatory genes related to plant type, early maturity, stress resistance, etc. for editing, such as flowering-related genes (AP1), high oleic acid regulatory genes (FAD2), plant type regulatory factors (TCP18), stress response genes (AFP3), etc., to identify excellent mutant materials and provide new germplasm for creating excellent cotton varieties. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the CRISPR / Cas12a gene editing system in cotton multi-gene editing, which creates a new cotton germplasm with early maturity, high oleic acid and improved plant type.

[0006] In order to achieve the above-mentioned object, the technical solution of the present invention is to provide a cotton multi-gene editing vector pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3, the vector

[0007] pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 contains the coding sequence shown in SEQ ID NO.1-4.

[0008] On the other hand, the technical solution of the present invention is to provide a cotton multi-gene editing vector

[0009] pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3, the vector

[0010] pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 contains the sequence shown in SEQ ID NO.5.

[0011] On the other hand, the technical solution of the present invention is to provide a carrier

[0012] The construction method of pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 selected AP1, FAD2, TCP18 and AFP3 as target genes, designed one sgRNA on the exons of these genes respectively; synthesized tandem sgRNA fragments and subcloned them into the pKSE401-CRISPR / Cas12a vector to obtain the cotton knockout vector, named

[0013] pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3.

[0014] On the other hand, the technical solution of the present invention is to provide a carrier

[0015] The method for creating new cotton germplasm with pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 is characterized in that the vector pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 is transformed into Agrobacterium; and the cotton is genetically transformed by using the Agrobacterium-mediated method.

[0016] On the other hand, the technical solution of the present invention is to provide a carrier

[0017] Application of pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 in cotton multi-genome editing.

[0018] On the other hand, the technical solution of the present invention is to provide a carrier

[0019] Application of pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 for simultaneously targeting multiple negative regulatory genes in the cotton genome.

[0020] On the other hand, the technical solution of the present invention is to provide a carrier as described

[0021] Application of pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 in cotton genetic transformation for simultaneous improvement of multiple traits.

[0022] On the other hand, the technical solution of the present invention is to provide an application of CRISPR / Cas12a in cotton multi-gene editing.

[0023] On the other hand, the technical solution of the present invention is to provide an application of CRISPR / Cas12a for simultaneously targeting multiple negative regulatory genes in the cotton genome.

[0024] On the other hand, the technical solution of the present invention is to provide an application of CRISPR / Cas12a in cotton genetic transformation to simultaneously improve multiple traits.

[0025] The specific application method is to select multiple target genes and design one sgRNA on the exons of these genes respectively; synthesize tandem sgRNA fragments and subclone them into the pKSE401-CRISPR / Cas12a vector to obtain the cotton knockout vector and transform Agrobacterium; use Agrobacterium-mediated method to carry out cotton genetic transformation.

[0026] Beneficial effects of the present invention:

[0027] The present invention provides an application of the CRISPR / Cas12a gene editing system in upland cotton gene editing. The present invention obtains CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 gene knockout plants by Agrobacterium-mediated cotton genetic transformation. Take leaves of T0 generation regenerated plants, extract genomic DNA, and verify the present invention.

[0028] In the event of pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3, at most three sites were successfully edited at the same time. The editing efficiency of site AP1 was 62%, the editing efficiency of site FAD2 was 54%, and the editing efficiency of site TCP18 was 8%. The efficiency of simultaneous editing of two sites was 46%, and the efficiency of simultaneous editing of three sites was 8%. A new germplasm with early maturity, high oleic acid and improved plant type was created. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the CRISPR / Cas12a editing vector structure. In the figure, LB is the left border; AtU6-26 is the promoter; DR is the direct repeat sequence; sgRNA1, sgRNA2, sgRNA3, and sgRNA4 are the sgRNAs corresponding to AP1, FAD2, TCP18, and AFP3 genes, respectively; 35S is the promoter; NOS is the terminator, and RB is the right border.

[0030] Figure 2 The electrophoresis diagram of PCR products of some T0 generation regenerated plants. DETAILED DESCRIPTION

[0031] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0032] Example 1: Obtaining pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 vector

[0033] The present invention selects AP1, FAD2, TCP18 and AFP3 as target genes, uses the CRISPR target design website CRISPRREGN tools (http: / / www.rgenome.net / cas-designer / ), and selects high-specificity targets with a GC content between 40% and 60% according to the CDS sequences of AP1, FAD2, TCP18 and AFP3 genes (the gene number of AP1 is Gh_A04G1264, the gene number of FAD2 is Gh_A01G2094, the gene number of TCP18 is Gh_D11G0061, and the gene number of AFP3 is Gh_A11G1291), and designs one sgRNA on the exons of these genes respectively. The coding sequence of sgRNA is as follows:

[0034] sgRNA-AP1: 5'-CAAAAGAAGAGCTGGTTTATTGA-3' (SEQ ID NO.1)

[0035] sgRNA-FAD2: 5'-TTGATTTCACTCAGAGTGAAGGG-3' (SEQ ID NO.2)

[0036] sgRNA-TCP18: 5'-TATTCCCCTTCTTCTCCGAATTA-3' (SEQ ID NO.3)

[0037] sgRNA-AFP3: 5'-TTTCTTTCTTAATGGGCTTTAAT-3' (SEQ ID NO.4)

[0038] Nanjing GenScript Biotechnology Co., Ltd. synthesized the tandem sgRNA fragments in the order of sgRNA-AP1-FAD2-TCP18-AFP3, and the pKSE401-CRISPR / Cas12a vector was digested with Bsa I. The tandem sgRNA fragments were then cloned into the digested pKSE401-CRISPR / Cas12a vector using the CloneEZ recombination cloning kit (the recombination system is shown in Table 1) to obtain the cotton knockout vector, named pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 (structure is shown in Table 1). Figure 1 , wherein the sequence of the crRNA expression cassette of the Cas12a multiple gene editing system is shown in SEQ ID NO.5). After obtaining the vector pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3, it was sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing analysis to further prove that the vector construction was successful, and the vector was transformed into Agrobacterium competent LBA4404 for standby use.

[0039] Table 1 Recombination system of sgRNA fragments and vectors

[0040]

[0041] The sequence of the crRNA expression cassette of the Cas12a multiplex gene editing system is:

[0042] cgacttgccttccgcacaatacatcatttcttcttagctttttttcttcttcttcgttcatacagtttttttttgtttatcagcttacattttcttgaaccgtagctttcgttttcttctttttaactttccattcggagtttttgtatcttgtttcatagtttgtcccaggattagaatgattaggcatcgaaccttcaagaatttgattgaataaaacatcttcattcttaagatatgaagataatcttcaaaaggcccctgggaatctgaaagaagagaagcaggcccatttatatgggaaagaacaatagtatttcttatataggcccatttaagttgaaaacaatcttcaaaagtcccacatcgcttagataagaaaacgaagctgagtttatatacagctagagtcgaagtagtgattgAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATAATTTCTACTAAGTGTAGATCAAAAGAAGAGCTGGTTTATTGATAATTTCTACTAAGTGTAGATTTGATTTCACTCAGAGTGAAGGGTAATTTCTACTAAGTGTAGATTATTCCCCTTCTTCTCCGAATTATAATTTCTACTAAGTGTAGATTTTCTTTCTTAATGGGCTTTAATAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAttttgcaaaattttccagatcgatttcttcttcctctgttcttcggcgttcaatttctggggttttctcttcgttttctgtaactgaaacctaaaatttgacctaaaaaaaatctcaaataatatgattcagtggttttgtacttttcagttagttgagttttgcagttccgatgagataaaccaata(SEQ ID NO.5) cgacttgccttccgcacaatacatcatttcttcttagctttttttcttcttcttcgttcatacagtttttttttgtttatcagcttacattttcttgaaccgtagctttcgttttcttctttttaactttccattcggagtttttgtatcttgtttcatagtttgtcccaggattagaatgattaggcatcgaaccttcaagaatttgattgaataaaacatcttcattcttaagatatgaagataatcttcaaaaggcccctgggaatctgaaagaagagaagcaggcccatttatatgggaaagaacaatagtatttcttatataggcccatttaagttgaaaacaatcttcaaaagtcccacatcgcttagataagaaaacgaagctgagtttatatacagctagagtcgaagtagtgattgAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATAATTTCTACTAAGTGTAGATCAAAAGAAGAGCTGGTTTATTGATAATTTCTACTAAGTGTAGATTTGATTTCACTCAGAGTGAAGGGTAATTTCTACTAAGTGTAGATTATTCCCCTTCTTCTCCGAATTATAATTTCTACTAAGTGTAGATTTTCTTTCTTAATGGGCTTTAATAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAttttgcaaaattttccagatcgatttcttcttcctctgttcttcggcgttcaatttctggggttttctcttcgttttctgtaactgaaacctaaaatttgacctaaaaaaaatctcaaataatatgattcagtggttttgtacttttcagttagttgagttttgcagttccgatgagataaaccaata(SEQ ID NO.5)

[0043] Example 2: Agrobacterium-mediated Genetic Transformation of Cotton

[0044] Cotton seeds (variety ZM24, No. 24 of Zhongmian Institute) were delinted with sulfuric acid, and then soaked in 70% (v / v) ethanol solution for 1 min, and then soaked in 0.1% mercuric chloride solution for 15 min to complete disinfection, and then washed 5 times with sterile water. The disinfected seeds were placed in a sterile plate covered with double-layer filter paper, and an appropriate amount of sterile water was added, and germinated in a 30°C plant incubator. After 24 hours, the seed shell and two cotyledons were peeled off to expose the apical meristem (explant). Pick a monoclonal Agrobacterium containing pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3, inoculate it into YEP liquid medium with 50 mg / L rifampicin and 50 mg / L kanamycin, and culture overnight at 28°C and 180r / min shaking. Take 1 mL of the above Agrobacterium culture solution and inoculate it into a new YEP liquid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin, and culture it at 28°C and 180 rpm until the OD 600 =0.6-0.8, centrifuged at 4000r / min for 5min, collected the bacteria and resuspended them in M1 resuspension for later use. Before infection, add 20mg / mL butyryl syringone (AS) solution with a final concentration of 0.1% (v / v) and surfactant Silwet-L77 with a final concentration of 0.2% (v / v) to the Agrobacterium resuspension to form the infection medium. Then, the explants were immersed in the infection medium, and ultrasonic and vacuum treatments were used to promote the integration of exogenous genes into the stem cells of the meristem (ultrasound time 40s, ultrasonic frequency 40KHz, infection time 50min); after infection, they were cultured in a 35°C co-culture medium for 3d, and then transferred to a recovery culture medium for 15d; then, positive buds were induced in the screening culture medium, and bud elongation was induced in the bud elongation culture medium; finally, the positive seedlings were induced to take root and transplanted into pots to obtain T0 generation regenerated plants.

[0045] In the present invention, the formula of the YEP liquid culture medium is: 10g beef extract, 10g yeast extract, 10g NaCl, water is added to make the volume to 1L, the pH value is adjusted to 7.0, and the medium is sterilized at 121°C for 20min.

[0046] The components of M1 resuspension are: 10ml / L CA mother solution, 30g / L glucose, 4.2g / L MES (morpholineethanesulfonic acid) (product of Sigma, item number V900336), 0.1ml / L B5 vitamin (product of PhytoTech, item number G219), 1mg / L 6-BA (6-benzyladenine), 0.1mg / L NAA (naphthaleneacetic acid), pH value 5.4.

[0047] The components of CA mother liquor are: 10 g / L magnesium sulfate, 5.36 g / L ammonium sulfate, 6 g / L sodium phosphate monohydrate, 6 g / L calcium chloride, 30 mg / L boric acid, 100 mg / L manganese sulfate, 20 mg / L zinc sulfate heptahydrate, 7.5 mg / L potassium iodide, 2.5 mg / L sodium molybdate dihydrate, 250 μg / L copper sulfate, 250 μg / L cobalt chloride hexahydrate, and 100 g / L potassium nitrate.

[0048] The components of the co-culture medium are: 10 ml / L CA mother solution, 30 g / L glucose, 4.2 g / L MES, 0.1 ml / L B5 vitamin (G219), 1 mg / L 6-BA, 0.1 mg / L NAA, 0.2 mM acetosyringone, 200 mg / mL cysteine ​​(CYS), pH 5.4.

[0049] The components of the recovery medium (R0) are: 4.4 g / L MS salt and B5 vitamin mixture (model M404), 20 g / L glucose, 1.29 g / L calcium gluconate, 4 g / L agar, 1 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L carbenicillin, 100 mg / L cephalosporin, pH 5.6.

[0050] The components of the screening medium are: 4.4 g / L MS salt and B5 vitamin mixture (model M404), 20 g / L glucose, 1.29 g / L calcium gluconate, 4 g / L agar, 1 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L carbenicillin, 100 mg / L cephalosporin, 100 mg / L spectinomycin, pH 5.6.

[0051] The components of the shoot elongation medium are: 4.4 g / L MS salt and B5 vitamin mixture (model M404), 20 g / L glucose, 1.29 g / L calcium gluconate, 4 g / L agar, 100 mg / L carbenicillin, 100 mg / L cephalosporin, 100 mg / L spectinomycin, pH 5.6.

[0052] Example 3: Genotyping of CRISPR / Cas12a gene editing

[0053] The leaves of the T0 generation regenerated plants transformed with pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 were taken, and the leaf genomic DNA was extracted by the CTAB method. As a template, PCR amplification was performed using Cas12a-F1 / R1 primers (primer concentration was 5 μM). The PCR reaction system is shown in Table 2, and the PCR reaction procedure is shown in Table 3. The PCR amplification product was 351 bp, and it was judged to be a positive plant. Figure 2 The electrophoresis diagram of PCR amplification products of some T0 generation regenerated plants.

[0054] The detection primers are as follows:

[0055] Cas12a-F1: 5'-GCTGTTGGTGGAAGATGAA-3' (SEQ ID NO.6)

[0056] Cas12a-R1: 5'-GTGAATGCTGTTGTGAATCC-3' (SEQ ID NO.7)

[0057] Table 2 PCR reaction system

[0058]

[0059] Table 3 PCR reaction program

[0060]

[0061] Note: The 54°C extension time is set according to the length of the amplified gene fragment. Generally, 1000 bp is amplified in 1 minute, and the calculation is based on the proportion.

[0062] Then, specific primers of AP1, FAD2, TCP18 and AFP3 were used for PCR amplification and sent to Zhejiang Shangya Company for sequencing. The detection primers are as follows:

[0063] Editing detection-AP1-F: 5'-CACATAGACACACACACAAAC-3' (SEQ ID NO. 8)

[0064] Editing detection-AP1-R: 5'-TTTACCAGGAATCAGTGGA-3' (SEQ ID NO.9)

[0065] Editing detection-FAD2-F: 5'-ATGTCGGTTCCAACGAGT-3' (SEQ ID NO. 10)

[0066] Editing detection-FAD2-R: 5'-GGTGTTAGAATGGTGACGC-3' (SEQ ID NO.11)

[0067] Editing detection-TCP18-F: 5'-TCCTTCAAACAGCAATGG-3' (SEQ ID NO.12)

[0068] Editing detection-TCP18-R: 5'-GACGACAATCAGGCAAGT-3' (SEQ ID NO.13)

[0069] Editing detection-AFP3-F: 5'-ACAGTGGGATTATGAGGTTA-3' (SEQ ID NO. 14)

[0070] Editing detection-AFP3-R: 5'-TTCGCTTCTTGTCTCCTT-3' (SEQ ID NO. 15)

[0071] Statistical analysis of amplification and sequencing results revealed (Table 4):

[0072] In the event of pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3, at most only three sites were successfully edited at the same time. The editing efficiency of site AP1 was 62%, the editing efficiency of site FAD2 was 54%, and the editing efficiency of site TCP18 was 8%. The efficiency of simultaneous editing of two sites was 46%, and the efficiency of simultaneous editing of three sites was 8%.

[0073] Table 4 Statistics of sequencing results

[0074]

[0075] Note: √ indicates successful editing, × indicates unedited, and — indicates negative seedlings.

[0076] The present invention uses the CRISPR / Cas12a system to design experiments, and connects four genes in series through DR. The vector structure is: pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3. By analyzing the editing of positive plants, it is found that up to three genes are edited at the same time, creating new germplasm with early maturity, high oleic acid and improved plant type. The present invention provides a theoretical basis for using Cas12a to simultaneously target multiple negative regulatory genes to create excellent materials with improved multiple traits at the same time.

Claims

1. A cotton multi-gene editing vector pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3, characterized in that: The vector pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 contains the coding sequence shown in SEQ ID NO.1-4.

2. A cotton multi-gene editing vector pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3, characterized in that: The vector pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 contains the sequence shown in SEQ ID NO.

5.

3. A method for constructing the vector pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 as claimed in claim 1, characterized in that: AP1, FAD2, TCP18 and AFP3 were selected as target genes, and one sgRNA was designed on the exons of each of these genes; tandem sgRNA fragments were synthesized and subcloned into the pKSE401-CRISPR / Cas12a vector to obtain a cotton knockout vector named pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3.

4. A method for creating new cotton germplasm using the vector pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 according to claim 1, characterized in that: The vector pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 was transformed into Agrobacterium; and cotton genetic transformation was carried out using the Agrobacterium-mediated method.

5. An application of the vector pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 as claimed in claim 1 in cotton multi-genome editing.

6. An application of the vector pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 as claimed in claim 1 for simultaneously targeting multiple negative regulatory genes in the cotton genome.

7. An application of the vector pKSE401-CRISPR / Cas12a-AP1-FAD2-TCP18-AFP3 as claimed in claim 1 for simultaneously improving multiple traits in cotton genetic transformation.

8. An application of CRISPR / Cas12a in cotton multi-gene editing.

9. An application of CRISPR / Cas12a to simultaneously target multiple negative regulatory genes in the cotton genome.

10. An application of CRISPR / Cas12a in cotton genetic transformation to simultaneously improve multiple traits.

Images