Preparation method of non-transgenic CRISPR mutant

A non-transgenic, mutant technology, applied in the field of preparation of non-transgenic CRISPR mutants, can solve problems such as inability to pass phenotypic judgment, inability to use antibiotic screening, inability to visually determine phenotypic changes, etc.

Inactive Publication Date: 2018-10-02
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

However, whether the Cas9 gene is located in the T-DNA region of Agrobacterium can be successfully expressed transiently in crop cells and achieve site-directed mutation of the target crop gene by the CRISPR-Cas9 protein has not been reported yet.
[0006] On the other hand, the site-directed mutation of target crop genes by CRISPR-Cas9 technology occurs randomly and automatically when the target plant repairs the DNA cut site of Cas9, which can cause different types of gene mutations in the target segment, possibly deleting a few A few nucleotides, it may also lead to the addition of a few nucleotides or the replacement of the original nucleotides, the situation of the mutant plants of different independent lines is different
Most of the gene mutations cannot be visually determined at the young seedling stage, that is, it is impossible to judge whether it is a mutant plant by phenotype
And because what is desired is a plant in which the exogenous gene has not been integrated into the genome, such plants are usually not resistant to antibiotics and cannot be screened with antibiotics, resulting in the obtained regenerated shoots containing both mutant plants and plants without mutations. wild type plants

Method used

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  • Preparation method of non-transgenic CRISPR mutant
  • Preparation method of non-transgenic CRISPR mutant
  • Preparation method of non-transgenic CRISPR mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1. Construction of Cas9 mutation carrier

[0061] 1. Carrier sequence with 15 bases at the 5' end of the 5'-SpeI restriction site-SpeI restriction site-Cas9 gene sequence containing 1 intron-ApaI restriction site-ApaI restriction site Point the 15-base carrier sequence at the 5' end-the sequence at the 3' end, and directly synthesize a Cas9 gene (SEQ ID NO.1) containing a complete intron. Using restriction endonucleases SpeI and ApaI to double digest and linearize the CRISPR base vector pK7WGF2::hCas9 (Addgene plasmid #46965) at 37°C overnight, use the Infusion method to connect the Cas9 gene containing introns into the vector to obtain Intronic Cas9 vector. 20 μl common PCR amplification and sequencing to verify whether the vector connection is correct. The PCR reaction system is shown in Table 1. The PCR reaction conditions are: 94°C for 30s, 55°C for 30s, 72°C for 1min, repeating 30 cycles.

[0062] 2. According to the sequence of the fourth exon of th...

Embodiment 2

[0068] Example 2. Transformation of Agrobacterium

[0069] The PDS-Cas9 mutant vector was transformed into Agrobacterium EHA105 competent cells by freeze-thaw method. The specific steps are as follows: Add 0.1-1 μg (5-10 μl) of plasmid DNA to 50 μl of Agrobacterium competent cells, and ice-bath for 30 minutes; put it in liquid nitrogen for 5 minutes (or 1 minute), and then immediately put it in a 37°C water bath for 5 minutes ;Take out the centrifuge tube, put it on ice for 2min immediately, then add 900μl of LB without any antibiotics, shake and culture at 28℃, 220rpm for 3-5h; take out the bacterial liquid, centrifuge at 3000 rpm for 3min, then remove 850μl of supernatant , leaving 100 μl of the supernatant to aspirate the suspended bacterial pellet with a gun, and then evenly spread it on an LB plate containing 100 mg / L spectinomycin. Colonies were visible in about 2 days. Pick a single colony, inoculate it in 5 ml of LB liquid medium containing 100 mg / L spectinomycin, an...

Embodiment 3

[0070] Example 3. Transient Infestation of Tobacco

[0071] 1. Agrobacterium preparation: Take the Agrobacterium preserved at -80°C in Example 2, streak it on an LB plate containing 100 mg / L spectinomycin, and culture it at 28°C for 2 days. Pick a single colony and inoculate it in 5ml LB liquid medium containing the same concentration of antibiotics, and shake at 200rpm at 28°C for 16h. Take 5ml of the bacterial solution and add it to a new 50ml LB medium, shake and culture at 200rpm 28°C for about 6-8h to make the OD600 value 0.6-0.8. Centrifuge at 5000rpm for 15min at 4°C, and use 50ml of resuspension (MS+ 100 μM AS) at 180 rpm at 28° C. for 1 hour before use.

[0072] 2. Agrobacterium infection and co-cultivation: Take the healthy leaves of the aseptic tobacco seedlings and cut them into about 0.5cm 2 size, immersed in the ready-to-use Agrobacterium suspension for 20 minutes. After the end, the excess suspension was sucked off with filter paper, transferred to MS solid m...

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Abstract

The invention provides a preparation method of a non-transgenic mutant based on a CRISPR-Cas9 technology. Specifically, the preparation method comprises a construction method and a screening method. The construction method is characterized in that CRISPR-Cas9 and sgRNA are preassembled and are located at T-DNA of agrobacterium tumefaciens; site-directed change of a target gene of a target plant can be realized by infection of the target plant by the agrobacterium tumefaciens and coculture without integrating the sequences of the CRISPR-Cas9 and the sgRNA into a genome of the target plant, so that an obtained site-directed mutant material of the target gene does not need the processes of sexual reproduction, segregation posterity, population screening and the like or does not contain any exogenous gene sequence. The obtained regeneration seedlings are screened by a high-throughput sequencing and high-resolution melting curve technology provided by the invention; mutant plants of which target genes are mutated can be obtained by identifying the regeneration seedlings efficiently and quickly at the current generation of transgene, even if low-proportion mutants of which the proportionis 1 / 100 of a mixed population can be screened. The screening method provided by the invention still can ensure excellent accuracy and sensitivity.

Description

technical field [0001] The invention belongs to the technical field of modern molecular breeding, and relates to the construction and screening of crop mutants using gene editing technology without introducing foreign genes, specifically a method for preparing non-transgenic CRISPR mutants. Background technique [0002] Transgenic technology provides a powerful tool for crop improvement. However, the public has concerns about food safety and gene drift that may be caused by exogenous genes in GM crops and their expression, which restricts the application of GM technology. In recent years, genome editing mediated by CRISPR-Cas9 technology has developed rapidly. Its precision and efficiency make site-directed mutagenesis based on it have great application potential in crop improvement, and it is even expected to replace traditional transgenic technology. [0003] Currently the most widely used CRISPR-Cas9 technology in plants relies on the stable integration of Cas9 endonucle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00
CPCC12N15/8205C12N2800/80C12N2810/10
Inventor 丁静陈龙正李威洛伦佐·卡廷-格拉齐尼李义
Owner NANJING AGRICULTURAL UNIVERSITY
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