Method for quickly and efficiently screening SgRNA (short guide RNA) targeted DNA sequences

A DNA sequence and screening method technology, applied in the field of cell biology, can solve the problems of limited number of detection sites, long time period, cumbersome operation, etc., and achieve the effect of improving efficiency, high success rate, and strong operability

Active Publication Date: 2018-08-03
AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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  • Application Information

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Problems solved by technology

[0004] Before the application of the CRISPR-Cas9 system, the cell line should be stably overexpressed Cas9 protein, and then transferred to the sgRNA with guiding cleavage, the active site of sgRNA targeted cleavage needs to be screened. fails, becoming the most important and time-consuming step in ap

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  • Method for quickly and efficiently screening SgRNA (short guide RNA) targeted DNA sequences
  • Method for quickly and efficiently screening SgRNA (short guide RNA) targeted DNA sequences
  • Method for quickly and efficiently screening SgRNA (short guide RNA) targeted DNA sequences

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Embodiment 1

[0031] Example 1 A method for rapid and efficient screening of sgRNA targeting DNA sequences

[0032] see figure 1 , is a technical roadmap of a method for quickly and efficiently screening sgRNA targeting DNA sequences of the present invention, comprising the following steps:

[0033] 1. Obtain general template DNA-1 and 2 by PCR method

[0034] The pLVX-hU6-SgRNA vector (purchased from Addgene) was used as a template, and universal PCR amplification was performed with universal primers 1F and 1R, and universal primers 2F and 2R designed for the test detection.

[0035] The amplification primers are:

[0036] 1F: CTTTGGCGCCGGCTCGAGTGTACA (SEQ ID No: 1);

[0037] 1R: CGGTGTTTCGTCCTTTCC (SEQ ID No: 2);

[0038] 2F: GTTTTAGAGCTAGAAATAGCA (SEQ ID No: 3);

[0039] 2R: CACCGGTTAGCGCTAGCTAATGCC (SEQ ID No: 4)

[0040]The general PCR program is: pre-denaturation at 95°C for 3min; 35 cycles of 95°C for 30s, 60°C for 30s, and 72°C for 40s; final total extension at 72°C for 7min; ...

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Abstract

The invention discloses a method for quickly and efficiently screening SgRNA (short guide RNA) targeted DNA sequences. The method comprises the following steps: designing a plurality of SgRNA targeting primers based on a SgRNA targeted DNA sequence framework; then, obtaining DNA fragments containing hU6 promoter sequences and targeting SgRNA sequences by PCR (polymerase chain reaction) with an SgRNA support as a template; next, transfecting the DNA fragments into cells capable of stably expressing Cas9, and extracting genomes in 48 hours; obtaining genomic DNA fragments containing SgRNA targets by PCR amplification, and screening SgRNA target DNA sequence cleavage active sites after two-way sequencing analysis. According to the screening method, gene target screening time is greatly shortened, and the operation difficulty of a Crispr-Cas9 system is reduced; the method has the advantages of high operability, good repeatability, high success rate, simple and convenient use and routable operation, and is suitable for research and development of kits.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and more specifically, the invention relates to a method for rapidly and efficiently screening sgRNA targeting DNA sequences. Background technique [0002] In recent years, CRISPR-Cas9 gene editing technology has become a powerful tool for functional genomics research. It can achieve precise gene editing in animal and cell models to better study gene function. Compared with the traditional gene editing system, this system is easy to operate and has high knockout efficiency, and has been widely and maturely used in gene editing research. [0003] The CRISPR-Cas9 system is a new generation of gene editing technology, and its essence is a defense system in bacteria against foreign DNA such as viruses. The working principle of this system is that clustered, regularly interspaced short palindromic repeat sequence crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA) through b...

Claims

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Application Information

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IPC IPC(8): C12Q1/6811
CPCC12Q1/6811C12Q2531/113C12Q2535/122C12N15/1093C12Q2521/301
Inventor 朱翠白银山蒋宗勇陈庄
Owner AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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