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

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...

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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 applying this system

[0005] Existing screening methods for sgRNA-targeted DNA sequences de novo synthesize sgRNA sequences or construct stable transfection vectors, and then carry out transgenic operations. The time period is long, the operation is cumbersome, and the number of detection sites is limited.

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