Method for capturing genome target sequence based on Crispr/cas9 and application thereof in high-throughput sequencing

Abstract

The invention discloses a method for capturing a genome target sequence based on Crispr / cas9 and application thereof in high-throughput sequencing. The method for capturing a target gene sequence from a genome DNA on the basis of a Crispr / cas9 system comprises the steps that 1, a plurality of gRNAs are designed and synthesized according to the target gene sequence; 2, the multiple gRNAs and a cas9 enzyme are adopted for performing a cleavage reaction on the genome DNA, and a cleavage product containing multiple 100-300 bp fragmentation products is obtained; 3, the multiple 100-300 bp fragmentation products in the cleavage product are captured. On the basis of the CRISPR gene, the target sequence is edited and captured, and the method can be used for high-throughput sequencing and is applicable to screening of specific population with family heredity, the public physical examination market and the like.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a method for capturing genome target sequences based on Crispr / cas9 and its application in high-throughput sequencing. Background technique [0002] CRlSPR / Cas (Clusteredregularly interspaccd short palindromic repeats / CRlSPR-associatednuclease) technology is a new DNA targeted editing tool developed in 2013, and was named one of the top ten scientific breakthrough technologies in 2013 by the American Science Journal. Cas9 nuclease, S.pyogenes, is an RNA-mediated nuclease that can catalyze the cleavage of double-stranded DNA at specific sites. The cutting site is located at 3 bases of the upstream sequence of NGG PAM (Protospacer Adjacent Motif). The NGG of the PAM sequence must be connected behind the target and located on the strand of DNA complementary to the gRNA. Its DNA-specific recognition is achieved by a short hairpin RNA structure, while DNA cutting is performed by C...

AI Technical Summary

Problems solved by technology

[0004] Based on liquid-phase capture high-throughput sequencing technology, the market mainly includes Agilent's whole-exon capture kit, Roche's solid-phase hybridization method and liquid-phase hybridization method. Although the DNA sequences captured by these products are only the whole genome Compared with whole genome sequencing, it has great advantages in terms of cost and time spent in some areas, but the price of each sample is still more than several thousand yuan, which is beyond the affordability of ordinary people and is not suitable for large-scale screening. Detection of monogenic genetic diseases

Moreover, the disadvantage of these technologies is that a large number of long fragments (150-200bp) of DNA need to be synthesized, amplified by PCR, and transcribed into biotin-modified RNA.

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