Targeted DNA capture method mediated by CRISPR/Cas9 system

Abstract

The invention discloses a DNA targeted capture method mediated by the CRISPR / Cas9 system. A sgRNA is designed for the upstream and downstream of the target DNA region respectively, and different capture sequences and a segment of the sgRNA are added to the 3' ends of the two sgRNA sequences. The reverse complementary sequence constitutes the pegRNA. When the pegRNA-Cas9 protein complex is targeted to bind the target DNA, through pegRNA-mediated site-specific insertion, different capture sequences are inserted in the upstream and downstream of the target DNA region, and then through the capture sequence. The primers are used for PCR amplification, targeted enrichment and isolation of target DNA from the DNA library or the whole genome, which can be directly used for subsequent capture sequencing. Compared with other enrichment methods, the method provided by the invention is simple, fast, specific and sensitive, and has broad application advantages in DNA sequence analysis.

Description

technical field [0001] The invention belongs to the technical field of gene sequencing, and in particular relates to a DNA target capture method mediated by a CRISPR / Cas9 system. Background technique [0002] In recent years, the DNA sequence information of a specific region has received extensive attention. DNA sequence analysis can discover genetic and epigenetic changes in the genome, such as single nucleotide variation (SNV), copy number variation (CNV), susceptibility position and methylation. With the advancement of DNA sequencing technology, next-generation sequencing (NGS) provides a powerful tool to decode DNA information at genome scale and single-base resolution. NGS-based DNA analysis has diverse applications such as precision medicine in humans and SNP breeding in animals. [0003] CRISPR-Cas9 is an adaptive immune defense formed during the long-term evolution of bacteria and archaea, which can be used to fight against invading viruses and foreign DNA. The CR...

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Problems solved by technology

[0004] At present, there are many targeted sequencing methods for the target region, but all of them have certain limitations. For example, the hybrid capture method first uses ultrasound to disrupt the genome, hybridizes through DNA-RNA, and then captures the target DNA with probes and enriches it by PCR. , the main disadvantage of hybridization-based sequencing methods is that genomic fragments that rely on sonication will generate random-sized DNA fragments, which will have a certain preference in the process of PCR enrichment, and short fragments will be preferentially amplified, resulting in uneven sequencing reads , partial sequence information loss in long fragments, etc.; there are also capture methods based on the CRISPR-Cas9 system, such as the CRISPR-DS technology developed by Nachmanson D, which can achieve high-sensitivity detection, but its limitation is that after the capture is completed, the Need to add A tail and ligation reaction, and then perform PCR enrichment, the experimental process is complicated. In addition, the success rate of A tail and ligation steps may affect the success rate of capture

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