A CRISPR/Cas9-enriched sequencing approach for large-scale screening of cancer genes

Abstract

The invention relates to a CRISPR / Cas9 enrichment sequencing method applied in large-scale screening of cancer genes. Firstly, an sgRNA library is established; then the sgRNA library is packaged by lentiviruses, and viruses are collected; the sgRNA library is screened in a cancer cell line, the obtained cells are extracted and screened, and genome DNAs of precancerous cells are screened; finally, enrichment of sgRNAs in the genome DNAs is carried out. When the provided method is compared with the prior art, the screening process of CRISPR / Cas9 cells is improved, the virus infection efficiency is determined and the virus MOI value is determined through a simple method and by utilization of puromycin resistance of infected cells, importantly, the restriction enzyme cutting method and a high flux method are combined, correct chromatin fragments can be obtained effectively, false positive caused by non-specific PCR amplification can be lowered, a DNA template of sgRNAs can be amplified efficiently, and a library establishment efficiency is raised.

Description

technical field [0001] The invention belongs to the technical field of high-throughput sequencing, and in particular relates to a CRISPR / Cas9 enrichment sequencing method applied to large-scale screening of cancer genes. Background technique [0002] Both oncogenes and tumor suppressor genes are potential targets of cancer gene therapy, so their identification has great application prospects in cancer treatment. Currently, drugs for the treatment of cancer have been developed targeting oncogenes EGFR, Alk and other genes. However, the current effective cancer gene-targeted drugs are far from meeting the needs of clinical treatment. Large-scale functional gene screening technology based on CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / clustered regularly interspaced short palindromic repeats associated protein 9), as an emerging technology in genetic engineering in recent years, can screen cancer-related genes at the genome-wide level, thereby providi...

AI Technical Summary

Problems solved by technology

[0003] At present, CRISPR / Cas9screen technology is only applied in a few laboratories at home and abroad, and has not been promoted

Moreover, this method requires high-throughput sequencing during use, because the template of sgRNA only accounts for a small part of genomic DNA. In order to achieve sufficient sgRNA coverage, a large amount of DNA needs to be built. Therefore, the existing library The method is time-consuming and laborious, and requires a large amount of PCR enzymes, and the economic burden is relatively large