CRISPR/Cas9 High Throughput Screening System

First,construct sgRNA library aimed at candidate genes possible related to viral pathogenesis.This library will be used to infect porcine cell lines which stably express Cas9 protein.The genomic DNA of the pooled surviving cells from the library,as well as the original library of cells before the toxin treatment,will be extracted and used for PCR amplification of the sgRNA-coding regions,before being subjected to deep-sequencing analysis.To determine whether the screening revealed novel genes involved in viral pathogenesis,we will pick several candidate genes from the top of sgRNA enrichment lists for further validation.