CRISPR screening of porcine sgRNA library identifies host factors associated with Japanese encephalitis virus replication

We design a porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library containing 85,674 single guide RNAs targeting 17,743 protein-coding genes, 11,053 long ncRNAs, and 551 microRNAs. Subsequently, we use the PigGeCKO library to identify key host factors facilitating JEV infection in porcine cells. Overall design: We initially used CRISPR-offinder (v1.2, BiooTools.com) to design 85,674 specific and predicted high-efficiency single guide RNAs (sgRNAs), that collectively targeted 17,743 protein-coding genes, 11,053 long ncRNAs (lncRNAs), and 551 microRNAs (miRNAs) in the porcine genome, as well as 1,000 negative control sgRNA constructs predicted not to target any porcine genome loci. These designed sgRNA constructs were synthesized as an oligo array, which was employed as the template for PCR amplification of the sgRNA oligos that were subsequently cloned into lentiviral vectors using Gibson assembly. We then developed a screening strategy to identify host genes required for successful JEV infection.