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CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection

Title
CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection
Author
배상수
Keywords
CRISPR; Cas; host-pathogen interaction; poliovirus; RNA virus; sialic acid; CRISPR screen; Enterovirus D68; whole-genome sequencing
Issue Date
2017-06
Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Citation
JOURNAL OF BIOLOGICAL CHEMISTRY, v. 292, no. 25, page. 10664-10671
Abstract
Several groups have used genome-wide libraries of lentiviruses encoding small guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids and target gene identification via whole-genome sequencing (WGS) confirmation of authentic mutations rather than statistical estimation through targeted amplicon sequencing. We used 30,840 pairs of individually synthesized oligonucleotides to construct the genome-scale sgRNA library, collectively targeting 10,280 human genes (i.e. three sgRNAs per gene). These plasmid libraries were co-transfected with a Cas9-expression plasmid into human cells, which were then treated with cytotoxic drugs or viruses. Only cells lacking key factors essential for cytotoxic drug metabolism or viral infection were able to survive. Genomic DNA isolated from cells that survived these challenges was subjected to WGS to directly identify CRISPR/Cas9-mediated causal mutations essential for cell survival. With this approach, we were able to identify known and novel genes essential for viral infection in human cells. We propose that genome-wide sgRNA screens based on plasmids coupled with WGS are powerful tools for forward genetics studies and drug target discovery.
URI
http://www.jbc.org/content/292/25/10664https://repository.hanyang.ac.kr/handle/20.500.11754/114389
ISSN
0021-9258; 1083-351X
DOI
10.1074/jbc.M117.782425
Appears in Collections:
COLLEGE OF NATURAL SCIENCES[S](자연과학대학) > CHEMISTRY(화학과) > Articles
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