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J Biol Chem. 2017 Jun 23;292(25):10664-10671. doi: 10.1074/jbc.M117.782425. Epub 2017 Apr 26.

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

Author information

1
From the Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea.
2
the Department of Chemistry, Seoul National University, Seoul 151-747, South Korea.
3
the Center for Convergent Research of Emerging Virus Infection and.
4
the Department of Chemistry, Hanyang University, Seoul 04763, South Korea, and.
5
Virus Research and Testing Center, Korea Research Institute of Chemical Technology, Daejeon 34114, South Korea.
6
ToolGen, Inc., Byucksan Kyoungin Digital Valley 2-Cha, Geumcheon-Gu, Seoul 153-023, South Korea.
7
the Center for Convergent Research of Emerging Virus Infection and chonskim@krict.re.kr.
8
From the Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea, jskim01@snu.ac.kr.

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.

KEYWORDS:

CRISPR screen; CRISPR/Cas; Enterovirus D68; RNA virus; host-pathogen interaction; poliovirus; sialic acid; whole-genome sequencing

PMID:
28446605
PMCID:
PMC5481571
DOI:
10.1074/jbc.M117.782425
[Indexed for MEDLINE]
Free PMC Article

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