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dc.contributor.author배상수-
dc.date.accessioned2019-11-26T01:24:59Z-
dc.date.available2019-11-26T01:24:59Z-
dc.date.issued2017-06-
dc.identifier.citationJOURNAL OF BIOLOGICAL CHEMISTRY, v. 292, no. 25, page. 10664-10671en_US
dc.identifier.issn0021-9258-
dc.identifier.issn1083-351X-
dc.identifier.urihttp://www.jbc.org/content/292/25/10664-
dc.identifier.urihttps://repository.hanyang.ac.kr/handle/20.500.11754/114389-
dc.description.abstractSeveral 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.en_US
dc.description.sponsorshipThis work was supported by IBS Grant IBS-R021-D1 (to J. S. K.), Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT, and Future Planning Grant 2016R1C1B2009585, and National Research Council of Science and Technology by the Korean government Grant CRC-16-01-KRICT (to C. K.). The authors declare that they have no conflicts of interest with the contents of this article.en_US
dc.language.isoen_USen_US
dc.publisherAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INCen_US
dc.subjectCRISPRen_US
dc.subjectCasen_US
dc.subjecthost-pathogen interactionen_US
dc.subjectpoliovirusen_US
dc.subjectRNA virusen_US
dc.subjectsialic aciden_US
dc.subjectCRISPR screenen_US
dc.subjectEnterovirus D68en_US
dc.subjectwhole-genome sequencingen_US
dc.titleCRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infectionen_US
dc.typeArticleen_US
dc.relation.no25-
dc.relation.volume292-
dc.identifier.doi10.1074/jbc.M117.782425-
dc.relation.page10664-10671-
dc.relation.journalJOURNAL OF BIOLOGICAL CHEMISTRY-
dc.contributor.googleauthorKim, Heon Seok-
dc.contributor.googleauthorLee, Kyungjin-
dc.contributor.googleauthorBae, Sangsu-
dc.contributor.googleauthorPark, Jeongbin-
dc.contributor.googleauthorLee, Chong-Kyo-
dc.contributor.googleauthorKim, Meehyein-
dc.contributor.googleauthorKim, Eunji-
dc.contributor.googleauthorKim, Minju-
dc.contributor.googleauthorKim, Seokjoong-
dc.contributor.googleauthorKim, Chonsaeng-
dc.relation.code2017002188-
dc.sector.campusS-
dc.sector.daehakCOLLEGE OF NATURAL SCIENCES[S]-
dc.sector.departmentDEPARTMENT OF CHEMISTRY-
dc.identifier.pidsangsubae-
dc.identifier.researcherIDE-5324-2017-
dc.identifier.orcidhttp://orcid.org/0000-0003-3615-8566-
Appears in Collections:
COLLEGE OF NATURAL SCIENCES[S](자연과학대학) > CHEMISTRY(화학과) > Articles
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