Arrayed CRISPR screen with image-based assay reliably uncovers host genes required for coxsackievirus infection

Cited 0 time in webofscience Cited 0 time in scopus
418 Viewed 29 Downloaded
Title
Arrayed CRISPR screen with image-based assay reliably uncovers host genes required for coxsackievirus infection
Author(s)
Heon Seok Kim; Kyungjin Lee; Seong-Jun Kim; Sungchan Cho; Hye Jin Shin; Chonsaeng Kim; Jin-Soo Kim
Publication Date
2018-06
Journal
GENOME RESEARCH, v.28, no.6, pp.859 - 868
Publisher
Cold Spring Harbor Laboratory Press
Abstract
Pooled CRISPR screens based on lentiviral systems have been widely applied to identify the effect of gene knockout on cellular phenotype. Although many screens were successful, they also have the limitation that genes conferring mild phenotypes or those essential for growth can be overlooked, as every genetic perturbation is incorporated in the same population. Arrayed screens, on the other hand, incorporate a single genetic perturbation in each well and could overcome these limitations. However, arrayed screens based on siRNA-mediated knockdown were recently criticized for low reproducibility caused by incomplete inhibition of gene expression. To overcome these limitations, we developed a novel arrayed CRISPR screen based on a plasmid library expressing a single guide RNA (sgRNA) and disrupted 1514 genes, encoding kinases, proteins related to endocytosis, and Golgi-localized proteins, individually using 4542 sgRNAs (three sgRNAs per gene). This screen revealed host factors required for infection by coxsackievirus B3 (CVB3) from Picornaviridae, which includes human pathogens causing diverse diseases. Many host factors that had been overlooked in a conventional pooled screen were identified for CVB3 infection, including entry-related factors, translational initiation factors, and several replication factors with different functions, demonstrating the advantage of the arrayed screen. This screen was quite reliable and reproducible, as most genes identified in the primary screen were confirmed in secondary screens. Moreover, ACBD3, whose phenotype was not affected by siRNA-mediated knockdown, was reliably identified. We propose that arrayed CRISPR screens based on sgRNA plasmid libraries are powerful tools for arrayed genetic screening and applicable to larger-scale screens. © 2018 Kim et al
URI
https://pr.ibs.re.kr/handle/8788114/4490
ISSN
1088-9051
Appears in Collections:
Center for Genome Engineering(유전체 교정 연구단) > Journal Papers (저널논문)
Files in This Item:
1806_김헌석_Genome Res.-2018-Kim-gr.230250.117.pdfDownload

qrcode

  • facebook

    twitter

  • Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.
해당 아이템을 이메일로 공유하기 원하시면 인증을 거치시기 바랍니다.

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Browse