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09월 03일 23시 이후 : 초록수정 불가능, 일정확인 및 검색만 가능
제122회 대한화학회 학술발표회, 총회 및 기기전시회 안내
CRISPR screening identifies host factors essential for viral infection
2018년 9월 2일 14시 19분 10초
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금 09시 : 25분
Life Chemistry -
Recent Trends in Genome Editing Technique
Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Korea
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 1,514 genes, encoding kinases, proteins related to endocytosis, and Golgi-localized proteins, individually using 4,542 sgRNAs (3 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.
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