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Next-generation libraries for robust RNA interference-based genome-wide screens

Cited 57 time in Web of Science Cited 58 time in Scopus
Authors
Kampmann, Martin; Horlbeck, Max A.; Chen, Yuwen; Tsai, Jordan C.; Bassik, Michael C.; Gilbert, Luke A.; Villalta, Jacqueline E.; Kwon, S. Chul; Chang, Hyeshik; Kim, V. Narry; Weissman, Jonathan S.
Issue Date
2015-06
Citation
Proceedings of the National Academy of Sciences of the United States of America, Vol.112 No.26, pp.E3384-E3391
Keywords
functional genomicsshRNAgenetic screenpooled screenmicroRNA
Abstract
Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity.
ISSN
0027-8424
URI
https://hdl.handle.net/10371/171958
DOI
https://doi.org/10.1073/pnas.1508821112
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College of Natural Sciences (자연과학대학)Dept. of Biological Sciences (생명과학부)Journal Papers (저널논문_생명과학부)
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