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Next-generation libraries for robust RNA interference-based genome-wide screens
Cited 70 time in
Web of Science
Cited 73 time in Scopus
- Authors
- Issue Date
- 2015-06
- Publisher
- National Academy of Sciences
- Citation
- Proceedings of the National Academy of Sciences of the United States of America, Vol.112 No.26, pp.E3384-E3391
- 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
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