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- 저자 : Kampmann, Martin (검색결과 1 건)
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Next-generation libraries for robust RNA interference-based genome-wide screens
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. National Academy of Sciences 2015 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.112 No.26
<P><B>Significance</B></P><P>Genetic screening is a classic approach to identify genes acting in a biological process of interest. In mammalian cells, screens are commonly based on RNA interference (RNAi), in which a short interfering RNA (siRNA) or short-hairpin RNA (shRNA) triggers degradation of cellular messenger RNAs. RNAi approaches are prone to false-positive results because of siRNA/shRNA off-target effects and false-negative results because of siRNAs/shRNAs lacking activity. We previously established that these problems can be minimized with ultracomplex shRNA libraries. Here, we present next-generation shRNA libraries targeting the human and mouse genomes, for which we improved several features to increase shRNA activity. In a pilot screen, the new library yields complementary results to clustered regularly interspaced short palindromic repeats interference (CRISPRi), an orthogonal approach we developed recently.</P><P>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.</P>
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