title: Next-generation libraries for robust RNA interference-based genome-wide screens.
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.
authors.affiliation: Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA 94158; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158; Martin.Kampmann@ucsf.edu jonathan.weissman@ucsf.edu.
authors.forename: Martin
authors.initials: M
authors.lastname: Kampmann
authors.affiliation: Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA 94158; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158;
authors.forename: Max A
authors.initials: MA
authors.lastname: Horlbeck
authors.affiliation: Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA 94158; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158;
authors.forename: Yuwen
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA 94158; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158;
authors.forename: Jordan C
authors.initials: JC
authors.lastname: Tsai
authors.affiliation: Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA 94158; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158;
authors.forename: Michael C
authors.initials: MC
authors.lastname: Bassik
authors.affiliation: Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA 94158; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158;
authors.forename: Luke A
authors.initials: LA
authors.lastname: Gilbert
authors.affiliation: Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA 94158; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158;
authors.forename: Jacqueline E
authors.initials: JE
authors.lastname: Villalta
authors.affiliation: Center for RNA Research, Institute for Basic Science, Seoul 151-742, South Korea; School of Biological Sciences, Seoul National University, Seoul 151-742, South Korea.
authors.forename: S Chul
authors.initials: SC
authors.lastname: Kwon
authors.affiliation: Center for RNA Research, Institute for Basic Science, Seoul 151-742, South Korea; School of Biological Sciences, Seoul National University, Seoul 151-742, South Korea.
authors.forename: Hyeshik
authors.initials: H
authors.lastname: Chang
authors.affiliation: Center for RNA Research, Institute for Basic Science, Seoul 151-742, South Korea; School of Biological Sciences, Seoul National University, Seoul 151-742, South Korea.
authors.forename: V Narry
authors.initials: VN
authors.lastname: Kim
authors.affiliation: Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA 94158; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158; Martin.Kampmann@ucsf.edu jonathan.weissman@ucsf.edu.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Weissman
mesh_terms: D000818:Animals; D001185:Artificial Intelligence; D016678:Genome; D006801:Humans; D051379:Mice; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D034741:RNA, Small Interfering
keywords: functional genomics; genetic screen; microRNA; pooled screen; shRNA