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

TitleNext-generation libraries for robust RNA interference-based genome-wide screens
Authors
KeywordsFunctional genomics
shRNA
microRNA
Genetic screen
Pooled screen
Issue Date2015
Citation
Proceedings of the National Academy of Sciences of the United States of America, 2015, v. 112, n. 26, p. E3384-E3391 How to Cite?
Abstract© 2015, National Academy of Sciences. All rights reserved. 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.
Persistent Identifierhttp://hdl.handle.net/10722/285885
ISSN
2023 Impact Factor: 9.4
2023 SCImago Journal Rankings: 3.737
PubMed Central ID
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorKampmann, Martin-
dc.contributor.authorHorlbeck, Max A.-
dc.contributor.authorChen, Yuwen-
dc.contributor.authorTsai, Jordan C.-
dc.contributor.authorBassik, Michael C.-
dc.contributor.authorGilbert, Luke A.-
dc.contributor.authorVillalta, Jacqueline E.-
dc.contributor.authorKwon, S. Chul-
dc.contributor.authorChang, Hyeshik-
dc.contributor.authorKim, V. Narry-
dc.contributor.authorWeissman, Jonathan S.-
dc.date.accessioned2020-08-18T04:56:54Z-
dc.date.available2020-08-18T04:56:54Z-
dc.date.issued2015-
dc.identifier.citationProceedings of the National Academy of Sciences of the United States of America, 2015, v. 112, n. 26, p. E3384-E3391-
dc.identifier.issn0027-8424-
dc.identifier.urihttp://hdl.handle.net/10722/285885-
dc.description.abstract© 2015, National Academy of Sciences. All rights reserved. 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.-
dc.languageeng-
dc.relation.ispartofProceedings of the National Academy of Sciences of the United States of America-
dc.subjectFunctional genomics-
dc.subjectshRNA-
dc.subjectmicroRNA-
dc.subjectGenetic screen-
dc.subjectPooled screen-
dc.titleNext-generation libraries for robust RNA interference-based genome-wide screens-
dc.typeArticle-
dc.description.naturelink_to_OA_fulltext-
dc.identifier.doi10.1073/pnas.1508821112-
dc.identifier.pmid26080438-
dc.identifier.pmcidPMC4491794-
dc.identifier.scopuseid_2-s2.0-84937856311-
dc.identifier.volume112-
dc.identifier.issue26-
dc.identifier.spageE3384-
dc.identifier.epageE3391-
dc.identifier.eissn1091-6490-
dc.identifier.isiWOS:000357079400011-
dc.identifier.issnl0027-8424-

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