Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 112, Issue 26, Pages E3384-E3391Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1508821112
Keywords
functional genomics; shRNA; genetic screen; pooled screen; microRNA
Categories
Funding
- Howard Hughes Medical Institute
- National Cancer Institute/NIH [U01 CA168370]
- National Cancer Institute/NIH Grant Pathway [K99 CA181494]
- Institute for Basic Science from the Ministry of Science, Information and Communication Technology, and Future Planning of Korea [IBS-R008-D1]
- University of California, San Francisco Medical Scientist Training Program Grant [T32 GM007618]
- Leukemia and Lymphoma Society
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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.
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