Format

Send to

Choose Destination
Proc Natl Acad Sci U S A. 2015 Jun 30;112(26):E3384-91. doi: 10.1073/pnas.1508821112. Epub 2015 Jun 15.

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

Author information

1
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.
2
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;
3
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.

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.

KEYWORDS:

functional genomics; genetic screen; microRNA; pooled screen; shRNA

PMID:
26080438
PMCID:
PMC4491794
DOI:
10.1073/pnas.1508821112
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center