Functional Identification of Optimized RNAi Triggers Using a Massively Parallel Sensor Assay

Fellmann, C., Zuber, J., McJunkin, K., Chang, K. , Malone, C. D., Dickins, R. A., Xu, Q., Hengartner, M. O., Elledge, S. J., Hannon, G. J., Lowe, S. W. (March 2011) Functional Identification of Optimized RNAi Triggers Using a Massively Parallel Sensor Assay. Molecular Cell, 41 (6). pp. 733-746. ISSN 1097-2765

URL: http://www.ncbi.nlm.nih.gov/pubmed/21353615
DOI: 10.1016/j.molcel.2011.02.008

Abstract

Summary Short hairpin RNAs (shRNAs) provide powerful experimental tools by enabling stable and regulated gene silencing through programming of endogenous microRNA pathways. Since requirements for efficient shRNA biogenesis and target suppression are largely unknown, many predicted shRNAs fail to efficiently suppress their target. To overcome this barrier, we developed a "Sensor assay" that enables the biological identification of effective shRNAs at large scale. By constructing and evaluating 20,000 RNAi reporters covering every possible target site in nine mammalian transcripts, we show that our assay reliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. Our unbiased analyses reveal that potent shRNAs share various predicted and previously unknown features associated with specific microRNA processing steps, and suggest a model for competitive strand selection. Together, our study establishes a powerful tool for large-scale identification of highly potent shRNAs and provides insights into sequence requirements of effective RNAi.

Item Type: Paper
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNAi
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > miRNA
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > miRNA

organism description > animal > mammal
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > shRNA
CSHL Authors:
Communities: CSHL Cancer Center Shared Resources > Bioinformatics Service
CSHL Cancer Center Shared Resources > DNA Sequencing Service
CSHL Cancer Center Shared Resources > Flow Cytometry Service
CSHL labs > Hannon lab
CSHL labs > Hengartner lab
CSHL labs > Lowe lab
Watson School > Publications
CSHL Cancer Center Program > Cancer Genetics
Depositing User: CSHL Librarian
Date: 18 March 2011
Date Deposited: 09 Nov 2011 16:32
Last Modified: 14 Oct 2015 15:58
PMCID: PMC3130540
Related URLs:
URI: http://repository.cshl.edu/id/eprint/15630

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