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RNAi screening comes of age: improved techniques and complementary approaches

Nature Reviews Molecular Cell Biology volume 15, pages 591–600 (2014)Cite this article

Subjects

Key Points

  • RNAi has been used for genome-wide screening and other studies that aim to uncover the function of genes and gene networks.

  • Sequence-specific RNAi off-target effects (OTEs) must be taken into consideration when interpreting RNAi data. New experimental and computational strategies such as the detection of microRNA-like seed sequence matches and the use of C911 RNAi controls help to address OTEs and improve data quality.

  • Innovations in RNAi screening include new applications for high-content imaging, screens for synthetic interactions using sensitized cell backgrounds, screening in three-dimensional tissue cultures, parallel screening in different species or using different approaches followed by result integration, and new strategies for in vivo RNAi screening.

  • RNAi and the genome-editing CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 system are complementary technologies, and using these two techniques together should result in improved assay development, screening and validation of screen results.

  • With careful attention to reagent and assay design, data analysis and experimental follow-ups, improved genome-wide RNAi screens are uncovering gene function in all areas of biology.

Abstract

Gene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells and in vivo in model organisms. These screens have resulted in the identification of new cellular pathways and potential drug targets. Considerable progress has been made to improve the quality of RNAi screen data through the development of new experimental and bioinformatics approaches. The recent availability of genome-editing strategies, such as the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 system, when combined with RNAi, could lead to further improvements in screen data quality and follow-up experiments, thus promoting our understanding of gene function and gene regulatory networks.

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Figure 1: Gene silencing by RNAi.
Figure 2: Strategies for validating RNAi screen results.
Figure 3: Genome-engineering approaches offer new opportunities for assay development, screening and validation.

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Acknowledgements

The Drosophila RNAi Screening Center is supported by US National Institutes of Health (NIH) NIGMS R01 GM067761 (N.P.). S.E.M. receives additional support from the Dana Farber/Harvard Cancer Center, which is supported in part by NCI Cancer Center Support Grant NIH 5 P30 CA06516. C.E.S. and J.A.S. are supported by funds from Harvard Medical School. R.A.N. is supported by a Human Frontier Science Program long-term fellowship. N.P. is an Investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Drosophila RNAi Screening Center, Harvard Medical School, Boston, MA 02115, Massachusetts, USA

    Stephanie E. Mohr & Norbert Perrimon

  2. Department of Genetics, Harvard Medical School, Boston, 02115, Massachusetts MA, USA

    Stephanie E. Mohr, Ralph A. Neumüller & Norbert Perrimon

  3. ICCB-Longwood Screening Facility, Harvard Medical School, Boston, 02115, Massachusetts MA, USA

    Jennifer A. Smith & Caroline E. Shamu

  4. Howard Hughes Medical Institute, Boston, 02115, Massachusetts MA, USA

    Norbert Perrimon

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Glossary

Genetic screens

Identification of the organisms or cells that display a mutant phenotype of interest following large-scale disruption of genes; for example, by using mutagens.

Pooled format RNAi screens

Screens in which pools of reagents are introduced together into the cell population; for mammalian cell screening this is typically carried out with the goal of integrating just one of the short hairpin RNAs into each cell. Positive reagents are identified by comparing the abundance of any given reagent in the cell population before and after selection.

Arrayed format RNAi screens

Screens in which each reagent (or a small pool of reagents directed against the same gene) is introduced separately into cells.

miRNA mimics

Short, double-stranded RNAs that, when introduced into cells, mimic endogenous microRNAs by activating post-transcriptional repression of target genes.

Seed region

Base pairs 2–8 of the fully processed siRNA. It can mediate microRNA-like effects even in the absence of perfect complementarity between the remainder of its sequence and that of the transcript.

Multiplicity of infection

(MOI). During a viral infection, the ratio of the number of infectious virions to the number of targeted cells.

Gene-trap retrovirus approach

Screening using mutagenic retroviral integration into genes. The integrated retrovirus can be used as a trap for identifying the genes disrupted in each resulting phenotype.

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Mohr, S., Smith, J., Shamu, C. et al. RNAi screening comes of age: improved techniques and complementary approaches. Nat Rev Mol Cell Biol 15, 591–600 (2014). https://doi.org/10.1038/nrm3860

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