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The art and design of genetic screens: RNA interference

Key Points

  • Examining the phenotypic consequences of gene mutations — the basis of genetic screening — provides unparalleled information on gene function.

  • RNAi is a simple and rapid method to harness the power of loss-of-function genetics — applying RNAi in a high-throughput genome-wide context is revolutionizing genetic screening.

  • Key to the success of an RNAi screen is a specific and robust biological assay.

  • A major advantage of an RNAi screen is that the identity of every screened gene is known, avoiding the need for post-screen cloning and allowing more sophisticated bioinformatic analyses.

  • RNAi screens have drawbacks of potential nonspecificity of reagents and variability of the level of knockdown.

  • Future development of databases and analysis methods are needed and will improve RNAi screens.

Abstract

The remarkable gene knockdown technique of RNAi has opened exciting new avenues for genetic screens in model organisms and human cells. Here we describe the current state of the art for RNAi screening, and stress the importance of well-designed assays and of analytical approaches for large-scale screening experiments, from high-throughput screens using simplified homogenous assays to microscopy and whole-animal experiments. Like classical genetic screens in the past, the success of large-scale RNAi surveys depends on a careful development of phenotypic assays and their interpretation in a relevant biological context.

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Figure 1: Approaches for genome-wide RNAi screens in different organisms.
Figure 2: Examples of RNAi phenotypes.
Figure 3: Analysis and validation approaches.

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Acknowledgements

We thank anonymous reviewers for comments. We are grateful to M. Zerial, E. Sali and H. Hutter for images and sharing unpublished data. J. A. is funded by a Wellcome Trust Senior Research Fellowship (054523). Work in the laboratory of M. B. is supported by grants from the European Commission, the Helmholz Association and the German Research Council.

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FURTHER INFORMATION

The Ahringer laboratory

The Boutros laboratory

CellHTS software package

CellProfiler

Drosophila RNAi Screening Center

EBImage

Elegenics

E-RNAi

FLIGHT

Flybase

Genome RNAi

MIARE (minimum information for an RNAi experiment)

National Institutes of Genetics Fly Stocks (NIG-FLY)

RNAiDB (RNAi database)

RNAi Codex

Vienna RNAi Screening Center

Wormbase

Glossary

Forward genetics

The selection of mutants displaying a phenotype after random mutagenesis. The mutated gene is identified by positional cloning or by a candidate-gene approach.

Reverse genetics

Genetic analysis that begins with a gene sequence followed by targeted inactivation. The function of the gene is inferred from the resulting phenotype.

Synthetic phenotype

The phenotype that is seen only when two gene products are simultaneously inhibited, for example, in double mutants or using RNAi or drugs.

Spermatheca

An organ of the hermaphrodite reproductive tract, which receives and stores semen.

High-content assay

The phenotypic scoring of multiple detailed characters that are usually spatially or temporally resolved.

Patch-clamp assay

A technique for recording changes in electrical potentials of individual neurons. It is used as a way of recording neuronal activity.

P element

Transposable element used in Drosophila for transformation and mutagenesis.

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Boutros, M., Ahringer, J. The art and design of genetic screens: RNA interference. Nat Rev Genet 9, 554–566 (2008). https://doi.org/10.1038/nrg2364

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