Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells   

Benjamin E. HousdenBenjamin E. Housden* Hilary  E. NicholsonHilary E. Nicholson*Norbert  PerrimonNorbert Perrimon  (*contributed equally to this work)
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Original research article

A brief version of this protocol appeared in:
Science Signaling
Sep 2015


A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden et al., 2015).

Keywords: Synthetic lethality, Screening, RNAi, Drosophila, S2R+ cells, Cell culture, CRISPR


Knowledge of genetic interactions such as synthetic lethality can be invaluable for determining the functional relationships between genes. For example, large scale genetic interaction screens in yeast were recently used to assemble a global ‘wiring diagram of cellular function’ (Costanzo et al., 2016). Alternatively, specific types of genetic interaction such as synthetic lethal interactions can be used to identify drug targets for diseases including cancer (Kaelin, 2005).

Identification of synthetic interactions requires combinatorial disruption of two genes. A previous method to achieve this in Drosophila cell culture was to deliver multiple dsRNA reagents simultaneously (e.g., Fisher et al., 2015). However, RNAi reagents have limitations including off-target effects and incomplete target knockdown, which are compounded when multiple reagents are delivered together. By combining CRISPR mutagenesis with single dsRNA treatments, these issues are avoided, leading to simpler interpretation of screen results and robust identification of ‘hits’.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Housden, B. E., Nicholson, H. E. and Perrimon, N. (2017). Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells. Bio-protocol 7(3): e2119. DOI: 10.21769/BioProtoc.2119.

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