Abstract
Over the past two decades, the concept of synthetic lethality (SL) that queries genetic relationships between gene pairs has gradually emerged as one of the best strategies to selectively eliminate cancer cells. Some of the most successful approaches to identify synthetic lethal interactions (SLIs) were largely dependent on pooled screening formats that require heavy validation in order to mitigate false positives. Here, we describe a high-throughput method to identify SLIs using CRISPR-based strategy that covers, high-throughput production of plasmid DNA preparations, lentiviral production, and subsequent cellular transduction using single guide RNAs (sgRNAs). This method could be adopted to query hundreds of SLIs. As an example, we describe the methods associated with building an interaction map for DNA damage and repair (DDR) genes. The use of multiwell plates and image-based quantification allows a comparative measurement of SLIs at a high-resolution on a one-by-one basis. Furthermore, this scalable, arrayed CRISPR screening method can be applied to multiple cancer cell types, and genes of interest, resulting in new functional discoveries that can be exploited therapeutically.
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Acknowledgments
We thank members of the Vizeacoumar and Freywald laboratories for their feedback on this chapter. This work is supported by funding support from College of Medicine, University of Saskatchewan to M.J.M. and F.S.V.
Author contribution: F.S.V. and M.J.M. conceived and planned the strategies discussed in the chapter. M.J.M., O.A., and F.S.V. wrote or contributed to the writing of the manuscript.
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MacAuley, M.J., Abuhussein, O., Vizeacoumar, F.S. (2021). Identification of Synthetic Lethal Interactions Using High-Throughput, Arrayed CRISPR/Cas9-Based Platforms. In: Vizeacoumar, F.J., Freywald, A. (eds) Mapping Genetic Interactions. Methods in Molecular Biology, vol 2381. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1740-3_7
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