Abstract
CRISPR-Cas9 technology provides a simple way to introduce targeted mutations into mammalian cells to induce loss-of-function phenotypes. The CRISPR-Cas9 system has now successfully been applied for genetic screens in many cell types, providing a powerful tool for functional genomics with manifold applications. Genome-wide guide-RNA (gRNA) libraries allow facile generation of a pool of cells, each harboring a gene knockout mutation that can be used for the study of gene function, pathway analysis or the identification of genes required for cellular fitness. Furthermore, CRISPR genetic screens can be applied for the discovery of genes whose knockout sensitizes cells to drug treatments or mediates drug resistance. Here, we provide a detailed protocol discussing the necessary steps for the successful performance of pooled CRISPR-Cas9 screens.
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Acknowledgments
We would like to thank members of the Moffat lab for helpful comments. This work was supported by the Canadian Institutes for Health Research (CIHR#342551) to JM. MA holds a postdoctoral fellowship award from the Swiss National Science Foundation, MC holds an Ontario Graduate Scholarship and JM is a Canada Research Chair in Functional Genetics Tier 2. Michael Aregger and Megha Chandrashekhar contributed equally to this work.
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Aregger, M., Chandrashekhar, M., Tong, A.H.Y., Chan, K., Moffat, J. (2019). Pooled Lentiviral CRISPR-Cas9 Screens for Functional Genomics in Mammalian Cells. In: Singh, S., Venugopal, C. (eds) Brain Tumor Stem Cells. Methods in Molecular Biology, vol 1869. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8805-1_15
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