Abstract
A specific targeting nuclease is a powerful tool for mediating genome alternative expression with high precision. The RNA sequence-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate genome engineering in cells by using a 20-nt targeting sequence. In this chapter, we describe a set of tools for Cas9-mediated genome editing via non-homologous end joining (NHEJ) or homology-directed repair (HDR) in the generation of modified cell lines for downstream functional studies. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency, and analysis of off-target activity. Beginning with target design, we will cover gene modifications and modified clonal cell lines.
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Acknowledgments
National Cancer Institute Core (5P30CA013696), Foundation Fighting Blindness (TA-NMT-0116-0692-COLU), and New York State (C029572).
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Stephen H. Tsang receives grant support from Abeona Therapeutics, Inc and Emendo. He is also the founder of Rejuvitas and is on the scientific and clinical advisory board for Nanoscope Therapeutics and Medical Excellence Capital.
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Chang, YJ., Ryu, J., Cui, X., Tsang, S.H. (2023). CRISPR-Mediated Genome Engineering in Cell Lines. In: Tsang, S.H., Quinn, P.M. (eds) Retinitis Pigmentosa. Methods in Molecular Biology, vol 2560. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2651-1_25
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DOI: https://doi.org/10.1007/978-1-0716-2651-1_25
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