CRISPR-Mediated Genome Engineering in Cell Lines.

Ya-Ju Chang, Joseph Ryu, Xuan Cui, Stephen H Tsang

Journal: Methods in molecular biology (Clifton, N.J.) 2022;2560():267-278

PMID: 36481903

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.

Address: Edward S. Harkness Eye Institute, Department of Ophthalmology, Columbia University Irving Medical Center, New York, NY, USA.; Jonas Children's Vision Care and Bernard & Shirlee Brown Glaucoma Laboratory, New York, NY, USA.; Department of Pathology and Cell Biology, Columbia University, New York, NY, USA.; Departments of Ophthalmology, Pathology & Cell Biology, Graduate Programs in Nutritional & Metabolic Biology and Neurobiology & Behavior, Columbia Stem Cell Initiative, New York, NY, USA. [email protected].

DOI: 10.1007/978-1-0716-2651-1_25

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https://doi.org/10.1007/978-1-0716-2651-1_25

MeSH Terms: Cell Line