Generating Single Cell-Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9

Giuliano, C. J., Lin, A., Girish, V., Sheltzer, J. M. (September 2019) Generating Single Cell-Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9. Curr Protoc Mol Biol, 128 (1). e100. ISSN 1934-3639 (Print)1934-3647

Abstract

CRISPR/Cas9 technology enables the rapid generation of loss-of-function mutations in a targeted gene in mammalian cells. A single cell harboring those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of biological reagents. However, the successful derivation of knockout clones can be technically challenging and may be complicated by multiple factors, including incomplete target ablation and interclonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation of true knockout clones and are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells. © 2019 The Authors.

Item Type:	Paper
Additional Information:	1934-3647 Giuliano, Christopher J Lin, Ann Girish, Vishruth Sheltzer, Jason M DP5 OD021385/OD/NIH HHS/United States Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Curr Protoc Mol Biol. 2019 Sep;128(1):e100. doi: 10.1002/cpmb.100.
Uncontrolled Keywords:	Animals *CRISPR-Cas Systems Cell Line *Clone Cells Gene Knockout Techniques/*methods HEK293 Cells Humans Mammals Plasmids RNA, Guide Transfection *CRISPR/Cas9 *cell lines *knockout *mammalian
CSHL Authors:	Sheltzer, Jason Giuliano, Christopher Lin, Ann Girish, Vishruth
Communities:	CSHL labs > Sheltzer lab
Depositing User:	Matthew Dunn
Date:	September 2019
Date Deposited:	14 Dec 2020 16:04
Last Modified:	14 Dec 2020 16:04
PMCID:	PMC6741428
Related URLs:	Publisher
URI:	https://repository.cshl.edu/id/eprint/39708

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