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

Giuliano, C. J., Lin, A., Girish, V., Sheltzer, J. M. (August 2019) Generating Single Cell–Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9. Current Protocols in Molecular Biology, 128 (1). Art number e100. ISSN 19343639 (ISSN)

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DOI: 10.1002/cpmb.100


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. © 2019 The Authors.

Item Type: Paper
Subjects: Investigative techniques and equipment > CRISPR-Cas9
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > loss of function
organism description > animal > mammal
CSHL Authors:
Communities: CSHL labs > Sheltzer lab
CSHL Cancer Center Program > Cancer Genetics and Genomics Program
Depositing User: Matthew Dunn
Date: August 2019
Date Deposited: 03 Sep 2019 20:58
Last Modified: 05 Nov 2020 19:27
PMCID: PMC6741428
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