Generating Single Cell-Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9.
CRISPR/Cas9
cell lines
knockout
mammalian
Journal
Current protocols in molecular biology
ISSN: 1934-3647
Titre abrégé: Curr Protoc Mol Biol
Pays: United States
ID NLM: 8908160
Informations de publication
Date de publication:
09 2019
09 2019
Historique:
entrez:
11
9
2019
pubmed:
11
9
2019
medline:
10
6
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 31503414
doi: 10.1002/cpmb.100
pmc: PMC6741428
mid: NIHMS1038380
doi:
Substances chimiques
RNA, Guide
0
Types de publication
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.
Langues
eng
Sous-ensembles de citation
IM
Pagination
e100Subventions
Organisme : NIH HHS
ID : DP5 OD021385
Pays : United States
Organisme : NCI NIH HHS
ID : P30 CA045508
Pays : United States
Informations de copyright
© 2019 The Authors.
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