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Cited by 130CRISPR and Genetic EngineeringRNA Interference and Gene DeliveryRNA regulation and disease

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

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Giuliano, Christopher J., Lin, Ann, Girish, Vishruth, Sheltzer, Jason M.·2019·Current Protocols in Molecular Biology
DOI 10.1002/cpmb.100PMC PMC6741428
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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.

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  • CRISPR technology has drawbacks including Cas9 off-target activity, cost, and a labor-intensive process of guide RNA design, viral packaging and infection, isolation of single cells, expansion of clones, and verification of correct targeting.

    “While complete gene knockout is often preferred, there are some drawbacks of CRISPR technology including Cas9 off-target activity [46], as well as the cost and labor-intensive process of guide RNA design, viral packaging and infection, isolation of single cells, expansion of clones, and verification of correct targeting [47].”

    From Regulation of Enterocyte Brush Border Membrane Primary Na-Absorptive Transporters in Human Intestinal Organoid-Derived Monolayers (2024) · cites this paper as [47]

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Cited by

Sorted by citations

Authors
Giuliano, C. J., Lin, A., Girish, V., & Sheltzer, J. M.
Affiliations
Cold Spring Harbor Laboratory (US)
Corresponding author
Sheltzer, Jason M.
ORCID
Venue
Current Protocols in Molecular Biology
Publisher
Wiley
Published
2019-07-26
Volume
128
Issue
1
Pages
e100-e100
ISSN
  • 1934-3639 (ISSN-L)
  • 1934-3647
DOI
10.1002/cpmb.100
PMC
PMC6741428
OpenAlex
W2918689988
Cited by
130
References
1
Citations by year
2026: 9 · 2025: 20 · 2024: 14 · 2023: 31 · 2022: 22 · 2021: 14 · 2020: 18 · 2019: 2
FWCI
4.63
Type
journal article
Language
en
Open access
Licence
Locations
  • Current Protocols in Molecular Biology (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • Cold Spring Harbor Laboratory Institutional Repository (Cold Spring Harbor Laboratory) (acceptedVersion)
  • Cold Spring Harbor Laboratory Institutional Repository (Cold Spring Harbor Laboratory) (submittedVersion)
Funders
  • National Science Foundation (1DP5OD021385)
  • Cold Spring Harbor Laboratory (5P30CA045508)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Keywords
CRISPR/Cas9, loss-of-function mutations, guide RNA design, CRISPR delivery, knockout validation, mammalian cell lines, gene function
MeSH terms
RNA, Guide, CRISPR-Cas Systems, Animals, Cell Line, Clone Cells, Humans, Mammals, Plasmids, Transfection, RNA, Guide, Kinetoplastida, Gene Knockout Techniques, HEK293 Cells, CRISPR-Cas Systems

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