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 |
| Subjects: |
bioinformatics
Investigative techniques and equipment > CRISPR
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing
bioinformatics > genomics and proteomics
Investigative techniques and equipment
organism description > animal
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > cell line
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > cell line
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > cell line
organs, tissues, organelles, cell types and functions > cell types and functions > cell types
organs, tissues, organelles, cell types and functions > cell types and functions > cell types
organs, tissues, organelles, cell types and functions > cell types and functions > cell types
organs, tissues, organelles, cell types and functions > cell types and functions
Investigative techniques and equipment > CRISPR-Cas9
organism description > animal > mammal > primates > hominids
organism description > animal > mammal > primates > hominids > human
organism description > animal > mammal
organs, tissues, organelles, cell types and functions
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > plasmid
organism description > animal > mammal > primates |
| CSHL Authors: |
|
| Communities: |
CSHL labs > Sheltzer lab |
| Depositing User: |
Matthew Dunn
|
| Date: |
September 2019 |
| Date Deposited: |
14 Dec 2020 16:04 |
| Last Modified: |
02 Feb 2024 15:20 |
| PMCID: |
PMC6741428 |
| Related URLs: |
|
| URI: |
https://repository.cshl.edu/id/eprint/39708 |
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