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Updated: Jul 29, 2025
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Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing
Published on: June 16, 2022
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CRISPR/Cas9-Mediated Gene Knockout in Cells and Tissues Using Lentivirus
Jiaoyang Lu1,2,3, Shaohe Wang1,4
1Cell Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland.
Current Protocols
|May 24, 2023
Summary
This study presents a user-friendly CRISPR-Cas9 gene editing protocol for newcomers. It simplifies gene knockout in human cells and mouse tissues using lentivirus, making CRISPR technology more accessible.
Area of Science:
- Molecular Biology
- Gene Editing Technologies
Background:
- CRISPR-Cas9 is a powerful gene editing tool.
- Newcomers find CRISPR applications challenging due to multi-step protocols.
- Variations in protocol steps add to the complexity.
Purpose of the Study:
- To provide a reliable, stepwise, and newcomer-friendly protocol for gene knockout.
- To simplify the application of CRISPR-Cas9 for researchers.
- To generate stable gene knockout cells and tissue explants.
Main Methods:
- sgRNA design using CRISPOR.
- Golden Gate cloning for an "all-in-one" vector (sgRNA and Cas9).
- Streamlined lentivirus production and cell transduction.
- Lentiviral transduction of ex vivo mouse embryonic salivary epithelial explants.
Main Results:
- A comprehensive protocol for gene knockout in human fibroblasts was established.
- Efficient lentivirus production achieved within one week post-cloning.
- Successful generation of knockout cell pools and tissue explants.
Conclusions:
- The protocol facilitates CRISPR-Cas9 application for generating stable gene knockouts.
- It is particularly useful for new researchers in molecular biology.
- The method enables gene editing in both cell cultures and tissue explants.
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