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Author Spotlight: Efficient CRISPR/Cas9 Genome Editing in Bone Marrow-Derived Macrophages for Precise Gene Disruption
Published on: August 4, 2023
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High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes
Julia Craft1, Tina Truong1, Bennett H Penn2
1Department of Internal Medicine, University of California, Davis.
Journal of Visualized Experiments : Jove
|August 17, 2023
Summary
This study presents an efficient CRISPR/Cas9 genome editing protocol for mouse bone marrow-derived macrophages (BMDMs). The method enables precise gene disruption, facilitating research into macrophage biology.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Bone marrow-derived macrophages (BMDMs) are crucial for studying tissue macrophage biology.
- Primary BMDMs offer superior in vivo physiological modeling compared to cell lines.
- Genetic manipulation in BMDMs is essential but technically challenging.
Purpose of the Study:
- To establish an efficient protocol for CRISPR/Cas9 genome editing in mouse BMDMs.
- To enable targeted gene function disruption through frameshift mutations.
- To provide a streamlined method for genetic modification of primary macrophages.
Main Methods:
- Synthesis of single-guide RNA (sgRNA)-Cas9 ribonucleoprotein complexes (RNPs).
- Delivery of RNPs into BMDMs via electroporation.
- Monitoring editing efficiency using Sanger sequencing and an online analysis tool.
Main Results:
- Achieved high editing efficiencies ranging from 85% to 95%.
- Protocol is rapid, completable within one week.
- Does not require plasmid construction, simplifying the workflow.
Conclusions:
- The developed protocol offers an efficient and accessible method for CRISPR/Cas9 genome editing in BMDMs.
- This technique facilitates functional genetic studies in primary macrophages.
- The protocol enhances the utility of BMDMs as a model system for macrophage research.
