Related Experiment Video
Updated: Feb 23, 2026
10:32
A Universal Protocol for Large-scale gRNA Library Production from any DNA Source
Published on: December 6, 2017
11.7K
Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library.
Brooke A Napier1, Denise M Monack1
1Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA.
Bio-Protocol
|September 5, 2017
Summary
This study details creating a CRISPR-Cas9 genome-wide library in murine macrophages. This tool screens gene functions and identifies novel mediators in cellular pathways, like caspase-11 cell death.
Area of Science:
- Molecular Biology
- Genetics
- Immunology
Background:
- CRISPR-Cas9 technology enables targeted gene knockout in mammalian cells.
- Understanding gene function is crucial for elucidating cellular pathways.
- Genome-wide libraries facilitate high-throughput screening of gene effects on cellular phenotypes.
Purpose of the Study:
- To describe a method for creating a CRISPR-Cas9 genome-wide library in RAW264.7 murine macrophage cells.
- To enable unbiased, high-throughput screening of gene functions in macrophage biology.
- To identify novel mediators in cellular pathways, such as the caspase-11 cell death pathway.
Main Methods:
- Construction of a CRISPR-Cas9 genome-wide library.
- Application of the library in a transformed murine macrophage cell line (RAW264.7).
- Utilizing guide RNA (gRNA) and Cas9 endonuclease for targeted gene knockout.
Main Results:
- Successful creation of a CRISPR-Cas9 genome-wide library in RAW264.7 cells.
- Demonstrated utility of the library in identifying novel mediators of the caspase-11 cell death pathway.
- Established a versatile tool for screening gene importance in various murine macrophage pathways.
Conclusions:
- The developed CRISPR-Cas9 library is an effective tool for functional genomics in murine macrophages.
- This method facilitates the discovery of new components in critical cellular signaling pathways.
- The library has broad applicability for investigating macrophage biology and related diseases.
