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Updated: Jul 24, 2026
Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
Viral Packaging and Cell Culture for CRISPR-Based Screens
Tim Wang1, Eric S Lander2, David M Sabatini1
1Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142; Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, Massachusetts 02139; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
This protocol details CRISPR screening using lentivirus and high-throughput sequencing. It covers tissue culture, library preparation, and data analysis for genetic screens.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- CRISPR-Cas9 technology enables genome-wide genetic screens.
- Efficient library preparation and sequencing are crucial for robust screening.
- Standardized protocols are needed for reproducible CRISPR-based screens.
Purpose of the Study:
- To provide a detailed protocol for CRISPR-based screening.
- To outline methods for tissue culture and high-throughput sequencing library preparation.
- To guide downstream analysis of sequencing data.
Main Methods:
- Preparation of pantropic lentivirus from a single guide RNA (sgRNA) plasmid pool.
- Application of lentivirus to target cells, followed by antibiotic selection.
- Culture of cells under screening conditions for 14 population doublings.
- Amplification of sgRNA barcode sequences and high-throughput sequencing.
Main Results:
- Successful integration and amplification of sgRNA barcodes.
- Generation of sequencing data reflecting the sgRNA distribution in cell populations.
- Establishment of a scalable method for CRISPR screening.
Conclusions:
- The described protocol enables efficient CRISPR-based screening.
- High-throughput sequencing provides quantitative readout of genetic perturbations.
- This method facilitates the identification of genes involved in specific biological processes.
