Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening
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Abstract
Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation.
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The CRISPR-Cas9 library system has been used to identify genes essential for cancer cell survival, proliferation, and targeted drug resistance in vitro and in vivo.
“It has been used to identify genes that are essential for cancer cell survival, proliferation, and targeted drug resistance in vitro and in vivo [21–24].”
From Genome-wide CRISPR-cas9 knockout screening identifies GRB7 as a driver for MEK inhibitor resistance in KRAS mutant colon cancer (2022) · cites this paper as [24]
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Self-citation
- Authors
- Joung, J., Konermann, S., Gootenberg, J. S., Abudayyeh, O. O., Platt, R. J., Brigham, M. D., Sanjana, N. E., & Zhang, F.
- Affiliations
- Broad Institute (US)
- Corresponding author
- Zhang, Feng
- ORCID
- Venue
- Nature Protocols
- Published
- 2017-03-23
- Volume
- 12
- Issue
- 4
- Pages
- 828-863
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC5526071
- OpenAlex
- W2601885162
- Cited by
- 1,243
- References
- 75
- Citations by year
- 2026: 142 · 2025: 153 · 2024: 184 · 2023: 173 · 2022: 168 · 2021: 138 · 2020: 119 · 2019: 85 · 2018: 58 · 2017: 21 · 2016: 1
- FWCI
- 30.81
- Type
- journal article
- Language
- en
- Update notices
- correction (2018-10-22)
- Open access
- green
- doi.org
- Licence
- Locations
- Nature Protocols (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
- Repository for Publications and Research Data (ETH Zurich) (submittedVersion, cc-by-nc-nd)
- PMC
- ETH Zürich Research Collection
- Funders
- Howard Hughes Medical Institute
- National Institutes of Health
- McGovern Institute for Brain Research, Massachusetts Institute of Technology
- National Institute of Mental Health (DP1MH100706, R01MH110049)
- National Human Genome Research Institute (R00HG008171)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01DK097768)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
- Keywords
- CRISPR/Cas9, guide RNA library, lentiviral vectors, forward genetic screen, RNA-guided endonuclease, library design, candidate gene validation, functional genomics
- MeSH terms
- Animals, Genetic Vectors, Humans, Phenotype, Transcriptional Activation, Gene Library, Lentivirus, Genomics, Mice, Gene Knockout Techniques, CRISPR-Cas Systems
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