Replicon-based genome-wide CRISPR knockout screening for the identification of host factors involved in viral replication
- Nat Commun. 2025 Dec 10;16(1):11028. doi: 10.1038/s41467-025-65979-3.
- 1. Chan Zuckerberg Biohub, San Francisco, CA, USA.
- 2. New York University Grossman School of Medicine, New York, NY, USA.
- 3. Washington University School of Medicine, St. Louis, MO, USA.
- 4. Stanford University, Palo Alto, CA, USA.
- 5. Medical College of Wisconsin, Milwaukee, WI, USA.
- 6. Integrated Research Facility at Fort Detrick, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, Frederick, MD, USA.
- 7. Chan Zuckerberg Biohub, San Francisco, CA, USA. [email protected].
We describe a viral replicon-based CRISPR knockout (KO) screening approach to specifically identify host factors essential for viral replication which are often missed in live virus screens. We benchmark the replicon screening using a stable fluorescent Dengue Virus type 2 (DENV-2) replicon cell line and successfully identify host genes known to be required for viral DENV-2 replication (e.g., endoplasmic reticulum membrane complex and oligosaccharyltransferase complex components), along with additional genes that have not been reported in prior CRISPR KO screens with DENV-2. We extend this replicon screening approach to chikungunya virus (CHIKV), a positive-sense RNA virus, and Ebola virus (EBOV), a negative-sense RNA virus, and identify distinct sets of genes required for replication of each virus. Our findings indicate that viral replicon-based CRISPR screens are a useful approach to identify host factors essential for replication of diverse viruses and to elucidate potential novel targets for host-directed medical countermeasures.