biostudies-literatureUnknown16(1)Cheng KWWe 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.Nature communications11028https://www.ebi.ac.uk/biostudies/studies/S-EPMC12696002biostudies-literatureReplicon-based genome-wide CRISPR knockout screening for the identification of host factors involved in viral replication.PMC12696002Travisano KAMarkhard ALAnaya APuschnik ASKuhn JHMedicielo JVBhatt KDAnantpadma MKistler ALPeng DBhave MLembirik SNjoya LCheng KWfalseReplicon-based genome-wide CRISPR knockout screening for the identification of host factors involved in viral replication.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.2025-01-01T00:00:00Z2025 Dec2026-06-06T00:27:27.772Z2026-05-24T03:11:17.219ZS-EPMC126960024137212110.1038/s41467-025-65979-3