Harnessing snakehead rhabdovirus (SHRV) for gene editing by installment of CRISPR/Cas9 in viral genome

●Recombinant SHRVs harboring CRISPR/Cas9 system in genome were rescued.●rSHRV having sgRNA for BHK-21 cell's IRF9 gene led to indels of the target gene.●rSHRVs could be removed from BHK-21 cells by temperature up-shift to 37 °C.●This is the first report on the use of piscine RNA virus for gene editing. As there is no risk of viral genome integration into host chromosome, cytoplasmic RNA viruses can be a safer vehicle to deliver CRISPR/Cas system. Snakehead rhabdovirus (SHRV) is a piscine RNA virus belonging to the family Rhabdoviridae, and, in the present study, we evaluated the availability of SHRV as a tool for CRISPR/Cas9 delivery in mammalian cells. SHRV was grown well in baby hamster kidney (BHK-21) cells at 28 °C, and the replication ability was greatly reduced by temperature up-shift to 37 °C. We rescued a recombinant SHRV that harboring not only the interferon regulatory factor 9 (IRF9) gene-targeting single-guide RNA (sgRNA) but also Cas9 gene in the genome using the reverse genetic technology. The IRF9 gene of BHK-21 cells was knocked-out by the infection with the IRF9 gene-targeting rSHRV. Moreover, the rSHRVs were sharply disappeared in the cells by elevating temperature to 37 °C, suggesting the possible regulation of knockout efficiency before virus infection-caused cell damage. Although further optimization researches are needed to enhance the editing efficiency using the recombinant SHRV, to our knowledge, this is the first report on the possible applicability of piscine RNA virus for the gene editing in mammalian cells.