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.

由于没有病毒基因组整合到宿主染色体中的风险，细胞质 RNA 病毒可以成为传递 CRISPR/Cas 系统的更安全的载体。蛇头弹状病毒 (SHRV) 是属于弹状病毒科的一种鱼类 RNA 病毒。并且，在本研究中，我们评估了 SHRV 作为哺乳动物细胞中 CRISPR/Cas9 递送工具的可用性。SHRV 在 28 °C 时在幼仓鼠肾 (BHK-21) 细胞中生长良好，并且随着温度升高到 37 °C，复制能力大大降低。我们使用反向遗传技术拯救了一种重组 SHRV，该重组 SHRV 不仅含有干扰素调节因子 9 (IRF9) 基因靶向单向导 RNA (sgRNA)，还含有基因组中的 Cas9 基因。BHK-21 细胞的 IRF9 基因被 IRF9 基因靶向 rSHRV 感染敲除。此外，通过将温度升高到 37 °C，细胞中的 rSHRV 急剧消失，这表明可能在病毒感染引起的细胞损伤之前调节敲除效率。