Oh Hyung Suk, Diaz Fernando M, Zhou Changhong, Carpenter Nicholas, Knipe David M
Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA.
Curr Res Virol Sci. 2022;3. doi: 10.1016/j.crviro.2022.100023. Epub 2022 Jun 20.
Recombinant herpes simplex virus strains can be constructed by several methods, including homologous recombination, bacterial artificial chromosome manipulation, and yeast genetic methods. Homologous recombination may have the advantage of introducing fewer genetic alterations in the viral genome, but the low level of recombinants can make this method more time consuming if there is no screen or selection. In this study we used complementing cell lines that express Cas9 and guide RNAs targeting the parental virus to rapidly generate recombinant viruses. Analysis of the progeny viruses indicated that CRISPR-Cas9 both promoted recombination to increase recombinant viruses and selected against parental viruses in the transfection progeny viruses. This approach can also be used to enrich for recombinants made by any of the current methods.
重组单纯疱疹病毒株可通过多种方法构建，包括同源重组、细菌人工染色体操作和酵母遗传学方法。同源重组可能具有在病毒基因组中引入较少基因改变的优势，但如果没有筛选或选择，重组体水平较低会使该方法更耗时。在本研究中，我们使用了表达Cas9和靶向亲本病毒的引导RNA的互补细胞系来快速产生重组病毒。对子代病毒的分析表明，CRISPR-Cas9既促进重组以增加重组病毒，又在转染子代病毒中对抗亲本病毒进行选择。这种方法也可用于富集通过任何现有方法产生的重组体。
