Four strategies for inserting exogenous EGFP gene into HSV-2genome using CRISPR/Cas9 technology were designed (1) conventional homology-directed repair circular two homology armdonor-mediated gene knock-in; (2) linearized single homology armdonor-mediated gene knock-in; (3) homology-independent targeted integration; (4) conventional homology-directed repair-mediated by cell lines stably expressing Cas9 and sgRNA.
Results:
The recombinant virusHSV-2-EGFP was successfully constructed based on the second, the third and the fourth strategies. The second strategy was the most efficient, followed by the third and the fourth strategies. The purified recombinant virus could stably express green fluorescent protein in seven passages and sharedsimilargrowth characteristics in Vero cells to the parental virus.