Generation and basic properties of gene drive viruses. A, Schematic representation of the genetic organization of plasmids and the resultant recombinant HSV1 viruses. B, Plaque sizes of HSV-1 viruses on Vero cells at 48 hours post infection. Twenty plaques from each virus were randomly picked, and the area for each plaque was calculated and plotted. The mean with 95% CI was marked for each virus. * P<0.05. C, One-step growth curve of HSV-1 viruses. D, Expression of GFP and HSV1 antigens at different time points after BHK cells were infected with HSV1 viruses (MOI=2).

Spread of gene drive in HSV1 viruses. A and B, WT (GFP-, gD+) and gene drive viruses (GFP+, gD+) coinfection of vero cells were performed at MOI=5 with an initial ratio of WT: gene drive viruses close to 4:1, and passaged for 2 generations. The ratio of each virus was measured before coinfection (P0) and after each passage by infecting fresh BHK cells for 12 hours and labeling them with antibodies. Representative flow cytometry graphs (A) and statistical summary (B) of 3 experiments are shown. * p<0.05 compared with P0. C and D, V19 (mCherry+) and gene drive virus (GFP+) coinfection of Vero cells were conducted as in A and B. Representative flow cytometry graphs (C) and statistical summary (D) of 3 experiments are shown. E, Schematic diagram showing the process and consequence of the spread of gene drive viruses with WT or V19 viruses. F-H, V19 (mCherry+) and V15 (GFP+) coinfection of Vero cells were conducted as in A and B, but for 3 passages. Flow cytometry graphs (F) and the average ratio of each strain (H) from two independent experiments was shown. H, P1 supernatant of V19:V15 coinfection was diluted and plated on Vero cells for 48 hours. Plaques were imaged for the expression of fluorescent proteins. Scale bar, 500 µM.

The transmission dynamics of the HSV1 gene drive viruses and the rise of resistant clones. A, Schematic diagram showing the procedure and possible consequences of V10 (gD+ gE-) and gene drive viruses (gD+ GFP+ gE+) coinfection. V10 can be converted by newly generated gene drive viruses (gD+ GFP+gE-) at late stage of coinfection, and may produce variants resistant (gD+ gE-) to Cas nuclease. B, V10 and gene drive coinfection of Vero cells was conducted at MOI=5 with the initial ratio of V10: gene drive =4:1 or 19:1. The ratio of each virus after each passage was determined by infecting and staining BHK cells, and summarized here. The blue, green and yellow bars represent V10, the input gene drive and the new gene drive viruses, respectively. C, NGS sequencing results for the UL3-UL4 intergenic region amplified from input viruses (P0) and coinfection supernatants. The scaled cycle showed the relative ratio for three types of reads, and inside each cycle were the total number of amplicon reads for each sample. D-G. Distribution and frequency of mutations accumulated at the UL3-UL4 intergenic region for coinfection V10:V23 (D), V10:V24 (E), V10:V25 (F) and V10:V28 (G). The locations of the sgRNA target sequences and the PAM are marked with blue‒red bars.