Fig. 1

Cytokine-armed oncolytic virus construction based on CRISPR-Cas9 gene editing. A Schematic of US12 locus in Human herpesvirus 1 strain 17 (NCBI Reference Sequence: NC_001806.2). B The molecular construct of mouse IL15Rα/IL15 fusion protein. The three-dimensional model structures of this fusion proteins were predicted using Al-phaFold2 and colored by predicted Local-Distance Difference Test or different domain. C-D Schematic of CRISPR/Cas9-mediated HDR for replacing US12 with mCherry reporter gene (C) or replacing mCherry reporter with therapeutic cytokine genes (D) The target sites of gRNA (US12 1.1 or mCherry 1.1 + mCherry 1.2) were indicated by the black arrowhead. Primer pairs flanking the region of replacement were indicated by a black arrowhead. E Flowcharts of the gene editing of oncolytic HSV-1 genome and strategy for isolation of the potential recombinant virus (mCherry-). F Representative images of isolation of mCherry- recombinant virus after CRISPR/Cas9-mediated gene replacement. mCherry- recombinant virus clone was indicated by red arrowhead. Pictures were taken at 200X magnification, scale bars 100 μm. G-I PCR detection of NHEJ and HDR events in the mCherry- isolates in the construction of OV-IFNG (G), OV-IL15Fu (H) and OV-GMCSF (I), respectively. Potential isolates with target replacement (HDR) were indicated by red arrowhead