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. 2020 Nov 3;3:643. doi: 10.1038/s42003-020-01374-6

Fig. 3. Cas9 reduces VACV titre irrespective of cleavage site or presence of HDR template.

Fig. 3

a Micrographs show examples of cytopathic effect 48 h after VACV-mCh (MOI 0.05) infection and Cas9/gRNA transfections. b Amounts of infectious progeny at 48 h from replicates of the experiment in (a) were determined by plaque assay (n = 4, ns p > 0.05, *p = 0.0131, **p = 0.0099). c The experiment in (a) was repeated with the inclusion of cultures transfected with Cas9/A23R-gRNA (n = 3, ns p > 0.05, *p = 0.0115, **p = 0.0059). d Experiments similar to (a) but using the VACV F5-GFP, A3-GFP or B5-GFP (as shown) and transfected with Cas9/eGFP-gRNA complexes; Ctrl cultures had no Cas9/gRNA (n = 3, *p = 0.0319, **p = 0.0032, ***p = 0.0003). Plaque assays were used to titrate virus from cultures infected with VACV F5-GFP, A3-GFP or B5-GFP and transfected with Cas9/eGFP complexes. e–h Micrographs (e, g) and virus production (f, h) from VACV-infected cultures 48 h after transfection with Cas9/TK-gRNA and pSC11-mCh (e, f) or Cas9-Spi2-gRNA and pBII-ΔR (g, h). Scale bars = 300 μm. Graphs display means and SEM from independent experiments (b, c, f) or replicate cultures (d, h). Significance determined by one-way ANOVA (b, c, d) or Student’s unpaired t-test (f, h) (ns p > 0.05, *p = 0.0458, **p = 0.0071).