Vaccinia virus (VACV) is a large dsDNA virus that replicates in the cytoplasm and recombinant VACVs are widely used and have potential applications as vaccines, cancer immunotherapies and oncolytic therapies. CRISPR/Cas9 genome editing has rapidly become the method of choice for making changes in cell lines and has been adapted readily to nuclear-replicating viruses such as herpes simplex virus. By contrast, the few reports of CRISPR/Cas9 editing of VACV in the literature have been difficult to replicate, potentially because of the cytoplasmic site of VACV replication. Further, while achieving localisation of Cas9 to the cytoplasm is trivial, this may compromise assembly with a nuclear-transcribed gRNA that is generated in many CRISPR/Cas9 protocols. We tried two possible solutions to this problem: 1) Co-transfection of a plasmid expressing cytoplasmic-localised Cas9 and a synthetic gRNA and 2) transfection of in vitro assembled Cas9 and gRNA. To test these, we used an mCherry-expressing VACV and targeted this gene for inactivation. We found strong Cas9 activity against the virus when an mCherry, but not a control sgRNA were used. Red fluorescence was reduced by 26% and 34%, and the amount of virus recovered by 53% and 76% in cells transfected with plasmid/sgRNA and in vitro assembled Cas9/sgRNA, respectively. However, surprisingly the remaining virus in each case was mCherry positive. These data suggest that cytoplasmic CRISPR/Cas9 is effective at inhibiting VACV replication, possibly because non-homologous end joining (NHEJ), which repairs dsDNA breaks in the nucleus, is not effective in the cytoplasm. We are currently testing whether these CRISPR/Cas9 methods can improve the efficiency of homologous recombination between VACV and an appropriately designed transfer construct.