In plants, DNA-free genome editing using Cas9/sgRNA RNPs transfected protoplasts has the advantage of avoiding transgene integration and off-target effects. These methods differ in gene editing efficiency depending on how many RNPs are introduced into the protoplasts, so optimization of the protoplast transfection conditions is necessary. In this study, we tested the protoplast transfection efficiency by cultivation conditions with photoperiod and continuous darkness. Lettuce and Chinese cabbage were cultivated under three different growth conditions: 7 days with a 16-h light/8-h darkness photoperiod (7P); 4 days 16-h light/8-h darkness photoperiod and 3 days continuous darkness (4P3D); and 3 days continuous darkness and 4 days 16-h/8-h darkness photoperiod (3D4P). To examine the transfection efficiency for each protoplast, we transfected the GFP expression plasmid by PEG-mediated protoplast transfection. The highest frequency of protoplasts expressing GFP was observed up to 25% in lettuce and 44% in Chinese cabbage under 4P3D conditions. Furthermore, we transfected LsPDS gene-targeted RNPs into three different lettuce protoplasts. Protoplasts were measured with the highest gene editing efficiency up to 25% under 4P3D conditions. These results suggest that continuous darkness pretreatment cultivation can promote protoplast-based DNA-free gene editing efficiency.