Etiolation promotes protoplast transfection and genome editing efficiency.

In plants, DNA-free genome editing using preassembled clustered regularly interspaced short palindromic repeats (CRISPR)-ribonucleoprotein (RNP) has the advantage of avoiding transgene integration and limiting off-target effects. The efficiency of this gene editing strategy can vary, so optimization of protoplast transfection conditions is necessary to achieve maximum yield. In this study, we examined the effects of etiolation, or increased exposure to darkness during cultivation, on the transfection efficiency of protoplasts from lettuce and Chinese cabbage. Seedlings were grown under three different conditions: non-etiolated, etiolated, and de-etiolated. First, we tested PEG-mediated transfection after etiolation using a plasmid DNA for green fluorescent protein (GFP)-expression. Etiolated protoplasts had the highest percentage of GFP-expressing cells, with a 3.1-fold and 4.8-fold improvement in lettuce and Chinese cabbage, respectively, compared with non-etiolated protoplasts. We also assessed gene editing of endogenous genes after etiolation using CRISPR-RNP. Using targeted deep sequencing, we observed the highest editing efficiency in etiolated protoplasts from both plant species, for the LsPDS and LsFT genes in lettuce, this led to an 8.7-fold and 4.4-fold improvement compared with non-etiolated protoplasts, respectively. These results suggest that etiolation during seedling growth can improve transfection efficiency and DNA-free gene editing in protoplasts.