Heritable DNA-free genome editing of canola ( Brassica napus L.) using PEG-mediated transfection of isolated protoplasts

Application of new genome editing technologies for plant improvement depends on efficient delivery of editing components into plant cells. For editing of canola, which is an important oil crop, CRISPR/Cas9 and Agrobacterium -mediated transformation of hypocotyl or stem segments have been the primary methods used. Here, we present the development of an efficient and high-throughput system for protoplast isolation, DNA-free editing, and production of edited canola plants. Protoplast polyethylene glycol (PEG)–mediated transfection was conducted with ribonucleoproteins (RNPs) comprising LbCas12a and a single guide RNA. Edits were found at a frequency of approximately 40% in protoplasts cultured for 2 d, in callus colonies derived from these protoplasts, and in regenerated plants. Out of 30 plants that were regenerated from the protoplasts, 12 were edited. Six of them were with biallelic mutations and another six had monoallelic mutations. Targeted deletions from 2 to 9 nucleotides were identified in the T 0 edited plants. Analysis of T 1 progeny confirmed that the RNP-based, DNA-free edits using this canola protoplast system are heritable.