CRISPR/Cas9 nuclease system is getting popular in precise genome editing of both eukaryotic and prokaryotic systems due to its accuracy, programmability, and relative ease of use. CRISPR/Cas systems can be delivered into live cells via plasmid DNA, RNA, and ribonucleoprotein (RNP). Of these, the RNP method is of special interest due to enzymatic action in shorter time and controllability over their activity. In addition, because RNP does not involve DNA, none of unwanted DNA footprints are left in the host genome. Previously, we demonstrated that plant protoplasts can be transfected with functional RNPs and the whole plants can be regenerated from an engineered protoplast. Relative to the published methods, the revised protocols described here should help increase the success rate of whole plant regeneration by reducing damages to the naked protoplast cells.
Keywords: CRISPR/Cas; Genome editing; Lettuce; Protoplast; Ribonucleoprotein (RNP); Tissue culture.