Genome editing using a DNA-free clustered regularly interspaced short palindromic repeats-Cas9 system in green seaweed Ulva prolifera

Although the green seaweed Ulva is one of the most common seaweeds in the coastal regions with well-studied ecological characteristics, few reverse genetic technologies have been developed for it. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is a simple genome-editing technology based on a ribonucleoprotein (RNP) complex composed of an endonuclease and programmable RNA to target particular DNA sequences. Genome editing makes it possible to generate mutations on a target gene in non-model organisms without established transgenic technologies. In this study, we applied the CRISPR-Cas9 RNP genome-editing system to the green seaweed Ulva prolifera, using polyethylene glycol (PEG)-mediated transfection. Our experimental system disrupts a single gene (UpAPT) encoding adenine phosphoribosyl transferase (APT) and generates a resistant phenotype for gametophytes cultured in a medium with toxic compound 2-fluoroadenine. The PEG-mediated transfection used for gametes resulted in 2-fluoroadenine-resistant strains containing short indels or substitutions on UpAPT. Our results showed that the CRISPR-Cas9 system with PEG-mediated transfection was efficient for genome editing in Ulva