Genome editing using a <scp>DNA</scp>‐free clustered regularly interspaced short palindromic repeats<scp>‐Cas9</scp> system in green seaweed <scp><i>Ulva prolifera</i></scp>
Kensuke Ichihara, Tomokazu Yamazaki, Shigeyuki Kawano
Abstract
SUMMARY 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 .