Application of CRISPR-Cas9 genome editing by microinjection of gametophytes of Saccharina japonica (Laminariales, Phaeophyceae)

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated genome editing has been used for reverse genetics studies in many organisms. However, application to commercially important species of seaweeds is still limited. The genetics and breeding technologies for species of the Laminariales will be advanced by developing a genome editing tool. In this study, we attempted to edit a counter-selectable marker, the adenine phosphoribosyl transferase ( APT ) gene using CRISPR-Cas9 ribonucleoprotein (RNP) complexes delivered by microinjection into gametophytes of Saccharina japonica . After injection of CRISPR-Cas9 RNP, 2-fluoroadenine (2-FA) was added to the medium (10–40 μM) to select APT mutants. Twenty-three female and 12 male 2-FA resistant gametophytes had mutations in their APT genes. Genome editing efficiency for the injection trials was 8.64% and 4.46% for the female and male gametophytes, respectively. The apt mutant gametophytes were able to produce sperm or eggs. Sporophytes derived by crossing sperm and eggs from apt mutant gametophytes were resistant to 40 μM 2-FA. Sporophytes derived from crosses with one wild type and one mutant parent also showed resistance to high concentrations (20–40 μM) of 2-FA, while wild-type sporophytes died in 10 μM 2-FA. This is the first report showing the validity of gene editing of S. japonica using the CRISPR-Cas9 RNP complex and microinjection method.