Abstract

CRISPR/Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until now only been applied to targeted gene disruption, whereas scar-less knock-in transgenesis has generally been considered infeasible. We have developed highly efficient homology-directed knock-in mutagenesis in cell-walled strains of Chlamydomonas. Our method allows scarless integration of fusion tags and sequence modifications of near arbitrary proteins without need for a preceding mutant line.