Genome editing of epigenetic transgene silencing in Chlamydomonas reinhardtii

Eukaryotic microalgae are steadily advancing as promising green cell factories for sustainable modern biotechnology. However, one of the greatest hurdles to such use are the efficient transgene silencing mechanisms that drastically limit transgene expression levels and stability in engineered microalgal strains. Here, we used CRISPR/Cas9 to target multiple genes involved in epigenetic regulation in the model green microalga Chlamydomonas reinhardtii to identify key factors in epigenetic transgene silencing. Disruption of 11 candidate genes and subsequent systematic combination in double and triple knockout (KO) mutants, enabled a distinct reduction in transgene silencing and improved the stability of transgene expression compared with previously established strains. In addition, a split selectable marker system utilizing the Nostoc punctiforme DnaE split intein for dual-targeted genome editing was established. In summary, this work distinctly advances the biotechnological potential of C. reinhardtii and establishes a valuable mutant collection for further investigation of epigenetic regulation in green microalgae and potentially other eukaryotes.