Targeting of Photoreceptor Genes in via Zinc-Finger Nucleases and CRISPR/Cas9.

The fast-growing biflagellated single-celled chlorophyte is the most widely used alga in basic research. The physiological functions of the 18 sensory photoreceptors are of particular interest with respect to Chlamydomonas development and behavior. Despite the demonstration of gene editing in Chlamydomonas in 1995, the isolation of mutants lacking easily ascertained newly acquired phenotypes remains problematic due to low DNA recombination efficiency. We optimized gene-editing protocols for several Chlamydomonas strains (including wild-type CC-125) using zinc-finger nucleases (ZFNs), genetically encoded CRISPR/associated protein 9 (Cas9) from and , and recombinant Cas9 and developed protocols for rapidly isolating nonselectable gene mutants. Using this technique, we disrupted the photoreceptor genes , (encoding channelrhodopsin 1 [ChR1]), (encoding ChR2), , , , , , and and created the and double mutants. Characterization of the , , and mutants confirmed the value of photoreceptor mutants for physiological studies. Genes of interest were disrupted in 5 to 15% of preselected clones (∼1 out of 4000 initial cells). Using ZFNs, genes were edited in a reliable, predictable manner via homologous recombination, whereas Cas9 primarily caused gene disruption via the insertion of cotransformed DNA. These methods should be widely applicable to research involving green algae.