3 years ago

Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-finger Nucleases and CRISPR/Cas9.

Hegemann, Sizova, Greiner, Kreimer, Evers, Kelterborn
The fast-growing biflagellated single celled chlorophyte Chlamydomonas reinhardtii is the most widely used alga in basic research. The physiological functions of the 18 sensory photoreceptors are of particular interest with respect to C. reinhardtii development and behavior. Despite the demonstration of gene editing in C. reinhardtii 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 Staphylococcus aureus and Streptococcus pyogenes, and recombinant Cas9 and developed protocols for rapidly isolating non-selectable gene mutants. Using this technique, we disrupted the photoreceptor genes COP½, COP3 (encoding channelrhodopsin-1 [ChR1]), COP4 (encoding ChR2), COP5, PHOT, UVR8, VGCC, MAT3 and aCRY and created the chr1 chr2 and uvr8 phot double mutants. Characterization of the chr1, chr2 and mat3 mutants confirmed the value of photoreceptor mutants for physiological studies. Genes of interest were disrupted in 5-15% 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 co-transformed DNA. These methods should be widely applicable to research involving green algae.

Publisher URL: http://doi.org/10.1105/tpc.17.00659

DOI: 10.1105/tpc.17.00659

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