Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation.

The green unicellular alga Chlamydomonas reinhardtii with two photoreceptors called channelrhodopsins is a model organism that gave birth to a new scientific field of biomedical studies, optogenetics. Although channelrhodopsins are helping to decipher the activity of the human brain, their functionality has never been extensively studied in the organism of origin, mainly due to the difficulties connected to reverse genetic interventions. In this study, we present a CRISPR-Cas9-based technique that enables a precise in vivo exchange of single amino acids in a selected gene. To shed light on the function of channelrhodopsins ChR1 (C1) and ChR2 (C2) in vivo, we deleted both channelrhodopsins independently in the wild-type strain and introduced point mutations in the remaining channel, causing modified photocycle kinetics and ion selectivity. The mutated strains, ΔC1C2-E123T, ΔC1C2-E90R and ΔC1C2-E90Q, showed about 100-fold decrease in photosensitivity, a reduced photophobic response and faster light adaptation rates due to accelerated photocycle kinetics and reduced Ca2+ conductance. Moreover, the ΔC1C2-E90Q with an additionally reduced H+ permeability produced an electrical response only in the presence of Na+ ions, highlighting a contribution and importance of H+ conductance to photocurrents in the wild-type algae. Finally, in the ΔC1C2-E90R strain with the channelrhodopsin selectivity converted to anions, no photo-responses were detected. We conclude that the precise photocycle kinetics and the particular ion selectivity of channelrhodopsins are the key parameters for efficient phototaxis in low light conditions.

Gene Editing in Green Alga Chlamydomonas reinhardtii via CRISPR-Cas9 Ribonucleoproteins.

With the establishment of the CRISPR-Cas9 molecular tool as a DNA editing system in 2012, the handling of gene editing experiments was strongly facilitated pushing reverse genetics approaches forward in many organisms. These new gene editing technologies also drastically increased the possibilities for design-driven synthetic biology. Here, we describe a protocol for gene editing in the green algae Chlamydomonas reinhardtii using preassembled CRISPR-Cas9 ribonucleoproteins.The three sections of the protocol guide through a complete gene editing experiment, starting with the experimental design and the choice of suitable CRISPR target sites and how to perform a Cas9 in vitro test digestion. The second part covers the transformation of algal cells with Cas9 RNPs using electroporation. In the last part, the PCR-based screening for mutants and isolation of clones is explained.

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

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 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 Staphylococcus aureus and Streptococcus pyogenes, and recombinant Cas9 and developed protocols for rapidly isolating nonselectable gene mutants. Using this technique, we disrupted the photoreceptor genes COP1/2, 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 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.