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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.

These authors contributed equally to this work. SUMMARYThe unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide range of tools for genetic manipulation have been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases (ZFNs). Our approach includes (i) design of gene-specific ZFNs using available online tools, (ii) evaluation of the designed ZFNs in a Chlamydomonas in situ model system, (iii) optimization of ZFN activity by modification of the nuclease domain, and (iv) application of the most suitable enzymes for mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs to target the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional aminoglycoside 3′-phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin-resistant (Pm-R) clones with expressing nucleases. Of these Pm-R clones, 1% also contained a modified COP3 locus. In cases where cells were co-transformed with a modified COP3 template, the COP3 locus was specifically modified by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas.

Homologous DNA recombination (HR) allows the deletion (knockout), repair (rescuing), and modification of a selected gene, thereby rendering a functional analysis of the gene product possible. However, targeting of nuclear genes has been an inefficient process in most eukaryotes, including algae, plants, and animals, due to the dominance of integration of the applied DNA into nonhomologous regions of the genome. We have shown for the green alga Chlamydomonas reinhardtii by repairing a previously introduced truncated aminoglycoside 3-phosphotransferase gene, aphVIII, that single-stranded DNA can recombine with a homologous endogenous DNA region of interest. Nonhomologous DNA integration appeared to be more than 100-fold reduced compared with the use of double-stranded DNA, thus allowing isolation of the homologous recombinants. We propose that this method will be applicable to direct targeting of nuclear C. reinhardtii genes.

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