Nuclear gene targeting inChlamydomonasusing engineered zinc-finger nucleases

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The unicellular green algaChlamydomonas reinhardtiiis 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 forChlamydomonasthat 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 aChlamydomonas in situmodel 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 theCOP3gene 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 shortCOP3target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and anaphVIIIDNA 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 modifiedCOP3locus. In cases where cells were co-transformed with a modifiedCOP3template, theCOP3locus was specifically modified by homologous recombination betweenCOP3and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes inChlamydomonas.

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