Generation of endogenous gene knockout in Chlamydomonas reinhardtii using zinc-finger nucleases made by Oligomerized Pool ENgineering (OPEN)
Xiaohong Li1, Lai-Wa Tam2, Feng Zhang1, Paul A. Lefebvre2, and Daniel F. Voytas1
1) Dept. of Genetics, Cell Biology and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA
2) Dept. of Plant of Biology, University of Minnesota, St. Paul, MN 55108,USA
Zinc-finger nucleases (ZFNs) can introduce double-strand DNA breaks (DSBs) into specific genomic loci, thereby stimulating targeted gene mutagenesis and gene replacement. Each ZFN consists of a non-specific cleavage domain of FokI endonuclease fused to a zinc finger array which can be designed to recognize specific DNA sequences of interest. Recently the first rapid, highly effective, and publicly available method for engineering zinc finger arrays was described by the Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology. The Consortium has used this new method (known as OPEN for Oligomerized Pool ENgineering) to generate high quality ZFN pairs that function in human, zebrafish and plant cells. However, targeted mutagenesis using ZFN in Chlamydomonas is not available so far. In this study, we used the OPEN method to generate ZFN pairs that target the endogenous Chlamydomonas nitrate reductase gene (NITI). NITI is an attractive gene for targeted mutagenesis since mutants of nitrate reductase cannot survive on growth medium that has nitrate as the sole nitrogen source. NIT1 mutants are also chlorate resistant. By using OPEN selections, we successfully generated several zinc finger arrays (ZFA) with high binding affinity to target sequences in the first two coding exons of NITI. The cleavage activity of Eight FokI-fused zinc finger pairs in yeast were tested and the results showed all the ZFNs have detectable activity, some of them are comparable to activity observed for a control ZFN engineered from the well-characterized ZFA, Zif268. The success of this study will improve gene targeting in Chlamydomonas and thus, improve the utility of Chlamydomonas for basic and applied research.
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