Construction of zinc finger nucleases for targeting of the Chlamydomonas ChR1 gene
Irina Sizova1,2 and Peter Hegemann2
1) St. Petersburg Nucl.Phys. Inst., Gatchina/St. Petersburg, Russia
2) Inst. für Biologie, Experimentelle Biophysik, Humboldt Universität zu Berlin, Berlin, Germany
Zinc finger nucleases (ZFNs) can be designed to create double-stranded DNA breaks (DSB) at specific DNA sequences and stimulate gene targeting (GT) and site-directed mutagenesis. ZFNs have the potential to open new perspectives for efficient and precise manipulation of the Chlamydomonas genome. By using of the model system we have found that in Chlamydomonas ZFNs are able to induce mutations at a predetermined site in the genome which are long deletions (>59 bp) in most of cases and, probably, originated on mechanism of microhomolohy-mediated end joining. To extend our studies on photoreception it is necessary to inactivate and create desired mutations in the Chlamydomonas sensory photoreceptor genes Channelrhodopsin I (CHRI ) which were unsuccessful using the single stranded DNA method that we have recently applied to the PHOT gene (Zorin et al. 2009). Taking into account published data about ZFN activity in vivo and in vitro (Maeder et al., 2009; Sander et al., 2009; Segal et al., 1999; and other) we generated 2 dimer ZFNs against a distinct position of ChR1 with the highest probability to yield functional zinc finger proteins and produce active ZFNs. We have found that one of two designed ZFN pairs is active in Chlamydomonas cells being able to insert DSBs and stimulate GT. The active ZFN pair has been composed of monomers, one of which contains the DNA recognizing ZF protein selected by the method OPEN (Maeder et al., 2008). Another has been made by joining Barbas ZF moduls. Activity of this ZFN pair was measured as the ability to stimulate restoration of a resident defective aphVIII gene, which was inactivated by insertion of ZFN recognition site of ChR1, by homologous recombination with donor DNA. Activity of the designed enzyme has proved to be similar to that of the control Zif268-FokI. By the use of this ZFN pair we have performed mutagenesis of two Chlamydomonas strains and are currently analyzing potential mutants on the ChR 1 gene.
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