Zinc finger nuclease-induced double-strand breaks mediate targeted mutagenesis in Caenorhabditis elegans

Zinc finger nucleases (ZFNs) are chimeric proteins composed of a DNA-binding domain comprised of several tandem Cys2His2 zinc fingers and a nonspecific endonuclease domain from the Type IIs restriction enzyme FokI. When expressed in the nematode Caenorhabditis elagans, these molecules bind it's DNA at a sequence specified by the zinc fingers and produces double-strand breaks (DSBs) at this target site in somatic tissues. These breaks activate the nematode DNA repair mechanisms, which attempt to repair the lesions either by homologous recombination (HR), using an unbroken homologous DNA template, or by nonhomologous end joining (NHEF), by which the broken ends are simply rejoined without regard for sequence conservation. The latter process will, at some frequency, produce targeted mutations at the break site. The work present characterizes ZFN-mediated mutagenesis in the somatic tissue of the nematode. Expression of sets of ZFNs, designed to specifically cleave either a synthetic site on an extrachromosomal array or a genomic target, resulted in a spectrum of NHEJ-medicated target site mutations at a frequency of about 20% of each locus. Elimination of the canonical NHEJ pathway in the nematode significantly altered the frequency and types of mutations observed, indicating that this process is fundamental in repairing ZFN-induced DSBs. It is hoped that the information gained from these studies will serve as a guide in the use of ZFNs for germline mutagenesis and gene targeting in the nematode. Use of this technology together with an introduced altered DNA template to direct HR-mediated DSB repair will herald the arrival of a long-sought method of efficient gene targeting of C. elagans.