There is currently no useful technique available to create permanent targeted mutations in the genome of the nematode, Caenorhabtidis elegans. Traditional mutagenesis protocols cannot target a specific gene, and suppression by RNA interference is not always completely effective. Thus, a method to produce a targeted mutation in the nematode genome would be a powerful tool in the world of C. elegans genetics. We have made some progress toward adapting the ZFN-based approach to this popular experimental organism. We have achieved high frequencies of targeted mutagenesis in somatic cells by induction of ZFNs, but it remains to translate this to the germline.
Our initial experiments made use of synthetic targets for a ZFN of known specificity. Both a heat-inducible gene for this protein, called QQR, and paired copies of its target were introduced into worms on an extrachromosomal array. After heat shock, sequence alterations were recovered (by PCR) in 26% of the targets. This showed that at least one ZFN could find and cleave its target in worm somatic cells.
We then designed a new pair of ZFNs to attack a genomic target, which we call Nowhere (Nw) because it is in an intergenic region, quite distant from any annotated gene. This site consists of two (different) ZF binding sites surrounding a recognition site for the restriction enzyme HindIII. It was chosen because of its ease of analysis after PCR - i.e., ZFN-induced sequence alterations would destroy the HindIII site. The heat-inducible ZFN genes were introduced on an array. Upon induction, altered targets were recovered at a frequency of 18%, showing that an endogenous genomic target could be cleaved by designed ZFNs with good efficiency.
With both the QQR and Nw targets, the altered products had sequence changes expected for inaccurate nonhomologous end joining (NHEJ) after cleavage. We explored this further by characterizing repair products from worms that lacked a key component of the NHEJ apparatus, DNA ligase IV. Altered targets were still recovered after ZFN induction, but at a much lower level. In addition, the nature of the products was altered, suggesting a greater reliance on homologous repair in the lig-4 worms and the presence of an alternative end-joining pathway. These conclusions are similar to ones drawn for other cells and organisms, and they show the power of ZFN-induced cleavage as a tool for studies of double-strand break repair, as well as for gene targeting.
Morton et al. (2006) PNAS 103: 16370-16375.
For more details, contact J. Morton.