New ‘open’ method for targeted gene modifications

29 July 2008

Zinc-fingers are small DNA-binding proteins that mediate DNA-protein interactions within cells, playing an important role in regulation of DNA expression. Zinc-finger nucleases (ZFNs) are synthetic enzymes that combine zinc-finger peptide domains (which target the protein to attach to specific DNA sequences) and a nuclease domain that will cut double-stranded DNA at the point where the protein is bound. Importantly, ZFNs can be used to create specific genomic modifications via these sequence-specific DNA breaks, encouraging the insertion of new DNA sequences or targeting the region for inactivation or repair via normal cellular mechanisms.

Research teams from six different US and German institutions, all members of the international Zinc Finger Consortium, have now published a new and efficient method for producing ZFNs in the journal Molecular Cell. The new technique, which they have dubbed OPEN (for Oligomerized Pool Engineering) is significantly more efficient than current methods, as well as being much easier and faster to perform [Maeder ML et al. (2008) Mol. Cell 31, 294-301, doi:10.1016/j.molcel.2008.06.016]. Target DNA sequences are used to select ZFNs from an archive of collections (or ‘pools’) of different zinc fingers. The method was used to produce ZFNs that were able to introduce modifications at sites in three different human genes (VEGF-A, HoxB13, and CFTR; mutations in the latter are associated with cystic fibrosis) within human cells, as well as a plant gene and an isolated human EGFP ‘reporter’ gene, with efficiencies ranging from 1%–50%. However, the paper reports that the desired genetic modifications were not introduced at some sites for which ZFNs were produced. It is suggested that this may have been the result of the state of the DNA at a target site (for example, active/unwound or inactive/supercoiled) or the stability of the ZFNs in the cell.

The researchers are now working to create more ‘pools’ of different ZFNs. Crucially, they are making them publicly available via non profit-making distributors for a modest charge, so that other researchers will be able to create special targeted zinc finger nucleases by the new method. Previously, methods for constructing ZFNs have been hugely labour-intensive, and the primary commercial provider has made them available only for selected academic collaborative projects; although they reportedly plan to offer a service to produce custom-made ZFNs, the expected charges will be very high (see Science news).

It is hoped that the availability of this novel method will facilitate all sorts of genome-based research, including the development of gene therapies directed against the mutations that cause single-gene diseases such as cystic fibrosis; the research was supported by the Cystic Fibrosis Foundation, among other bodies.Senior author J. Keith Joung of the Massachusetts General Hospital in Boston commented: “Our method will enable academic researchers to rapidly create high quality ZFNs for genes of interest and will stimulate use of this technology in biological research and potentially gene therapy” (see press release).

Comment: Research news is often dominated by discoveries or achievements, but new and effective methods can have the greatest impact of all if they facilitate rapid progress in many different areas of research simultaneously, as this latest technique may do for genomic research.

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