Cystic fibrosis (CF) affects 1 in 4000 individuals. Most CF patients have the ΔF508 mutation in the cftr gene which disrupts an apical membrane chloride channel. Virus vector-mediated gene replacement therapy has not succeeded. Attempts to repair, rather than replace the defective gene, have been thwarted by the extremely low efficiency of homologous recombination (1 in 100.000 cells). In 2005, Urnov and colleagues designed a pair of zinc finger nucleases (ZFNs) to induce a double stranded break at a unique genomic site, and showed that co-transfection of the ZFNs with a donor repair sequence resulted in homology-directed repair (HDR) of a target allele in up to 20% of transfected cells (1). This level of HDR has been independently confirmed (2), and the system can be delivered by virus vectors (3). We describe a pair of ZFNs (ZFN1 and ZFN2) that target the cftr gene, as a first step towards cftr gene correction. Two ZF proteins were designed and synthesised to independently recognise two 9 bp sequences, separated by a 4 bp spacer. The combined sequences corresponds to a 22 bp region of intron 9 in the cftr gene, close to the ΔF508 site. The DNA sequence of each ZF protein was fused inframe with the nuclease domain of FokI (4) in pcDNA 3.1+ (InVitrogen). Recombinant proteins (ZFN1 and ZFN2) were produced in a coupled transcription-translation reaction (Promega). Two 9 bp target sequences (t1 and t2) were assembled together and cloned into pGL3 (Promega) to mimic the cftr gene sequence (pTarget1/2), or duplicated separately (pTarget1/1 and pTarget 2/2) to test efficacy and specificity of individual ZFNs. Target DNA (1000ng) was mixed with 10, 50 and 100ng ZFN protein and incubated for 1 hr at 37°C in enzyme buffer #2 (New England Biolabs). ZFN1 and ZFN2 together linearised pTarget1/2 at all amounts tested. ZFN2 alone cleaved pTarget2/2 but not pTarget1/1. In contrast, ZFN1 alone cleaved pTarget1/1, but not pTarget2/2. Thus, initial data suggested the ZFNs were behaving as predicted. However, some non-specific cleavage was observed when high levels of ZFN were used: 100ng ZFN1 could partially cleave pTarget1/2. Recent studies have suggested that non-specific binding and cleavage can be prevented by mutation of the nuclease domains (5). Introduction of these mutations (QuikChange site-directed mutagenesis; Stratagene) into ZFN1 (to create ZFN1⁺⁺) and ZFN2 (to create ZFN2^—) prevented non-specific cleavage. In summary, a pair of ZFNs can specifically bind and cleave an 18 bp sequence in the cftr gene close to the ΔF508 site. They will be used with a suitable donor sequence to determine the efficiency of ZFN-mediated homology directed repair in a ΔF508^-/- cell line.