targeted gene knockout by direct delivery of zinc - finger nuclease proteins

nature methods | ADVANCE ONLINE PUBLICATION | purify in quantities sufficient for analysis in cell culture (data not shown). Following these results, and based on the observation that ZFP DNA-binding domains carry a net positive charge (Fig. 1a), we hypothesized that ZFNs might penetrate the cell in the absence of additional modification. We expressed in Escherichia coli ZFNs designed to target the CCR5 gene and lacking any transduction domain and then purified them to homogeneity from either the soluble or the insoluble fractions (Supplementary Fig. 1). In vitro analysis confirmed that functional ZFN proteins with similar DNA cleavage profiles could be obtained by either method (Supplementary Fig. 2 and Supplementary Note). To determine the ability of ZFN proteins to penetrate cells and stimulate mutagenesis, we generated a fluorescence-based reporter system to measure ZFN-induced DSBs (Fig. 1b). This system uses an integrated EGFP gene whose expression has been interrupted by a frameshift mutation introduced by a strategically placed ZFN cleavage site. ZFN proteins that penetrate reporter cells can induce DSBs at this target site and drive the introduction of small insertions and deletions in the EGFP locus by NHEJ. Because NHEJ is a stochastic process, approximately one-third of these mutational events (+2, +5, +8,... bp or −1, −4, −7,... bp) will restore the frame and EGFP function. Direct application of ZFN proteins to reporter cells resulted in a dose-dependent increase in EGFP fluorescence, with maximum activity (6% EGFP-positive cells) achieved after treatment with 2 μM ZFN proteins (Fig. 1c). By comparison, transient transfection of ZFN expression plasmids under saturating conditions resulted in ~7% EGFP-positive cells (Supplementary Fig. 3). We observed no difference in activity between ZFN proteins purified from the soluble fraction or inclusion bodies (Supplementary Fig. 4). At all ZFN concentrations evaluated, the use of transient hypothermic culture conditions9 enhanced the efficiency of mutagenesis nearly twofold (Fig. 1c,d). Extended periods of incubation (>60 min) did not increase the frequency of genome editing (Supplementary Fig. 5). Consecutive protein treatments, however, did increase the percentage of EGFP-positive cells (Fig. 1d,e). Notably, repeated treatment with ZFN proteins over 3 d using transient hypothermic conditions yielded ~12% EGFP-positive cells (Fig. 1e). Sequence analysis of isolated EGFPpositive cells verified targeted mutagenesis, confirming the presence of the anticipated ZFN-induced insertions and deletions in the EGFP locus (Fig. 1f). To determine the contribution of each ZFN component to cellular penetration, we incubated cells with fluorescently labeled ZFN or FokI cleavage domain proteins (Supplementary Fig. 6). We observed fluorescence in cell lysate following treatment with ZFN—in the presence or absence of a nuclear localization targeted gene knockout by direct delivery of zincfinger nuclease proteins