The OPEN reagents and methods we describe in this report provide a rapid, highly effective, and publicly available platform for engineering zinc-finger arrays. With OPEN, we created 37 ZFN pairs which mediate highly efficient modification at four different sites within a chromosomally integrated EGFP reporter gene, six different sites within three endogenous human genes (VEGF-A, HoxB13, and CFTR), and one site within an endogenous plant gene (SuRA). The absolute rates of gene modification induced by our OPEN ZFNs ranged from 1 to 50%, and we were able to alter as many as two alleles in a single plant cell and four alleles in a single polyploid human cell.
We found OPEN to be more effective than previously described modular assembly approaches for making zinc-finger arrays. In our studies targeting the EGFP
gene, only two of 11 (~18%) modularly assembled ZFN pairs tested showed activity in human cells, in contrast to 15 of 20 (75%) OPEN ZFN pairs. In addition, at the one target site for which both methods were successful, OPEN ZFNs showed significantly more activity than modularly assembled ZFNs. The low efficacy rate observed with modular assembly is consistent with the results of a large-scale assessment of this method recently conducted by our groups (Ramirez et al., 2008
). The higher success rate of OPEN is likely attributable to its consideration of context-dependent effects on DNA-binding among neighboring zinc-fingers (Elrod-Erickson et al., 1996
; Isalan et al., 1997
; Wolfe et al., 2001
; Wolfe et al., 1999
) which are largely ignored by modular assembly.
With the set of zinc finger pools described in this report, we estimate that OPEN can be used to engineer three-finger proteins for ~4.1% ((23*21*22)/(64*64*64)) of all possible 9 bp target sites or ~0.16% (4.1%*4.1%) of all possible 18 bp ZFN sites. Because ZFNs can bind to sites in which two “half-sites” are separated by “spacer sequences” of five, six or seven base pairs (Bibikova et al., 2001
; Porteus and Baltimore, 2003
) (K. Wilson and M.H.P., unpublished data), one should be able to find approximately five full ZFN sites in any given kb of random sequence (0.0016 *1000* 3). Thus, with the pools described in this report, one should be able to target multiple ZFN sites within a typical size gene. Important goals for future work will be to generate additional finger pools that expand the targeting range of OPEN and to test whether the approach can also be used to generate arrays composed of more than three-fingers.
While using ZFNs to modify human genes, we observed two limitations that have not been emphasized in previous reports. First, not all zinc-finger arrays that possess sequence-specific DNA-binding activities (as measured in the well-established B2H method) will function as ZFNs in human cells. ZFNs for one of the four sites targeted in the HoxB13 locus, for three of the five sites targeted in the VEGF-A locus, and for one of the five sites targeted in an integrated EGFP reporter gene failed to induce mutagenic NHEJ repair in human cells. In addition, some ZFNs we made to the HoxB13 gene were active in 293 cells () but not in K562 cells (data not shown). We speculate that the transcriptional status and chromatin configuration of a target site may influence ZFN access to target sites: HoxB13 is transcriptionally active in 293 cells (data not shown) but bears chromatin marks consistent with a repressive state in K562 cells (B. Bernstein, personal communication). Additional studies will be needed to determine whether lack of ZFN activity results from chromatin effects on DNA accessibility or other reasons such as ZFN expression/stability or target site methylation. Second, although the use of vinblastine increased the frequency of gene targeting, DNA sequencing reveals that many alleles still underwent insertions or deletions caused by error-prone NHEJ and that some alleles underwent both a gene targeting event and an insertion. These findings demonstrate limitations in relying solely on PCR- or Southern blot-based assays and suggest that DNA sequencing should always be performed to verify ZFN-induced gene targeting events.
Because OPEN is rapid, reliable, and publicly available, it will foster wider usage and larger-scale applications of engineered zinc-finger technology. OPEN selections are performed in E. coli
and do not require specialized equipment. Although our ZFN validation experiments were performed in plant and human cells, OPEN should also be useful for generating ZFNs that function well in other organisms such as zebrafish, mosquitos, Drosophila
, and C. elegans
. The rapidity and effectiveness of OPEN should enable genome-scale ZFN projects (e.g. developing ZFNs for all human kinase genes or for every zebrafish gene). We note that this report doubles the number of endogenous mammalian genes described in the published literature -- from three (IL2Rg
, and DHFR
) (Lombardo et al., 2007
; Santiago et al., 2008
; Urnov et al., 2005
) to six -- that have been successfully modified using ZFNs. In conclusion, our publicly available OPEN platform will enable scientists to perform the research and development required to move ZFN technology forward for applications in biological research and gene therapy.