In this report, we describe a general and simple method for converting ZFNs into ZFNickases. Introduction of a previously described D450A mutation into one monomer of a ZFN pair can generate a ZFNickase. This result parallels recent work from Halford and colleagues in which they used a similar approach to convert the wild-type FokI restriction enzyme into a nickase. Our qualitative in vitro
data demonstrate that each ZFNickase preferentially cuts one DNA strand at a position either identical or within 1
nt of the cut positions of its matched ZFN. Furthermore, the data show that each ZFN monomer cuts the DNA strand to which it makes most of its DNA base contacts, providing direct experimental support for the model of binding and cleavage illustrated in . Testing in two different human cell-based reporter systems revealed that ZFNickases can induce HDR-mediated repair, albeit at lower levels than matched ZFNs from which they were derived. Of the eight ZFNickases we tested (two pairs each derived from ZFNs targeted to four different target sites; data presented in and ), six induced statistically significant levels of HDR. The levels of HDR we observed with the ZFNickases ranged widely, from between 2-fold and >100-fold lower than those observed with the corresponding ZFNs from which they were derived. However, for at least some of the ZFNickases we tested (e.g. HX735 −/+, VF2468 −/+ and +/− and CCR5 −/+ and +/−), the levels of HDR induced were of sufficiently high frequency (≥0.1%) to be useful for research applications and some potential therapeutic strategies. Our observations that ZFNickases can induce HDR events and that HDR efficiency is positively correlated with the concentration of donor present in cells are consistent with the findings of others using homing endonucleases engineered to induce nicks (22–27
). However, to our knowledge, our findings are the first to report that nickases derived from ZFNs can be used to induce HDR events.
Although absolute rates of HDR were lower for ZFNickases than ZFNs in our human cell-based reporter assays, we also observed a consistent reduction in mutagenic NHEJ rates in the TLR assay. This reduction is not entirely surprising given that nicks are typically repaired without causing mutations (17
). However, we do not know the origin of the residual NHEJ-mediated events we observed with some of the ZFNickases we tested. Possible explanations include conversion of a nick into a DSB that may occur with replication fork collapse (see below) or weak residual homodimerization of the active ZFNickase monomer that may lead to cleavage at the intended target site. Use of improved second-generation FokI heterodimer variants (3
) may reduce activity due to the latter mechanism [we used first-generation FokI heterodimer variants (4
) for this study].
Importantly, for five of the six ZFNickases we tested in the TLR assay, the ratio of HDR to NHEJ events was increased compared with the three matched ZFNs from which they were derived. These results demonstrate that ZFNickases can induce HDR events with relatively lower rates of NHEJ-mediated mutations created at the nick site. We do not currently know the mechanism of the ZFNickase-mediated HDR or the improved HDR:NHEJ ratios we observe. One possibility for the improved HDR:NHEJ ratios is that a nick in the path of a DNA replication fork may be converted to a DSB leading to fork collapse, the repair of which would be expected to lead to repair by either NHEJ or HDR. A potential hypothesis for why we observe a preferential shift from NHEJ to HDR with ZFNickases may be the more frequent repair of nick-induced replication fork collapse by HDR (41
), in part due to the availability of repair factors for homologous recombination during DNA replication in S-phase (42
). Interestingly, for every target site we tested in our human cell-based assays, one ZFNickase combination consistently outperformed the other with respect to absolute HDR rates and, for those assayed using the TLR assay, improved HDR:NHEJ ratios. This reproducible difference does not appear to be correlated with whether the nicked strand is transcribed or not, and there were no strand cleavage preferences discernible from the in vitro
data. It is possible that strand-dependent differences in HDR activity arise due to different DNA-binding affinities of zinc finger domains in each monomer and how this may affect asymmetric accessibility to the break by cellular repair machinery. Regardless of the precise mechanism, our results suggest that testing both potential ZFNickases for a given target site is worthwhile to identify the most active nickase possible.
Our work demonstrates that ZFNickases with predictable strand nicking activities can be easily derived from ZFNs and that these enzymes can be used in cells to induce HDR with improved HDR:NHEJ ratios. It will be of interest in future experiments to test whether ZFNickase-induced HDR rates can be further increased by using improved FokI heterodimer frameworks and hyperactive FokI variants (3
). Our observation of reduced mutagenic NHEJ events at the target nicking site suggest that ZFNickases will also likely induce fewer mutations at potential off-target sites elsewhere in the genome, a prediction that can easily be tested for ZFNs with known off-target sites (15
). In addition, site-specific nickases may generally be of interest for the study of biological phenomena such as replication fork dynamics. Our results suggest ZFNickases may provide a means to induce HDR with reduced mutagenesis caused by NHEJ and that additional optimization of this platform should be an important goal for future investigation.