CCR5 is the major co-receptor used by HIV-1 and individuals homozygous for a 32bp deletion in CCR5 are profoundly resistant to HIV-1 infection. Using engineered zinc finger nucleases (ZFNs), we were able to disrupt the CCR5 gene in human hematopoietic stem/progenitor cells (HSC) at a mean frequency of 17% of total alleles in a population. This procedure produces both mono and bi-allelically disrupted cells. ZFN-treated HSC retained the ability to engraft NOD/SCID/IL2rγnull mice and gave rise to polyclonal multi-lineage progeny with the CCR5 gene permanently disrupted. Control mice receiving untreated HSC and challenged with CCR5-tropic HIV-1 displayed profound CD4+ T cell loss. In contrast, mice transplanted with ZFN-modified HSC underwent rapid selection for CCR5-negative cells, had significantly lower HIV-1 levels and preserved human cells throughout their tissues. The demonstration that a minority of CCR5-modified HSC can populate an infected animal with HIV-1-resistant, CCR5-negative progeny suggests the use of ZFN-modified autologous HSC as a clinical approach to treating HIV-1.

Zinc-finger nucleases (ZFNs) allow gene editing in live cells by inducing a targeted DNA double-strand break (DSB) at a specific genomic locus. However, strategies for characterizing the genome-wide specificity of ZFNs remain limited. We show that nonhomologous end-joining captures integrase-defective lentiviral vectors at DSBs, tagging these transient events. Genome-wide integration site analysis mapped the actual in vivo cleavage activity of four ZFN pairs targeting CCR5 or IL2RG. Ranking loci with repeatedly detectable nuclease activity by deep-sequencing allowed us to monitor the degree of ZFN specificity in vivo at these positions. Cleavage required binding of ZFNs in specific spatial arrangements on DNA bearing high homology to the intended target site and only tolerated mismatches at individual positions of the ZFN binding sites. Whereas the consensus binding sequence derived in vivo closely matched that obtained in biochemical experiments, the ranking of in vivo cleavage sites could not be predicted in silico. Comprehensive mapping of ZFN activity in vivo will facilitate the broad application of these reagents in translational research.

Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome. Subsequent repair of this break via the nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene addition, respectively. Here, we report that ZFNs can be engineered to induce a site-specific DNA single-strand break (SSB) or nick. Using the CCR5-specific ZFNs as a model system, we show that introduction of a nick at this target site stimulates gene addition using a homologous donor template but fails to induce significant levels of the small insertions and deletions (indels) characteristic of repair via NHEJ. Gene addition by these CCR5-targeted zinc finger nickases (ZFNickases) occurs in both transformed and primary human cells at efficiencies of up to ∼1%–8%. Interestingly, ZFNickases targeting the AAVS1 “safe harbor” locus revealed similar in vitro nicking activity, a marked reduction of indels characteristic of NHEJ, but stimulated far lower levels of gene addition—suggesting that other, yet to be identified mediators of nick-induced gene targeting exist. Introduction of site-specific nicks at distinct endogenous loci provide an important tool for the study of DNA repair. Moreover, the potential for a SSB to direct repair pathway choice (i.e., HDR but not NHEJ) may prove advantageous for certain therapeutic applications such as the targeted correction of human disease-causing mutations.

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