Target Practice ZFNs, which are conjugates of zinc finger peptides (yellow spheres) with a nuclease (blue sphere), interact in pairs (top) with specific sequences of genomic DNA (parallel lines). Off-target interactions also occur, but with lower affinity because of sequence mismatches (bottom, red).

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Researchers have devised the first techniques that directly assess “off-target effects” of gene-cutting zinc finger nucleases (ZFNs). The work could lead to ZFNs that more precisely target specific genes for use in gene therapy or as research tools.

ZFNs are designed zinc-containing protein-nuclease conjugates that act in pairs to bind adjacent sequences on genomic DNA and cut between them. The cuts can be repaired with specific DNA bases to modify genes or control gene expression. Sangamo BioSciences, in Richmond, Calif., dominates ZFN commercialization and has a ZFN in clinical trials for HIV and AIDS.

Although ZFN pairs are intended to recognize unique genomic sequences, they sometimes bind off target. The off-target genomic sequences could previously be identified only indirectly, such as by analyzing ZFNs’ binding specificity.

Now, two groups have devised direct ways to identify off-target genomic sequences that ZFNs cleave. Chemical biologist David R. Liu of Harvard University, ZFN guru J. Keith Joung of Massachusetts General Hospital, and coworkers tested DNA libraries containing all possible DNA sequences seven or fewer mutations away from ZFN target sequences for off-target cleavages and then confirmed those cuts in living cells (Nat. Meth., DOI: 10.1038/nmeth.1670).

And Luigi Naldini of the San Raffaele Telethon Institute for Gene Therapy, in Milan, Italy; Christof von Kalle of the National Center for Tumor Diseases, in Heidelberg, Germany; and coworkers used viruses that tag ZFN-induced DNA breaks to identify ZFN off-target cuts in living cells (Nat. Biotechnol., DOI: 10.1038/nbt.1948).

Both studies identified off-target breaks that were not previously known. Therefore, drug developers “should proceed with caution—but still, I would argue, proceed,” Liu says.

“There could have been off-target effects at huge numbers of genomic sites, but it turns out that there are relatively few,” says gene regulation specialist Scot Wolfe of the University of Massachusetts Medical School. The studies “suggest that there is great potential for ZFNs as therapeutic agents to correct single-gene disorders. The question is now how to tune things to maximize on-target and minimize off-target effects.”

One idea, Liu suggests, would be to design ZFNs with intentional sequence mismatches that don’t coincide with any off-target sites.

Ultimately, “one could envision sequencing entire genomes and comparing those” to find off-target ZFN-induced genome modifications even more definitively, comments ZFN specialist David Segal of the University of California, Davis. But that would take considerable time and effort, and the new techniques represent significant advances that are more practical today, he says.