The off-target effects of a genome-editing technology called zinc-finger nucleases (ZFNs) are reported online this week in Nature Biotechnology and Nature Methods. Both studies provide insights into the precision of this tool.
ZFNs are enzymes that can make modifications at selected sites in the genome and are therefore promising tools for both basic research and gene therapy. But precision, in such a tool, is everything. Off-target effects, where the ZFN modifies a genomic position other than the one it is designed to target, can cause problems for many applications but have not been studied experimentally across the entire genome.
Two independent studies describe methods to carry out such experimental tests and report their findings. In Nature Methods, David Liu and colleagues use an in vitro, deep sequencing-based assay to study the cleavage specificities of two ZFN pairs. They observe cleavage of off-target sequences that are present in the human genome and see evidence that these sites are cleaved when the ZFN pairs are expressed in cells.
In Nature Biotechnology, Luigi Naldini, Christof von Kalle and colleagues identify the cleavage sites of four ZFN pairs by using a virus that integrates into the genome where breaks have occurred. Although most cuts are at the expected locations, the team notes that off-target events are found at other locations that were not predicted by computational methods.
Whereas both studies focus on a ZFN that is in clinical trials, neither is done in the clinically relevant cell type. The results of such analyses are likely to vary with cell type and with different expression levels of the ZFN. These reports strongly indicate that additional study of ZFN off-target effects in clinically relevant cells is warranted.