28 | VOL.9 NO.1 | JANUARY 2012 | NATURE METHODS SPECIAL FEATURE | COMMENTARY METHOD OF THE YEAR Moira A. McMahon, Meghdad Rahdar and Matthew Porteus are at Stanford University, Stanford, California, USA. e-mail: mporteus@stanford.edu the most striking results so far have been obtained with ZFNs, genome editing is pos- sible with all three nuclease types. It is pos- sible that TALENs will supplant ZFNs as the genome-editing tool of choice, because they are easier than ZFNs to assemble and have near-equal activity. But ZFNs and modified homing endonucleases will probably con- tinue to have an important role as well. Targeted gene mutation Gene disruption or knockdown is crucial for understanding proteins and pathways of interest (Fig. 1). In mammalian cells, gene disruption is now typically done using short interfering RNA (siRNA) 10,11 . But siRNA knockdown can be incomplete and variable, and one must control for off-target effects. Engineered nucleases Gene editing: not just for translation anymore Moira A McMahon, Meghdad Rahdar & Matthew Porteus Engineered nucleases have advanced the field of gene therapy with the promise of targeted genome modification as a treatment for human diseases. Here we discuss why engineered nucleases are an exciting research tool for gene editing and consider their applications to a range of biological questions. Precise genome editing is a powerful tool for studying biological processes. Yeast, for example, has been an incredibly use- ful system in part because researchers are able to precisely and efficiently change its genome by homologous recombination. Gene targeting by homologous recombina- tion in mouse embryonic stem cells, in turn, revolutionized mouse genetics, as was rec- ognized by the awarding of the Nobel Prize in Medicine to Mario Capecchi and Oliver Smithies for developing this technique. But precise genome editing in other systems has been limited, primarily by its low efficiency. This is now changing. Driven in part by clinical interest, efficient genome editing has been developed for a broader range of experimental systems over the past decade and is now poised to become a standard experimental strategy for gene manipula- tion in the research laboratory. Development of gene editing Genome editing in mammalian cells was greatly enhanced by the discovery, made by Jasin and her colleagues, that a gene- specific double-stranded break (DSB) in DNA could stimulate gene targeting by homologous recombination by at least three orders of magnitude in mammalian cells 1 . The researchers used the I-SceI hom- ing endonuclease in their work, but engi- neered nucleases have a similar effect 2 . This tremendous stimulation in gene targeting results from harnessing the cell’s natural homologous recombination machinery to repair DSBs. Carroll and his colleagues demonstrated that engineered nucleases could generate mutations and disrupt genes 3 by using the cell’s own mutagen- esis-prone nonhomologous end-joining (NHEJ) repair pathway. So, the develop- ment of gene editing has been the result of using engineered nucleases to create DSBs at desired locations in the genome in con- junction with harnessing the cell’s endog- enous mechanisms to repair the induced break. In principle, both of these cellular- repair pathways (homologous recombi- nation and NHEJ) could be exploited to alter almost any gene of interest. There are now three different platforms for creating designer nucleases: re-engineered hom- ing endonucleases 4 , zinc-finger nucleases (ZFNs) 2,5 and transcription activator–like effector nucleases (TALENs) 6–9 . Although Chromosome + single engineered nuclease pair Gene-specific double-strand break Tag Mutation or small change Endogenous tag addition Gene addition Promoter mutation Cell lines Fertilized oocyte Transgenic animals Primary cells Promoter addition Figure 1 | Genome editing using a single pair of engineered nucleases. An array of genome modifications can be designed to result after creating a double-strand break. Gene editing can be done in cell lines, in primary cells (including somatic and pluripotent stem cells) and in fertilized oocytes for the generation of transgenic animals. © 2012 Nature America, Inc. All rights reserved.