Targeted gene knockout by direct delivery of zinc-finger nuclease proteins

Zinc-finger nucleases (ZFNs) are versatile reagents that have redefined genome engineering. Realizing the full potential of this technology requires the development of safe and effective methods for delivering ZFNs into cells. We demonstrate the intrinsic cell-penetrating capabilities of the standard ZFN architecture and show that direct delivery of ZFNs as proteins leads to efficient endogenous gene disruption in a variety of mammalian cell types with minimal off-target effects. Zinc-finger nucleases (ZFNs) are fusions of the non-specific cleavage domain from the FokI restriction endonuclease with custom-designed Cys2-His2 zinc-finger proteins (ZFPs)1. These chimeric nucleases induce sequence-specific DNA double-strand breaks (DSBs) that can be repaired by error-prone non-homologous end joining (NHEJ) to yield small alterations at targeted genomic loci. This strategy has enabled highly efficient gene disruption in numerous cell types2, 3 and model organisms4, 5 and has facilitated the progress of targeted gene therapy in humans6, 7. Despite these advances and more recent methodological improvements8–10, there remains a need for new methods that can improv