Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in ‘all-in-one’ lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.
Altering the genetic code of a living organism to produce certain desirable outcomes is the goal of genetic engineering. The field builds on a long history of human attempts to alter genetics, from selective breeding of crops and livestock to genetically modified organisms and gene therapies. Researchers routinely use gene editing to create ‘knock-out’ mice in which a particular gene is turned off: the researchers can learn more about the function of this gene by watching what happens when it is absent.
As gene editing techniques have grown more sophisticated, they have become an increasingly promising tool for treating diseases that are caused by gene mutations. The aim of this work is to replace faulty genes with genes that work properly. However, it has been difficult to adapt genetic engineering techniques so that they can be used safely in humans.
Scientists have created customized enzymes called nucleases that can remove specific genes, but it has been a challenge to get these nucleases into cells in the first place. A virus can be used to deliver the genes that encode these nucleases into the DNA of a cell, but this approach can lead to the production of too many nucleases and to the removal of more genes than intended.
Now Cai et al. have developed a ‘hit-and-run’ method for getting the nucleases into cells and making them active only for a short period of time. This method involves using a virus to deliver two different nucleases to a cell. Once inside the cell, the viruses released the nucleases, which were able to remove up to one-quarter of their gene targets, with relatively few errors, in the time that they were active.
Next, Cai et al. added gene patches—new genes to replace those removed by the nucleases—to the viruses. This ‘cut and patch’ strategy was successful in up to 8% of the treated cells. The results also suggest that this approach is safer than other gene-editing techniques.