Genome editing with engineered zinc finger nucleases

Abstract

Targeted genetic engineering in many important model systems and in human tissue culture cells has historically been challenging. This has changed dramatically over the past 5 years with the development of zinc finger nuclease (ZFN) technology. A ZFN is an artificial endonuclease that consists of a designed zinc finger protein (ZFP) fused to the cleavage domain of the FokI restriction enzyme. A ZFN may be redesigned to cleave new targets by developing ZFPs with new sequence specificities. For genome engineering, a ZFN is targeted to cleave a chosen genomic sequence. The cleavage event induced by the ZFN provokes cellular repair processes that in turn mediate efficient modification of the targeted locus. If the ZFN-induced cleavage event is resolved via non-homologous end joining, this can result in small deletions or insertions, effectively leading to gene knockout. This approach has now been used to establish facile and efficient reverse genetics (that is, reverse genetics that does not require selection) in Drosophila melanogaster, zebrafish, rats, Arabidopsis thaliana and mammalian somatic cells. If the break is resolved via a homology-based process in the presence of an investigator-provided donor, small changes or entire transgenes can be transferred, often without selection, into the chromosome; this is referred to as 'gene correction' and 'gene addition', respectively. This approach has been used to make novel alleles in D. melanogaster, mammalian cells and tobacco, and has been used to drive targeted integration in maize, tobacco and human embryonic stem and induced pluripotent stem cells. Therapeutic application of ZFN technology requires the engineering of ZFNs that are highly specific in their action. Three clinical trials with ZFNs are underway, including one in which T cells are isolated from a patient infected with HIV, treated with ZFNs that disrupt the chemokine (C-C motif) receptor type 5 (CCR5) gene to make them resistant to virus infection, and transferred back to the patient.