Targeted genome engineering via zinc finger nucleases

Abstract

With the development of next-generation sequencing technology, ever-expanding databases of genetic information from various organisms are available to researchers. However, our ability to study the biological meaning of genetic information and to apply our genetic knowledge to produce genetically modified crops and animals is limited, largely due to the lack of molecular tools to manipulate genomes. Recently, targeted cleavage of the genome using engineered DNA scissors called zinc finger nucleases (ZFNs) has successfully supported the precise manipulation of genetic information in various cells, animals, and plants. In this review, we will discuss the development and applications of ZFN technology for genome engineering and highlight recent reports on its use in plants.

Fig. 1

Structure of ZFNs. Schematic representation of ZFNs bound to their target sites. These nucleases are created by fusing an engineered ZF DNA binding domain to the DNA cleavage domain of the FokI type IIS restriction enzyme. An array of 3 ZFs (shaded ovals) for each ZFN protein will recognize a target sequence on DNA (shaded boxes). The binding of two ZFNs on the target DNA with a spacer (5/6 bp) in the middle will trigger the dimerization of the ZFN nuclease domains and the cleavage of the target DNA

Fig. 2

ZFN-mediated targeted genome engineering via NHEJ. a Gene knockout and DNA insertion. A DSB created by a pair of ZFNs stimulates the error-prone nonhomologous end joining (NHEJ) pathway, and the target gene can be disrupted by small indels induced by NHEJ repair (left pathway). Small DNA fragments delivered into the cell can be captured into the target site of a DSB induced by ZFNs via NHEJ (right pathway). b Gene deletion. The NHEJ repair of two concurrent DSBs flanking a target gene is induced by two ZFN pairs, which can give rise to targeted deletions of genes between the two sites

Fig. 3

ZFN-mediated targeted genome engineering via HR. The repair of ZFN-induced DSBs via the HR pathway can be exploited to induce gene correction, gene knockout, and gene addition using properly designed donor DNA