Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells

Abstract

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting—the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys2His2) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for ‘directed mutagenesis’ and targeted ‘gene editing’ of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.

What history tells us XL. The success story of the expression ‘genome editing’

“…'Genome editing' with this new meaning was first used at the beginning of the 2000s (Balbas and Gosset 2001;Stark and Akoplan 2003;Gruenert et al 2003), and the expression progressively became more widespread in subsequent years. The main researchers involved in the development of these new tools, such as Dana Carroll and Srinivasan Chandrasegaran (Durai et al 2005;Carroll 2008), were far from being the strongest supporters of the new vocabulary.…”

Transformation as a Tool for Genetic Analysis in Populus

“…ZFNs have been developed to: (i) increase the extremely low rate of homologous gene targeting (GT) in plants and most mammalian systems (excluding mice) and (ii) introduce mutations in a target locus by the imperfect repair of the cleaved target DNA by the ZFNs (Durai et al, 2005). ZFNs are synthetic proteins that can introduce double-stranded breaks (DSB) in DNA.…”

“…ZFNs are synthetic proteins that can introduce double-stranded breaks (DSB) in DNA. The protein consist of a DNA binding domain (DBD) composed of 3-4 (Cis2-His2) zinc fingers fused to the DNA-cleavage domain of a FokI restriction endonuclease (Durai et al, 2005). The zinc fingers provide targeting and binding to a particular specific sequence, and the cleavage domain introduces a DSB.…”

“…The process of matching finger-to-sequence is still imperfect, and two approaches are widely used. One is based on a selection process, where different zinc finger combinations are screened for affinity to target sequence via library (phage or bacterial two-hybrid system) screening procedures (Durai et al, 2005). An alternative strategy is using computer-generated matches that use a domain library (http://www.scripps.edu/mb/barbas/zfdesign/zfdesignhome.php).…”

Targeted genetic modification of cell lines for recombinant protein production

“…The discovery that HR could be greatly enhanced by introducing a DSB at the target locus prompted an interest in creating chimeric enzymes that could do so in a sequence specific manner (Durai et al 2005). Indeed the ability to target other enzymatic or functional activities to predetermined sites has led to the emergence of a specialist field in designer zinc finger (ZF) proteins (Mandell and Barbas 3rd 2006).…”