Genome Engineering With Zinc-Finger Nucleases
Abstract
Zinc-finger nucleases (ZFNs) are targetable DNA cleavage reagents that have been adopted as gene-targeting tools. ZFN-induced double-strand breaks are subject to cellular DNA repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies. This article briefly reviews the history of ZFN development and summarizes applications that have been made to genome editing in many different organisms and situations. Considerable progress has been made in methods for deriving zinc-finger sets for new genomic targets, but approaches to design and selection are still being perfected. An issue that needs more attention is the extent to which available mechanisms of double-strand break repair limit the scope and utility of ZFN-initiated events. The bright prospects for future applications of ZFNs, including human gene therapy, are discussed.
Genetics is driven by the ability to connect genotype with phenotype. The classical approach is to identify a novel phenotype, whether occurring spontaneously or derived by mutagenesis, to identify the responsible gene(s) and to discover why mutations at that locus have the observed effect. A more modern approach, sometimes called reverse genetics, is to identify a gene from a genomic sequence to make mutations specifically in that gene and to characterize the resulting phenotype.
Two types of gene-specific manipulations can be envisioned (Figure 1). In one, which we can call “targeted gene replacement,” the goal is to make localized sequence changes, often ones that will create a null mutation. In targeted gene replacement, the goal is to replace an existing sequence with one designed in the laboratory. The latter allows the introduction of both more subtle and more extensive alterations.
Illustration of two types of genome engineering. In the top portion, the horizontal line represents a genome segment, and the open rectangles, two individual genes. The jagged arrow on the left indicates an unspecified mutagenic agent targeted to one gene. The shaded rectangle on the right is a manipulated version of the second gene that has been supplied by the experimenter. The outcomes below are targeted mutagenesis, resulting in a localized sequence alteration (“x”), and targeted gene replacement, produced by homologous recombination between the original and exogenous gene copies.
Making directed genetic changes is often called “gene targeting.” It sounds simple enough, but targeting a single gene within a large genome presents a substantial challenge. Procedures for gene replacement in baker’s yeast, Saccharomyces cerevisiae, have been available for several decades (Scherer and Davis 1979; Rothstein 1983). Success in this case depends on several features: the ability to manipulate segments of yeast DNA in the laboratory, the ability to introduce DNA into yeast cells, interaction between donor and target DNA by homologous recombination, the near absence of competing reactions that would integrate the donor into alternative sites in the genome, and the ability to apply strong selection for the desired product. These properties are shared by some other fungi and many bacteria, but not by the majority of eukaryotic organisms.
Making targeted gene replacements has also become standard practice in mice, thanks to the availability of embryonic stem (ES) cells that can be manipulated in culture and the development of powerful selection procedures (Capecchi 2005). Like targeting in yeast, the process in mice depends on homologous recombination between the donor and the target. In addition, selection must be applied against the more common products of random integration. This is accomplished by placing a positive selectable marker inside the donor homology and a negative selectable marker outside the homology (Mansour et al. 1988). Double selection yields the desired replacements, and the pluripotency of the ES cells allows them to populate all cell lineages after injection into early embryos.
In both yeast and mouse cells, the absolute frequency of homologous recombination between donor and target sequences is quite low—on the order of one in every 10 to 10 cells. Selection in culture allows the recovery of the rare cells that have enjoyed the desired event. With other experimental organisms, ES cells are not available, screening or selection procedures are not adequate, and development of useful gene-targeting approaches is impeded by the low frequency of recombination.
TM refers to targeted mutagenesis by nonhomologous end joining TGR is targeted gene replacement by homologous recombination. In addition to the examples shown here, I have heard reliable, but unpublished, reports of successful ZFN-induced targeting in several other organisms. The list of references is not exhaustive, but provides guidance to key publications.
Acknowledgments
I am grateful to the people who have worked with me over the years on ZFNs, including those who, through collaboration, have opened the door to organisms previously unfamiliar to me. I also acknowledge the outstanding scientists working on ZFN development and applications, in both the public and private sectors, who have pushed the technology forward and freely shared their progress with me. Research in my lab has been supported by National Institutes of Health awards GM58504 and {"type":"entrez-nucleotide","attrs":{"text":"GM078571","term_id":"221388715","term_text":"GM078571"}}GM078571, by a contract from Dow AgroSciences, and by core facilities funded in part by the University of Utah Cancer Center Support Grant.