A potential route for introducing precise changes into the genome was suggested by the discovery of homologous recombination and its application in creating transgenic and knockout mice, for which the Nobel Prize in Physiology or Medicine was awarded in 2007. This method, however, was only efficient in mouse embryonic stem cells and not immediately applicable to cells from other species or even other mouse cell types.
But in 1994 a breakthrough study showed that creating a double-strand break at a specific site in the genome could stimulate the cellular DNA repair pathway and increase the frequency of homologous recombination at the break point by many orders of magnitude. Critically, this study also suggested a direct approach for editing virtually any gene sequence in many different cell types and species using homologous recombination—but only if DNA breaks could be targeted to specific sequences.
The re-engineering of molecular machinery to recognize new sequences in complex genomes is a daunting task. However, in 1991, the crystal structure of Zif268, a naturally occurring zinc finger protein, provided insight into how Nature solved this problem. This structure led to the discovery that zinc finger proteins, among the most common class of DNA-binding proteins across all domains of life, recognize DNA using independent and modular domains that make specific contacts with three base pairs of DNA (Figure 1). This work suggested that these domains could be redesigned to recognize new base pair combinations and linked together to form new proteins.
Subsequent research by several laboratories led to the development of technologies for making custom synthetic zinc finger proteins that can be targeted to a broad range of sites in almost any genome. This constituted the first technology for targeting and regulating specific endogenous genes.
In the late-1990s, the catalytic domain of the FokI endonuclease, which nonspecifically cleaves DNA, was fused to custom zinc finger proteins to generate the first zinc finger nuclease (ZFN). Because the DNA-binding specificity of zinc finger proteins could be reprogrammed, new ZFNs could be rapidly formed and used to introduce targeted double-strand breaks to almost any gene in the genome.
Critically, because FokI acts as a dimer, it’s necessary to engineer two ZFNs that target opposite strands of DNA in a head-to-head configuration (Figure 1). Thus, when two FokI catalytic domains assemble together at the targeted DNA site, a double-strand break is created and genome editing is initiated.