Abstract
Genome editing for therapeutic applications often requires cleavage within a narrow sequence window. Here, to enable such high-precision targeting with zinc-finger nucleases (ZFNs), we have developed an expanded set of architectures that collectively increase the configurational options available for design by a factor of 64. These new architectures feature the functional attachment of the FokI cleavage domain to the amino terminus of one or both zinc-finger proteins (ZFPs) in the ZFN dimer, as well as the option to skip bases between the target triplets of otherwise adjacent fingers in each zinc-finger array. Using our new architectures, we demonstrate targeting of an arbitrarily chosen 28 bp genomic locus at a density that approaches 1.0 (i.e., efficient ZFNs available for targeting almost every base step). We show that these new architectures may be used for targeting three loci of therapeutic significance with a high degree of precision, efficiency, and specificity.
Highlights
Genome editing for therapeutic applications often requires cleavage within a narrow sequence window
A key motivation for these studies was the realization that a modest amount of diversification in the linkages between the zinc-finger proteins (ZFPs) and FokI cleavage domain, as well as between adjacent fingers, could yield a large increase in the number of distinct zinc-finger arrays enabling cleavage at a chosen genomic site
The option to insert such linkers would allow for exploration of diverse alternative ZFPs, to identify the bestperforming zinc-finger nucleases (ZFNs) pairs for any given target
Summary
Genome editing for therapeutic applications often requires cleavage within a narrow sequence window To enable such high-precision targeting with zinc-finger nucleases (ZFNs), we have developed an expanded set of architectures that collectively increase the configurational options available for design by a factor of 64. Even strategies that target larger regions, such as open reading frames, can be constrained by factors such as the need to cleave a common exon among multiple splice variants, or to avoid cleaving highly similar pseudogenes or homologs Given these considerations, fully realizing the promise of therapeutic genome engineering will require a high degree of “targeting precision”, i.e., the ability to design a nuclease for efficient cleavage at any chosen base position. The generation of ZFNs for a chosen target has typically been accomplished via modular assembly[31] of one- and two-finger units with pre-characterized DNA-binding preferences[32,33]
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