Native homing endonucleases can target conserved genes in humans and in animal models

Abstract

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assays.

Tapping natural reservoirs of homing endonucleases for targeted gene modification

“…The concept of tapping the reservoir of natural diversity within the LHE family has been recently described (42) as an attractive alternative to extensive protein engineering. In that study, the tendency of LHEs (in particular, those associated with inteins) to tolerate base substitutions in their DNA target sites that correspond to degenerate or "wobble" positions in their host genes (which lead to synonymous or neutral mutations in the host gene product) was documented.…”

Genome editing and the next generation of antiviral therapy

“…Unlike other targeted endonuclease platforms, HEs use a single protein domain for target site binding and cleavage, and it was suggested that any off-target activity would be more limited and predictable because potential off-target sites need to be substrates for both binding and cleavage. Although off-target cleavage by HEs can been observed (Barzel et al 2011), the levels seen depend on the particular HE and in general tend to be low. Subsequently, newer HE platforms have been developed that increase enzyme specificity, and subsequently the levels of HE-mediated off-target cleavage can be significantly minimized (Boissel et al 2014; Wolfs et al 2014).…”

Targeted gene therapies: tools, applications, optimization

“…To date, hundreds of HEs have been identified in Archaea, Eubacteria and Eukarya and their viruses (Barzel et al ., 2011). HEs are classified into five families based on their conserved active site core motif.…”

“…To take advantage of the reservoir of natural diversity, the enormous public sequence databases were searched to create a library containing an estimated 416 unique target ranges for 684 HEs that can be freely accessed at http://homebase-search.tau.ac.il/ (Barzel et al ., 2011). Structural conservation permits well-studied HEs to serve as scaffolds onto which specificity determinants identified in such libraries may be grafted, thereby generating novel specificities.…”