A New TALE for Targeting Genes

In addition to its widely-anticipated potential to provide highly-effective therapies for genetic disorders, somatic gene therapy is an essential enabling technology for the repair or obviation of several  of the cellular and molecular lesions driving age-related disease and dysfunction (notably the accumulations of mutations in mitochondrial and nuclear DNA). One of the most promising routes to somatic gene therapy is zinc finger nucleases (ZFNs),  engineered DNA-binding proteins consisting of a FokI restriction enzyme catalytic core bookmarked into a dimer of zinc finger array DNA binding domains. The choice of zinc finger domains allows the engineer to target twinned 9-18 base-pair sequences in the recipient genome, separated from each other by a (typically) 5-7 base pair spacer. Upon binding, the restriction enzyme dimerizes, creating a double-strand break at the spacer locus; the engineer then takes advantage of the native DNA repair machinery to insert an user-supplied DNA repair template through Non-Homologous End Joining (NHEJ).

One significant barrier to the engineering of specific ZFNs has been the difficulty in engineering new target sites within the host genome. To facilitate the  targeting of new sites, a collaboration between Drs. Dan Voytas of the University of Minnesota Center for Genome Engineering and Adam J. Bogdanove of the Department of Plant Pathology at Iowa State University, engineered a new system for the targeted introduction of DSBs in host genomes by inserting the ZFN endonuclease into the homing system used by  transcription activator-like effectors (TALEs), a family of plant pathogen virulence factors. TALEs bind to host-cell DNA and transcriptionally activate host genes that enhance pathogen virulence (or, in some cases, host defense). As Dr. Bogdanove had discovered, the TALE targeting system consists of central repetitive regions of varying repeat number and sequence but with specificity determined by  two adjacent “repeat variable diresidues” (RVDs), primarily at repeat positions 12 and 13, with direct correspondence to host genome target site nucleosides.(1) By exploiting the simple, predictable target specificities of engineered TALEs, they looked to target the ZFN restriction enzyme to novel sites for facile genome editing.

The authors  inserted the ZFN endonuclease into either the AvrBs3 TALE from the pepper-plant pathogen Xanthomonas campestris pv. vesicatoria or the PthXo1 TALE from the rice pathogen X. oryzae pv. oryzae at a restriction fragment unencumbered by the transcriptional activation domain. They then performed preliminary experiments with the newly-christened “TALE Nuclease (TALEN)” system with an initial, exploratory choice of a 15 base pair spacer size, which fortuitously turned out to be a shared optimum spacer length between the two TALENs (in addition to other, pathogen-specific optima) and yielded robust gene-targeting activity in a yeast lacZ reporter assay.

The researchers then went on to demonstrate the ability of engineered TALENs to target novel host target genes, using genes previously used to demonstrate site-directed mutagenesis  by ZFNs: ADH1 in Arabidopsis, and more intriguingly the zebrafish Gridlock gene. They selected RVDs suitable to target coding regions within these genes with sequences consistent with the TALE targeting system parameters, removed a repeat domain from the native TALE genome, replaced it with these RVDs in proper order for gene targeting, and finally inserted the ZFN restriction enzyme into the TALE BamHI restriction fragment.

 The resulting TALEN was tested against the target genes in the yeast lacZ assay, using the same DNA binding sites on either side of the gene. While artificial, this assay demonstrated the ability of the TALENs to specifically target these selected genes for site-directed mutagenesis, with robust activity in 2 such TALENs and more modest activity in a third.

The authors note how much work remains to be done to make practical use of the TALEN system:

the failure of some custom TALENs suggests that yet unknown rules govern the assembly of functional repeat domains. For example, repeat composition may influence protein stability, or interactions among repeat domains may affect DNA binding activity as has been observed for finger-finger interactions in zinc finger arrays . Alternatively, the spacer lengths we used may have prevented dimerization of [the ZFN endonuclease], as appeared to be the case for some spacers with AvrBs3. Clearly, it will be important to gain a better understanding of the relationship of spacer length to function for TALENs with different repeat domains. Ascertaining the minimal DNA binding domain might help accomplish this; however, we believe the repeats alone are not sufficient for adequate DNA binding, as TALENs constructed with just the repeat domain did not function in the yeast assay (data not shown). In the short-term, we will test whether custom TALENs can be created that recognize and cleave endogenous chromosomal targets, and we will evaluate the efficiency with which custom TALENs create genome modifications by non-homologous end-joining and homologous recombination. Such experiments will be key to assessing the full utility of these reagents for eukaryotic genome engineering.

The authors are duly cautious too about the ultimate use of their new system, not suggesting the possibility of its eventual use in mammalian systems, let alone for therapeutic use. But should they be able to demonstrate the targeting of native genes in situ, utilizing the binding sites actually present in the host genome, this paper will be remembered as a landmark along the way to precise repair of defective genes in inherited disease, and of renovation of the genome for the rejuvenation of aging tissues.