Success and failure stories associated with gene editing approaches based on zinc finger nucleases and transcription activator-like effector nucleases (TALENs) have been making headlines at an increasing frequency lately. This largely stems from hopes that the technique will allow high-precision and high-efficiency modifications in a target gene sequence to produce basic research tools and to enable dramatically improved gene therapy.
The sequence specificity of these new agents is based on the precise recognition of DNA base pairs by conserved transcription activator-like (TAL) protein domains. A hybrid protein composed of series of TAL units assembled to target a specific DNA sequence, in combination with nuclease variant, can bind the specific genomic DNA stretch and effect a double-strand break in the sequence. In turn, the double-strand break is typically repaired through nonhomologous end-joining or homology-directed repair with the end result being a modified sequence in the target of interest.
In principle, the large-scale generation of candidate gene editing tools should enable rapid testing and identification of the best agent to be used for a particular target (i.e., the one that edits at the highest efficiency and with minimal off-target effects). However, the majority of methods that permit the combinatorial assembly of these multidomain proteins are low throughput and at present the highest throughput platforms have largely remained proprietary. Here, a readily available high-throughput solid-phase oligonucleotide synthesis and DNA processing platform, based on the capture and manipulation of nucleic acids attached to magnetic beads, is being used to create a prototype for assembly of TALENs (see Figure).
The authors* generated a collection of 376 plasmids coding for one, two, three, or four TAL effector repeats comprising all possible combinations of base pair binders (see Figure). Automated assembly was made possible by the iterative use of the magnetic beads–based platform to run protocol steps of serial restriction digest, purification, and ligation. The authors further optimized the platform to process 96-well plates in combination with a pipeting robot; with this streamlined platform, 96 combinations of TAL repeats could be produced in 1 day. Scale-up validation was performed to generate plasmids encoding 48 TALEN pairs targeted to different sites in the EGFP reporter gene, and the resulting TALENs were tested in human cells for their ability to disrupt the EGFP gene; all 48 TALEN pairs were found to exhibit significant activities.
During the pilot studies the authors noticed that shorter-length TALENs, while showing similar activity compared with longer-length TALENs, were more cytotoxic in human cells. Although the phenomenon was not studied in detail, the toxicity of the shortmers may well have been due to off-target effects, and this finding should serve as a useful guideline for future TALEN designs.
Lastly, to test the applicability to a wide range of gene targets, the authors used the platform to manipulate 78 genes associated with cancer and 18 genes implicated in epigenetic regulation. Analysis of the outcome indicated an overall success rate of 88%, with TALEN efficiencies ranging between 2.5% and 55.8% (Table 1 in the article).*
While the present study did not touch upon the pertinent subject of off-target effects of the newly constructed TALENs, the technology's efficiency should enable multiple research teams to generate medium- to large-size collections of effectors to study this aspect of gene editing.
*Abstract from Nature Biotechnology
Engineered transcription activator–like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published, and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the fast ligation-based automatable solid-phase high-throughput (FLASH) system, a rapid and cost-effective method for large-scale assembly of TALENs.
We tested 48 FLASH-assembled TALEN pairs in a human cell–based EGFP reporter system and found that all 48 possessed efficient gene-modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with other genome modification technologies.