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.