As a kid there was no greater feeling on a weekend shopping trip than being set loose on the Pick ‘N’ Mix, carefully choosing from the wealth of candy to create the perfect selection. Lucky for you synthetic biology promises the same thrill, by providing a selection of ready-to-roll interchangeable parts to choose from allowing you to build complex synthetic gene circuits. One sticking point has held up this Pick ‘N’ Mix approach: the lack of well-characterized and orthogonal transcriptional repressors.
You may be familiar with classical synthetic transcriptional repressors, which typically involve the fusion of transcriptional repression domain to an engineered DNA binding protein domain, such as a Zinc finger protein, transcription activator-like effector (TALE) protein or deactivated Cas9 nuclease in a CRISPR system. The use of the transcriptional repressor domain has its problems, including that they tend to cause epigenetic modifications on the target promoter region, thereby interfering with temporal response due to delayed transcriptional reactivation.
To overcome this shortcoming, Li and colleagues decided to harness the technology that employs steric hindrance rather than repression domains. Specifically, the team constructed cell-type specific, microRNA-controlled transcription activator-like effector repressor (TALER) promoters that are transcriptionally inactivated upon TALER binding.
The TALER promoter consists of five upstream activation binding sites and a minimal cytomegalovirus (CMV) promoter flanked by two TALER binding sites. Mechanistically, constitutively expressed GalVP16 interacts with the upstream binding site and elicits the activation of the gene encoding the red fluorescence reporter mKate2. If a TALER protein is present, the repressor binds its 5’ and 3’ recognition sites situated at the ends of the minimal CMV promoter and thus suppressing transcription.
Using this system the researchers found:
- 23 of the 26 engineered TALER promoter-repressor pairs exhibited greater than 90% transcription repression efficiency in a co-transfection experiment in human embryonic kidney (HEK293) cells.
- 16 of the 23 TALERs blocked their target promoters by 100-fold more than the controls.
- RNA-seq analysis revealed that TALERs are highly specific for their intended targets.
- Having both the 5’ and 3’ binding sites available for TALERs and positioning TALER binding sites closer to the minimal CMV promoter increases the level of repression.
The authors concluded that the library of synthetic TALER circuit modules will have far-reaching biomedical and biotechnological applications, such as “precise cell-type classification for a mixed-cell population and sensing multi-input cell-type specific microRNA profile”.
To view the selection run on over to Nature Chemical Biology, February 2015.