Many systems have been generated to turn gene expression on and off in an inducible way, but so far each has shown shortcomings such as leakiness or irreversibility. Now, by coupling RNAi to an inducible system that utilizes repressor proteins, James Collins and colleagues have obtained a genetic switch that guarantees tight and tunable control of gene expression.

The authors engineered a synthetic gene network (LTRi) involving the bacterial LacI and TetR transcriptional repressors and an RNAi component coding for a short-hairpin RNA (shRNA). The target of the shRNA is a complementary 19-bp sequence placed in the 3′ UTR of the gene of interest.

The default state of the switch is off: here, LacI represses the transcription of the gene of interest and also of the TetR repressor so that TetR cannot suppress shRNA transcription. Should any leakage occur, the shRNA molecule further knocks down the gene, post-transcriptionally. The addition of isopropyl-β-thiogalactopyranoside (IPTG), which inhibits LacI repressor function, turns the gene on by releasing its LacI-mediated repression and also LacI-mediated repression of TetR, which can then suppress transcription of the shRNA.

Given the modular structure of the switch, the authors were able to test each module independently; this was done by transiently transfecting human cells, using EGFP as the gene of interest. Adding the RNAi module or the LacI-repressor module alone achieved an 80% or 85% reduction of EGFP expression, respectively; by contrast, combining the modules altogether attained a greater than 99% repression of the transgene.

Stable transfections of LTRi–EGFP in different human cell lines showed that, in each cell type, the switch can be repeatedly and reversibly flipped on and off by IPTG addition or removal, and gene expression levels can be finely tuned by using intermediate concentrations of IPTG.

The authors confirmed the tightness of the switch by showing that it can completely suppress the expression of the highly toxic α-chain of diphtheria toxin — of which just a molecule suffices to kill a cell — and of the cre recombinase in primary mouse fibroblasts. Moreover, the tunable property of the switch makes it a unique tool to study phenotypes that depend on threshold levels of gene expression, as the authors showed for BAX-driven apoptosis of primary mouse fibroblasts.

Spanning from basic research to disease study and gene therapy, the potential applications of this tunable switch seem to be immense.