Robust gene expression control in human cells with a novel universal TetR aptamer splicing module

Abstract

Fine-tuning of gene expression is desirable for a wide range of applications in synthetic biology. In this context, RNA regulatory devices provide a powerful and highly functional tool. We developed a versatile, robust and reversible device to control gene expression by splicing regulation in human cells using an aptamer that is recognized by the Tet repressor TetR. Upon insertion in proximity to the 5′ splice site, intron retention can be controlled via the binding of TetR to the aptamer. Although we were able to demonstrate regulation for different introns, the genomic context had a major impact on regulation. In consequence, we advanced the aptamer to develop a splice device. Our novel device contains the aptamer integrated into a context of exonic and intronic sequences that create and maintain an environment allowing a reliable and robust splicing event. The exon-born, additional amino acids will then be cleaved off by a self-cleaving peptide. This design allows portability of the splicing device, which we confirmed by demonstrating its functionality in different gene contexts. Intriguingly, our splicing device shows a high dynamic range and low basal activity, i.e. desirable features that often prove a major challenge when implementing synthetic biology in mammalian cell lines.

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

“…Switches based on this mechanism promoted 2-4-fold suppression of gene expression in response to signaling molecules such as TNF-α or LTD4. The bacterial TetR protein has also been adapted for use in splicing regulation, with TetR aptamers providing tetracycline-mediated control over splice site accessibility and allowing regulation of gene expression in human cells [73]. As with other non-self protein-mediated expression control systems however, TetR immunogenicity and the size of its expression cassette may limit use in AAV.…”

Inducible nuclear import by TetR aptamer-controlled 3′ splice site selection

“…After addition of doxycycline and the resulting detachment of TetR from the aptamer, usage of the canonical 3 ′ SS was restored. Since RNA-based synthetic devices are highly dependent on genetic context and stability of the regulatory RNA element (Vogel et al 2018;Groher et al 2019;Mol et al 2019), the TetR aptamer was optimized for both the lengths of the closing stem P1 and of the Py-tract (Supplemental Fig. S3; B Suess, unpubl.…”

“…allows control over intracellular miRNA levels and, consequently, their gene-silencing properties. The latest approach (Mol et al 2019) exploits the TetR-binding aptamer for the control of translation initiation and pre-mRNA splicing. For this, the TetR aptamer was inserted into the 5 ′ untranslated region in such a way that it interferes with ribosomal scanning when bound by TetR.…”

“…Moreover, upon insertion in proximity to the 5 ′ SS, intron retention was controlled via the binding of TetR to the aptamer. Again, in both approaches TetR binding is fully relieved by the addition of doxycycline (Mol et al 2019).…”

Synthetic polycistronic sequences in eukaryotes

“…In metabolic engineering and synthetic biology, viral 2 A sequences in combination with other tools—such that in vivo assembly to generate large constructs—permitted the production of complex molecules, in heterologous eukaryotic chassis or the implementation of novel transcriptional regulation systems [ [120] , [121] , [122] , [123] ] Enzymatic systems requiring several cofactors have been repurposed, by means of 2 A peptides, both in Schizosaccharomyces pombe and S. cerevisie . [ [113] , [114] , [115] ], The baker's yeast, moreover, is commonly treated as a cell factory to host heterologous pathways (such as that for β-carotene biosynthesis [ 60 , 112 ]) and as a chassis for synthetic networks that make use of the interactions between transcription factors derived from nuclease-deficient Cas protein and their antagonist anti -CRISPR macromolecules (see Fig.…”