Ribozyme-Enabled Tissue Specificity (RETS): A System for Precise Gene Expression without Specialized Promoters

Tissue- or cell-type-specific expression of transgenes
is often
essential for interrogation of the biological phenomenon or predictable
engineering of multicellular organisms but can be stymied by cryptic
enhancers that make identification of promoters that generate desired
expression profiles challenging. In plants, the months-to-years long
timeline associated with prototyping putative tissue-specific promoters
in transgenic lines deepens this challenge. We have developed a novel
strategy called Ribozyme-Enabled Tissue Specificity
(RETS) that leverages the knowledge of where and when genes are expressed
derived from transcriptomic studies to enable tissue-specific expression
without needing characterized promoters. It uses a split self-splicing
ribozyme based on a group I intron from Tetrahymena
thermophila to enable the conditional reconstitution
of a transgene mRNA in the presence of a secondary tissue-specific
mRNA of choice. We elucidate the design features that enable flexible
swapping of transgenes and targets, enhancing transgene expression,
and circumventing host RNA interference responses. We then show that
these innovations enable tissue-specific and dose-dependent expression
of transgenes in Arabidopsis thaliana. Finally, we demonstrate the utility of RETS both for creating genetically
encoded biosensors to study the spatiotemporal patterns of gene expression in planta and for engineering tissue-specific changes in
organ size. RETS provides a novel avenue to study expression patterns
of native loci with nondestructive imaging, complementing the weakness
of existing approaches. Additionally, the spatiotemporal control of
transgene expression afforded by RETS enables precision engineering
of plant phenotypes, which will facilitate enhancing crops without
the trade-offs associated with constitutive expression.