bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2019.12.15.867150

PERSIST: A programmable RNA regulation platform using CRISPR endoRNases

DiAndreth, B.·Wauford, N.·Hu, E.·Palacios, S.·Weiss, R.
View original source ↗Export metadata ↓

Abstract

Regulation of transgene expression is becoming an integral component of gene therapies, cell therapies and biomanufacturing. However, transcription factor-based regulation upon which the majority of such applications are based suffers from complications such as epigenetic silencing, which limits the longevity and reliability of these efforts. Genetically engineered mammalian cells used for cell therapies and biomanufacturing as well as newer RNA-based gene therapies would benefit from post-transcriptional methods of gene regulation, but few such platforms exist that enable sophisticated programming of cell behavior. Here we engineer the 5 and 3 untranslated regions of transcripts to enable robust and composable RNA-level regulation through transcript cleavage and, in particular, create modular RNA-level OFF- and ON-switch motifs. We show that genomically introduced transgenes exhibit resistance to silencing when regulated using this platform compared to those that are transcriptionally-regulated. We adapt nine CRISPR-specific endoRNases as RNA-level "activators" and "repressors" and show that these can be easily layered and composed to reconstruct genetic programming topologies previously achieved with transcription factor-based regulation including cascades, all 16 two-input Boolean logic functions, positive feedback, a feed-forward loop and a putative bistable toggle switch. The orthogonal, modular and composable nature of this platform as well as the ease with which robust and predictable gene circuits are constructed holds promise for their application in gene and cell therapies.

Explore related subjects

synthetic biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

DiAndreth, B., Wauford, N., Hu, E., Palacios, S., Weiss, R.. 2019-12-16. PERSIST: A programmable RNA regulation platform using CRISPR endoRNases. https://doi.org/10.1101/2019.12.15.867150

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Creation of genomic chimeras via megabase scale genome expansion

An organism's physiology is controlled by its genomic content. Building genomic chimeras would enable melding of the diverse functions and properties of life. However, prior arts in genome synthesis are limited to reconstituting pre-existing functions within a singular genome rather than combining diverse genomic functions across multiple distinct genomes. Existing methods are also prohibitively expensive, labor intensive, and time consuming, and thus fundamentally not scalable for creating large synthetic chimeric genomes. To address all these limitations, we invented Additive Conjugative-CAST Engineering (ACE) combining conjugation with CRISPR-associated transposition (CAST) to deliver and integrate up to half a genome per step from a donor cell into a precisely defined position in the recipient's genome. We have demonstrated ACE's capability to integrate at least a 2-megabase donor genomic segment in a single step and at least 3 megabase in two consecutive steps. Importantly, we confirmed ACE's generality through the creation of multiple genomic chimeras across species, genus, and at least order barriers. With ACE, we further demonstrate such chimeric organisms, or what we call genome expanded organisms (GEOs), can be stably forged from at least three starting bacterial strains, and can be stably maintained to express all their acquired genomic parts in a single chimera. ACE offers a new paradigm of creating artificial lifeforms to combine desired properties and potentially create novel functions beyond the constraints of nature. ACE also offers a unique perspective to probe unexplored plasticity in genome architecture and expression patterns of novel, massively expanded genomes.

synthetic biology↗
bioRxiv2026

Functional analysis of small-molecule and protein binding domains using monomeric streptavidin-based two-component biosensors

Protein biosensors are important analytical and diagnostic tools that convert molecular recognition into measurable activity. Most common two-component biosensors require two non-competing recognition domains, yet binding affinity and specificity alone do not reliably predict their compatibility with the rest of the biosensor. We report a modular platform that exploits monomeric streptavidin (mSA2) as a surrogate recognition domain, enabling rapid functional evaluation of candidate binders in the context of biosensor scaffold without requiring a second ligand-specific binding protein. We established a chromatography-free protocol for recovering functional mSA2-based biosensor components from Escherichia coli. Using biotinylated small molecules methotrexate and vitamin D3, as well as heart failure protein biomarkers galectin-3 and S100A7 we demonstrate ligand-dependent reporter biosensor activation. The developed platform provides a scalable approach for rapid binder evaluation and biosensor prototyping, streamlining biosensor development using binding domains generated by directed evolution or computational protein design.

synthetic biology↗
bioRxiv2026

Developing synthetic auxin-based trans-kingdom signaling

Synthetic trans-kingdom signaling systems with tunable transduction properties provide a unique avenue to explore how communication between organisms coordinate behaviors without the confounding complexity of natural systems. However, existing trans-kingdom signaling systems suffer from either toxic signal molecules, a need for rare precursors for signal generation, or transduction machinery which is either species-specific or cannot be easily tuned. This limits their capacity to be broadly applied across kingdoms and their utility as a model system to study trans-kingdom interactions. In this work we elucidate the design principles for a trans-kingdom signaling system based on the plant phytohormone, Indole 3-Acetic Acid (auxin), that overcomes these challenges. We demonstrate how auxin biosynthesis can be engineered into a model sender organism, the cyanobacteria Synechococcus elongatus PCC 7942, and how this signal can be tuned with low leak and high dynamic range. We also show these signals can be connected to gene expression using tunable synthetic auxin-responsive signaling systems across multiple kingdoms. We demonstrate that S. elongatus generated auxin signals can tune gene expression in co-cultured Saccharomyces cerevisiae via auxin-induced degradation. Additionally, we show that when the S. elongatus are associated with the roots of the model plant, Arabidopsis thaliana, the auxin they generate can control gene expression via auxin-responsive synthetic transcription factors expressed from the plants genome. Taken together, our results demonstrate the design principles for a tunable auxin-based trans-kingdom signaling system that can be broadly applied across species.

synthetic biology↗