icRNA: in vivo circular RNAs for efficient expression and control of genes and polyproteins

Potent, stable and controllable gene expression is a long-standing goal of synthetic biology. Thanks to Watson-Crick base pairing, RNA-based controllers have the potential to be more programmable and predictable, and to impose less metabolic burden to host cells compared to protein-based controllers. In microbes, however, RNA-based systems typically offer limited dynamic range and require high-levels of RNA expression due to the short half-lives of linear RNAs. This project aims to develop an RNA-based controller with a high dynamic range and low host burden by using in vivo circularisation and linearisation of mRNA. Circular RNAs (cRNAs) lack 5' and 3' ends, which eliminates end-dependent degradation by exoribonucleases and several endoribonucleases, causing cRNAs to exhibit far longer half-lives compared to linear mRNAs. However, due to a lack of efficient methods for in-vivo cRNA production, the use of cRNAs has been limited so far.

This project will focus on the development of a novel in-vivo cRNA expression system in E. coli (and potentially in S. cerevisiae, too), adapting the Tornado RNA circularisation system to circularise long mRNAs. The main task of the project will be to rationally engineer the sequence of the ribozymes and transcripts to optimise cleavage and ligation. To further increase the dynamic range of the circuit, we will implement an RBS-repairing strategy where the RBS is initially split in two parts placed at the ends of the linear mRNA and gets repaired only when the RNA is circularised. In parallel with the experimental work, computational models accurately describing RNA circularisation and linearisation kinetics and circular mRNA translation dynamics will be built to understand and predict the dynamic behaviour of these RNA-circularisation systems.

This cRNA control system will be used as a platform for controlling gene expression with applications in production of materials, therapeutics, and potentially vaccines. Additionally, by removing the stop codon from the coding sequence in a circular mRNA, rolling circle translation can be achieved, which can be used for the efficient production of valuable multi-unit repeat proteins with useful properties for the production of novel biomaterials.

The 2016 UK Roadmap Bio-design for the Bio-economy highlighted the substantial impact that synthetic biology can bring to the UK and global economies by developing: frontier science and technology; establishing a healthy innovation pipeline; a highly skilled workforce and an environment in which innovative science and businesses can thrive. Synthetic biology promises to transform the UK Bio-economy landscape, bringing bio-sustainable and affordable manufacturing routes to all industrial sectors and will ensure society can tackle many contemporary global Grand Challenges including: Sustainable Manufacturing, Environmental Sustainability Energy, Global Healthcare, and Urban Development. Whilst synthetic biology is burgeoning in the UK, we now need to build on the investments made and take a further lead in training next generation scientists to ensure sustained growth of a capable workforce to underpin the science base development and growth in an advanced UK bio-economy.
This training provided by this CDT will give students from diverse backgrounds a unique synthesis of computational, biomolecular and cellular engineering skills, a peer-to-peer and industrial network, and unique entrepreneurial insight. In so doing, it will address key EPSRC priority areas and Bioeconomy strategic priorities including: Next-generation therapeutics; Engineered biomaterials; Renewable alternatives for fuels, chemicals and other small molecules; Reliable, predictable, and scalable bioprocesses; Sustainable future; Lifelong health & wellbeing.
Advances created by our BioDesign Engineering approach will address major societal challenges by delivering new routes for chemical/pharma/materials manufacture through to sustainable energy, whilst providing clean growth and reductions in energy use, greenhouse gas emissions and carbon footprints. Increased industry awareness of bio-options with better civic understanding will drive end-user demand to create market pull for products. The CDT benefits from unrivalled existing academic-industry frameworks at the host institutions, which will provide direct links to industrial partners and a direct pathway to early economic and industrial impact.

This CDT will develop 80-100 next-generation scientists and technologists (via the funded cohort and wider integration of aligned students at the three institutions) as adept scientists and engineers, instilled with technical leadership, who as broadly trained individuals will fill key skills gaps and could be expected to impact internationally through leadership roles in the medium term. Importantly the CDT addresses key skill-gaps identified with industry, which are urgently required to create and support high value jobs that will enable the UK to compete in global markets. Commercialisation and entrepreneurship training will equip the next generation of visionaries and leaders needed to accelerate and support the creation of new innovative companies to exploit these new technologies and opportunities.

The UK government identified Synthetic Biology as one of the "Eight Great Technologies" that could be a key enabler to economic and societal development. This CDT will be at the forefront of research that will accelerate the clean growth agenda and the development of a resilient circular bioeconomy, and will align with key EPSRC prosperity outcomes including a productive, healthy and resilient nation. To foster wider societal impact, the CDT will expect all students to contribute to public outreach and engagement activities including: open days, schools visits, and science festival events: students will participate in an outreach programme, with special focus on widening participation.

This CDT will contribute to the development of industrial strategy through the Synthetic Biology Leadership Council (SBLC), Industrial Biotechnology Leadership Forum (IBLF), and wider Networks in Industrial Biotechnology and Bioenergy and Professional Institutes.