Developing a CRISPR toolbox for efficient gene editing in crops

This project aims to develop better gene editing tools for crop plants using enhanced molecular
and computational tools. We will prototype these in E. coli as a more feasible test platform for
rapid implementation of the design-build-test-learn (DBTL) cycle.

Efficient gene editing in plants remains a challenge, due to the complexity of the experimental
system (genome size, polyploidy) and the molecular properties of the tools used to make the
modifications. We will use directed evolution and machine learning to improve the nucleases with
a focus on stability, activity, size of the protein, and its gene expression burden in host cells.
The project will focus on MAD7 (ErCas12a) nuclease, a CRISPR-associated protein that can be used
for gene editing in a wide variety of organisms. It has practical advantages over alternatives
because it can be used for commercial product development without incurring royalties.

However, the stability and activity of MAD7 at room temperature make it difficult to work with
compared to standard nucleases such as Cas9 and Cpf1 (LbCas12a). This is particularly true for
non-transgenic plant gene editing protocols where the nuclease is supplied as a purified
ribonucleoprotein to improve the efficiency and reduce off-target effects. The wild-type MAD7
nuclease has ~2-3% efficiency of target DNA cleavage at 27C in plant cells (unpublished data,
Phytoform Labs).

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.