Engineering molecular RNA sensors for controlling CRISPR activity in vivo

Brief Introduction:

Current ways of restricting the activity of CRISPR systems to cell types of interest include tissue-specific promoters or targeted delivery approaches. However, such strategies are optimised for a limited subset of cell types and tissues. The development of CRISPR-based RNA sensors that are conditionally activated upon detection of endogenous RNA biomarkers that reflect different cell identities, differentiation and disease status or exposure to environmental factors is poised to open new opportunities in research and therapeutics. Here, we use the sgRNA engineering approach to control the activity of Streptococcus pyogenes Cas9 (SpCas9) in response to such endogenous inputs. sgRNAs are engineered to fold into complex secondary structures that, in the ground state, inhibit their activity. Upon recognising complementary RNA biomarkers, these sgRNAs become activated, enabling Cas9 to perform its function.

Specific aims:

1. Testing if engineered sgRNAs can detect RNA
2. Optimising engineered sgRNA designs
3. Characterising activation of engineered sgRNA designs using different CRISPR effectors
4. Characterising the mechanism of activation of engineered sgRNAs
5. Generating a computational pipeline that allows users to design engineered sgRNA designs for their custom CRISPR applications
6. Generating transgenic zebrafish lines for testing engineered sgRNAs in vivo
7. Testing engineered sgRNA designs in zebrafish embryos

This project falls within the EPSRC Synthetic Biology research area and was sponsored by EvOX Therapeutics between 1st of October 2018 and 1st of October 2021.

The emerging and dynamic field of Synthetic Biology has the potential to provide solutions to some of the key challenges faced by society, ranging across the healthcare, energy, food and environmental sectors. The UK government has recently a "Synthetic Biology Roadmap", which presents a vision and direction for Synthetic Biology in the UK. The report projects that the global Synthetic Biology market will grow from $1.6bn in 2011 to $10.8bn by 2016. It highlights that there is an urgent need for the UK to develop the interdisciplinary skills required to take advantage of the opportunities provided by Synthetic Biology.

The challenge to the academic and industrial research communities is to develop new translational approaches to ensure that these potential benefits are realised. These new approaches will range across the design and engineering of biologically based parts, devices and systems as well as the re-design of existing, natural biological systems across all scales from molecules to organisms. The techniques will encompass not only individual cells, but also self-assembled biomimetic systems, engineered microbial communities and multicellular organisms, combining multiple perspectives drawn from the engineering, life and physical sciences.

Realising these goals will require a new generation of skilled interdisciplinary scientists, and the training of these scientists is the primary goal of the SBCDT. Our programme will give the breadth of coverage to produce a "skilled, energized and well-funded UK-wide synthetic biology community", who will have "the opportunity to revolutionise major industries in bio-energy and bio-technology in the UK" (David Willetts, Minister for Universities and Science) in their future careers. This will be made possible through genuine inter-institutional collaboration in partnership with key industrial, academic and public facing institutions.

The potential impact of the SBCDT, and its potential national importance, are very therefore high, and the potential benefits to society are significant.