CRISPR Screen Multiplexing for Uncharacterised Region Function (SMURF)

A preliminary stage of the drug development process requires a dependency on a potential cellular drug target, such as a protein, to be validated in a specific disease setting. This development stage is termed 'target validation' and it provides evidence that modulation of a specific target may confer therapeutic benefit to patients. However current methods in target validation frequently identify false positive targets which are then pursued further for drug development. As a result, candidate drug compounds are developed that may confer little or no therapeutic benefit. This incurs substantial cost and time delays to the development process and leaves patients without appropriate treatment options. One approach to reducing the number of flawed target hypotheses is to improve the structural and functional data on targets during their validation.

The CRISPR-Cas gene editing system has been derived from bacteria and archaea where its native function is as an immune defence against viral infection. This has now been adapted to edit the genome of mammalian cells and has proven invaluable for use within both research and therapeutic settings. Recently published CRISPR-based screening data has demonstrated that certain sections of a gene sequence are not critical to protein function and are therefore unlikely to confer any therapeutic benefit as specific drug target sites. The focus of this project is to determine whether the CRISPR system is amenable to high-throughput screening formats and capable of producing reliable data relating to a target's structure and function. This would allow CRISPR-Cas to be utilised on a larger scale within drug discovery for the validation of novel uncharacterised drug targets that have clinical potential.

This project has two distinct aims. Firstly, precise DNA editing will be undertaken within a target protein to induce differential sensitivity to specific inhibitor compounds. Secondly, larger randomised gene knockout screens will be conducted to identify those nucleotide sequences that are critical to the function of a target protein. Following the design and development of stable cell lines which express the requisite CRISPR-Cas components, protocols will be validated using cell-based assays to identify perturbed protein function. Genetic sequencing of the target will also be required to illustrate the exact DNA changes that have been introduced. Initially these studies will be conducted in a simplified cell line system before progressing to more complex human cancer cell lines.

Cyclin-dependent kinase 2 will initially be used as a model target since its structure and function have been well characterised in the literature. Alternative model targets will also undergo analysis during the course of the project. If successful, this research may provide the pharmaceutical industry with improved methods for target validation by aiding the identification of relevant drug target sites on proteins. Ultimately, this approach may facilitate the identification of effective drugs for life-threatening diseases.

The CDT has five primary beneficiaries:
The CDT cohort
Our students will receive an innovative training experience making them highly employable and equipping them with the necessary knowledge and skillset in science and enterprise to become future innovators and leaders. The potential for careers in the field is substantial and students graduating from the CDT will be sought after by employers. The Life Sciences Industrial strategy states that nearly half of businesses cite a shortage of graduates as an issue in their ability to recruit talent. Collectively, the industrial partners directly involved in the co-creation of the proposal have identified recruitment needs over the next decade that already significantly exceed the output of the CDT cohort.
Life science industries
The cohort will make a vital contribution to the UK life sciences industry, filling the skills gap in this vital part of the economy and providing a talented workforce, able to instantly focus on industry relevant challenges. Through co-creation, industrial partners have shaped the training of future employees. Additional experience in management and entrepreneurship, as well as peer-to-peer activities and the beginning of a professional network provided by the cohort programme will enable graduates to become future leaders. Through direct involvement in the CDT and an ongoing programme of dissemination, stakeholders will benefit from the research and continue to contribute to its evolution. Instrument manufacturers will gain new applications for their technologies, pharmaceutical and biotech companies will gain new opportunities for drug discovery projects through new insight into disease and new methods and techniques.
Health and Society
Research outputs will ultimately benefit healthcare providers and patients in relevant areas, such as cancer, ageing and infection. Pathways to such impact are provided by involvement of industrial partners specialising in translational research and enabling networks such as the Northern Health Science Alliance, the First for Pharma group and the NHS, who will all be partners. Moreover, graduates of the CDT will provide future healthcare solutions throughout their careers in pharmaceuticals, biotechnology, contract research industries and academia.
UK economy
The cohort will contribute to growth in the life sciences industry, providing innovations that will be the vehicle for economic growth. Nationally, the Life Sciences Industrial Strategy Health Advanced Research Programme seeks to create two entirely new industries in the field over the next ten years. Regionally, medicines research is a central tenet of the Northern Powerhouse Strategy. The CDT will create new opportunities for the local life sciences sector, Inspiration for these new industries will come from researchers with an insight into both molecular and life sciences as evidenced by notable successes in the recent past. For example, the advent of Antibody Drug Conjugates and Proteolysis Targeting Chimeras arose from interdisciplinary research in this area, predominantly in the USA and have led to significant wealth and job creation. Providing a cohort of insightful, innovative and entrepreneurial scientists will help to ensure the UK remains at the forefront of future developments, in line with the aim of the Industrial Strategy of building a country confident, outward looking and fit for the future.
Institutions
Both host institutions will benefit hugely from hosting the CDT. The enhancement to the research culture provided by the presence of a diverse and international cohort of talented students will be beneficial to all researchers allied to the theme areas of the programme, who will also benefit from attending many of the scientific and networking events. The programme will further strengthen the existing scientific and cultural links between Newcastle and Durham and will provide a vehicle for new collaborative research.