CRISPR Screen Multiplexing for Uncharacterised Region Function (SMURF)

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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.