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Genomics


(Cas9) to the desired location in the genome. Once the nuclease has made a double-strand cut, the cell’s own repair mechanisms will fix the break. This either leads to a loss of function of the gene or replacement with an alternative sequence, depend- ing on the particular reagents present. By contrast, CRISPRa and CRISPRi work by


guiding a nuclease-dead Cas9 (‘dCas9’) coupled to either a transcriptional activator or repressor to a specific promoter sequence, where it activates or inhibits gene transcription. Not only does this begin to provide insights into the potential impact of alterations in non-coding regulatory elements that control gene expression levels, it also more realistically mimics the physiological effect of a drug in the body, which might reduce but not com- pletely remove the target. Initially we plan to focus on the exome, altering the function of protein cod- ing genes, expanding to encompass more esoteric targets such as microRNAs and key regions of the non-coding genome in the future. Our initial screens are being carried out using


cancer cell lines and primary tumour cells, for example from CRISPR-derived cellular or animal models of disease, and from patient samples. However, we are aiming to move from simple cell culture to more realistic situations that incorporate elements of the tumour microenvironment, such as hypoxia or immune cell infiltration. Manipulating culture conditions may also reveal phenotypes that


are only expressed under certain physiological con- ditions, such as oxidative stress or increased acidi- ty. Carrying out screening or target validation in more physiologically-relevant systems such as organoids, co-cultures, patient samples, animal models or organ-on-a-chip technology is more like- ly to provide results that will bear up in clinical testing, greatly increasing the chances of success. CRISPR offers a completely new approach for


the discovery and validation of novel targets. It is efficient – assaying 20,000 genes simultaneously – and we can skip straight from a CRISPR ‘hit’ to the underlying biological target. This approach also has wider applications outside of oncology, and we are applying the concept of large-scale, high- throughput CRISPR screening to identify and vali- date novel targets across a wide range of therapeu- tic areas including respiratory, cardiovascular, metabolic and renal diseases.


AI approaches for rational discovery While we anticipate that manipulating individual genes across the genome will reveal novel targets, cancer cells are complex systems, with multiple genes working together in pathways that can be genetically or epigenetically ‘rewired’ in response to treatment. It is therefore likely that we will need to investigate the impact of altering combinations of multiple genes at the same time, in multiple cells types and with multiple drug combinations.


Cambridge Biomedical Campus, UK


Drug Discovery World Spring 2019


25


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