DNA damage response

genome across a wide range of cell types to see how it affects growth and function or contributes to therapeutic resistance.

From gene to function Our initiative builds on the approach that has been used by geneticists for more than a century as they seek to uncover the link between genotype and phenotype: create a change in the genome then observe the effect. Initially this was done by mutagenesis with chemicals or X-rays, becoming more sophisticated through the use of genetic engineering techniques such as transgenic (‘knock- out’) cell and animal models. Both these approaches have significant drawbacks, including the challenge of tracing a resulting phenotype back to the underlying gene fault in the case of mutagenesis, or the high cost and low efficiency of generating knockouts. Things began to change around a decade ago

with the development of TALENs and zinc finger genome editing tools, which could be used to cre- ate highly-targeted DNA alterations at specific sequences. The field made a quantum leap in 2012 with the demonstration that the bacterial DNA cleavage system CRISPR/Cas9 (usually known as CRISPR) could be targeted to any location in the genome of human cells with much higher specifici- ty, allowing researchers to manipulate sequences relatively quickly, cost-effectively and with high efficiency. Since its discovery, CRISPR has proved to be a powerful tool for unpicking the complex connections between genotype and phenotype, par- ticularly in cancer research5.


At AstraZeneca, we were quick to adopt gene

editing technologies into our drug discovery pro- grammes, altering specific genes to generate more physiological cellular and animal models of dis- ease. Moreover, it is now possible to use these tools at scale, using genome-wide libraries of CRISPR guide RNAs to alter every gene in the human genome in normal or cancer cells, screening 20,000 genes in a single experiment. Running these assays in combination with high-throughput robotics and phenotypic analysis techniques allows for massive- ly parallel screening, dramatically accelerating the identification of novel genes involved in biological processes.

Up, down and out We will initially use genome-wide CRISPRn libraries generated by our collaborators at the Wellcome Sanger Institute in Cambridge, UK, to completely knock out all 20,000 genes across a wide range of cell and tumour types. Future libraries will allow us to upregulate (CRISPR acti- vation or CRISPRa) or downregulate (CRISPR interference, CRISPRi) these genes. We can then quantify a wide range of phenotypic responses, such as cell proliferation or death, RNA or protein expression, metabolism, morphology and resis- tance to therapy, using this information to develop valuable new biological insights for cancer drug discovery. It is notable that we are looking at the impact of

up- or down-regulating each gene as well as knock- ing out its function. CRISPRn usually relies on a specific guide RNA to direct a DNA nuclease

Drug Discovery World Spring 2019

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