Drug Discovery


How phenotypic CRISPR screening is revolutionising drug discovery


Research in drug discovery and biotechnology increasingly exploits gene editing at industrial scale in order to identify and validate new biological targets for precision medicines. The discovery of CRISPR-Cas9 systems has fuelled a rapid expansion of gene editing adoption with particular interest in the increasingly prevalent area of functional genomic screening. As these technologies have matured and evolved, so have the applications to which they are deployed. While early efforts were heavily enriched for studies in cancer biology, the advent of more complex phenotypic analyses, coupled to CRISPR-based discovery efforts, have substantially broadened the impact of these tools to provide discovery opportunities in myriad disease areas with greater power for hit ID and fewer off-target effects than other screening tools.


B


roadly speaking, functional genomic screen- ing requires three elements: an ability to perturb gene function, an assay by which to


measure phenotypic response and a mechanism to couple these two observations together (see Figure 1)1. In the case of CRISPR, the most common per- turbations are provided by the canonical knock- out based approach (CRISPRko) or by transcrip- tional control using the catalytically-inactivated and repurposed CRISPR modalities of CRISRPi (interference) and CRISPRa (activation). For gene knock-outs, the benefits are in the high penetrance of the perturbation, where the frequency of homozygous deletion can drive the response close to maximum in the cell system and allows excellent reproducibility between samples and replicates. Recent observations have indicated that some model systems might be unduly influenced by gene copy-number artefacts with this approach2, but while this might be a concern for some cancer tar-


Drug Discovery World Fall 2018


get ID applications, it is likely that in many cases the benefits of the clean and robust CRISPRko gene editing tool outweighs this consideration. CRISPRi and CRISPRa provide both ortholo-


gous and complementary tools for discovery, aug- menting a researcher’s ability to find, validate and exploit novel hits and elucidate new biological understanding. Inspired by modified versions of both ZnF and TALE nucleases3, these tools utilise a nuclease-dead version of Cas9 (dCas9) which in CRISPRi, rather than mutating specific genomic loci, allows disruption of gene transcription sim- ply by the binding of proximal sequences at the promoter region of the gene4-8. By covalently link- ing KRAB transcriptional repressor domain to dCas9, enhanced transcriptional silencing is achieved9,10. Using the same repurposed dCas9, it is also possible to create a transcriptional activa- tion tool, termed CRISPRa, by fusing dCas9 to the VP64 and p65 activation domains11-13,8. Effective


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By Dr Steffen Lawo and Dr Carlos le Sage


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