Drug Discovery


Figure 1


Functional genomic screening links genotype to phenotype on a very large scale, up to a genome-wide level to ask


questions such as which genes influence a particular disease physiology


gene activation with CRISPRa has been accom- plished by several approaches: the SunTag array, which uses multiple VP64s recruited on to a pep- tide array14; VPR, a synergistic tripartite activa- tion method using a fusion of VP64, p65 and Rta15; and the Synergistic Activation Mediator complex (SAM)16, which uses a dCas9-VP64 and recruitment of p65 and HSF1 via RNA binding protein components. These adapted CRISPR tools provide clear new opportunities to study gain-of- function mutations in genome-wide screens16,17.


Assays for phenotypic screening: a pooled solution Selecting the right assay is crucial for functional genomic screening success, and the biology to be measured influences key aspects of screen design. For example, in cancer biology it is often sufficient to evaluate the survival of a given cancer cell line model in order to robustly predict the response of the disease state. This can be done either in vitro or in vivo, but a simple measure of cell health is often not sufficient to evaluate more complex diseases or


present a complete picture of cell behaviour for high fidelity target ID. This is where phenotypic approaches with higher resolution yield substantial benefits, notwithstanding that cell health is itself a valuable phenotype for predictive biology. To date, most CRISPR-based functional


genomic screens have been conducted in a pooled format. This approach uses a viral-based system to tag the edited cells with a unique barcode, delineating their edited genotype. The identifying sequences can then be identified by amplicon deep sequencing, which in turn provides quantita- tive data about genotype abundance to inform biological interpretations. This technique, initial- ly developed for shRNA screening, was rapidly adopted for CRISPR approaches18-20, and pro- vides an immensely powerful dataset. Variable abundance of genotypes in differently-treated samples can be used to explore cell health in the context of drug responses for chemotherapeutic agents or to investigate genetic interactions between panels of cells of different origin. This latter example can be applied to explore genetic


Figure 2: Pooled phenotypic CRISPR screening uses high-throughput FACS to determine cell phenotypes based on a biomarker signal. This can be via immunostaining approaches or by using fluorescence-linked reporter cell systems and can be used to explore cell surface, intracellular or even secreted biomarkers


54 Drug Discovery World Fall 2018


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