Jochen Hartner, Horizon Discovery Ltd, UK
Functional genomic screening with CRISPR–Cas9 has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. Rapid development of pooled lentivirus and deep-sequencing-led approaches have allowed us and others to exploit this technology in target ID, target validation, drug MOA analysis and patient stratification.
To date, the majority of screens have been conducted using loss-of-function perturbation driven by CRISPR-Cas9 enacted gene knock-out. Whilst powerful, this approach does not allow for the examination of activating gene function, leaving a salient gap in the functional genomic analysis. To address this, Horizon has developed two new paired pooled screening platforms employing catalytically inactivated Cas9 (dCas9) to control gene expression in both loss- and gain-of-function cell re-programming. Validation analysis of both tools revealed outstanding performance and sensitivity, with greater than ten-fold improvement in detection rates compared to existing platforms.
Screening for drug resistance with this dual platform yields unambiguous target discovery and the simultaneous evaluation of both activating and inhibiting perturbations reveals direct and opposing phenotypic effects within complex gene networks. Thus, in contrast to loss-of-function-only analysis, these tools can switch the response of affected cells to either sensitisation or resistance allowing the discovery of key genes which sit in the centre of the hit nexus. Bi-directional functional genomic screening provides a powerful new tool and truly novel discovery opportunities
