Next-Generation Pooled and Arrayed CRISPR-Cas9 screens to identify and prioritise drug targets

CRISPR screening is a transformative technology that utilises the power and precision of CRISPR-Cas9 gene editing to reveal and validate novel drug targets or to study underlying causes of disease and to explore the effect of genetic mutations on drug resistance and patient responsiveness.

Horizon Discovery broadly employs two CRISPR screening formats in target ID and validation studies: pooled and arrayed, which can be utilised to answer a plethora of research questions.

Pooled screens involve introducing a ‘pool’ or mixture of sgRNA into a single population of cells which enables large genome-wide screens. However, these are most commonly performed using CRISPR-knockout, which limits the exploration of biology inaccessible to knockout screening. To address this limitation, we have evolved our pooled CRISPRko screening platform to include CRISPRi (interference) and CRISPRa (activation), which can be used to down- or up-regulate endogenous gene expression, respectively. Moreover, CRISPRi and CRISPRa can be combined to deliver dual loss-of-function or dual direction screening. This strategy enables researchers to explore drug mechanisms of action, enables the identification of novel biomarkers, and can provide compelling targets for the development of combination therapies. Additionally, Horizon has recently demonstrated the amenability of our platform for conducting next generation in vivo pooled CRISPR screens in patient-derived tumor xenograft PDX mouse models, screens in spheroids/organoids and screens that utilise single-cell analysis.

In arrayed CRISPRko screening, only one gene is targeted per well within multi-well plates using CRISPR libraries containing guide RNAs using the latest sgRNA design algorithms. Arrayed screens are key for functional genomic screening with multiplexed phenotypic endpoints, complex co-culture assay and for the confirmation of hits identified in pooled screens. To address the challenges of scaling arrayed screens, we have developed a range of automated workflows to perform screens in both primary and secondary cells.

Finally, CRISPR-based functional studies in primary immune cells has recently received a lot of attention as it opens the doors to significant scientific discovery in crucial areas of research, specifically in immuno-oncology. However, widespread application of these screens has been limited due to their extremely technically challenging nature. To address this, Horizon has developed an innovative approach to support the robust screening of primary human immune cells including gene modified B and T cells. Any potential new target or biological behaviour identified in these screens could translate more effectively and predictively from bench to bedside.

Here we present a range of these next generation pooled and arrayed CRISPR screening platforms and illustrate how each type of screen can be used throughout the drug development pipeline to speed the identification of new therapeutics and reduce the chance of late-stage failures in the clinic.