Enabling Scalable CRISPR Screening in 3D Patient-derived Organoids by Using Microfluidics-on-Chip

Abstract: Patient-derived organoids hold great promise for personalised medicine as they are disease-relevant models which preserve the molecular features, tissue architecture and cell function of the tissue-of-origin. Proof-of-principle experiments have shown that 3D patient-derived organoids are amenable for drug screening and functional genomics approaches aimed at target identification and validation, including introducing genetic perturbations mediated by CRISPR-Cas9. However, compared with 2D in vitro cell line models, scalability remains a major challenge, which limits the applications of patient-derived organoid models in the drug discovery pipeline. 

Here we report a novel miniaturisation technology that has the potential to enable the use of 3D patient-derived organoids at scale for functional genomics approaches. To this end, we performed proof-of-principle experiments using a microfluidics-on-chip device, which is currently under development by Okomera. Featuring 8 chips, each one containing 78 traps encapsulating individual organoids, this system enables multiplexing of small libraries of lentiviral particles encoding sgRNAs and drug treatments, where each library is uniquely labelled with fluorescent barcodes.
Specifically, taking advantage of this automated microfluidics-on-chip device, we will show how we performed on the same chips i) first, injection of single cells, which led to organoid formation on the chip, using different media conditions; ii) second, delivery of a library of lentiviral particles encoding for sgRNA targeting a single gene per individual organoid; iii) third, delivery of drugs to test the sensitization effect of a genetic knock-out to specific compounds. Importantly, we have integrated this system with our AI-based imaging analyses to score organoid size and viability in response to genetic knock-out and drug treatment. By scoring multiple phenotypes across multiple individual organoids per condition, we demonstrate the ability to perform robust statistical analyses despite the inherent heterogeneity of organoids cultures and their responses to genetic and compound perturbations.

In summary, our pilot experiments demonstrated the feasibility of CRISPR and drug screens at scale in patient-derived organoids for target identification and validation. Notably, due to the miniaturisation of the screening process which requires very low volumes of media and extracellular matrix, and the ability to multiplex conditions, this microfluidics system represents a significant advance toward scalability and sustainability of CRISPR and drug screens in organoids. Together, this novel miniaturised methodology holds great promise for the use of 3D patient-derived organoids in functional genomics and drug discovery pipeline.