Abstract: Drug developers are constantly seeking strategies to improve clinical relevance of drug discovery workflows. Genetic screens, in which large numbers of perturbations are made to the genomes of biologically relevant preclinical models, are a powerful strategy to accelerate early discovery. Arrayed genetic screening, as opposed to a pooled approach, offers better coverage of the genome by examining the effects of individual edits, enabling complex assay configurations and multi-parametric high throughput readouts for richer data. Unfortunately, arrayed screens are difficult to perform with translatable cell models, as they are prohibitively expensive and require large cellular inputs which necessitates pooling samples from multiple donors, thereby masking human diversity.
To address these issues and facilitate genome-wide arrayed CRISPR screening in precious patient derived cells, we have developed an automated microfluidic platform that miniaturizes non-viral transfection at scale. This innovative platform builds upon existing electroporation technologies by utilizing specific droplet geometries that enable gentle but efficient delivery of various payloads to cells. Our microliter-sized droplets drastically reduce the number of cells required per edit, from millions to a few thousand while maintaining high efficiency and high viability in primary cells. We have proven delivery across a wide range of payloads, including mRNA, RNP, DNA and beyond, achieving delivery efficiencies on par to existing delivery platforms, with no impact on cell viability or function. Our platform is cell type agnostic, achieving efficient editing in primary immune cell subsets, stem cells and even 3D structure such as organoids.
Herein, we present our custom 48-plex editing cartridge and associated workflow for integration with automation work cells. We will share the protocols developed to make our SBS-format consumable compatible with existing acoustic liquid handling technologies, enabling deposition of 48 independent guide RNAs across the cartridge. Next, we will present our integration efforts which have made our platform fully compatible with standard liquid handlers and robotic grippers, allowing for on-deck operation of our microfluidics system with robotic loading and offloading of reagents thereby facilitating the execution of thousands of edits per day without human intervention. Lastly, we will present the user-friendly interface which allows the user to program parameters for each independent electroporation site and monitor experimental success through our custom feedback system.
By driving rapid low cost, high throughput discovery at the single patient resolution, this platform will reduce the attrition of drug candidates during clinical trials and ensure that better therapies are developed for each and everyone of us.