(1142-C) CRISPR knockout screening for drug target identification and validation using CRISPR-Ready ioMicroglia
Tuesday, February 6, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: Functional genomics screening plays a crucial role in drug target discovery by systematically assessing the impact of genetic perturbations on cellular functions. Methods such as CRISPR/Cas9-based knockout screening enable both the identification of potential therapeutic targets, and the validation of these targets by elucidating their role in disease pathways. The use of physiologically relevant model systems is critical in these screening approaches to ensure the future clinical success of identified targets. Human induced pluripotent stem cell (hiPSC) derived cell types have emerged as a powerful tool to advance our understanding of complex human biology and accelerate drug discovery programs.
Leveraging bit.bio’s precision reprogrammed hiPSC derived ioMicroglia™, we introduce CRISPR-Ready ioMicroglia for CRISPR/Cas9-based knockout screening. CRISPR-Ready ioMicroglia have been engineered to constitutively express Cas9, which is functional from day 1 post thaw. Using single guide RNA (sgRNA) targeting beta-2 microglobulin (B2M), delivered by lentiviral transduction, we demonstrate >86% knockout efficiency at protein level by flow cytometry analysis. Importantly, CRISPR-Ready ioMicroglia share the same features as ioMicroglia, expressing key markers including CD45, CD14, P2RY12, CX3CR1, CD11b and IBA1, and have comparable cytokine secretion profiles. Constitutive Cas9 expression does not impact the reprogramming potential, transcriptional profile or functionality of CRISPR-Ready ioMicroglia.
Using these cells, a pooled CRISPR/Cas9-based knockout screen was performed in which 110 genes involved in the modulation of innate immune signalling were perturbed and the phenotypic consequences were profiled using a targeted single-cell RNA sequencing readout. Additionally, a lipopolysaccharide (LPS)-induced activation signature was identified using bulk RNA sequencing and 258 genes were nominated and used as a benchmark to identify modulators of LPS-induced activation. Through subsequent bioinformatic analysis, 17 genes were identified that, when knocked out, altered responses to LPS stimulation and led to a reduction in microglial activation. Top screening hits will be brought forward for further validation using functional assays for cytokine secretion and phagocytosis activity.
In Conclusion, CRISPR/Cas9-based knockout screening using CRISPR-Ready ioMicroglia enables researchers to systematically interrogate healthy and diseased cell states for drug target discovery and validation using a physiologically relevant human cell type.