CRISPR-Cas9 genome editing and induced pluripotent stem cells (iPSCs) are both Nobel Prize-winning technologies that represent a powerful tool for functional genomic screening and drug discovery.
Physiologically relevant model systems, like iPSCs, have a crucial advantage in enabling researchers to interrogate disease states more accurately, leading to more efficient and successful drug target identification and validation. However, editing iPSCs can be extremely time-consuming, inefficient and often unsuccessful.
This poster describes the use of CRISPR-ready iPSCs, which are engineered to constitutively express Cas9, enabling the rapid generation of high-efficiency gene knockouts and CRISPR screens in a functional, physiologically relevant cell background.
Download this poster to discover how to:
- How CRISPR-ready cells were developed to ensure constitutive expression of Cas9
- How to use them to interrogate the relationship between genotype and phenotype in a physiologically relevant iPSC-derived cell type
- How to generate high-efficiency knockouts and perform large scale screens for drug target identification and validation
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 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 targeting beta-2 microglobulin (B2M), we demonstrate high
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.
We performed a pooled CRISPR/Cas9-based knockout screen targeting 110 genes involved in the modulation of
innate immune signaling and the phenotypic consequences were profiled using a targeted single-cell RNA sequencing
readout. 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
physiologically relevant human cell types.
Summary & conclusions
Functional
CRISPR-Ready
ioMicroglia
Well characterised
and functional
human microglia
with constitutive
Cas9 expression.
High Knockout
Efficiency
Deliver gRNA by
lipid-based
tra
