(1143-D) CRISPR-Ready ioGlutamatergic Neurons: a functional genomics tool to easily generate disease-specific models for drug discovery and development
Tuesday, February 6, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: Gene knockouts have become indispensable tools for understanding gene functions and the complex interplay of genes in various diseases. In this context, human induced pluripotent stem cell (iPSC)-derived neurons have emerged as groundbreaking assets, offering physiologically relevant cell models pivotal for both biological research and drug discovery. However, neurons are notoriously difficult to transfect, and this has pushed scientists to perform gene knockout at the iPSC stage. This method has its drawbacks, as the consequences of gene disruption can manifest differently between iPSCs and their mature neuron counterparts. An alternative route is the creation of Cas9-ready cells. While this method sidesteps the challenges of direct neuron transfection, it demands a significant time commitment. Generating these cells requires meticulous engineering and a rigorous characterization to ensure they function as desired in research experiments. CRISPR-Ready ioGlutamatergic Neurons™ are precision reprogrammed human iPSC-derived neurons containing a constitutively expressed Cas9 nuclease. This product has been designed for scientists looking to generate gene knockouts and perform CRISPR screens in a physiologically relevant human cell, without the need to spend months engineering and characterising their own Cas9 stable iPSC lines. Cas9 is expressed and functional from day 1 in CRISPR-Ready ioGlutamatergic Neurons. Using guide RNAs (gRNAs) delivered by either lentiviral transduction or lipid-based transfection, we show successful knockout of SOX11, evidenced by more than 80% indel formation and immunofluorescence staining. A pooled CRISPR screen of 100 known genes involved in neurodegenerative diseases shows clustering in UMAPs of aminoacyl-tRNA synthetase (aaRSs) knockouts, genes associated with Charcot–Marie–Tooth neuropathy (CMT). CRISPR-Ready ioGlutamatergic Neurons form structural neuronal networks and express pan-neuronal markers (MAP2, TUBB3) and glutamatergic neuron specific markers (VGLUT2) by day 11. These cells share the same user benefits as bit.bio’s well-established ioGlutamatergic Neurons, offering users a well-defined and characterised human neuron that shows a rapid gain of functional activity. Their high lot-to-lot consistency, and their simplicity in handling and culturing support experimental scalability. In conclusion, CRISPR-Ready ioGlutamatergic Neurons offer easy disease-specific model generation for drug development. Using bit.bio’s simple cell culturing protocol with optimised gRNA delivery, researchers can achieve high gene knockout efficiencies and obtain functional experimental readouts from 4 days post-thaw. The cells arrive ready to use and offer the opportunity to perform single gene knockouts, and scale to high throughput pooled or arrayed CRISPR screens.