Abstract: CRISPR/Cas9 gene knockouts are essential for dissecting gene function but can be challenging to implement effectively in neural cell types due to low transfection efficiency and difficulties generating stable Cas9-expressing lines. To address these limitations, we combined our deterministic cell programming technology (opti-ox™) with CRISPR/Cas9 to develop CRISPR knockout (CRISPRko)-Ready ioCells™ - human induced pluripotent stem cell (iPSC)-derived ioMicroglia, ioOligodendrocyte-like cells, and ioMotor Neurons constitutively expressing Cas9. The integration of opti-ox and CRISPR technologies provides reproducible, scalable, and physiologically relevant neural cells, enabling efficient gene editing workflows via synthetic or lentiviral guide RNA (gRNA) delivery one day post thaw and at later experimental timepoints. Cas9 functionality has been rigorously validated across all cell types, consistently achieving knockout efficiencies above 70%. Importantly, each of the CRISPRko-Ready ioCells retains the phenotypic and functional characteristics of its wild-type counterpart, ensuring reliable experimental outcomes. The optimised performance of these cells positions them ideally for CRISPR screening workflows. As a proof-of-concept, a pooled single cell CRISPR knockout screen in ioMicroglia successfully identified genetic modulators of microglial polarisation between homeostatic (M0) and activated (M1) states, illustrating the practical utility of these cells for functional genomics. Overall, CRISPRko-Ready ioCells provide researchers with a robust and flexible research tool to accelerate gene function studies, streamline target identification, and advance drug discovery in neuroscience.
