Abstract: CRISPR technology has transformed the study of gene function, enabling precise manipulation through multiple modalities: knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa). While CRISPRko provides robust gene disruption, CRISPRi and CRISPRa offer nuanced, transcriptional modulation - downregulating or upregulating gene expression without genome editing. Implementing these approaches in post-mitotic human neurons remains challenging due to low transfection efficiency and the complexity of stable line generation. To overcome these barriers, we developed CRISPR-Ready ioGlutamatergic Neurons, human induced pluripotent stem cell (hiPSC)-derived neurons stably expressing either active Cas9 (CRISPRko) or catalytically inactive Cas9 (dCas9) fused to transcriptional effectors. These cells enable efficient, rapid gene perturbations via lentiviral or lipid-based guide RNA delivery immediately after thawing. Crucially, these hiPSC-derived neurons maintain key neuronal markers (MAP2, TUBB3, VGLUT2), form functional networks within days, and exhibit high lot-to-lot consistency. Leveraging our CRISPRko-Ready ioGlutamatergic Neurons, we established a single-cell CRISPR screening platform to identify genes involved in neurological diseases. A proof-of-concept pooled scCRISPR screen targeting neurodegeneration-associated genes identified genes implicated in Charcot-Marie-Tooth Disease. Furthermore, CRISPRi/a-Ready ioCellsTM allow precise perturbation of gene networks, facilitating comprehensive analyses of complex regulatory interactions. CRISPR-Ready ioGlutamatergic Neurons simplify experimental workflows, accelerate target identification and validation for drug discovery, and broaden possibilities for functional genomic studies in neurological disorders.
