CRISPR/Cas9-mediated

Introduction: PHOX2B is a master transcriptional regulator of autonomic nervous system development whose mutations cause neurocristopathies including neuroblastoma and congenital central hypoventilation syndrome (CCHS). However, the identification and functional relevance of PHOX2B target genes remain incompletely defined, partly due to the lack of robust cellular models. Methods: Through CRISPR/Cas9-engineering we established a PHOX2B functional knockout IMR32 neuroblastoma model, and combined transcriptomic, chromatin-binding, electrophysiological, and live-cell imaging analysis to investigate PHOX2B-dependent pathways. We then assessed the developmental relevance of the obtained results in iPSC-derived sympathetic neurons from CCHS patients carrying different PHOX2B mutations. Results: PHOX2B functional knockout (PHOX2B-KO) cells exhibited morphological and molecular features indicative of enhanced neuronal maturation. RNA-seq and ChIP-seq analyses highlighted an unexpected direct role for PHOX2B in regulating the expression of ion channels, including Discussion: Together, our data establish the PHOX2B functional knockout neuroblastoma model as a valuable platform for functional genomics, enabling systematic analysis of PHOX2B targets and highlighting its critical role in coordinating transcriptional programs that shape neuronal excitability and maturation.