Investigation of molecular mechanisms behind the neurodevelopmental disorder PURA syndrome using CRISPR/Cas9-generated cell models

The purine-rich element-binding protein A (PURA), is a small DNA and RNA binding protein, located in the cytoplasm and involved in multiple cellular molecular pathways, albeit the actual molecular function is still unclear. De novo heterozygous mutations on PURA, cause the rare neurodevelopmental disorder PURA syndrome, characterized by moderate to severe intellectual disability, hypotonia, epilepsy, feeding difficulties, next to many other symptoms that vary between patients. Dissecting PURA’s molecular function in a cellular context is fundamental to understand its role in PURA syndrome and so to develop potential treatments. In this study, an isogenic control (isoCTRL) cell line was successfully generated from patient induced Pluripotent Stem Cells (iPSC) carrying the most common mutation F233del using the CRISPR/Cas9 technology. Subsequent differentiation of both isoCTRL and patient iPSC into Neural Progenitor Cells (NPCs) revealed that the F233del does not impair early neural differentiation based on NPC morphology and expression of neuronal markers like PAX6, MAP2 and FOXG1. Furthermore, it was shown that PURA and FMRP colocalize in NPCs and that this colocalization was not disrupted in the patient cells. In addition, a PURA overexpressing cell line for BioID analysis to investigate PURA’s interactome and a PURA Knock Out (KO) cell line were generated from Human Neural Stem Cells (HNSCs), which are fundamental to explore potential neural markers interacting with PURA or regulated by PURA, respectively. This comprehensive approach provides the cellular bases and molecular insights to further study PURA’s molecular mechanism and its implications in PURA syndrome.