02076nas a2200325   4500000000100000000000100001008004100002260001500043653001500058653002100073653001100094653002600105653004000131100001100171700001800182700003500200700001800235700001900253700002300272700001900295700001700314700002000331700002100351245014500372856004700517300001200564490000800576520115200584022001401736       2024                            d  c2024-06-0110aCRISPR-Cas10adisease modeling10aHA-tag10aMembrane localization10aStem cell–derived sensory neurons1 aYi Liu1 aRachna Balaji1 aMarcelo A. Szymanski de Toledo1 aSabrina Ernst1 aPetra Hautvast1 aAylin B. Kesdoğan1 aJannis Körner1 aMartin Zenke1 aAnika Neureiter1 aAngelika Lampert00aThe pain target NaV1.7 is expressed late during human iPS cell differentiation into sensory neurons as determined in high-resolution imaging  uhttps://doi.org/10.1007/s00424-024-02945-w  a975-9920 v4763 aHuman-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel NaV1.7, which is a validated pain target. NaV1.7 deficiency leads to pain insensitivity, whereas NaV1.7 gain-of-function mutants are associated with chronic pain. During differentiation, the sensory neurons start spontaneous action potential firing around day 22, with increasing firing rate until day 40. Here, we used CRISPR/Cas9 genome editing to generate a HA-tag NaV1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface NaV1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust NaV1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes NaV1.7 surface expression. A clinically effective NaV1.7 blocker is still missing, and we expect this iPS cell model system to be useful for drug discovery and disease modeling.  a1432-2013