668 CRISPR-based cellular models with endogenous expression of HiBiT-tagged wild-type and mutant cystic fibrosis transmembrane conductance regulator enables high-throughput biology studies and screening for new transporter modulators

membrane.It is recognized as a membrane domain insertase and plays a critical role in mediating the biosynthesis of multi-transmembrane proteins [1,2], especially the ion channels, but whether EMC regulates CFTR biogenesis is not clear.Methods: In this study, we generated intestinal epithelial cell-specific EMC subunit 3 (EMC3) knockout (KO) mice by crossing EMC3 flox mice with Villin-Cre mice.Intestinal crypts were harvested and analyzed by reverse transcription polymerase chain reaction, mass spectrometry and western blotting.Intestinal organoids were used to study CFTR function through cyclic adenosine monophosphate (cAMP)-and calcium-dependent pathways and to monitor calcium flux between wild-type (WT) and KO mice.Results: Although EMC3 KO mice were viable after birth, they were smaller than their WT littermates, indicating a functional impairment of intestinal epithelium in the EMC3 KO mice.Molecular analysis of the protein profile collected by mass spectrometry of intestinal crypts from these mice revealed downregulation of the EMC complex, CFTR, and many other ion transporters.Western blot confirmed significant lower CFTR protein in EMC3 KO villi, whereas CFTR transcription was not altered.Forskolin-and cpt-cAMP-stimulated intestinal organoid fluid secretion was greatly reduced and delayed (Figure 1A).We found that EMC3 deficiency completely inhibited carbachol-mediated intestinal organoid fluid secretion (Figure 1B), whereas muscarinic receptors (carbachol receptors) were upregulated at transcriptional and translational levels in EMC3 KO villi.These data indicate that downstream signaling of muscarinic receptors was compromised in EMC3 KO epithelial cells, and muscarinic receptorspecific agonist Oxotremorine M failed to induce intracellular calcium flux or fluid secretion in EMC3 KO organoids.Pathway analysis of the above mass spectrometry data supported these observations.Conclusions: We conclude that EMC plays a critical role in vivo in CFTR biogenesis and activation, especially Ca 2+ -mediated CFTR activation.Targeting the EMC could enhance mutant CFTR expression and function.