Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.

Ubiquitylation (ubi) by the E3-ligases Mindbomb1 (Mib1) and Neuralized (Neur) is required for activation of the DSL ligands Delta (Dl) and Serrate (Ser) to activate Notch signalling. These ligases transfer ubiquitin to lysines of the ligands' intracellular domains (ICDs), which sends them into an Epsin-dependent endocytic pathway. Here, we have tested the requirement of ubi of Dl for signalling. We found that Dl requires ubi for its full function, but can also signal in two ubi-independent modes, one dependent and one independent of Neur. We identified two neural lateral specification processes where Dl signals in an ubi-independent manner. Neur, which is needed for these processes, was shown to be able to activate Dl in an ubi-independent manner. Our analysis suggests that one important role of DSL protein ubi by Mib1 is their release from cis-inhibitory interactions with Notch, enabling them to trans-activate Notch on adjacent cells.