CRISPR/Cas9 and Transposon-Based Conversion of a Pathogenic Biallelic Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes.

Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived -mutated iPSCs edited using the CRISPR/Cas9 and technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its "stemness". The correction of the disease-causing missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic mutations on nervous system development and physiology.