Generation of corrected hiPSC clones from a Cornelia de Lange Syndrome (CdLS) patient through CRISPR-Cas-based technology.

backgroundCornelia de Lange syndrome (CdLS) is a rare multisystem genetic disorder which is caused by genetic defects involving the Nipped-B-like protein (NIPBL) gene in the majority of clinical cases (60-70%). Currently, there are no specific cures available for CdLS and clinical management is needed for life. Disease models are highly needed to find a cure. Among therapeutic possibilities are genome editing strategies based on CRISPR-Cas technology.

methodsA comparative analysis was performed to test the most recent CRISPR-Cas technologies comprising base- and prime-editors which introduce modifications without DNA cleavages and compared with sequence substitution approaches through homology directed repair (HDR) induced by Cas9 nuclease activity. The HDR method that was found more efficient was applied to repair a CdLS-causing mutation in the NIPBL gene. Human-induced pluripotent stem cells (hiPSCs) derived from a CdLS patient carrying the c.5483G > A mutation in the NIPBL were modified through HDR to generate isogenic corrected clones.

resultsThis study reports an efficient method to repair the NIPBL gene through HDR mediated by CRISPR-Cas and induced with a compound (NU7441) inhibiting non-homologous end joining (NHEJ) repair. This sequence repair method allowed the generation of isogenic wild-type hiPSCs clones with regular karyotype and preserved pluripotency.

conclusionsCdLS cellular models were generated which will facilitate the investigation of the disease molecular determinants and the identification of therapeutic targets. In particular, the hiPSC-based cellular models offer the paramount advantage to study the tissue differentiation stages which are altered in the CdLS clinical development. Importantly, the hiPSCs that were generated are isogenic thus providing the most controlled experimental set up between wild-type and mutated conditions.