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

Abstract:

Background Cornelia 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.

Methods We tested CRISPR-Cas technologies including most recent base- and prime editors which introduce modifications without DNA cleavages and compared with sequence substitution approaches through homology directed (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.We generated isogenic corrected clones from human induced pluripotent stem cells (hiPSCs) obtained from a CdLS patient carrying the c.5483G>A mutation in the NIPBL gene.

Results We discovered that the most efficient method to repair the NIPBL gene is HDR mediated by CRISPR-Cas and induced with a compound (NU7441) inhibiting non-homologous end joining (NHEJ) repair. This sequence repair method allowed us to generate isogenic wild-type hiPSCs clones with regular karyotype and preserved pluripotency.

Conclusions With this study we generated a CdLS cellular model which will facilitate the investigation of the disease molecular determinants and the identification of therapeutic targets. In particular the hiPSCs cellular models offer the paramount advantage to study the tissue differentiation stages which are altered in the CdLS clinical development. Importantly, the hiPSCs that we have generated are isogenic thus providing the most controlled experimental set up between wild-type and mutated conditions. Finally, the efficient and precise gene replacement strategy developed in this study can be extended to the modification of other genomic loci to produce hiPSCs preserving staminal properties.