CRISPR-Cas strategies for the correction of Cornelia de Lange Syndrome and Cystic Fibrosis mutations

CRISPR-Cas nucleases are revolutionizing the field of life sciences, allowing the editing of any site into the genome. Following the technological development of CRISPR-Cas systems, two main editing strategies can be used to introduce modifications in the DNA: induction of a Cas-dependent DNA double strand break (DSB) or DSB-free approaches, which rely on functional modules, such as a deaminase or a reverse transcriptase, fused to a catalytically impaired nuclease. The advancements of new CRISPR-Cas genome editing tools highly facilitated the generation of more refined disease models and is paving the way towards new perspectives for gene therapy, offering the opportunity of a permanent cure for monogenic disorders. In the first part of this thesis, diverse CRISPR-Cas-based technologies were tested to repair a mutation in the NIPBL gene (c.5483G>A), which causes the Cornelia de Lange syndrome (CdLS). An efficient strategy to correct this mutation through homology directed repair (HDR) was identified. This method included the use of a CRISPR-Cas nuclease combined with a specific donor DNA and a compound (NU7441) inhibiting non-homologous end joining (NHEJ) repair. The technology allowed to efficiently engineer pluripotent stem cells (hiPSCs) obtained from a CdLS patient resulting in the production of isogenic hiPSCs. The corrected hiPSCs were shown to have a regular karyotype and preserved pluripotency phenotype. In the second part, genome editing strategies based on HDR, base editing and prime editing were used to correct a class I cystic fibrosis (CF) mutation, the CFTR splicing defect 1717-1G>A. To this aim CF HEK293 cell models mimicking the splicing mutation were generated. Up to 42% of correction at the target adenine were obtained using adenine base editing. Nevertheless, high secondary modifications (bystander edits) potentially interfering with the corrected CFTR protein were detected with this approach. Conversely, the exploitation of alternative CRISPR-Cas technologies, including HDR and prime editing, resulted in efficient correction (over 40% of precise edits) in the absence of unwanted secondary edits. Overall, these results demonstrate that the advancement of CRISPR-Cas technology through the development of a large variety of tools can be applied differently depending on the genetic defect, target locus and cell type providing valuable experimental models such as hiPSC as well as novel potential therapeutic strategies.