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Functional restoration of a CFTR splicing mutation through RNA delivery of CRISPR adenine base editor

Journal Contribution - Journal Article

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The 2789+5G>A CFTR mutation is a quite frequent defect causing an aberrant splicing and a non-functional CFTR protein. Here we used a CRISPR adenine base editing (ABE) approach to correct the mutation in the absence of DNA double-strand breaks (DSB). To select the strategy, we developed a minigene cellular model reproducing the 2789+5G>A splicing defect. We obtained up to 70% editing in the minigene model by adapting the ABE to the PAM sequence optimal for targeting 2789+5G>A with a SpCas9-NG (NG-ABE). Nonetheless, the on-target base correction was accompanied by secondary (bystander) A-to-G conversions in nearby nucleotides, which affected the wild-type CFTR splicing. To decrease the bystander edits, we used a specific ABE (NG-ABEmax), which was delivered as mRNA. The NG-ABEmax RNA approach was validated in patient-derived rectal organoids and bronchial epithelial cells showing sufficient gene correction to recover the CFTR function. Finally, in-depth sequencing revealed high editing precision genome-wide and allele-specific correction. Here we report the development of a base editing strategy to precisely repair the 2789+5G>A mutation resulting in restoration of the CFTR function, while reducing bystander and off-target activities.
Journal: MOLECULAR THERAPY
ISSN: 1525-0016
Issue: 6
Volume: 31
Pages: 1647 - 1660
Publication year:2023
Accessibility:Open