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

Cystic fibrosis (CF) is a genetic disease caused by a variety of mutations in the CF transmembrane conductance regulator (CFTR) gene. The 2789+5G>A CFTR mutation is among the most common defects causing an aberrant splicing and resulting in the production of a non-functional CFTR protein. Here we used a CRISPR adenine base editing (ABE) approach to precisely correct the mutation and restore CFTR channel function in the absence of DNA double-strand breaks (DSB). To select the most efficient and accurate 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 relevant secondary (bystander) A>G conversions in nearby nucleotides, which affected the wild-type CFTR splicing. To maximise the 2789+5G>A on-target editing over the bystander edits we used a specific ABE type (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 obtain recovery of CFTR function. Finally, in depth sequence analysis showed genome-wide high editing precision as well as allele-specific correction, preventing unwanted edits of the second mutant CFTR allele.Here we report the development of a base editing strategy to repair the 2789+5G>A mutation allowing the reconstitution of CFTR function, while preventing bystander and off-target activities.