Functional correction of the CFTR 1717-1G>A mutation using a precise adenine base editor

The 1717-1G>A is a prevalent splicing mutation causing cystic fibrosis (CF) for which no pharmacological treatments have been approved. This mutation disrupts a canonical 3' AG splice acceptor site in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to severe RNA mis-splicing which prevents the correct synthesis of the encoded protein. In this study we developed an adenine base editing (ABE) strategy to efficiently reverse the 1717-1G>A mutation. By using ABE9 with the PAM-relaxed Cas9 variant SpRY, we obtained up to 30% editing with minimal bystander effects in a HEK293-based cellular model. Through a systematic optimization of the ABE system, delivered as mRNA-sgRNA, we demonstrated genetic and functional repair of the 1717-1G>A in airway epithelial cells and intestinal organoids derived from patients with CF. These results indicate SpRY-ABE9 as a potential genome editing strategy to permanently restore the 1717-1G>A-CFTR function.