Functional correction of the untreatable CFTR 1717-1G>A mutation through mRNA- and sgRNA-optimized base editing

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 missplicing, which prevents the correct synthesis of the encoded protein. In this study, we developed an adenine base editing (ABE) strategy to efficiently correct the 1717-1G>A mutation. By using the ABE9 base editor with the protospacer adjacent motif–relaxed Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) variant SpRY, we obtained up to 30% editing with limited bystander effects in a human embryonic kidney (HEK) 293–based cellular model. Through systematic optimizations of the ABE system, delivered by electroporation of base editor messenger RNA (mRNA) and single guide RNA (sgRNA), we demonstrated genetic repair of the 1717-1G>A mutation in airway epithelial cells and intestinal organoids derived from people with CF. Functional analysis was performed by measuring short-circuit current in air-liquid interface (ALI) culture and by assessing forskolin-induced swelling (FIS) in intestinal organoids, which revealed restoration of CFTR channel activity. These results highlight SpRY-ABE9 as a potential genome editing strategy to permanently correct the CFTR 1717-1G>A mutation and restore CFTR function.