CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank

Adenine base editing (ABE) enables enzymatic conversion from A-T into G-C base pairs. ABE holds promise for clinical application, as it does not depend on the introduction of double-strand breaks, contrary to conventional CRISPR/Cas9-mediated genome engineering. Here, we describe a cystic fibrosis (CF) intestinal organoid biobank, representing 664 patients, of which ~20% can theoretically be repaired by ABE. We apply SpCas9-ABE (PAM recognition sequence: NGG) and xCas9-ABE (PAM recognition sequence: NGN) on four selected CF organoid samples. Genetic and functional repair was obtained in all four cases, while whole-genome sequencing (WGS) of corrected lines of two patients did not detect off-target mutations. These observations exemplify the value of large, patient-derived organoid biobanks representing hereditary disease and indicate that ABE may be safely applied in human cells. [Display omitted] •664 patients and 154 CFTR mutations represented in an organoid biobank•Adenine base editors enable efficient repair of nonsense mutations in CFTR•xCas9 increases the target scope of CFTR repair in our biobank•Adenine base editors cause no detectable off-target effects during repair Here, we show the generation of an extensive cystic fibrosis patient-derived intestinal organoid biobank. We use this biobank to study gene correction by adenine base editors and show genetic repair of four selected nonsense mutations in CFTR without any genome-wide off-target effects on canonical and non-canonical PAMs.