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

Abstract

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.

Keywords: CFTR mutations; CRISPR/Cas9; Cas9 off-target analysis; adenine base-editing; cystic fibrosis; evolved Cas9 proteins; genome editing; human intestinal organoids; organoid biobank; patient-derived adult stem cells.