CRISPR-based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank.

Adenine base editing (ABE) enable enzymatic conversion from A-T into G-C base pairs. ABE hold 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 646 patients, of which ~20% can theoretically be repaired by ABE. We apply the SpCas9-ABE (PAM recognition sequence: NGG) and the 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.
Whole genome sequencing of Cas9 repaired cystic fibrosis organoids
Three SpCas9-ABE (R785X/R785X) and three xCas9-ABE-repaired organoid clones (F508del/R553X) and their respective unrepaired control organoids were paired-end whole genome sequenced using Illumina Novaseq 6000 system. This WGS confirmed the Cas9 repair worked in these organoids and showed no off-target hits nor no mutational hotspots created by the repair system.EGA study EGAS00001003951