Cell Stem Cell
Volume 26, Issue 4, 2 April 2020, Pages 503-510.e7
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Clinical and Translational Report
CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank

https://doi.org/10.1016/j.stem.2020.01.019Get rights and content
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Highlights

  • 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

Summary

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

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

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These authors contributed equally

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These authors contributed equally

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