@article{6caa131794e34b4c81995df709ea9d0e,
title = "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 \textasciitilde{}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 = "adenine base-editing, Cas9 off-target analysis, CFTR mutations, CRISPR/Cas9, cystic fibrosis, evolved Cas9 proteins, genome editing, human intestinal organoids, organoid biobank, patient-derived adult stem cells",
author = "Geurts, \{Maarten H.\} and \{de Poel\}, Eyleen and Amatngalim, \{Gimano D.\} and Rurika Oka and Meijers, \{Fleur M.\} and Evelien Kruisselbrink and \{van Mourik\}, Peter and Gitte Berkers and \{de Winter-de Groot\}, \{Karin M.\} and Sabine Michel and Danya Muilwijk and Aalbers, \{Bente L.\} and Jasper Mullenders and Boj, \{Sylvia F.\} and Suen, \{Sylvia W.F.\} and Brunsveld, \{Jesse E.\} and Janssens, \{Hettie M.\} and Mall, \{Marcus A.\} and Graeber, \{Simon Y.\} and \{van Boxtel\}, Ruben and \{van der Ent\}, \{Cornelis K.\} and Beekman, \{Jeffrey M.\} and Hans Clevers",
note = "Funding Information: This work was supported by the NWO building blocks of life project: Cell dynamics within lung and intestinal organoids (737.016.009), CRUK Specificancer (C6307/A29058), and grants of the Dutch Cystic Fibrosis Foundation (NCFS), the Netherlands as part of the HIT-CF Program; the Dutch Health Organization ZonMw, the Netherlands. Furthermore, we would like to thank the FACS facility of the Princes Maxima Centre, Utrecht, the Netherlands, for allowing us to perform sorting and analysis experiments. S.Y.G. is a participant in the BIH-Charit{\'e} Clinician Scientist Program funded by the Charit{\'e} – Universit{\"a}tsmedizin Berlin and the Berlin Institute of Health. Conceptualization, M.H.G. E.d.P. J.M.B. and H.C.; Base-Editing Experiments, M.H.G. and E.d.P.; Writing – Original Draft, M.H.G. and E.d.P.; Writing – Review \& Editing, M.H.G. E.d.P. J.M. H.M.J. K.M.d.W.-d.G. J.M.B. and H.C.; Supervision, J.M.B. and H.C.; Fluid Secretion \& Biochemical Assays, M.H.G. E.d.P. and F.M.M.; Whole-Genome Sequencing Experiments and Analysis, M.H.G. E.d.P. R.O. and R.v.B.; Tissue Harvesting and Biobanking, P.v.M. G.B. K.M.d.W.-d.G. S.M. D.M. B.L.A. J.M. S.F.B. H.M.J. M.A.M. S.Y.G. and C.K.v.d.E.; Tissue Culturing, M.H.G. E.d.P. E.K. S.W.F.S. and J.E.B.; Funding Acquisition, J.M.B. and H.C. J.M.B. is an inventor on (a) patent(s) related to the FIS assay and received financial royalties from 2017 onward. J.M.B. reports receiving (a) research grant(s) and consultancy fees from various industries, including Vertex Pharmaceuticals, Proteostasis Therapeutics, Eloxx Pharmaceuticals, Teva Pharmaceutical Industries, and Galapagos outside the submitted work. H.C. holds several patents on organoid technology. Their application numbers, followed by their publication numbers (if applicable), are as follows: PCT/NL2008/050543, WO2009/022907; PCT/NL2010/000017, WO2010/090513; PCT/IB2011/002167, WO2012/014076; PCT/IB2012/052950, WO2012/168930; PCT/EP2015/060815, WO2015/173425; PCT/EP2015/077990, WO2016/083613; PCT/EP2015/077988, WO2016/083612; PCT/EP2017/054797, WO2017/149025; PCT/EP2017/065101, WO2017/220586; PCT/EP2018/086716, n/a; and GB1819224.5, n/a. Funding Information: This work was supported by the NWO building blocks of life project: Cell dynamics within lung and intestinal organoids ( 737.016.009 ), CRUK Specificancer ( C6307/A29058 ), and grants of the Dutch Cystic Fibrosis Foundation (NCFS), the Netherlands as part of the HIT-CF Program; the Dutch Health Organization ZonMw, the Netherlands. Furthermore, we would like to thank the FACS facility of the Princes Maxima Centre, Utrecht, the Netherlands, for allowing us to perform sorting and analysis experiments. S.Y.G. is a participant in the BIH-Charit{\'e} Clinician Scientist Program funded by the Charit{\'e} – Universit{\"a}tsmedizin Berlin and the Berlin Institute of Health . Publisher Copyright: {\textcopyright} 2020 Elsevier Inc.",
year = "2020",
month = apr,
day = "2",
doi = "10.1016/j.stem.2020.01.019",
language = "English",
volume = "26",
pages = "503--510.e7",
journal = "Cell stem cell",
issn = "1934-5909",
publisher = "Cell Press",
number = "4",
}