Prime editing for functional repair in patient-derived disease models

• Published in: Nature communications Vol. 11; no. 1; pp. 5352 - 8
• Main Authors: Schene, Imre F., Joore, Indi P., Oka, Rurika, Mokry, Michal, van Vugt, Anke H. M., van Boxtel, Ruben, van der Doef, Hubert P. J., van der Laan, Luc J. W., Verstegen, Monique M. A., van Hasselt, Peter M., Nieuwenhuis, Edward E. S., Fuchs, Sabine A.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 23-10-2020; Nature Publishing Group; Nature Portfolio
• Subjects: 13; 13/100; 42/109; 42/41; 45; 45/23; 631/1647/1511; 631/1647/1513/1967/3196; 631/532/2118/2437; 631/61/201/2110; beta Catenin - genetics; Cell culture; Cell Line; Cell Proliferation; Copper-Transporting ATPases - genetics; CRISPR-Cas Systems; Deoxyribonuclease I - metabolism; Diacylglycerol O-Acyltransferase - genetics; Gene Editing - methods; Gene sequencing; Genetic modification; Genome editing; Genomes; HEK293 Cells; Hepatolenticular Degeneration - genetics; High-Throughput Nucleotide Sequencing; Homology; Humanities and Social Sciences; Humans; Intestine; Liver; Liver cancer; Mimicry; multidisciplinary; Mutation; Organoids; Organoids - metabolism; Recombinational DNA Repair; Repair; RNA-directed DNA polymerase; Science; Science (multidisciplinary); Stem Cells; Subgroups; Targeted Gene Repair - methods; Whole genome sequencing; Wilson's disease; Wnt protein
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-19136-7

Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding β‐catenin (
CTNNB1
) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (
ATP7B
). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.
Prime editing uses Cas9 nickase fused to a reverse transcriptase to edit genetic information. Here, the authors prime edit primary adult stem cells in 3D organoid cultures to show functional correction of pathogenic mutations without genome-wide off-target effects.