Abstract
Prime editing is a novel genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. 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 ß-catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers diseasecausing 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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵# These authors jointly supervised this work
All main figures have been revised in order to better represent the data. Figure 1h has been added to provide an in-depth comparison of prime-editing efficiency and byproducts in 2D grown cell lines and 3D grown primary organoid cells. Figure 3 has been added and provides a whole-genome-sequencing analysis of prime-edited cells, showing that prime editing does not result in any predicted off-target effects or genome-wide mutational signature.