Comparison of In-Frame Deletion, Homology-Directed Repair, and Prime Editing-Based Correction of Duchenne Muscular Dystrophy Mutations
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Recent progress in CRISPR gene editing tools has substantially increased the opportunities for curing devastating genetic diseases. Here we compare in-frame deletion by CRISPR-based non-homologous blunt end joining (NHBEJ), homology-directed repair (HDR), and prime editing (PE, PE2, and PE3)-based correction of two Duchenne Muscular Dystrophy (DMD) loss-of-function mutations (c.5533G>T and c.7893delC). To enable accurate and rapid evaluation of editing efficiency, we generated a genomically integrated synthetic reporter system (VENUS) carrying the DMD mutations. The VENUS contains a modified enhanced green fluorescence protein (EGFP) gene, in which expression was restored upon the CRISPR-mediated correction of DMD loss-of-function mutations. We observed that the highest editing efficiency was achieved by NHBEJ (74–77%), followed by HDR (21–24%) and PE2 (1.5%) in HEK293T VENUS reporter cells. A similar HDR (23%) and PE2 (1.1%) correction efficiency is achieved in fibroblast VENUS cells. With PE3 (PE2 plus nicking gRNA), the c.7893delC correction efficiency was increased 3-fold. Furthermore, an approximately 31% correction efficiency of the endogenous DMD: c.7893delC is achieved in the FACS-enriched HDR-edited VENUS EGFP+ patient fibroblasts. We demonstrated that a highly efficient correction of DMD loss-of-function mutations in patient cells can be achieved by several means of CRISPR gene editing.
Originalsprog | Engelsk |
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Artikelnummer | 870 |
Tidsskrift | Biomolecules |
Vol/bind | 13 |
Udgave nummer | 5 |
Antal sider | 15 |
ISSN | 2218-273X |
DOI | |
Status | Udgivet - 2023 |
Bibliografisk note
Funding Information:
This research was funded by Novo Nordisk Foundation (NNF21OC0072031 to Y.L.).
Publisher Copyright:
© 2023 by the authors.
ID: 352953372