Journal Article

Comparison of In-Frame Deletion, Homology-Directed Repair, and Prime Editing-Based Correction of Duchenne Muscular Dystrophy Mutations

Zhao, Xiaoying, Bolund, Lars, Lin, Lin, Luo, Yonglun, Qu, Kunli, Curci, Benedetta, Yang, Huanming

2023

Overview

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.

• Publisher: MDPI AG,MDPI
• Subject: Care and treatment / Cas9 / Communication / CRISPR / CRISPR-Cas Systems - genetics / DMD / DNA repair / Duchenne muscular dystrophy / Duchenne's muscular dystrophy / Dystrophin - genetics / E coli / Efficiency / Fibroblasts / Flow cytometry / Gene deletion / gene editing / Gene mutations / Gene therapy / Genetic disorders / Genetic research / Genetic Therapy / Genome editing / gRNA / HEK293 Cells / Homology / Humans / Methods / Muscular dystrophy / Muscular Dystrophy, Duchenne - genetics / Muscular Dystrophy, Duchenne - metabolism / Muscular Dystrophy, Duchenne - therapy / Mutation / prime editing / Proteins