In vivo genome editing restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers [GUIDE-seq]

Duchenne muscular dystrophy (DMD) is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. In this study we developed a novel strategy for reframing DMD mutations by CRISPR-mediated large-scale excision of exons 46–54. We compared this approach to other DMD rescue strategies using DMD patient-derived primary muscle-derived stem cells (MDSCs) and found that it showed the highest efficiency in terms of restoring of dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cpf1)-mediated genome editing could correct DMD mutation with higher specificity than CRISPR-associated protein 9 (Cas9). Furthermore, A patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Dystrophin expression levels were increased by 10%–30% in human DMD muscle fibers. The restored dystrophin in vivo was functional, as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. This study provides a sensitive indicator for in vivo efficacy of gene editing and lays the foundation for a clinical trial of DMD treatment with gene editing technology. The GUIDE-seq (Genome-wide, unbiased identification of DSBs enabled by sequencing) was applied to capture all the DNA double-stranded breaks (DSBs) generated by Cas9 and Cas12a. When the gRNA library is constructed, two libraries are generated of each gRNA sample, one is forward and the other is backward, which respectively correspond to N_R and P_R sequencing files. At the same time, each library will generate a corresponding index file, corresponding to the file containing N_I and P_I respectively