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Dual CRISPR-Cas3 system for inducing multi-exon skipping in DMD patient-derived iPSCs

Kita, Yuto Okuzaki, Yuya Naoe, Youichi Lee, Joseph Bang, Uikyu Okawa, Natsumi Ichiki, Akane Jonouchi, Tatsuya Sakurai, Hidetoshi Kojima, Yusuke Hotta, Akitsu 京都大学 DOI:10.1016/j.stemcr.2023.07.007

2023.09.12

概要

To restore dystrophin protein in various mutation patterns of Duchenne muscular dystrophy (DMD), the multi-exon skipping (MES) approach has been investigated. However, only limited techniques are available to induce a large deletion to cover the target exons spread over several hundred kilobases. Here, we utilized the CRISPR-Cas3 system for MES induction and showed that dual crRNAs could induce a large deletion at the dystrophin exon 45–55 region (∼340 kb), which can be applied to various types of DMD patients. We developed a two-color SSA-based reporter system for Cas3 to enrich the genome-edited cell population and demonstrated that MES induction restored dystrophin protein in DMD-iPSCs with three distinct mutations. Whole-genome sequencing and distance analysis detected no significant off-target deletion near the putative crRNA binding sites. Altogether, dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein via MES induction.

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