Researchers from Kyoto University demonstrate how a dual CRISPR RNA method restored dystrophin protein function in induced pluripotent stem cells derived from Duchenne muscular dystrophy (DMD) patients. The approach worked by removing large sections of the dystrophin gene, allowing the cells to skip faulty or misaligned sections of the genetic code. This yields truncated but still functional proteins for a wide variety of mutation patterns associated with DMD.

Their findings are published in Stem Cell Reports in an article titled, “Dual CRISPR-Cas3 system for inducing multi-exon skipping in DMD patient-derived iPSCs.”

“To restore dystrophin protein in various mutation patterns of DMD, the multi-exon skipping (MES) approach has been investigated,” wrote the researchers. “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.”

“Dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein via multi-exon skipping induction,” explained senior author Akitsu Hotta of Kyoto University. “We expect this study to enlighten new ways to treat DMD patients and other genetic disorders that require extensive deletions.”

Due to significant variations in the mutation patterns affecting the dystrophin gene, deleting a small section of the gene can only be used for a limited number of DMD patients. Few techniques are available to induce a large deletion to cover the target exons spread over several hundred kilobases.

To overcome this hurdle, Hotta and his team used CRISPR-Cas3 to induce a deletion of up to 340 kilobases at the dystrophin exon 45–55 region in various DMD mutation patterns. Because it was rare to observe a deletion of more than a hundred kilobases using a single CRISPR RNA, the researchers used a pair of CRISPR RNAs inwardly sandwiching the target genomic region.

The authors noted potential limitations of the dual CRISPR RNA system. First, there is variation in the deletion pattern, and the precise start and end points of the deletion cannot be fully controlled. This could be a drawback when a large but precise deletion is required. Second, the study did not demonstrate the functionality of the recovered dystrophin protein. Third, other methods should be developed to improve the overall genome editing efficiency of the Cas3 system.

“Our dual-Cas3 system might apply to future gene therapies once we’re able to deliver the dual-Cas3 components in vivo to skeletal muscle tissues safely and efficiently,” said Hotta. “The ability to induce several hundred kilobases of DNA deletion itself also has broad applicability for basic research when a large deletion is needed.”

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