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New CRISPR-Based Myoediting Technique Simplifies Correction of DMD Mutations

NEW YORK (GenomeWeb) – Researchers in the US and Germany have developed a CRISPR-based editing technique for Duchenne muscular dystrophy mutations that they say is simpler and more efficient than the editing methods currently being used to try to correct the underlying genetic basis of the disease.

"The large size and complicated structure of the DMD gene contribute to its high rate of spontaneous mutation," they wrote in their paper, which was published in Science Advances yesterday. "There are about 3,000 documented mutations in humans, which include large deletions or duplications (about 77 percent), small indels (about 12 percent), and point mutations (about 9 percent). These mutations mainly affect exons."

The new technique, called myoediting, uses a guide RNA (gRNA) that is capable of skipping 12 of the 79 encoded exons in the DMD gene. And myoediting uses only one gRNA instead of the two that are usually used for DMD corrections because the researchers found that it isn't necessary to excise large regions of the genome. Instead, they noticed that most patients' mutations can be categorized into groups, and if those groups of mutations are corrected through CRISPR editing, dystrophin expression can be restored in large numbers of patients even if individual exons are skipped.

"Human DMD mutations are clustered in specific 'hotspot' areas of the gene (exons 45 to 55 and exons 2 to 10) such that skipping one or two of 12 targeted exons within or nearby the hotspots (termed 'top 12 exons') can, in principle, rescue dystrophin function in a majority (about 60 percent) of DMD patients," the authors wrote.

The team identified these top 12 exons, and then screened pools of gRNAs to target the exons in the human DMD gene. The potential guides were then tested in human embryonic kidney 293 cells. Once the guides were selected, the researchers performed myoediting on iPSC lines from DMD patients with large deletion mutations (which account for about 60 percent to 70 percent of DMD-causing mutations), rare pseudo-exon mutations, and large duplication mutations (which account for about 10 percent to 15 percent of identified DMD-causing mutations). In each case, the myoediting technique corrected the mutations.

The researchers also tested the efficacy of myoediting on pools of patient-derived induced cardiomyocytes (iCMs), to determine whether the technique could restore the cells' dystrophin protein levels and function. They found that dystrophin protein expression levels of the corrected iCMs were estimated to be comparable to wild-type cardiomyocytes, and that contractile dysfunction was efficiently restored in corrected DMD engineered heart muscles (EHM) to a comparable level of wild-type EHM.

"The potential power of this approach lies in its simplicity and efficiency. Even relatively large and complex deletions can be corrected by a single cut in the DNA sequence that eliminates a splice acceptor or donor site without the requirement for multiple guide RNAs to direct simultaneous cutting at distant sites with ligation of DNA ends," the authors concluded. "Although exon-skipping mainly converts DMD to milder [Becker muscular dystrophy], for a subset of patients with duplication or pseudo-exon mutations, myoediting can eliminate the mutations and restore the production of normal dystrophin protein."

The researchers cautioned, however, that a comprehensive and extensive analysis of off-target effects was beyond the scope of this study, and that it will eventually be important to evaluate the possibilities of such effects before myoediting is used in potential therapeutic applications.