CRISPR/Cas 9 Used to Generate Mouse Model of DMD

The mutation occurs on the X-chromosome, and the disease effects about one of every 3,500 boys whose muscle function is so degraded that they die usually before reaching the age of 30. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame.

CRISPR/Cas9 is regarded as the cutting edge of molecular biology technology. CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. Since 2013, the CRISPR/Cas9 system has been used in research for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation. By delivering the Cas9 enzyme and appropriate guide RNAs (sgRNAs) into a cell, the organism's genome can be cut at any desired location. The conventional CRISPR/Cas9 system is composed of two parts: the Cas9 enzyme, which cleaves the DNA molecule and specific RNA guides that shepherd the Cas9 protein to the target gene on a DNA strand. Efficient genome editing with Cas9-sgRNA in vivo has required the use of viral delivery systems, which have limitations for clinical applications.

Investigators at the University of Texas Southwestern Medical Center (Dallas, USA) used CRISPR/Cas9 gene editing to generate a DMD mouse model by deleting exon 50. These mice displayed severe muscle dysfunction, which was characteristic of DMD.

The investigators described in the November 29, 2017, online edition of the journal Science Translational Medicine how this dysfunction was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. They optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of the model mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represented a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.

“We made a mouse model that more faithfully represents the human disease,” said senior author Dr. Eric Olson, professor of molecular biology at the University of Texas Southwestern Medical Center. “We think these advancements will be valuable for the field and can help us move closer to tackling this disease in humans.”

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University of Texas Southwestern Medical Center