A consolidated AAV system for single-cut CRISPR correction of a common Duchenne muscular dystrophy mutation.

Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene, is a lethal neuromuscular disease. Correction of DMD mutations in animal models has been achieved by CRISPR/Cas9 genome editing using Cas9 (Cas9) delivered by adeno-associated virus (AAV). However, due to the limited viral packaging capacity of AAV, two AAV vectors are required to deliver the Cas9 nuclease and its single guide RNA (sgRNA), impeding its therapeutic application. We devised an efficient single-cut gene-editing method using a compact Cas9 (Cas9) to restore the open reading frame of exon 51, the most commonly affected out-of-frame exon in DMD. Editing of exon 51 in cardiomyocytes derived from human induced pluripotent stem cells revealed a strong preference for exon reframing via a two-nucleotide deletion. We adapted this system to express Cas9 and sgRNA from a single AAV9 vector. Systemic delivery of this All-In-One AAV9 system restored dystrophin expression and improved muscle contractility in a mouse model of DMD with exon 50 deletion. These findings demonstrate the effectiveness of CRISPR/Cas9 delivered by a consolidated AAV delivery system in the correction of DMD , representing a promising therapeutic approach to correct the genetic causes of DMD.