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

Abstract

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 <i>Streptococcus pyogenes</i> Cas9 (<i>Sp</i>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 <i>Sp</i>Cas9 nuclease and its single guide RNA (sgRNA), impeding its therapeutic application. We devised an efficient single-cut gene-editing method using a compact <i>Staphylococcus aureus</i> Cas9 (<i>Sa</i>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 <i>Sa</i>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/<i>Sa</i>Cas9 delivered by a consolidated AAV delivery system in the correction of DMD <i>in vivo</i>, representing a promising therapeutic approach to correct the genetic causes of DMD.