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

Yu Zhang; Takahiko Nishiyama; Hui Li; Jian Huang; Ayhan Atmanli; Efrain Sanchez-Ortiz; Zhaoning Wang; Alex A Mireault; Pradeep P A Mammen; Rhonda Bassel-Duby; Eric N Olson

doi:10.1016/j.omtm.2021.05.014

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

Mol Ther Methods Clin Dev. 2021 Jun 4:22:122-132. doi: 10.1016/j.omtm.2021.05.014. eCollection 2021 Sep 10.

Authors

Yu Zhang^{1

2}, Takahiko Nishiyama^{1

2}, Hui Li^{1

2}, Jian Huang³, Ayhan Atmanli^{1

2}, Efrain Sanchez-Ortiz^{1

2}, Zhaoning Wang^{1

2}, Alex A Mireault^{1

2}, Pradeep P A Mammen^{2

3}, Rhonda Bassel-Duby^{1

2}, Eric N Olson^{1

2}

Affiliations

¹ Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
² Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
³ Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

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 Streptococcus pyogenes Cas9 (SpCas9) delivered by adeno-associated virus (AAV). However, due to the limited viral packaging capacity of AAV, two AAV vectors are required to deliver the SpCas9 nuclease and its single guide RNA (sgRNA), impeding its therapeutic application. We devised an efficient single-cut gene-editing method using a compact Staphylococcus aureus Cas9 (SaCas9) 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 SaCas9 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/SaCas9 delivered by a consolidated AAV delivery system in the correction of DMD in vivo, representing a promising therapeutic approach to correct the genetic causes of DMD.

Keywords: AAV; CRISPR/Cas; Duchenne muscular dystrophy; SaCas9; exon reframing; exon skipping; gene editing; induced pluripotent stem cells; sgRNA.

Abstract

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