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

No single guide RNA for wild-type SaCas9 efficiently reframes human DMD exon 51, but the KKH variant of SaCas9 — carrying PAM-interacting domain substitutions that relax PAM specificity — accepts a 5′-AACAGT-3′ PAM within exon 51 and generates a double-strand break 4 bp upstream of the 5′-TGA-3′ premature termination codon created when exons 48 to 50 are deleted. Relaxed PAM recognition is what makes this therapeutically useful target site accessible at all.

KKH SaCas9 paired with an sgRNA targeting human DMD exon 51 generates over 65% total indels in DMD ΔEx48–50 patient-derived iPSCs, of which more than 55% are productive 3n-2 deletions capable of restoring the exon 51 open reading frame. A single cut is therefore sufficient to reframe the exon, without a second nuclease, donor template or excision strategy.

The indel spectrum of a Cas9 cut can be biased toward a precise reframing outcome by local microhomology: the DMD exon 51 sgRNA positions the KKH SaCas9 break between the two halves of a 5′-CTCT-3′ tetranucleotide, leaving a 2-nt 5′-CT-3′ microhomology on each side of the break, and 45% of resulting indels are a precise deletion of that 5′-CT-3′ dinucleotide, which reframes the exon.

Cardiomyocytes differentiated from DMD ΔEx48–50 iPSCs after KKH SaCas9 single-cut editing express dystrophin at a level comparable to healthy control iPSC-derived cardiomyocytes even without clonal selection or expansion of the edited pool. Calcium transient kinetics, both the time to peak of calcium release and the decay constant tau of reuptake, are abnormally elevated in uncorrected DMD ΔEx48–50 cardiomyocytes and return to healthy control values after editing.

KKH SaCas9 editing of DMD exon 51 in patient-derived iPSC cardiomyocytes produces no significant genomic editing at the top eight computationally predicted off-target sites, despite the variant's relaxed PAM requirement.

An All-In-One AAV9 vector can carry a complete KKH SaCas9 editing system within AAV's packaging limit: the muscle-specific Creatine Kinase 8 (CK8) promoter drives KKH SaCas9 expression, restricting it to skeletal muscle and heart, and two copies of the same sgRNA cassette are driven by separate RNA polymerase III promoters, 7SK and U6. The sgRNA is rate limiting for in vivo DMD editing, which is why two guide cassettes are included rather than one.

Intraperitoneal delivery of All-In-One AAV9-packaged KKH SaCas9 to postnatal day 4 ΔEx50 DMD mice restores dystrophin dose-dependently and unevenly across muscles. At 2 × 10^14 vg/kg, dystrophin-positive myofibers reach 36% in tibialis anterior, 52% in triceps and 79% in diaphragm, with dystrophin protein at 12% and 26% of wild-type in tibialis anterior and triceps but over 45% in diaphragm and over 38% in heart; raising the dose to 4 × 10^14 vg/kg pushes tibialis anterior and triceps protein above 27%. Total genomic indels are 4%–10% at the low dose and 8%–12% at the high dose, with a −2-nt deletion predominant.

Restoring dystrophin by All-In-One AAV9 KKH SaCas9 editing rescues both muscle histology and contractile function in ΔEx50 DMD mice four weeks after systemic delivery: centrally nucleated regenerating myofibers fall from 25%–35% of fibers, fibrotic and necrotic area falls from 10%–15%, and serum creatine kinase drops by 66% and 81% at the low and high dose. At the high dose, forelimb and hindlimb grip strength improve by 86% and 67% over untreated littermates, specific force rises by 51% in slow-twitch soleus and 78% in fast-twitch extensor digitorum longus, and fatigue resistance returns to wild-type levels.