Figure 1.

AAV Vectors and Dystrophin Gene-Correction Strategy

(A) The dual-AAV6 vector CRISPR system comprised of a nuclease vector (N; left) expressing SaCas9 under control of the striated CK8e muscle-specific expression cassette (MSEC) and a target vector (T; right) containing two U6 promoter-driven single guide (sg)RNA expression cassettes and a mCherry reporter gene expression cassette under control of the CMV enhancer/promoter. (B) gRNAs produced from the target vector recruit SaCas9 to target sequences within introns 51 (i51) and 53 (i53) of the murine Dmd gene in striated muscle. Upon excision of the 45-kb intervening genomic region that contains the C to T nucleotide substitution in exon 53 of mdx^4cv mice (encoding a TAA nonsense mutation), a slightly truncated but functional dystrophin protein lacking the amino acids encoded on exons 52 and 53 (Δ5253) is produced. (C) Schematic of AAV vector expressing microdystrophin 5 (μDys5)⁷ under control of the CK8e MSEC.