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. 2020 Apr 2;9(4):869. doi: 10.3390/cells9040869

Figure 4.

Figure 4

“All-in-one” HC-AdV transduction of RNA-guided CRISPR-Cas9 nucleases (RGNs) for DMD reading frame restoration. (A) DMD correction via NHEJ-induced reading frame resetting or exon-skipping. DMD deletions spanning exons 48 through 50 (i.e., DMD deletions Δ48–50) yield mRNA transcripts with frame shifts downstream of the exon 47–51 junction installing a premature stop codon. These transcripts result in low or no dystrophin in differentiated DMD.Δ48–50 muscle cells. HC-AdV-mediated delivery of RGNs cleaving genomic DNA between the exon 51 splice acceptor site (SA) and the downstream non-sense mutation (STOP) can install DSB-derived indels that yield Becker-like dystrophins either through reading frame resetting or SA knockout-induced exon-skipping. SD: splice donor site. (B) DMD correction via NHEJ-mediated multi-exon deletion. Removal of DMD-causing mutations (i.e., DMD deletions Δ45–52 and Δ48–50) in the major mutational hotspot after HC-AdV-mediated delivery of dual RGNs targeting introns 43 and 54. The excision of exons 44 through 54 results in the splicing of exon 43 to exon 55 and the generation of an in-frame mRNA species coding for a 359-kDa Becker-like dystrophin. Grey oval: C-terminal domain; green pentagon: ZZ zinc-finger domain; blue pentagons: EFH1 and EFH2 hand-regions harboring cysteine-rich motifs that, among other proteins, interact with β-dystroglycan; orange oval: WW domain, green boxes, spectrin-like repeats; black hexagons: hinges; orange hexagons: CH1 and CH2 actin-binding domain; vertical arrowheads: junctions between amino acids in the indicated spectrin-like repeats (R) after DMD gene editing. The diagrams showing the structural organization of the Becker-like dystrophins assembled after single and dual RGN delivery were made using software available in the eDystrophin online database (http://edystrophin.genouest.org).