Figure 4: Deletion of exon 51 from the human genome using multiplex CRISPR/Cas9 gene editing. | Nature Communications

Figure 4: Deletion of exon 51 from the human genome using multiplex CRISPR/Cas9 gene editing.

From: Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy

Figure 4

(a) End-point genomic PCR across the exon 51 locus in human DMD myoblasts with a deletion of exons 48–50. The top arrow indicates the expected position of full-length PCR amplicons and the two lower arrows indicate the expected position of PCR amplicons with deletions caused by the indicated sgRNA combinations. (b) PCR products from a were cloned and individual clones were sequenced to determine insertions and deletions present at the targeted locus. The top row shows the wild-type unmodified sequence and the triangles indicate SpCas9 cleavage sites. At the right are representative chromatograms showing the sequences of the expected deletion junctions. (c) End-point reverse transcription–PCR analysis of dystrophin mRNA transcripts in CRISPR/Cas9-modified human Δ48–50 DMD myoblasts treated with the indicated sgRNAs. A representative chromatogram of the expected deletion PCR product is shown at the right. Asterisk: band resulting from hybridization of the deletion product strand to the unmodified strand. (d) Rescue of dystrophin protein expression by CRISPR/Cas9 genome editing was assessed by western blot for the dystrophin protein with GAPDH as a loading control. The arrow indicates the expected restored dystrophin protein band.

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