Fig. 2: Exon 45 skipping of human dystrophin using dual sgRNAs in DMD-iPS cells. | Nature Communications

Fig. 2: Exon 45 skipping of human dystrophin using dual sgRNAs in DMD-iPS cells.

From: Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice

Fig. 2: Exon 45 skipping of human dystrophin using dual sgRNAs in DMD-iPS cells.The alternative text for this image may have been generated using AI.

a Two sgRNAs were designed to induce human exon 45 skipping. hEx45 sgRNA #1 and hEx45 sgRNA #23 target the splice acceptor and donor site, respectively. The human DMD exon 45 sequence is highlighted in red. b Therapeutic strategy of genomic exon skipping by the dual sgRNA approach. Exon 45 skipping results in restoration of the dystrophin protein by adjusting the protein reading frame. c DMD-iPS cells were derived from a DMD patient lacking exon 44. Myogenic differentiation of the DMD-iPS cells was induced by the overexpression of MYOD1 (DMD-Myoblast). LNP-CRISPR, which is a mixture of LNP-Cas9 mRNA (1 µg mRNA) and LNP-sgRNA (total 1 μg of sgRNA: either 1 μg of #1, 1 μg of #23, or 0.5 μg each of #1 and #23), was administrated for 72 h. d Exon skipping efficiency was measured by RT-PCR and TapeStation. n = 1 experiment is shown, but similar exon skipping were observed in more than 3 experiments. e Dystrophin restoration was measured by Western blot. n = 1 experiment is shown, but similar Dystrophin recovery were obtained in more than 3 experiments.

Back to article page