Fig. 7
From: Direct reprogramming of urine-derived cells with inducible MyoD for modeling human muscle disease
CRISPR/Cas9-mediated gene editing in UDCs. a Targeting strategy to introduce the 521ΔT point mutation into control SGCG exon 6 through gRNA guided, Cas9 nuclease generated double strand DNA break (DSB) with ssODN-mediated HDR repair. A portion of the SGCG exon 6 coding region is shown; a single T was removed from the 5T sequence region (cyan box). The downstream gRNA target sequence is highlighted in blue, the PAM sequence in pink, and the Cas9 DSB indicated by a yellow triangle. Below it is a portion of the 142 bp ssODN HDR repair template encoding the single T deletion along with a downstream G > A synonymous mutation (green) that destroys the PAM sequence and introduces an Alu1 restriction site. b Summary of efficiency of CRISPR/Cas9 protocol. c Alu1 restriction digest of a PCR-amplified genomic region including SGCG exon 6 and the flanking intronic sequences. d Left, partial summary of the Sanger sequencing results from individual line 2 edited clones. Right, the DNA trace from an unedited control and a clone with a homozygous deletion of a single T from the 5T of SGCG exon 6, recreating the most common LGMD2C mutation