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. 2017 Jun 12;25(9):1999–2013. doi: 10.1016/j.ymthe.2017.05.015

Figure 3.

Figure 3

CRISPR-Cas9 Correction of the Deep Intronic IVS26 Mutation in CEP290 Using S. aureus Cas9

(A) Schematic representation of our paired guide strategy for the correction of the deep intronic IVS26 cryptic splice site mutation in CEP290. Two guides targeting upstream (sg3 and sg4) of the IVS26 mutation (red box) were each paired with two guides (sg5 and sg6) targeting downstream of the mutation to delete the exon X sequence and IVS26 mutation. (B) Representative gel image showing sg4/5-SaCas9-mediated IVS26 deletion in patient-derived iPSCs. Two lines of patient-derived iPSCs carrying homozygous IVS26 genotypes and those from a control unaffected individual were transfected with sg4/5-SaCas9-expressing plasmid (+) or Cas9 only (−). (C) Representative gel image demonstrating restoration of CEP290 transcript in sg4/5-SaCas9-treated iPSCs. POLR2A was amplified as a loading control. RET, human control retina cDNA. (D) Schematic representation of our CRISPR-Cas9/HDR strategy for the correction of the IVS26 cryptic splice site mutation (top; red box) in CEP290. Sg4/5RNA pair-expressing plasmid was co-delivered with a donor plasmid (middle; HDR cassette) containing 500 bp of homology flanking a floxed puromycin selection cassette and the wild-type IVS26 sequence resulting in the repaired allele (bottom). (E) Bright field image of a homozygous IVS26 patient-derived iPSC clone post-puromycin selection (note normal feeder-free iPSC morphology). Scale bar, 400 μM. (F) Representative gel image of genomic DNA from iPSC clones co-transfected with sg4/5-expressing plasmid and donor plasmid and cultured under puromycin selection. (G) Representative gel image demonstrating restoration of CEP290 transcript in sg4/5-SaCas9/donor-plasmid-treated iPSC clones. POLR2A was amplified as a loading control. WT, unaffected control iPSC cDNA; IVS, uncorrected patient-derived iPSCs carrying homozygous IVS26 alleles. (H) Western blot demonstrating restoration of full-length CEP290 protein in CRISPR/HDR-treated iPSC clones. Anti-α-Tub was used as a loading control.