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Image Search Results
Journal: The CRISPR journal
Article Title: CRISPR-AsCas12a Efficiently Corrects a GPR143 Intronic Mutation in Induced Pluripotent Stem Cells from an Ocular Albinism Patient.
doi: 10.1089/crispr.2021.0110
Figure Lengend Snippet: FIG. 1. CRISPR-SpCas9 targets and sgRNA screening in OA1 patient-derived iPSCs. (A) Sequence of the double- stranded GPR143 intron 7 surrounding the patient’s G > A mutation (highlighted in red). The sequence of the two tested sgRNAs is also shown, with the 3¢ PAM sequence underlined. (B) In vitro digestion assay for the positive control (C+) sgRNA targeting the CDC42BPB gene. The ND PCR product size is 478 bp, whereas the estimated sizes of the CRISPR-SpCas9-digested bands are 407 and 71 bp, with only the higher band visible in the agarose gel. (C) In vitro digestion assay for the sgRNAs targeting the GPR143 intronic mutation. The ND PCR product size is 576 bp, whereas the estimated sizes of the CRISPR-SpCas9-digested bands are *315 and *261 bp. (D) Surveyor assay of OA1 patient-derived iPSCs treated with the C+ sgRNA targeting the CDC42BPB gene, and (E) with the sgRNAs targeting the GPR143 intronic mutation. NT cells were used as control. S- and S+, samples incubated in the absence or presence of the Surveyor nuclease, respectively. (F) Percentage of indels in the CRISPR-SpCas9-treated patient-derived iPSCs estimated by the TIDE and the ICE analyses. iPSCs, induced pluripotent stem cells; MW, molecular-weight ladder; ND, non-digested; NT, non-treated; OA1, ocular albinism type 1; PAM, protospacer adjacent motif; PCR, polymerase chain reaction; sgRNA, single-guide gRNA; TIDE, Tracking of Indels by DEcomposition.
Article Snippet: Cells were observed using the FluoView FV1000 confocal laserscanning microscope (Olympus, JSEI Core facility). iPSC in vitro differentiation assay The corrected patient-derived iPSC clones were differentiated using the
Techniques: CRISPR, Derivative Assay, Sequencing, Mutagenesis, In Vitro, Positive Control, Agarose Gel Electrophoresis, Control, Incubation, Molecular Weight, Polymerase Chain Reaction