Fig. 2: Gene correction of SCA3-iPSCs using CRISPR/Cas9 system. | Translational Psychiatry

Fig. 2: Gene correction of SCA3-iPSCs using CRISPR/Cas9 system.

From: CRISPR/Cas9 mediated gene correction ameliorates abnormal phenotypes in spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cells

Fig. 2

a Schematic depicting the donor vector and Cre-loxP-based HR selection strategy used for targeting the ATXN3 locus, the targeting vector (pFlexible-DT) with left and right arms of 1.9 and 3.2 kb, respectively, and the 17 CAG repeats and Puro cassettes inserted upstream of the corrected region. The targeted fragment sizes were verified by P1–P2 and P3–P4 primers. b Quantification of the transfection efficiency of ATXN3-targeted sgRNAs, detected by flow cytometry, negative cell population (the left figure), 10% RFP positive cells (the middle figure), and 2.8% paired sgRNAs/Cas9n (sgRNA1 + sgRNA2) positive cells (the right figure). c Overview of the targeting workflow, including electroporation, selecting, and screening. d PCR-based screening for successfully targeted SCA3-iPSCs, the positive clones (C3, C11, C12, C13) contained 510 bp (31 CAG) and 3090 bp using P1–P2 primers, while the mutant bands (639 bp) indicated the 74 CAG repeats shown in red arrow. e Verification of successful correction at the ATXN3 locus in corrected SCA3-iPSCs by Western blotting, the C3 and C12 clones indicated no expanded polyQ tracts, the red arrow indicates the mutant ataxin-3 protein. f Summary of targeted efficiency of CRISPR/Cas9 genome editing HR was 1.7%.

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