Fig. 1: Analysis of ATXN3-sgRNAs/Cas9n cleavage activity in HEK293T cells. | Translational Psychiatry

Fig. 1: Analysis of ATXN3-sgRNAs/Cas9n cleavage activity in HEK293T cells.

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

Fig. 1

a SgRNA1 and sgRNA2 were designed to target CAG repeats located in the exon 10 of the ATXN3 NGG PAM sequences are highlighted in red. b Illustration depicting the CRISPR/Cas9 expression plasmid (PX330) co-express Cas9 protein with the mCherry reporter marker and sgRNA under the U6 promoter. c Sequence of sgRNA1 and sgRNA2 successfully cloned into PX330 plasmid. d The transfection efficiency of paired sgRNAs transfected into HEK293T cells were detected by immunofluorescence and flow cytometry, the transfection efficiency of sgRNA1, sgRNA2, and RFP was 38.5%, 43.5%, and 50.3%, respectively. Scale bar: 100 μm. e Analysis of sgRNA1 and sgRNA cleavage activity in HEK293T cells by T7EN1 assay. The length of the targeting PCR product is 630 bp (uncleaved band). Cleaved bands were marked with red arrow, sgRNA1 and sgRNA2 cleavage efficiency reached 19.3% and 22.6%, respectively. f After genome-editing with CRISPR/Cas9, the sequence trace after the break site comprised a mixture of signals derived from the unmodified and modified DNA.

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