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. 2019 Aug 16;2(4):209–222. doi: 10.1089/crispr.2019.0013

FIG. 3.

FIG. 3.

Detection of iCas9 function using an episomal deletion assay in human cells. (A) A dual-fluorescence plasmid systems contains an EF1α-HTLV promoter (gray arrow), iCas9 recognition sites (gray and orange rectangles) flanking mCherry (red), and a downstream GFP (green) reading frame. iCas9 targeting results in deletion of mCherry and generation of a GFP-only vector. (B) HEK293Ts were cotransfected with the GFP–mCherry reporter plasmid, iCas9, and guides targeting the recognition sites. Cotransfection of iCas9 and a sg(NT) resulted in no shift of GFP expression. However, targeting with 22, 30, and 40 bp sgRNA spacings spacing shifted GFP by 2.8 ± 0.7, 2.7 ± .0.4 and 1.3 ± 0.4 % respectively. *p < 0.05, **p < 0.01, ***p < 0.001, by one-way ANOVA with Tukey's multiple comparison test. NS, nonsignificant.