Fig. 3

Editing of IRF3, p65 and TLR3 genes in HeLa cells using the Cas9-T2A-CD95∆ construct and IZsCD95L as selection agent. Three different gRNAs per gene were tested and a control gRNA targeting GFP was used. Cells denoted as TLR3 gRNA-1* were not treated with IZsCD95L. a Sanger sequencing results. The frequency of indels in polyclonal cell lines was quantified from chromatograms using the TIDE analysis. Genome extraction, PCR and sequencing were performed twice. PCR1 was in addition sequenced with a second primer (p2). Mutation = 0 represents wild type sequence. The non-interpretable fraction (n.i.) relates to the correlation coefficient of the TIDE analysis with R² = 100 - n.i. b Representative agarose gels from three T7E1 assays. Genome extraction from polyclonal HeLa cell lines, PCR and T7E1 digest were repeated three times. c Predictions of the cleavage fraction were obtained by estimating the amount of heteroduplexed DNA by using equation (2) and data from the Sanger sequencing (green-striped). The cleavage fraction (yellow bars) was quantified from T7E1 assays by: cleaved DNA/(cleaved DNA + non-cleaved DNA). d Purple-striped bars show the predicted editing efficiency using equation (1) and data from the T7E1 assay. A different estimate of the editing efficiency was obtained by Sanger sequencing and the TIDE analysis (grey bars)