Estimation of MS2V5 _17X copy number in the genomic DNA of LiveArt clonal cells. (A) Representative images to show MUC4 labeling in four LiveArt clonal cells. Nuclear-localized BFP indicates the expression of sgRNA targeting MUC4. Arrows pointed to MUC4 loci labeled by dCas9-GFP_14X. (B) Histograms of MUC4 loci number quantified by CRISPR imaging. (C) qPCR standard curves were generated using plasmids pMUC4 and pMS2V5_17x as template. Regression curves of the 10-fold serial dilutions are presented with respect to the log of the DNA load (number of plasmid copies) added to the reaction mixture versus Ct (mean of triplicate samples). (D) Parameters obtained from standard curves in C were used to calculate and assess the PCR efficiency. The regression curves of the log of the plasmid load versus Ct were nearly linear (R² > 0.99). The efficiency (E) of the qPCR was calculated using the equation E = 10^1/−m −1, where m is the slope of the line. To pass validation, the efficiency must be >90%. (E) qPCR results and calculated copy numbers. n = 2 biological replicates. (F) Copy number of MS2 cassette in LiveArt clonal cell lines. Black line indicates the mean value.