Fluorescence image analysis for levels of expression

Fluorescence images to visualize expression of the gRNAs-eGFP and dCas9-KRAB-mCherry were adjusted for appropriate LUTs for comparable analysis. To quantify fluorescent images of dCas9 fluorescence time course (Fig. 2), with mCherry reporter, images were binarized and positive pixel area was quantified as a ratio of the total area. To quantify co-expression ratios in Fig. 4b, f and Supplementary Figure 4, images in each channel (eGFP or mCherry) were adjusted for contrast and binarized using a threshold 0.15–0.21 for the eGFP channel and 0.2–0.3 for the mCherry channel, where all possible values lie between 0–1. Expression ratios were defined by the number of positive pixels in a channel over the total pixels within a field of view or region of interest. For eGFP: n_eGFP+/n_tot; for mCherry: n_mCherry+/n_tot; for overlapping eGFP and mCherry: n_{(eGFP+ & mCherry+)}/n_eGFP+, where n_tot was the total number of pixels for the camera (512 × 512). These ratios were calculated and summarized in a violin plot (or box plot) using GraphPad Prism.