Quantitative CRISPR Imaging Protocols

CRISPR imaging data were acquired on a Nikon Ti-E inverted wide-field fluorescence microscope equipped with a ×100 NA 1.40 PlanApo oil immersion objective, an LED light source (Excelitas X-Cite XLED1), an sCMOS camera (Hamamatsu Flash 4.0), and a motorized stage (ASI) with stage incubator (Tokai Hit). Acquisitions were controlled by MicroManager. All images were taken as z stacks at 0.4 μm steps and with a total of 15 steps and were projected in maximum intensity. Long-term live cell imaging was performed on Andor Dragonfly (high-speed confocal microscopy) based on Nikon TI microscope with Nikon Perfect Focus system, ×60 NA 1.40 objective, an Andor iXon Ultra 888 EM-CCD camera. Images were taken as z stacks at 0.5 μm steps (7 steps) and at a frame rate of 5 Hz. Interval time was set as 10 min. Cells were imaged for 4–6 h. During image acquisition, cells ware maintained at constant temperature of 37 °C and 5% CO₂ within an incubation box. All the fluorescence imaging data were analyzed by Image J. Signal-to-noise ratio was defined as the ratio of the intensity of a fluorescent signal and the power of background noise as following formula: $$\mathrm{SNR}=\frac{P_{\mathrm{signal}}}{P_{\mathrm{noise}}}=\frac{\mathrm{Max}\mathrm{intensity}\mathrm{of}\mathrm{GFP}\mathrm{spot}-\mathrm{Mean}\mathrm{intensity}\mathrm{of}\mathrm{background}\mathrm{GFP}}{\mathrm{Std.}\mathrm{dev.}\mathrm{of}\mathrm{background}\mathrm{signal}}$$