EGFP Disruption Assay

HEK293T-EGFP cells generated by lentiviral transduction were transfected with a plasmid for a sgRNA targeting the EGFP gene along with the relevant Cas9 expression plasmid (WT Cas9, C-SMASh Cas9, or NC-SMASh Cas9) using Lipofectamine 3000. To compare the frequency of EGFP disruption under different conditions of ASV treatment, the fluorescence signal was either photographed under a Zeiss Axio Imager microscope (Carl Zeiss, San Diego, CA, USA) or analyzed using flow cytometry 6 days after transfection. For imaging, cells were re-plated on glass-bottom plates (Celltreat, Pepperell, MA, USA). Both differential interference contrast (DIC) images and EGFP fluorescence images were captured and analyzed using ZEN 2012 software. To quantify the loss of EGFP expression, transfected cells were trypsinized, resuspended in PBS containing 2% BSA, and analyzed on an Accuri C6 flow cytometer (BD Biosciences, Ann Arbor, MI, USA).