Ms2 p65 hsf1 gfp

For the experiment assessing Cas9 cleavage capacity at a synthetic promoter, HEK293FT cells were co-transfected with 200 ng gRNA, 200 ng Cas9 constructs, 50 ng reporter plasmid, and 25 ng enhanced blue florescent protein (EBFP) expressing plasmid as the transfection control. For the experiment assessing Cas9 transcriptional activation capacity at a synthetic promoter, HEK293FT cells were co-transfected with 50 ng gRNA, 70 ng Cas9 constructs, 100 ng MS2-P65-HSF1-GFP (Addgene plasmid ID: 61423), 200 ng reporter plasmid. Fluorescent reporter experiments were performed 48 h after transfection. Nonviable cells were excluded from the analysis using 7-AAD (7-amino-actinomycin D) conjugated with PerCP. Next, we selected cells expressing EBFP >2 × 10² A.U. or GFP >2 × 10² A.U. (transfection markers) in the cleavage and activation experiments, respectively to exclude untransfected cells (Supplementary Fig. 4). Flow cytometry was performed using a FACSCelesta flow cytometer (Becton Dickson) with HTS. Flow cytometry data were analyzed using FlowJo software. Untransfected controls were included in each experiment. Experiments underwent initial validation in duplicate and then were repeated in triplicate for the final manuscript.