Optimizing conditions for mRNA-Cas9 delivery. (A) Efficiency and toxicity profile of mRNA-Cas9 delivery in a panel of five mammalian cell lines. Cells were transfected in a 384-well format with a titration of mRNA and transfection reagent and fixed either 6 or 24 h later (pt, post-transfection). OptiMEM (vehicle) was used as a control. Charts show the frequency of Cas9-positive cells (immunostaining for HA tag on Cas9 with gating for low and high expression based on fluorescence intensity; bar graph, left axis, labeled in light and dark gray), and the total number of cells (dot plot, right axis, labeled in magenta) as an indication of cytotoxicity. Data points represent mean per cell intensity values aggregated at well level from two independent experiments (minimum of 100 cells from two wells analyzed per condition). Error bars, SD. The condition selected for the study was 1% (vol/vol) MessengerMAX with 40 ng mRNA per well of a 384-well plate. (B) Representative images of U-2 OS cells stained for Cas9 (HA) and DNA (Hoechst) after transfection of mRNA-Cas9 (40 ng/well of a 384-well plate) coupled with 1% (vol/vol) RNAiMAX or MessengerMAX. Scale bar, 200 µm. (C and D) Reduced cell number with the highest tested MessengerMAX concentration is caused by high mRNA transfection efficiency and is not specific to Cas9 expression. U-2 OS cells were transfected with a titration of either mRNA-Cas9 or mRNA-GFP coupled with 1.5 % (vol/vol) MessengerMAX and fixed 24 h after transfection. Linear regression analysis showed a negative relationship between both Cas9-HA intensity (C) and GFP intensity (D) and cell number. The data point represents mean per cell intensity values from two independent experiments (minimum of 100 cells from two wells analyzed per condition).