Cv7000 confocal microscope

HEK293T stoplight cells were plated at a quantity of 30,000 cells per well in a 96-well imaging plate (Greiner CellStar #655090). After 24 h of incubation, cells were treated with different concentrations of Cas-CPP and Cas-HDR-CPP nanocomplexes. After 24 h of incubation at 37 °C and 5% CO₂, cells were washed with 100 μL of high-glucose DMEM medium supplemented with 10% FBS. Following this, cells were incubated for another 24 h at 37 °C and 5% CO₂. Cell nuclei were stained by adding 2 µg/mL Hoechst 33342 in a complete cell culture medium as the final concentration and incubated for 30 min, after which cells were imaged using the Yokogawa CV7000 Confocal Microscope. (Yokogawa Corporation, Tokyo, Japan).

The endocytic uptake was monitored in ciPTEC following incubation for 1.5 h at 37°C with 50 μg/ml of either BSA‐AlexaFluor‐647 (A34785, Thermo Fisher Scientific) or DQ Red BSA (D12051, Invitrogen). The cells were then fixed and stained with Hoechst 33342 (1 µM) for 10 min and imaged using a CV7000 confocal microscope (Yokogawa Electric corporation, Tokyo, Japan). Data were quantified with Columbus™ Image Data Storage and analysis software (PerkinElmer, Groningen, the Netherlands). Data are expressed as the number of BSA/DQ‐BSA spots per cell.

HEK293T stoplight cells were plated at a density of 3 × 10⁵ cells/cm² on a 96-well black plate (Greiner CellStar #655090). The following day, the cells were treated with 10 μL of LNP-RNP supplemented with 1% antibiotic/antimycotic solution (Sigma-Aldrich, Zwijndrecht, The Netherlands). Cells were washed after 24 h with 100 μL of low-glucose DMEM medium supplemented with 10% FBS and 1% antibiotic/antimycotic solution. The cells were incubated for another 24 h at 37 °C and 5% CO₂. Following this, the cells were treated with 2 μg/mL Hoechst 33342 in complete cell culture medium for 15 min and imaged using a Yokogawa CV7000 Confocal Microscope (Yokogawa Corporation, Tokyo, Japan). The fluorescence image analysis was performed with the Columbus Software (Perkin Elmer, version 2.7.1), of which the analysis workflow is shown in Supplementary Figure S18. Gene editing efficiency was defined as the number of cells expressing EGFP divided by the number of cells expressing mCherry, as described previously [29 (link)]. LNP formulations were compared to a positive control, consisting of RNP delivered using ProDeliverIN CRISPR (Oz Biosciences, San Diego, CA, USA), as specified by the manufacturer, except that a 3.3 μL:1 μg ratio of reagent to protein was used.