Neon transfection system 10 μl kit

Cells were transfected with pcDNA3.1 V5-His A, pcDNA3.1+-C-(K)-DYK plasmids encoding either wild-type EBP or one of the three mutant EBPs (E80K, R147H, W196S), EGFP-hGal3, or PM-GFP plasmids using Neon Transfection System 10 μL Kit (Thermo Fisher Scientific, #MPK1025, Waltham, MA, USA) following the manufacturer’s protocol. In brief, cells were prepared and resuspended in buffer R at a concentration of 100,000 cells/10 µL buffer R. Then, 10 µL of cell suspension was added to each plasmid solution containing 0.5 µg or 1 µg of plasmid DNA. The cell-plasmid DNA mixtures were then subjected to electroporation using the Neon Transfection System and transferred into laminin-coated 24 well plates containing 500 µL of prewarmed standard NSC media in each well. Transfected cells were selected with G418 sulfate (ThermoFisher Scientific, #10131035, Waltham, MA, USA) at 250 µg/mL for 5 days and subsequently maintained in 100 µg/mL G418 sulfate.

293A cells (Invitrogen) were maintained in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37°C in an atmosphere of 5% CO₂. After introduction of the episomal vector encoding SpCas9 variants, AcrIIA4-Cdt1-T2A-SpCas9 variants, or AcrIIA5-Cdt1-T2A-SpCas9 variants using Lipofectamine 3000 (Thermo Fisher Scientific), cells were selected using 350 μg/mL Hygromycin solution (FUJIFILM) for 3 days when lipofection was used for introduction of sgRNA plasmid and for 4–5 days when electroporation was used for introduction of sgRNA plasmid and ssODN. A Neon Transfection System 10 μL Kit (Thermo Fisher Scientific) was used for electroporation. In the HDR assessment using ssODN as the template, 50 pmol of ssODN and 250 ng of sgRNA plasmid were used for 1 × 10⁵ cells and conditions for electroporation were as follows: a pulse voltage of 1,245 V, pulse width of 10 ms, and three pulses.

N/TERT-2G keratinocytes were electroporated using the NEON transfection system 10μL kit (ThermoFisher Scientific). N/TERT-2G keratinocytes were detached from culture plastic and washed twice with dPBS (without Ca²⁺ and Mg²⁺) as described above. Meanwhile, per electroporation condition, synthetic sgRNA (300ng) and Cas9 (1.5 μg) were incubated with 5 μL resuspension buffer R for 20 min before adding 1×10⁵ N/TERT-2G keratinocytes. After mixing the cell suspension, the cells were electroporated using 1 pulse of 1700V for a duration of 20ms before immediate seeding in a pre-warmed 6 well plate. DNA was isolated using the QIAamp DNA blood mini kit (51106, Qiagen, Hilden, Germany) according to manufacturer’s protocol after a couple of days and CRISPR/Cas9 induced editing efficiency was analyzed by PCR and separation of amplicon on 2% agarose gel containing 1:10.000 GelRed nucleic acid gel stain (41003, Biotium Inc., Fremont, CA, USA). Amplicons were purified by MinElute Gel extraction kit (28606, Qiagen) using the manufacturers protocol and sanger sequenced to assess editing efficiency. Sanger sequencing reads were analyzed using the Inference of CRISPR edits (ICE) webtool (ice.synthego.com, v3, Synthego Corporation, Menlo Park, CA, USA). See Table 4 for details on the PCR primers used.