Before transfecting, the sgRNA-Cas9 RNP complex was assembled by mixing 10 µl of P3 buffer from the P3 Primary Cell 4D-Nucleofector X Kit (Lonza no. V4XP-3032), 2 µl EnGen Spy Cas9 NLS (New England Biolabs, no. M0646M), and 2.5 µl of 60 µM folded sgRNA in a sterile microcentrifuge tube. The solution was gently mixed by swirling with a filtered micropipette tip and incubated at room temperature for 30 min to allow RNP complex formation. Cells were prepared for nucleofection during this incubation period. For co-transfection experiments, 5 µl of P3 buffer was mixed with 2 µl EnGen Spy Cas9 NLS and 2.5 µl of each sgRNA. The two mixtures were combined before being added to the cells.

Cells were harvested from the flask surface using a cell scraper, then homogenized with a micropipette. Cell density was determined using a Neubauer chamber. 1.5 × 10⁶ cells per transfection were centrifuged at 2000 × g for 5 min at room temperature. The supernatant was carefully removed, and the cell pellet was washed with 100 µl ice-cold 1X PBS. After a second centrifugation step, the supernatant was removed, and the pellet was resuspended in 6.5 µl P3 buffer per reaction. This solution was gently mixed with the RNP complex and quickly transferred to a well in the nucleofection strip from the Primary Cell 4D-Nucleofector X Kit.

For transfections including a repair template, 2 µl of 100 µM repair template (oligonucleotides no. 16–23, electronic supplementary material, table S1) were added to the RNP complex before nucleofection. Transfection was performed using the EN138 pulse on the Amaxa 4D-Nucleofector. Immediately after nucleofection, 80 µl of Marine Broth was added to the electroporation chamber to recover the cells. The entire volume was transferred to a 12-well plate (Corning no. 3513) containing 1 ml Marine Broth and incubated at 23°C for 24 h before proceeding to downstream experiments.