Neon

The T47D cell electroporation was performed using the Neon^TM electroporation system in 100 μL Neon^TM Electroporation tip (Thermo Fisher). In brief, T47D or HEK293T cells, in suspension, were electroporated with a plasmids mixture containing 9 μg pCMV-PE2, 3 μg of either TP53 pegRNA, or HEK3 C > G pegRNA, and 1 μg of respective PE3 nicking plasmids. The electroporation was performed in serum- and antibiotic-free media, and the electroporation parameters used were 1700 V pulse voltage, 20 ms pulse width, and 1 pulse number. For the HEK293T cells, the following parameters were used: 1100 V pulse voltage, 20 ms pulse width, and 2 pulse number. The media was replaced with complete media 24 h post-electroporation. To establish the single cell-derived clones for TP53 prime editing, the electroporated T47D and HEK293T cells were subjected to single cell dilution. The cells were seeded at a density of 1 cell/well on a 96-well plate and were allowed to grow for 3–4 weeks, with interval media replenishment. The formation of single cell-derived clones was monitored under the microscope and expanded for subsequent experiments.