They transfected mammalian cells with plasmids using jetOPTIMUS according to Polyplus recommendation. They kept the total DNA amount constant in all transient experiments to ensure reproducible complex formation and comparable results. They used 100 ng, 300 ng, and 2.4 μg of total DNA per well for 96-well, 48-well, and 6-well plate transfections, respectively. They used a 1:1 ratio of pegRNAs to prime editors and a 2:1 ratio of pegRNAs to auxiliary components like exonucleases, nicking gRNAs, and MLHdn. They plated the cells one day before transfection (25,000 cells per well in 100 μl for 96-well plates; 75,000 cells per well in 500 μl for 48-well plates; 600,000 cells per well in 3 ml for 6-well plates). They added 100 μl of fresh medium per well in 96-well transfections 24 hours after transfection. They removed 100 μl of medium per well and replaced it with fresh medium in 96-well transfections 48 hours after transfection.

Prime editing (PE) is a powerful gene-editing technique based on targeted gRNA-templated reverse transcription and integration of the de novo synthesized single-stranded DNA. To circumvent one of the main bottlenecks of the method, the competition of the reverse-transcribed 3' flap with the original 5' flap DNA, we generated an enhanced fluorescence-activated cell sorting reporter cell line to develop an exonuclease-enhanced PE strategy ('Exo-PE') composed of an improved PE complex and an aptamer-recruited DNA-exonuclease to remove the 5' original DNA flap. Exo-PE achieved better overall editing efficacy than the reference PE2 strategy for insertions ≥30 base pairs in several endogenous loci and cell lines while maintaining the high editing precision of PE2. By enabling the precise incorporation of larger insertions, Exo-PE complements the growing palette of different PE tools and spurs additional refinements of the PE machinery.