Nucleofection was performed in all experiments using K562, HeLa, and
U2OS cells. For PE conditions in these cell types, 800ng prime editor-expression
plasmid, 200ng PEgRNA-expression plasmid, and 83ng nicking plasmid was
nucleofected in a final volume of 20uL in a 16-well nucleocuvette strip (Lonza).
For HDR conditions in these three cell types, 350 ng nuclease-expression
plasmid, 150 ng sgRNA-expression plasmid and 200 pmol (6.6 μg) 100-nt
ssDNA donor template (PAGE-purified; Integrated DNA Technologies) was
nucleofected in a final volume of 20 μL per sample in a 16-well
Nucleocuvette strip (Lonza). K562 cells were nucleofected using the SF Cell Line
4D-Nucleofector X Kit (Lonza) with 5 × 105 cells per sample
(program FF-120), according to the manufacturer’s protocol. U2OS cells
were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with
3—4 × 105 cells per sample (program DN-100), according
to the manufacturer’s protocol. HeLa cells were nucleofected using the SE
Cell Line 4D-Nucleofector X Kit (Lonza) with 2 × 105 cells per
sample (program CN-114), according to the manufacturer’s protocol. Cells
were harvested 72 hours after nucleofection for genomic DNA extraction.
U2OS cells. For PE conditions in these cell types, 800ng prime editor-expression
plasmid, 200ng PEgRNA-expression plasmid, and 83ng nicking plasmid was
nucleofected in a final volume of 20uL in a 16-well nucleocuvette strip (Lonza).
For HDR conditions in these three cell types, 350 ng nuclease-expression
plasmid, 150 ng sgRNA-expression plasmid and 200 pmol (6.6 μg) 100-nt
ssDNA donor template (PAGE-purified; Integrated DNA Technologies) was
nucleofected in a final volume of 20 μL per sample in a 16-well
Nucleocuvette strip (Lonza). K562 cells were nucleofected using the SF Cell Line
4D-Nucleofector X Kit (Lonza) with 5 × 105 cells per sample
(program FF-120), according to the manufacturer’s protocol. U2OS cells
were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with
3—4 × 105 cells per sample (program DN-100), according
to the manufacturer’s protocol. HeLa cells were nucleofected using the SE
Cell Line 4D-Nucleofector X Kit (Lonza) with 2 × 105 cells per
sample (program CN-114), according to the manufacturer’s protocol. Cells
were harvested 72 hours after nucleofection for genomic DNA extraction.