N1 methylpseudouridine triphosphate

The prime editor plasmid was first amplified by PCR to add optimized 5′ UTR regions and a poly-A tail in 3′ (Table S2). The resulting PCR product was purified with the PCR Products Purification Kit (EZ-10 Spin Column) (Bio Basic, Toronto, ON, Canada) and served as a template for subsequent in vitro transcription. Prime editor mRNAs were transcribed from this template using the HiScribe T7 mRNA Kit with CleanCap Reagent AG (New England BioLabs Inc., Ipswich, MA, USA) with complete replacement of UTP with N1-Methylpseudouridine-triphosphate (TriLink Biotechnologies Inc., San Diego, CA, USA). The reaction mixture was incubated at 37 °C for 4 h. The reaction volume was then increased to 50 µL with nuclease-free water. Then, 2 µL of DNase I was added to the reaction mixture. The reaction mixture was incubated at 37 °C for 15 min. Transcribed mRNAs were purified with the Monarch RNA Cleanup Kit (500 µg) (New England BioLabs Inc., Ipswich, MA, USA), eluted in 1 mM Sodium Citrate pH 6.4 and quantified by BioDrop, Cambridge, UK. PE mRNAs were then stored at −80 °C.

Human embryonic kidney cells, HEK293T, were purchased from the American Type Culture Collection and cultured according to the recommended conditions. N1-methylpseudouridine-triphosphate was purchased from TriLink Biotechnologies. Transfection reagents Fugene 6, Fugene HD, ViaFect and Luciferase Assay System kit were obtained from Promega. Linear PEI (25 kDa) was purchased from Polysciences, rotenone from MP Biomedicals and Antimycin A from Sigma-Aldrich. Lipofectamine 2000, LF, Dulbecco’s modified Eagle’s medium, DMEM, Opti-MEM reduced serum medium, phosphate buffered saline, PBS, and MitoSOX were from ThermoFisher Scientific. The plasmid pTK305 used for in vitro transcription of firefly Luciferase mRNA was purchased from Addgene (plasmid # 66,812; http://n2t.net/addgene:66812; RRID:Addgene_66812).