We introduced MFN1 editing sites to the genome of HEK293T cells via Prime editing (38 (link)). The spacer and extension sequences were designed according to the guidelines provided previously (38 (link)). The designed oligos were cloned into the pU6-pegRNA-GG-acceptor (Addgene, 132777) to generate both pegRNA and nick guide RNA (gRNA) expressing constructs (oligo sequences were listed in table S4). Plasmids expressing pegRNA (250 ng), nick gRNA (83 ng), and prime editor (750 ng), namely, pCMV-PE2 (Addgene, 132775), were cotransfected into HEK293T (7500 cells per well in 48-well plates) with Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific, catalog no. L3000015) according to the manufacturer’s protocol. After 72 hours, genomic DNA was extracted, amplified by PCR, and sequenced via Sanger sequencing to confirm genome editing events. To optimize the editing efficiency, different combinations of pegRNAs and nick gRNAs (PE2, PE3, and PE3b) were tested. The prime binding site (PBS) and RT template were also optimized to 9 nt for PBS and 19 nt for RT template. The optimal condition (PE3b, 9-nt PBS, and 19-nt RT template) was used in scaled-up experiments to generate single-cell clones with MFN1 genome editing by serial dilution.