Orthogonal R-loop assay

Orthogonal R-loop assays to measure off-target editing were performed as previously described⁴⁹, with minor modifications. Under standard conditions, 200 ng of SpCas9 sgRNA plasmid, 200 ng of SaCas9 sgRNA plasmid, 300 ng of base editor plasmid, and 300 ng of dSaCas9 plasmid were co-transfected into HEK293T cells using 1.5 μL of Lipofectamine 2000. For these transfection experiments, cells were cultured for 3 d, then washed with 1x PBS (ThermoFisher Scientific), followed by genomic DNA extraction by addition of 100 μL freshly prepared lysis buffer (10 mM Tris-HCl, pH 7.5, 0.05% SDS, 25 μg/mL proteinase K (ThermoFisher Scientific)) directly into each transfected well. The mixture was incubated at 37 °C for 1 h then heat inactivated at 80 °C for 30 min. Genomic DNA lysate was used immediately for high-throughput sequencing (HTS).