Plasmid Cloning for Prime Editing

Plasmids expressing pegRNAs and epegRNAs were cloned either by Gibson assembly, Golden Gate assembly using either a previously described custom acceptor plasmid^{14 (link)} or newly designed custom acceptor plasmids that contain trimmed evopreQ₁ or mpknot (the use of which is described in Supplemental Note 1), or synthesized and cloned by Twist Biosciences. Plasmids expressing sgRNAs were cloned via Gibson or USER assembly. DNA amplification was accomplished by PCR with Phusion U or High Fidelity Phusion Green Hot Start II (New England Biolabs). Plasmids expressing pegRNAs were purified using PureYield plasmid miniprep kits (Promega) when transfecting HEK293T cells or Plasmid Plus Midiprep kits (Qiagen) when transfecting other cell types, while plasmids expressing prime editors were purified exclusively using Plasmid Plus Midiprep kits. Plasmids ordered from Twist Biosciences were resuspended in nuclease-free water and used directly. Primers and dsDNA fragments were ordered from Integrated DNA Technologies (IDT). Uncropped agarose and northern blot gels are provided in Supplementary Figs. 16 and 17.