a, Electrophoretic mobility shift assays with dCas9, 5′-extended pegRNAs and 5′-Cy5-labelled DNA substrates. pegRNAs 1–5 contain a 15-nt linker sequence (linker A for pegRNA 1, linker B for pegRNAs 2–5) between the spacer and the PBS, a 5-nt PBS sequence, and RT templates of 7 nt (pegRNAs 1 and 2), 8 nt (pegRNA 3), 15 nt (pegRNA 4), and 22 nt (pegRNA 5). pegRNAs are those used in e and f; full sequences are listed in Supplementary Table 2. b, In vitro nicking assays of Cas9(H840A) using 5′-extended and 3′-extended pegRNAs. Data in a, b are representative of n = 2 independent replicates. c, Cas9-mediated indel formation in HEK293T cells at HEK3 using 5′-extended and 3′-extended pegRNAs. Mean ± s.d. of n = 3 independent biological replicates. d, Overview of prime editing in vitro biochemical assays. 5′-Cy5-labelled pre-nicked and non-nicked dsDNA substrates were tested. sgRNAs, 5′-extended pegRNAs, or 3′-extended pegRNAs were pre-complexed with dCas9 or Cas9(H840A) nickase, then combined with dsDNA substrate, Superscript III M-MLV RT, and dNTPs. Reactions were allowed to proceed at 37 °C for 1 h before separation by denaturing urea PAGE and visualization by Cy5 fluorescence. e, Primer extension reactions using 5′-extended pegRNAs, pre-nicked DNA substrates, and dCas9 lead to substantial conversion to RT products. f, Primer extension reactions using 5′-extended pegRNAs as in b with non-nicked DNA substrate and Cas9(H840A) nickase. Product yields are greatly reduced by comparison to pre-nicked substrate. g, An in vitro primer extension reaction using a 3′-pegRNA generates a single apparent product by denaturing urea PAGE. The RT product band was excised, eluted from the gel, then subjected to homopolymer tailing with terminal transferase (TdT) using either dGTP or dATP. Tailed products were extended using poly-T or poly-C primers, and the resulting DNA was sequenced. Sanger traces indicate that three nucleotides derived from the pegRNA scaffold were reverse-transcribed (added as the final 3′ nucleotides to the DNA product). Note that pegRNA scaffold insertion is much rarer in mammalian cell prime editing experiments than in vitro (Extended Data Fig. 6), potentially owing to the inability of the tethered RT to access the Cas9-bound guide RNA scaffold, and/or cellular excision of mismatched 3′ ends of 3′ flaps containing pegRNA scaffold sequences. Data in e–g are representative of n = 2 independent replicates. For gel source data, see Supplementary Fig. 1.