Schematic detailing critical steps of prime editing mechanism of action broken down by Cas9 activity, RT activation and function, stochastic endogenous repair mechanisms, and potential editing outcomes. (i) Protospacer hybridization between pegRNA and target DNA sequence. (ii) Engineered SpCas9 creates single-strand break in strand opposite pegRNA hybridization. (iii) Hybridization between the protospacer binding sequence (PBS) of pegRNA and newly generated 3′ flap from nickase activity. (iv) Reverse transcriptase adds nucleotides to the new 3′ end of the nicked DNA strand as directed by reverse transcription template (RTT) found adjacent to the PBS sequence. (v) An equilibrium is achieved between the unedited and edited flaps, where only one is reinserted back into DNA via endogenous DNA repair mechanisms. (vi) Insertion of 3′ flap back into the DNA and pruning of the 5′ flap by exonucleases results in the formation of a heteroduplex, where mismatch repair mechanisms determine whether the unedited strand will be remodeled in response to the edit, or whether the edit will be undone with the unedited strand as template. This process can be shifted in favor of incorporating the edit by introducing an sgRNA that nicks the unedited strand, increasing mismatch repair and improving editing efficiencies. Adapted from da Costa et al. (30).