fig5

Figure 5. Mechanism of Prime Editing. (A) Prime editing initiates when the protospacer of the pegRNA binds to its cognate site within the genome before a nick in the phosphodiester backbone is created on the target strand; (B) Following nicking, the PBS anneals to the DNA flap with the resulting double-stranded DNA duplex acting to prime reverse transcription using the RTT as a template; (C) This produces a 3’ flap containing the desired edit which then undergoes flap equilibration with the 5’ flap lacking the edit. Degradation of the 5’ flap and ligation of the 3’ flap into the genome will result in the incorporation of the edit. Recognition of the mismatch between the 3’ flap containing the edit and the unedited by the MMR DNA repair machinery can prevent incorporation of the edit. A nick to the non-edited strand in the PE3 and PE5 systems and/or expression of a dominant negative mismatch repair protein MLH1 in PE4 and PE5 systems can bias DNA repair in favour of incorporation of the edit. RTT: Reverse transcription template; pegRNA: prime editing guide RNA; PBS: primer binding site; MMR: mismatch repair; WT: wildtype. Adapted from Doman et al.^[92], Created in BioRender. Torrance R (2025) https://BioRender.com/06c7els.