Fig. 2.

Schematic representation of prime editing system. (A) The basic construct of PE consists of nCas9(H840A) fused to reverse transcriptase (RT) via a linker. The prime guide RNA (pegRNA) in PE consists of gRNA, primer binding site (PBS) and RT template. Once nCas9(H840A) nicks the strand, RT uses PBS fused to 3′ flap as a primer to encode RT DNA template. (B) After reverse transcription, PE1 and PE2 systems involve flap equilibrium of edited 3′ and unedited 5′ flap. The unedited 5′ flap is then cleaved off by structure-specific endonucleases followed by ligation and DNA repair/replication leading to permanent editing (Green bases: pegRNA binding regions, Red bases: desired edits). (C) After 5′ flap cleavage, a heteroduplex of the edited and unedited strand is formed. PE3 and PE3b involve a 2nd nick in the unedited strand 14-116 nt away from the initial pegRNA nick. This increases the chances of generation of dsDNA with the desired edit by favouring the repair of unedited strand by repair/replication machinery (Black strand: original DNA, Red strand: edited strand, Yellow triangle: nick site).