Prime editing systems combine a SpCas9 protein with a reverse transcriptase and a prime editing guide RNA (pegRNA) to install a wide variety of genome edits, circumventing the need for DNA double-strand breaks or a DNA donor template. How a prime editor complex initiates and terminates pegRNA-guided reverse transcription remains poorly understood. Now, in Nature, Shuto et al. use cryoelectron microscopy (cryo-EM) to determine the spatial structure of a prime editor complex, providing insights into the stepwise mechanism of prime editing.
The authors assembled a primer editor complex of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukemia virus reverse transcriptase (M-MLV RT) together with pegRNA and target DNA and determined its structure at pre-initiation, initiation, elongation and termination states. Structural comparison between the different states revealed that the M-MLV RT stays in the same position relative to SpCas9 during reverse transcription, while the heteroduplex of pegRNA with synthesized DNA builds up along the surface of SpCas9. Importantly, the termination structure showed that M-MLV RT extends reverse transcription beyond the expected site, which can result in undesired edits at the target loci due to partial incorporation of the scaffold sequence. Developing strategies to eliminate these undesired inserts will be important for therapeutic applications of prime editing systems. The structural insights obtained here provide a valuable resource to guide the rational engineering of improved prime editors.
