Prime editing involves three major components:
* A prime editing guide RNA (pegRNA), capable of (i) identifying the target nucleotide sequence to be edited, and (ii) encoding new genetic information that replaces the targeted sequence. The pegRNA consists of an extended single guide RNA (sgRNA) containing a primer binding site (PBS) and a reverse transcriptase (RT) template sequence. During genome editing, the primer binding site allows the 3’ end of the nicked DNA strand to hybridize to the pegRNA, while the RT template serves as a template for the synthesis of edited genetic information.
* A fusion protein consisting of a Cas9 H840A nickase fused to a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase.
**Cas9 H840A nickase: the Cas9 enzyme contains two nuclease domains that can cleave DNA sequences, a RuvC domain that cleaves the non-target strand and a HNH domain that cleaves the target strand. The introduction of a H840A substitution in Cas9, through which the 840th amino acid histidine is replaced by an alanine, inactivates the HNH domain. With only the RuvC functioning domain, the catalytically impaired Cas9 introduces a single strand nick, hence the name nickase.
**M-MLV reverse transcriptase: an enzyme that synthesizes DNA from a single-stranded RNA template.
* A single guide RNA (sgRNA) that directs the Cas9 H840A nickase portion of the fusion protein to nick the non-edited DNA strand.