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Review
. 2021 Aug;28(7-8):396-401.
doi: 10.1038/s41434-021-00263-9. Epub 2021 May 24.

Prime editing - an update on the field

Janine Scholefield  1   2 , Patrick T Harrison  3
Affiliations
Review

Prime editing - an update on the field

Janine Scholefield et al. Gene Ther. 2021 Aug.
No abstract available

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Prime editing in five steps.
Prime editing has just two components, a Cas9 nickase fused to a modified reverse-transcriptase (referred to as PE2) and a multifunctional prime editing guide RNA (pegRNA). 1 The Cas9-H840A/pegRNA complex binds to the desired target region and creates a nick 3 bp upstream of the PAM site. The nick must be upsteam of the first variant site (in this case a TGA stop codon) and occurs on the same strand as the PAM liberating a 3’ flap. 2 This 3’ flap forms a sequence-specific interaction with the 14-16 nt “primer binding site” located at the 3’ end of the pegRNA. This RNA/DNA hybrid serves as the PRIMEr site for new DNA synthesis using the RNA “edit site” as a template; the modified RT polymerase copies the template thereby extending the 3’ flap. 3 The edited 3’ flap displaces the variant unedited 5’ flap, which is removed by a cellular nuclease FEN1. 4 In this example, this leaves two MisMatches to be resolved, one in the edited codon (G ≠ T), and one in the modified PAM (C ≠ C) which can be introduced as an option to prevent further editing to the corrected sequence. 5 MisMatch Repair resolves the DNA resulting in either precisely edited DNA (with no indels), or the original variant sequence. In the latter case where the PAM sequence has not been modified, the Cas9-H840A/pegRNA complex can bind to the variant sequence again and have another attempt at PRIME editing. Key: Cas9-H40A nickase shown in Green, Reverse Transcriptase in yellow. The PAM site is highlighted in a grey box; the pegRNA in blue; the 3’ edit site in red; the edited PAM site in bold; FEN1 in grey. For clarity, only part of the pegRNA is shown in step 1.
See this image and copyright information in PMC

References

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