The Icebox

Prime Editing

Prime editing is a more precise gene editing technique than CRISPR-Cas9, allowing specific edits to be made in vivo to a cell’s genome.1 Using a fusion Cas9-reverse transcriptase enzyme and a prime editing guide RNA (pegRNA), the target site can be identified, and the DNA replaced. This does not require ds breaks or a donor DNA template (due to the reverse transcriptase).1 Prime editing has been used in animal models, to correct pathogenic mutations in liver and eye diseases, successfully treating the pathogenic mutation and altering the phenotype. There were no detectable off-target effects in this study2.

The pegRNA is an extended version of the guide RNA (gRNA) used in CRISPR-based gene editing techniques. This extended guide RNA also contains a primer binding site (hybridising with the desired section of DNA) and a reverse transcriptase template (acting as a template for DNA synthesis of the edited sequence).1

Cas9 works in much the same way as in CRISPR-based approaches, forming single-strand breaks (nicks) in the DNA, at the specified locations. Reverse transcriptase is used to synthesise the complementary strand from the template on the pegRNA.1

Getting the pegRNA into the cells

To get the pegRNA and Cas9-RT protein into the target cells, they must be transfected. This could be by electroporation, a viral vector, or lipofection (such as lipid nanoparticles).1

How does prime editing compare to CRISPR-Cas9?

Prime editing allows different DNA repair pathways to be used. Instead of non-homologous end joining and homology directed repair, which are used in CRISPR-Cas9, prime editing uses DNA mismatch repair. DNA mismatch repair reduces the chance of insertions or deletions at the site being edited, increasing the precision of the technique.1,3

References:

  1. Anzalone, A.V., Randolph, P.B., Davis, J.R. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019). doi: 10.1038/s41586-019-1711-4↩

  2. Jang, H., Jo, D.H., Cho, C.S. et al. Application of prime editing to the correction of mutations and phenotypes in adult mice with liver and eye diseases. Nat Biomed Eng 6, 181–194 (2022). https://doi.org/10.1038/s41551-021-00788-9↩

  3. Sheridan, C. (2019) ‘Gene editing enters “prime” time’, Nature biotechnology. doi: 10.1038/d41587-019-00032-5.↩