Recent developments have led to significant improvements in the CRISPR-Cas9 technology for precise genome editing with the introduction of prime editing (PE).1 PE uses the Cas9 nickase fused to a reverse transcriptase to copy a DNA sequence into a specific locus from a PE guide RNA (pegRNA), allowing specific substitutions, small deletions, or insertions at targeted DNA sites without causing double-strand breaks.2
Prime editors are highly accurate and precise. However, PE efficiency is relatively low in cells because of the interference of DNA mismatch repair (MMR) proteins that localize to the sites of PE and directly counteract the installation of edits.3
To address this critical roadblock, in a recent study, Park et al.4 investigated the role of the MMR complexes MutSα (MSH2-MSH6 subunits) and MutSβ (MSH2-MSH3 subunits) in human embryonic stem cells (hESCs). Because MSH2, MSH3, and MSH6 gene expression is higher in hESCs compared to differentiated cell derivatives, the authors hypothesized that MutSα and MutSβ expression hinders the efficiency of PE in hESCs. To test this hypothesis, the investigators generated MSH2, MHS3, and MSH6 gene knockout (KO) hESC lines using CRISPR-Cas9 genome editing.
By using homozygous KO of the MSH2, MSH3, or MSH6 gene, they discovered that MutSα and MutSβ play a crucial role in determining the efficiency of PE in a size-dependent manner. In particular, when MSH2 is disrupted, both MutSα and MutSβ complexes are affected, resulting in a significant increase in PE efficiency from 1 up to 10 bases, with enhancement of up to 50-fold. Likewise, when MutSα is impaired through MSH6 KO, the editing efficiency improves from 1 to 3 bases, while defective MutSβ, caused by MSH3 KO, enhances efficiency from 3 to 10 bases. These findings demostrate that MutSα and MutSβ influence the size of the prime editing outcomes, underscoring the significance of MMR as a crucial determinant of PE efficiency in hESCs.
Although this study presents compelling evidence for the inhibitory role of MutSα and MutSβ in PE, there are some concerns regarding the authors’ approach. First, the long-term inhibition of MMR due to KO of MSH2, MSH3, and MSH6 can lead to increased mutational risk and genome instability. The authors explored the transient inhibition of MMR using small interfering RNAs (siRNAs), but their experiments did not show significant improvement of PE upon siRNA-mediated knockdown of MHS2 or MHS6. Further testing is necessary to determine whether transient inhibition of the MutSα and MutSβ complexes can effectively improve PE efficiency without the associated risks of gene KO. Second, the study focused solely on hESCs. It would be beneficial to test the approach in other primary cell lines to confirm the applicability of the findings in different cellular contexts. Finally, a comparison with existing methods for inhibiting MMR would provide more insight into the advantages and limitations of the proposed approach.
In summary, the findings of Park et al.4 provide valuable insights into the inhibitory role of MutSα and MutSβ complexes in PE outcomes. A better understanding of the inhibitory role of MMR can lead to strategies for overcoming the limitations in PE, opening new possibilities for precise and reliable genetic modifications.
Acknowledgments
Declaration of interests
The authors declare no competing interests.
References
- 1.Anzalone A.V., Randolph P.B., Davis J.R., Sousa A.A., Koblan L.W., Levy J.M., Chen P.J., Wilson C., Newby G.A., Raguram A., Liu D.R. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576:149–157. doi: 10.1038/s41586-019-1711-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Chen P.J., Liu D.R. Prime editing for precise and highly versatile genome manipulation. Nat. Rev. Genet. 2023;24:161–177. doi: 10.1038/s41576-022-00541-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Chen P.J., Hussmann J.A., Yan J., Knipping F., Ravisankar P., Chen P.F., Chen C., Nelson J.W., Newby G.A., Sahin M., et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell. 2021;184:5635–5652.e29. doi: 10.1016/j.cell.2021.09.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Park J.C., Kim Y.J., Han J.H., Kim D., Park M.J., Kim J., Jang H.K., Bae S., Cha H.J. MutSα and MutSβ as size-dependent cellular determinants for prime editing in human embryonic stem cells. Mol. Ther. Nucleic Acids. 2023;32:914–922. doi: 10.1016/j.omtn.2023.05.015. [DOI] [PMC free article] [PubMed] [Google Scholar]