We developed a prime editing (PE) strategy by incorporating a 5′–3′ exonuclease activity, which enhanced the efficacy and precision of ≥30-nucleotide DNA insertions without a secondary nick. Our optimization of the PE complex revealed that recruiting the exonuclease via an RNA aptamer outperformed direct protein fusions.
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References
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019). This study showed that combining a reverse transcriptase with a Cas9 nickase can be harnessed for genome editing using a 3′-extended pegRNA.
Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652.e29 (2021). This study reveals that DNA mismatch repair determines the outcome of PE events.
Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40, 402–410 (2022). This study showed that the intrinsic instability of pegRNAs can be improved by the 3′ addition of exonuclease-resistant motifs.
Liang, Z. et al. Addition of the T5 exonuclease increases the prime editing efficiency in plants. J. Genet. Genomics. 50, 582–588 (2023). The authors show that a fused T5 exonuclease enhances PE efficacy in plants.
Lin, Q. et al. High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat. Biotechnol. 39, 923–927 (2021). One of the first publications demonstrating that opposing paired pegRNAs can edit the DNA stretch between the two nicking sites.
Choi, J. et al. A time-resolved, multi-symbol molecular recorder via sequential genome editing. Nature 608, 98–107 (2022). This study reveals a versatile molecular DNA recorder that uses a PE4 strategy (PE2 plus mismatch repair inhibition).
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This is a summary of: Truong, D.-J. J. et al. Exonuclease-enhanced prime editors. Nat. Methods https://doi.org/10.1038/s41592-023-02162-w (2024).
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A prime editor that makes space for insertions. Nat Methods 21, 383–384 (2024). https://doi.org/10.1038/s41592-023-02163-9
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DOI: https://doi.org/10.1038/s41592-023-02163-9
