Enhanced prime editing systems by manipulating cellular determinants of editing outcomes
Chen, P.J. et al. (2021) Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652. DOI: 10.1016/j.cell.2021.09.018
This study presents a significant advancement in the precision and efficiency of prime editing, a versatile genome editing technique capable of introducing targeted substitutions, insertions, and deletions without creating double-stranded breaks. Through pooled CRISPR interference (CRISPRi) screens across 476 DNA repair genes, the researchers identified the mismatch repair (MMR) pathway as a major barrier to prime editing efficiency and fidelity.
The team developed enhanced editing systems, termed PE4 and PE5, by co-expressing a dominant-negative form of MLH1 (MLH1dn), an MMR-inhibitory protein. These systems boosted editing efficiency by an average of 7.7-fold (PE4) and 2.0-fold (PE5), while improving edit-to-indel ratios up to 3.4-fold, particularly in MMR-proficient cell types such as HeLa, K562, U2OS, and patient-derived cells.
Furthermore, the authors engineered a new prime editor protein, PEmax, incorporating codon optimization, linker design, and nuclear localization signals. PEmax, when combined with PE4 or PE5 and engineered pegRNAs (epegRNAs), significantly increased editing outcomes. For example, PE4max with epegRNAs improved editing efficiency by up to 72-fold in MMR-proficient cells compared to PE2.
Notably, the strategic incorporation of additional silent mutations adjacent to the edit site further enhanced editing by evading MMR recognition, even without MLH1dn. The systems were validated at disease-relevant loci, including those implicated in sickle cell anemia, prion disease, HIV resistance, and CDKL5 deficiency disorder, in various cell types including human induced pluripotent stem cells (iPSCs) and primary T cells. Importantly, transient expression of MLH1dn did not induce detectable microsatellite instability, underscoring its safety for therapeutic applications.
Keywords: prime editing, CRISPR, genome engineering, mismatch repair, MLH1dn, PE4, PE5, PEmax, epegRNA, indel suppression, gene therapy, iPSC, T cells, microsatellite instability, 2025 Breakthrough Prize, D.R. Liu
