Prime editing is a versatile genome-editing technology, but it suffers from low editing efficiency. In the present study, we introduce optimized prime editors with substantially improved editing efficiency. We engineered the Moloney-murine leukemia virus reverse transcriptase by removing its ribonuclease H domain and incorporated a viral nucleocapsid protein with nucleic acid chaperone activity. Each modification independently improved prime editing efficiency by ~1.8-3.4-fold in plant cells. When combined in our engineered plant prime editor (ePPE), the two modifications synergistically enhanced the efficiency of base substitutions, deletions and insertions at various endogenous sites by on average 5.8-fold compared with the original PPE in cell culture. No significant increase in byproducts or off-target editing was observed. We used the ePPE to generate rice plants tolerant to sulfonylurea and imidazolinone herbicides, observing an editing frequency of 11.3% compared with 2.1% using PPE. We also combined ePPE with the previously reported dual-prime editing guide (peg) RNAs and engineered pegRNAs to further increase efficiency.
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