A design optimized prime editor with expanded scope and capability in plants

The ability to manipulate the genome in a programmable manner has illuminated biology and shown promise in plant breeding. Prime editing, a versatile gene-editing approach that directly writes new genetic information into a specified DNA site without requiring double-strand DNA breaks, suffers from low efficiency in plants(1-5). In this study, N-terminal reverse transcriptase-Cas9 nickase fusion performed better in rice than the commonly applied C-terminal fusion. In addition, introduction of multiple-nucleotide substitutions in the reverse transcriptase template stimulated prime editing with enhanced efficiency. By using these two methods synergistically, prime editing with an average editing frequency as high as 24.3% at 13 endogenous targets in rice transgenic plants, 6.2% at four targets in maize protoplasts and 12.5% in human cells was achieved, which is two- to threefold higher than the original editor, Prime Editor 3. Therefore, our optimized approach has potential to make more formerly non-editable target sites editable, and expands the scope and capabilities of prime editing in the future.

Automated summary

This study demonstrated that using N-terminal reverse transcriptase-Cas9 nickase fusion and introducing multiple-nucleotide substitutions in the reverse transcriptase template can significantly enhance the efficiency of prime editing in plants and human cells. The optimized approach achieved higher editing frequencies compared to the original editor, Prime Editor 3, showing the potential to make previously non-editable target sites editable and expand the capabilities of prime editing in the future.