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

• Published in: Nature plants Vol. 8; no. 1; pp. 45 - 52
• Main Authors: Xu, Wen, Yang, Yongxing, Yang, Biying, Krueger, Christopher J., Xiao, Qianlin, Zhao, Si, Zhang, Lu, Kang, Guiting, Wang, Feipeng, Yi, Hongmei, Ren, Wen, Li, Lu, He, Xiaoqing, Zhang, Chuanmao, Zhang, Bo, Zhao, Jiuran, Yang, Jinxiao
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.01.2022; Nature Publishing Group
• Subjects: 45; 45/23; 631/449; 631/449/2169; Biomedical and Life Sciences; Corn; CRISPR-Cas Systems; Deoxyribonucleic acid; Design optimization; DNA; DNA damage; Efficiency; Gene Editing - methods; Genetic modification; Genomes; Letter; Life Sciences; Nucleotides; Oryza - genetics; Plant Breeding; Plant Sciences; Plants, Genetically Modified - genetics; Protoplasts; Rice; RNA-directed DNA polymerase; Transgenic plants
• ISSN: 2055-0278; 2055-0278
• DOI: 10.1038/s41477-021-01043-4

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. A study finds that N-terminal RT–Cas9 nickase fusion, versus the commonly applied C-terminal fusion, and multiple-nucleotide substitutions in prime editing guide RNAs can synergistically and greatly enhance the efficiency of prime editor 3 in rice and maize.