Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure

Xiaosa Li (), Lina Zhou, Bao-Qing Gao, Guangye Li, Xiao Wang, Ying Wang, Jia Wei, Wenyan Han, Zixian Wang, Jifang Li, Runze Gao, Junjie Zhu, Wenchao Xu, Jing Wu, Bei Yang, Xiaodong Sun (), Li Yang () and Jia Chen ()
Additional contact information
Xiaosa Li: Shanghai Jiao Tong University School of Medicine
Lina Zhou: ShanghaiTech University
Bao-Qing Gao: University of Chinese Academy of Sciences, Chinese Academy of Sciences
Guangye Li: ShanghaiTech University
Xiao Wang: ShanghaiTech University
Ying Wang: University of Chinese Academy of Sciences, Chinese Academy of Sciences
Jia Wei: University of Chinese Academy of Sciences, Chinese Academy of Sciences
Wenyan Han: ShanghaiTech University
Zixian Wang: ShanghaiTech University
Jifang Li: ShanghaiTech University
Runze Gao: ShanghaiTech University
Junjie Zhu: ShanghaiTech University
Wenchao Xu: ShanghaiTech University
Jing Wu: ShanghaiTech University
Bei Yang: Shanghai Clinical Research and Trial Center
Xiaodong Sun: Shanghai Jiao Tong University School of Medicine
Li Yang: Fudan University and Shanghai Key Laboratory of Medical Epigenetics, International Laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University
Jia Chen: ShanghaiTech University

Nature Communications, 2022, vol. 13, issue 1, 1-9

Abstract: Abstract Prime editor (PE), which is developed by combining Cas9 nickase and an engineered reverse transcriptase, can mediate all twelve types of base substitutions and small insertions or deletions in living cells but its efficiency remains low. Here, we develop spegRNA by introducing same-sense mutations at proper positions in the reverse-transcription template of pegRNA to increase PE’s base-editing efficiency up-to 4,976-fold (on-average 353-fold). We also develop apegRNA by altering the pegRNA secondary structure to increase PE’s indel-editing efficiency up-to 10.6-fold (on-average 2.77-fold). The spegRNA and apegRNA can be combined to further enhance editing efficiency. When spegRNA and apegRNA are used in PE3 and PE5 systems, the efficiencies of sPE3, aPE3, sPE5 and aPE5 systems are all enhanced significantly. The strategies developed in this study realize highly efficient prime editing at certain previously uneditable sites.

Date: 2022
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DOI: 10.1038/s41467-022-29339-9

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