Mini-PE, a prime editor with compact Cas9 and truncated reverse transcriptase

Ting Lan; Huangyao Chen; Chengcheng Tang; Yuhui Wei; Yang Liu; Jizeng Zhou; Zhenpeng Zhuang; Quanjun Zhang; Min Chen; Xiaoqing Zhou; Yue Chi; Jinling Wang; Yu He; Liangxue Lai; Qingjian Zou

doi:10.1016/j.omtn.2023.08.018

Mini-PE, a prime editor with compact Cas9 and truncated reverse transcriptase

Mol Ther Nucleic Acids. 2023 Aug 18:33:890-897. doi: 10.1016/j.omtn.2023.08.018. eCollection 2023 Sep 12.

Authors

Ting Lan^{1

2}, Huangyao Chen^{1

3

2}, Chengcheng Tang¹, Yuhui Wei², Yang Liu², Jizeng Zhou⁴, Zhenpeng Zhuang^{1

3

2}, Quanjun Zhang^{1

2}, Min Chen¹, Xiaoqing Zhou¹, Yue Chi¹, Jinling Wang¹, Yu He⁵, Liangxue Lai^{1

2}, Qingjian Zou¹

Affiliations

¹ Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Wuyi University, Jiangmen 529020, China.
² CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
³ University of the Chinese Academy of Sciences, Beijing 100049, China.
⁴ School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, China.
⁵ National Drug Clinical Trial Institution, Jiangmen Central Hospital, Jiangmen, Guangdong 529000, China.

Abstract

Prime editor (PE) is a versatile genome editing tool that does not need extra DNA donors or inducing double-strand breaks. However, in vivo implementation of PE remains a challenge because of its oversized composition. In this study, we screened out the smallest truncated Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) with the F155Y mutation to keep gene editing efficiency. We discovered the most efficient gene editing variants of MMLV RT with the smallest size. After optimization of the pegRNAs and incorporation with nick sgRNAs, the mini-PE delivered up to 10% precise editing at target sites in human and mouse cells. It also edited the mouse Hsf1 gene in the mouse retina precisely after delivery with adeno-associated viruses (AAVs), although the editing efficiency was lower than 1%. We will focus on improving the editing efficiency of mini-PE and exploiting its therapeutic potential against human genetic diseases.

Keywords: AAV; CjCas9; MT: RNA/DNA Editing; miniature; prime editor; truncated MMLV.