Mol Ther Nucleic Acids. 2023 Aug 17
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, and Qingjian Zou
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Research Field: gene editing; eye

AAV Serotype: AAV9

Dose: One microliter (~1×10^10 vg) for each AAV vector solution was mixed and injected into the vitreous cavity of the retina.

Routes of Administration: Intravitreal injections

Targeted organ: eye

Animal or cell line strain: the two-week-old mice

Abstract

Prime editor (PE) is a versatile genome-editing tool without needing extra DNA donors or inducing double-strand breaks. However, in vivo implementation of PE remains challenge because of its oversized composition. In this study, we screened out the smallest truncated Moloney-murine leukemia virus reverse transcriptase (M-MLV RT) with F155Y mutation to keep gene editing efficiency. The truncated M-MLV RT variant was further fused with Campylobacter jejuni Cas9 (CjCas9, H559A) to construct a mini-PE system with a reduced gene size (4.5 kb) right in the packaging capacity of adeno-associated virus. After optimizing of the pegRNAs and incorporating with nick sgRNAs, the mini-PE delivered up to 10% precise editing at target sites in human and mouse cells. It also edited mouse Hsf1 gene in mouse retina precisely after delivered with AAVs, although the editing efficiency was lower than 1%. We will focus on improving the editing efficiency of mini-PE, and exploiting it therapeutic potential against human genetic diseases.

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