Developing a multi-modular assembled prime editing (mPE) system improved precise multi-base insertion efficiency in dicots

文献类型： 外文期刊

第一作者： Lu, Pengjun

作者： Lu, Pengjun;Zuo, Erwei;Yan, Jianbin

作者机构：

关键词： Prime editing; CRISPR; Genome editing; Dicot; Arabidopsis thaliana; Nicotiana benthamiana

期刊名称：JOURNAL OF ADVANCED RESEARCH （ 影响因子：13.0； 五年影响因子：11.6 ）

ISSN： 2090-1232

年卷期： 2025 年 71 卷

页码：

收录情况： SCI

摘要： Introduction: The Prime Editing (PE) system is a precise and versatile genome editing tool with great potential in plant breeding and plant synthetic biology. However, low PE efficiency severely restricts its application, especially in dicots. PE can introduce small tags to trace target protein or cis-element to regulate gene transcription which is an expertise superior to other gene editing tools. Owing to low efficiency, PE adaption in stably transformed Arabidopsis is lacking. Objectives: This study aimed to investigate the issue of low PE efficiency in dicots and develop systematic solutions to improve it. Currently, PE in dicots is undetectable and inconsistent, and this study seeks to address it. Split PE into several parts showed better performance in some target sites in mammal cells. We plan to discover the optimal split PE combination in dicot. Methods: We conducted large-scale transformation experiments in dicot model plants Arabidopsis thaliana (At) and Nicotiana benthamiana (Nb) by Agrobacterium-mediated transformation with deep amplicon sequencing (0.2-0.5 million clean total reads). Results: The editing efficiency decreased upon using a fused reverse transcriptase (RT) or an extended pegRNA separately and further decreased dramatically when these were used together. With the help of the pol II strategy to express PE gRNA (pegRNA), we named the most effective split PE combination as a multi-modular assembled prime editing system (mPE). mPE exhibited improved precise editing efficiency on most gene sites with various editing types, ranging from 1.3-fold to 1288.5-fold and achieved PE on some sites that could not be edited by original PE2. Especially, mPE showed superiority for multibase insertion with an average improvement of 197.9-fold. Conclusion: The original PE architecture strongly inhibited the cleavage activity of Cas9. Split PE improved PE efficiency extensively and was in favor of introducing small insertions in dicot plants, indicating that different PE variants might have their own expertise. (c) 2024 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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