摘要
引导编辑是基于CRISPR/Cas技术发展的一种精准基因编辑技术,可以在不造成DNA双链断裂和无需外源DNA供体的情况下实现对碱基的任意替换和片段的精准删除与插入,因此,受到全球科研工作者的广泛关注.引导编辑的精准性对植物遗传变异的获得、基因功能的研究以及种质资源的创制都具有重要作用,从而推动其迅速发展和应用.本文首先阐述植物引导编辑系统的建立与发展:最早以水稻为受体,建立植物引导编辑系统,并发展至玉米、小麦等单子叶以及拟南芥、番茄等双子叶植物上,但编辑效率较低.接着,重点介绍了植物引导编辑器的改良和效率优化思路以及策略,主要包括对pegRNA和效应蛋白的构型改造.然后,总结了引导编辑工具在植物基因表达量的微调和功能研究、新位点挖掘、性状改良和作物育种上的应用.虽然,基于引导编辑的植物基因组长片段或染色体水平的变异操纵仍是挑战,其中阐明介导片段缺失、替换和易位等变异的修复机制和找出关键调控因子尤为重要.我们坚信引导编辑定能成为高效精确操纵植物基因组变异的强有力手段.它不仅能够促进基因功能等基础研究,而且也能为作物设计育种提供技术支撑.
The well-established CRISPR-Cas9 gene editing systems can introduce random insertions and/or deletions in eukaryotic genome targets.To achieve precise editing,prime editors(PEs)have been developed by combining Cas9 nickase and MMLV reverse transcriptase.These methods allow for type-free nucleotide substitutions and programmable installation of small indels in the genome without causing double-strand breaks or requiring exogenous DNA donors.Plant prime editing technology was initially developed for the rice genome and has since been extended to the genomes of various plant species,enabling the manipulation of all 12 types of base substitutions,as well as the insertion or deletion of small fragments.This review will focus on the establishment and development of PEs,along with improvements and optimizations,and on their applications in plant genome editing.The first reported plant prime editing system,known as PPE2,achieved an efficiency ranging from 2.6%to 21.8%in rice.PPE3 and PPE3b were also described in the same article;however,unlike their counterparts in mammalian cell systems,these systems did not significantly improve editing efficiency.Subsequently,prime editing systems were successfully established for monocots such as corn and wheat,as well as dicots such as Arabidopsis and tomato.To further expand the range of applications,an NNGRRT PAM-recognition prime editor was developed from a SaCas9 ortholog.The primary focus of plant PE engineering was efficiency optimization.Over the past four years,significant advancements have been made in this area.Efficiency can be greatly enhanced by preventing the degradation of pegRNAs through the incorporation of structured RNA motifs at the 3′terminus.Other strategies,such as adjusting the melting temperature of primer binding site(PBS)sequences,expressing pegRNAs with composite promoters,and employing twin pegRNAs for a single target,further optimize the activity of plant PEs.Moreover,the main elements of plant PE proteins have also been engineered to improve performance by increasing the activity of Cas nuclease through the introduction of mutations in key amino acids,enhancing reverse transcriptase activity by modifying the structure of M-MLV,and manipulating the mismatch repair(MMR)pathway by accumulating dominant-negative variants of MLH1.Comprehensive engineered versions of PE4,PE5,PE4max,and PE5max have shown promising editing efficiency at different sites in plant genomes.Taking advantage of their flexible editing ability,PEs have been utilized to install precise amino acid mutations to obtain various herbicide-resistant mutants of several crops and to install binding elements to generate broad-spectrum resistance to pathogens.Additionally,prime editing in plants has been employed for fine-tuning gene expression,protein in situ labeling,and direct evolution via saturation mutagenesis.Although robust nucleotide and short fragment prime editing have been achieved,precise long fragment manipulation or chromosomal-scale genome editing remains a major challenge for prime editing in plants.Novel editors or prime editing strategies are eagerly anticipated to overcome this issue.It is crucial to elucidate the distinct mechanisms of DNA mismatch repair involving nucleotides or long fragments,as well as the main repair pathway and key regulatory factors involved in fragment deletions,substitutions,and inversions.We envision and believe for the first time that the current powerful PEs and future optimized versions can provide revolutionary molecular approaches for efficiently and accurately manipulating plant genomes.This approach will not only boost the exploration of fundamental functional genome research but also lead to the development of novel germplasm resources for crop design and breeding.
作者
周苏淮
李芳
李娟
吴潇雅
吴丹丹
许蓉芳
魏鹏程
Suhuai Zhou;Fang Li;Juan Li;Xiaoya Wu;Dandan Wu;Rongfang Xu;Pengcheng Wei(College of Agronomy,Anhui Agricultural University,Hefei 230031,China;Rice Research Institute,Anhui Academy of Agricultural Sciences,Hefei 230031,China;Hefei Jian Gu Biotechnology Co.,Ltd,Hefei 230031,China)
出处
《科学通报》
北大核心
2025年第16期2414-2422,共9页
Chinese Science Bulletin
基金
国家自然科学基金(32000284)
安徽省自然科学基金(2308085Y20,2208085Y11)
国家阵型及生物育种技术优势企业项目(抗除草剂水稻种质创新技术研究与应用)资助。
关键词
引导编辑
精准编辑
植物
优化
育种
prime editing
precise editing
plant
optimization
breeding
