Abstract：
BACKGROUND: 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.
OBJECTIVE: 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 multi-base insertion with an average improvement of 197.9-fold.
CONCLUSIONS: 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.
OBJECTIVE: 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 multi-base insertion with an average improvement of 197.9-fold.
CONCLUSIONS: 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.
摘要：
背景：PrimeEditing（PE）系统是一种精确且通用的基因组编辑工具，在植物育种和植物合成生物学中具有巨大的潜力。然而,低PE效率严重制约了其应用,尤其是在双子叶植物中.PE可以引入小标签来跟踪靶蛋白或顺式元件以调节基因转录，这是优于其他基因编辑工具的专业知识。由于效率低,在稳定转化的拟南芥中缺乏PE适应。
目的：本研究旨在调查双子叶植物PE效率低的问题，并开发系统的解决方案来改善它。目前,双子叶植物中的PE是不可检测和不一致的，这项研究试图解决这个问题。将PE分成几个部分在哺乳动物细胞的一些靶位点显示出更好的性能。我们计划在双子叶中发现最优的分割PE组合。
方法：我们通过土壤杆菌介导的转化和深度扩增子测序在双子叶模型植物拟南芥（At）和Nicotianabenthamiana（Nb）中进行了大规模转化实验（0.2-0.5百万清洁总读数）。
结果：编辑效率在分别使用融合逆转录酶（RT）或延伸的pegRNA时降低，并且在一起使用时进一步显着降低。借助polII策略表达PEgRNA（pegRNA），我们将最有效的分割PE组合命名为多模块组装的主编辑系统（mPE）。mPE在大多数具有各种编辑类型的基因位点上表现出提高的精确编辑效率，范围从1.3倍到1288.5倍，并在一些无法由原始PE2编辑的网站上实现了PE。尤其是,mPE显示出多碱基插入的优势，平均提高了197.9倍。
结论：原始PE结构强烈抑制Cas9的切割活性。SplitPE广泛地提高了PE效率，并且有利于在双子叶植物中引入小插入物，表明不同的PE变体可能有自己的专业知识。
目的：本研究旨在调查双子叶植物PE效率低的问题，并开发系统的解决方案来改善它。目前,双子叶植物中的PE是不可检测和不一致的，这项研究试图解决这个问题。将PE分成几个部分在哺乳动物细胞的一些靶位点显示出更好的性能。我们计划在双子叶中发现最优的分割PE组合。
方法：我们通过土壤杆菌介导的转化和深度扩增子测序在双子叶模型植物拟南芥（At）和Nicotianabenthamiana（Nb）中进行了大规模转化实验（0.2-0.5百万清洁总读数）。
结果：编辑效率在分别使用融合逆转录酶（RT）或延伸的pegRNA时降低，并且在一起使用时进一步显着降低。借助polII策略表达PEgRNA（pegRNA），我们将最有效的分割PE组合命名为多模块组装的主编辑系统（mPE）。mPE在大多数具有各种编辑类型的基因位点上表现出提高的精确编辑效率，范围从1.3倍到1288.5倍，并在一些无法由原始PE2编辑的网站上实现了PE。尤其是,mPE显示出多碱基插入的优势，平均提高了197.9倍。
结论：原始PE结构强烈抑制Cas9的切割活性。SplitPE广泛地提高了PE效率，并且有利于在双子叶植物中引入小插入物，表明不同的PE变体可能有自己的专业知识。
