Data Sheet 1_A simple and efficient system for evaluating plant genome editing efficiency and its application in optimizing the ISAam1 TnpB nuclease.docx

Genome editing technology has revolutionized plant genetic breeding. However, Significant variability in editing activity has been observed across different genome editing systems and target sites, highlighting the importance of developing efficient evaluation systems for assessing genome editing efficiency in plants. In this study, we developed a simple, rapid, and efficient system based on hairy root transformation to evaluate somatic genome editing efficiency in plants. This system is easy to implement, does not require sterile conditions, and enables visual identification of transgenic hairy roots within two weeks. We first validated the system using the CRISPR/Cas9 genome editing platform, confirming its effectiveness. Subsequently, we applied this system to assess the somatic editing activity of the recently identified ISAam1 TnpB nuclease, which show considerable promise for plant genome editing applications. Furthermore, through protein engineering, we identified two variants, ISAam1(N3Y) and ISAam1(T296R), which exhibited a 5.1-fold and 4.4-fold enhancement in somatic editing efficiency, respectively. These findings demonstrate that the developed method provides an effective tool for optimizing genome editing system and screening potential target sites in plant genomes.

基因组编辑技术已彻底革新了植物遗传育种领域。然而，不同基因组编辑系统及靶位点的编辑活性存在显著差异，这凸显了开发高效评价体系以评估植物基因组编辑效率的重要性。本研究基于毛根转化技术，开发了一套简便、快速且高效的植物体细胞基因组编辑效率评价体系。该体系操作简便，无需无菌培养条件，且可在两周内实现转基因毛根的可视化鉴定。研究首先利用CRISPR/Cas9基因组编辑平台对该体系进行了验证，证实了其有效性。随后，利用该体系对新近发现的ISAam1 TnpB核酸酶的体细胞编辑活性进行了评估，该酶在植物基因组编辑应用中展现出可观的应用前景。此外，通过蛋白质工程技术，本研究筛选得到两个突变体ISAam1(N3Y)与ISAam1(T296R)，二者的体细胞编辑效率分别提升了5.1倍与4.4倍。上述研究结果表明，本研究所开发的方法可为优化植物基因组编辑体系及筛选基因组潜在靶位点提供一款高效工具。