Gong Zheng, Zhang Yan, Xia Di, Yoon Sohye, Crisp Peter Alexander, Botella José Ramón
Plant Genetic Engineering Laboratory, School of Agriculture and Food Sustainability, The University of Queensland, St Lucia, QLD 4072, Australia.
School of Agriculture and Food Sustainability, The University of Queensland, St Lucia, QLD 4072, Australia.
iScience. 2025 Apr 29;28(6):112350. doi: 10.1016/j.isci.2025.112350. eCollection 2025 Jun 20.
Accurately detecting and quantifying CRISPR edits with high sensitivity is crucial for developing new genome editing applications in plants. This is especially important when analyzing heterogeneous populations from transient expression-based approaches used for technology development as well as evaluating of guide RNA (gRNA) performance . However, current studies employ vastly different techniques to quantify genome editing outcomes, limiting the comparability and repeatability of results. In this study, we systematically evaluated and compared experimental techniques for quantifying plant genome editing across a wide range of efficiencies. We measured genome editing efficiency from 20 transiently expressed Cas9 targets using different techniques, including targeted amplicon sequencing (AmpSeq), PCR-restriction fragment length polymorphism (RFLP) assays, T7 endonuclease 1 (T7E1) assays, Sanger sequencing of amplicon products (deconvoluted and analyzed using three algorithms), PCR-capillary electrophoresis/InDel detection by amplicon analysis (PCR-CE/IDAA), and droplet digital PCR (ddPCR). We assessed methods based on their accuracy, sensitivity, and cost, benchmarked to AmpSeq. Furthermore, we discuss the advantages and drawbacks of each technique, the issues faced during optimization and the solutions we devised. This study will be useful to experienced and new researchers in the field, providing suggestions and guidelines to standardize data output and advice about the most suitable technique/s to quantify genome edits for different plant applications.
以高灵敏度准确检测和量化CRISPR编辑对于开发植物新的基因组编辑应用至关重要。在分析用于技术开发的基于瞬时表达方法的异质群体以及评估引导RNA（gRNA）性能时，这一点尤为重要。然而，目前的研究采用了截然不同的技术来量化基因组编辑结果，限制了结果的可比性和可重复性。在本研究中，我们系统地评估和比较了在广泛效率范围内量化植物基因组编辑的实验技术。我们使用不同技术测量了20个瞬时表达的Cas9靶点的基因组编辑效率，这些技术包括靶向扩增子测序（AmpSeq）、聚合酶链反应-限制性片段长度多态性（RFLP）分析、T7核酸内切酶1（T7E1）分析、扩增子产物的桑格测序（使用三种算法进行解卷积和分析）、聚合酶链反应-毛细管电泳/通过扩增子分析进行插入缺失检测（PCR-CE/IDAA）以及液滴数字聚合酶链反应（ddPCR）。我们根据准确性、灵敏度和成本对这些方法进行了评估，并以AmpSeq为基准。此外，我们讨论了每种技术的优缺点、优化过程中面临的问题以及我们设计的解决方案。本研究将对该领域经验丰富的研究人员和新研究人员有用，为标准化数据输出提供建议和指导，并就最适合不同植物应用量化基因组编辑的技术提供建议。
