Rapid and low-cost methods of detecting mutations and polymorphisms are crucial for genotyping applications including mutagenesis and gene editing. S1 family endonucleases such as T7E1, EndoV and CELI can potentially be used in enzymatic mismatch detection. Among them, CELI has been shown to be effective in detecting mutations in Targeting Induced Local Lesions IN Genomes (TILLING). However, current method of CELI purification from celery is laborious, and challenging for many non-biochemical laboratories, and the presence of post-translational modifications hinders efficient production of the enzyme in E. coli. Here, we report an efficient system for bulk production of enzymatically active CELI endonuclease through transient expression in a model plant Nicotiana benthamiana. We also optimized the reaction buffer, by additions of Mn2+ and DTT, with enhanced mismatch cleavage activity. Using the new CELI production and reaction system, we were able to routinely detect mismatches in 1/32 mixed mutant and wildtype DNA samples. We believe the newly established system has many applications in characterization of mutations occurred in natural variations, mutagenized populations and gene editing.

中文翻译：

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通过瞬时表达在本塞姆氏烟草中高效生产 CELI 核酸内切酶及其在突变和基因编辑事件检测中的应用


快速且低成本的突变和多态性检测方法对于突变和基因编辑等基因分型应用至关重要。 S1 家族核酸内切酶（例如 T7E1、EndoV 和 CELI）可潜在用于酶促错配检测。其中，CELI已被证明可以有效检测基因组中靶向诱导局部病变（TILLING）的突变。然而，目前从芹菜中纯化 CELI 的方法非常费力，对许多非生化实验室来说具有挑战性，而且翻译后修饰的存在阻碍了大肠杆菌中酶的有效生产。在这里，我们报告了一种通过在模型植物本塞姆氏烟草中瞬时表达来批量生产具有酶活性的 CELI 核酸内切酶的有效系统。我们还通过添加 Mn2+ 和 DTT 来优化反应缓冲液，增强错配裂解活性。使用新的 CELI 生产和反应系统，我们能够常规检测 1/32 混合突变体和野生型 DNA 样本中的错配。我们相信新建立的系统在自然变异、诱变群体和基因编辑中发生的突变表征方面有许多应用。