Danilo Benoit, Montes Émilie, Archambeau Héloïse, Lodé Maryse, Rousseau-Gueutin Mathieu, Chèvre Anne-Marie, Mazier Marianne
GAFL UR 1052 , INRAE PACA , Avignon, France.
IGEPP Université de Rennes , INRAE , Agrocampus Ouest, F-35650, Le Rheu, France.
Transgenic Res. 2022 Feb;31(1):87-105. doi: 10.1007/s11248-021-00287-2. Epub 2021 Oct 11.
Meganucleases are rare cutting enzymes that can generate DNA modifications and are part of the plant genome editing toolkit although they lack versatility. Here, we evaluated the use of two meganucleases, I-SceI and a customized meganuclease, in tomato and oilseed rape. Different strategies were explored for the use of these meganucleases. The activity of a customized and a I-SceI meganucleases was first estimated by the use of a reporter construct GFFP with the target sequences and enabled to demonstrate that both meganucleases can generate double-strand break and HDR mediated recombination in a reporter gene. Interestingly, I-SceI seems to have a higher DSB efficiency than the customized meganuclease: up to 62.5% in tomato and 44.8% in oilseed rape. Secondly, the same exogenous landing pad was introduced in both species. Despite being less efficient compared to I-SceI, the customized meganuclease was able to generate the excision of an exogenous transgene (large deletion of up to 3316 bp) present in tomato. In this paper, we also present some pitfalls to be considered before using meganucleases (e.g., potential toxicity) for plant genome editing.
归巢核酸酶是一种罕见的切割酶，可产生DNA修饰，是植物基因组编辑工具包的一部分，尽管其通用性不足。在此，我们评估了两种归巢核酸酶I-SceI和一种定制归巢核酸酶在番茄和油菜中的应用。探索了使用这些归巢核酸酶的不同策略。首先通过使用带有靶序列的报告构建体GFFP来估计定制归巢核酸酶和I-SceI归巢核酸酶的活性，结果表明这两种归巢核酸酶均可在报告基因中产生双链断裂和同源定向修复（HDR）介导的重组。有趣的是，I-SceI的双链断裂（DSB）效率似乎高于定制归巢核酸酶：在番茄中高达62.5%，在油菜中为44.8%。其次，在这两个物种中引入了相同的外源着陆平台。尽管与I-SceI相比效率较低，但定制归巢核酸酶能够切除番茄中存在的外源转基因（高达3316 bp的大片段缺失）。在本文中，我们还提出了在将归巢核酸酶用于植物基因组编辑之前需要考虑的一些问题（例如潜在毒性）。
