Kazama Tomohiko, Arimura Shin-Ichi
Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
Graduate School of Agriculture and Life Sciences, University of Tokyo, Tokyo, Japan.
Methods Mol Biol. 2023;2615:365-378. doi: 10.1007/978-1-0716-2922-2_25.
The ability to transform plant mitochondrial genomes has many benefits. Although delivery of foreign DNA to mitochondria is presently very difficult, it is now possible to knock out mitochondrial genes using mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs). Such knockouts have been achieved by a genetic transformation of mitoTALENs encoding genes into the nuclear genome. Previous studies have shown that double-strand breaks (DSBs) induced by mitoTALENs are repaired by ectopic homologous recombination. As a result of DNA repair by homologous recombination, a portion of the genome containing the mitoTALEN target site is deleted. The deletion and repair process cause the mitochondrial genome to become more complex. Here, we describe a method for identifying the ectopic homologous recombination events that occur following the repair of double-strand breaks induced by mitoTALENs.
改造植物线粒体基因组的能力具有诸多益处。尽管目前将外源DNA导入线粒体非常困难，但现在可以使用线粒体靶向的转录激活样效应物核酸酶（mitoTALENs）敲除线粒体基因。通过将编码mitoTALENs的基因遗传转化到核基因组中已实现了此类基因敲除。先前的研究表明，mitoTALENs诱导的双链断裂（DSBs）通过异位同源重组进行修复。同源重组导致的DNA修复结果是，包含mitoTALEN靶位点的基因组部分被删除。这种删除和修复过程使线粒体基因组变得更加复杂。在此，我们描述了一种鉴定mitoTALENs诱导的双链断裂修复后发生的异位同源重组事件的方法。
