Curing cytoplasmic male sterility via TALEN-mediated mitochondrial genome editing

Sequence-specific nucleases are commonly used to modify the nuclear genome of plants. However, targeted modification of the mitochondria! genome of land plants has not yet been achieved. In plants, a type of male sterility called cytoplasmic male sterility (CMS) has been attributed to certain mitochondria! genes, but none of these genes has been validated by direct mitochondria! gene-targeted modification. Here, we knocked out CMS-associated genes (orf79 and orf125) of CMS varieties of rice and rapeseed, respectively, using transcription activator-like effector nucleases (TALENs) with mitochondria localization signals (mitoTALENs). We demonstrate that knocking out these genes cures male sterility, strongly suggesting that these genes are causes of CMS. Sequencing revealed that double-strand breaks induced by mitoTALENs were repaired by homologous recombination, and that during this process, the target genes and surrounding sequences were deleted. Our results show that mitoTALENs can be used to stably and heritably modify the mitochondria! genome in plants.