Multiple inducible DNA double-strand breaks using a restriction endonuclease in Arabidopsis thaliana mitochondria

Plant mitochondrial genomes are associated with important agronomic traits, such as cytoplasmic male sterility, which would be a desirable trait to control. However, plant mitochondrial genomes are currently neglected as a target for genome editing in plants, due to their recalcitrance to CRISPR/Cas9 editing, and because the understanding of DNA repair mechanisms in plant mitochondrial genomes is still limited. To further study double-strand break repair (DSBR) and to advance the ability to make targeted DNA breaks in plant mitochondrial genomes, we developed an Arabidopsis thaliana line containing a nuclear encoded T-DNA expression cassette carrying a restriction enzyme that is targeted to the mitochondria and has its expression induced with application of β-estradiol. The restriction enzyme chosen, KasI, has 35 recognition sites throughout the A. thaliana mitochondrial genome, with cut sites being in essential coding regions, non-functional DNA, and within or near non-tandem repeats. Following a single induction of this nuclear-encoded, mitochondrial-targeted restriction enzyme, we were able to decipher the consequences on the structure of the mitochondrial genome, informing on required homology lengths for DNA repair, as well as the consequences on individual cut sites, comparing non-functional DNA, coding DNA, and repeat region repair choices. This research provides a unique tool to study DNA repair in plant mitochondrial genomes, following multiple simultaneous double-strand breaks in a plant with an intact repair system. This tool can also be used to further characterize DNA repair proteins that act in plant mitochondria.