Key determinants of CRISPR/Cas9 induced inversions in tomato

Inversions in chromosomes occur widely within plants and suppress meiotic recombination which can be beneficial or detrimental for plant breeders. Therefore, induction or reversion of inversions via CRISPR/Cas9 has been extensively researched recently. Extensive variation in inversion induction rates and sizes have been reported, from hundreds to several million base pairs. Here, we dissect the influential factors of inversion induction efficiency using CRISPR/Cas9. By using a fixed reference gRNA, we could directly correlate gRNA mutation frequency to inversion frequency and inversion size, of inversions up to 37.5 Mb in length in tomato. Our findings indicate that the least efficient gRNA is the bottleneck for inversion induction, with inversion size having no significant influence unless the inversions were larger than 1 Mb in size. For these huge inversions, the frequency dropped astoundingly, regardless of the gRNA cutting efficiencies. We hypothesize an in planta yet unknown variant of non-homologous-end-joining (NHEJ)-based repair which utilizes active transport of damaged chromosomal sections to dedicated repair sites in the cell nucleus, where repair is finalized. We propose that large inversions are formed less frequently because the transport of these segments to the repair sites may be hampered by their sheer size.