During the evolution of plant genomes, sequence inversions occurred repeatedly making the respective regions inaccessible for meiotic recombination and thus for breeding. Therefore, it is important to develop technologies that allow the induction of inversions within chromosomes in a directed and efficient manner. Using the Cas9 nuclease from Staphylococcus aureus (Sa Cas9), we were able to obtain scarless heritable inversions with high efficiency in the model plant Arabidopsis thaliana . Via deep sequencing, we defined the patterns of junction formation in wild‐type and in the non‐homologous end‐joining (NHEJ ) mutant ku70‐1 . Surprisingly, in plants deficient of KU 70, inversion induction is enhanced, indicating that KU 70 is required for tethering the local broken ends together during repair. However, in contrast to wild‐type, most junctions are formed by microhomology‐mediated NHEJ and thus are imperfect with mainly deletions, making this approach unsuitable for practical applications. Using egg‐cell‐specific expression of Cas9, we were able to induce heritable inversions at different genomic loci and at intervals between 3 and 18 kb, in the percentage range, in the T1 generation. By screening individual lines, inversion frequencies of up to the 10% range were found in T2. Most of these inversions had scarless junctions and were without any sequence change within the inverted region, making the technology attractive for use in crop plants. Applying our approach, it should be possible to reverse natural inversions and induce artificial ones to break or fix linkages between traits at will
