CRISPR–Cas9-mediated induction of heritable chromosomal translocations in Arabidopsis

Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) technology hasbeen applied in plant breeding mainly on genes for improving single or multiple traits$^{1-4}$. Here we show that this technologycan also be used to restructure plant chromosomes. Using the Cas9 nuclease from Staphylococcus aureus$^{5}$, we were able toinduce reciprocal translocations in the Mbp range between heterologous chromosomes in Arabidopsis thaliana. Of note,translocation frequency was about five times more efficient in the absence of the classical non-homologous end-joiningpathway. Using egg-cell-specific expression of the Cas9 nuclease and consecutive bulk screening, we were able to isolate heritable events and establish lines homozygous for the translocation, reaching frequencies up to 2.5% for individuallines. Using molecular and cytological analysis, we confirmed that the chromosome-arm exchanges we obtained betweenchromosomes 1 and 2 and between chromosomes 1 and 5 of Arabidopsis were conservative and reciprocal. The inductionof chromosomal translocations enables mimicking of genome evolution or modification of chromosomes in a directed manner,fixing or breaking genetic linkages between traits on different chromosomes. ... mehrControlled restructuring of plantgenomes has the potential to transform plant breeding. Given the challenges of feeding the rapidly growing human