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

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) technology has been applied in plant breeding mainly on genes for improving single or multiple traits(1-4). Here we show that this technology can also be used to restructure plant chromosomes. Using the Cas9 nuclease from Staphylococcus aureus(5), we were able to induce 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-joining pathway. 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 individual lines. Using molecular and cytological analysis, we confirmed that the chromosome-arm exchanges we obtained between chromosomes 1 and 2 and between chromosomes 1 and 5 of Arabidopsis were conservative and reciprocal. The induction of 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. Controlled restructuring of plant genomes has the potential to transform plant breeding. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) technology was used to induce heritable reciprocal chromosomal translocations in the Mb range in Arabidopsis, providing new possibilities to modify chromosomes and break linkage drag in plant breeding.