Cold acclimation diversity in Arabidopsis thaliana: CRISPR/Cas9 as a tool to fine analysis of Tandem Gene Arrays, application to CBF genes.

In this period of climate change, it is of major importance to increase knowledge about the mechanisms by whose plants adapt to their environment. Tandem gene arrays (TAG) are overrepresented in the pool of tandem duplicates involved in stress response and are consequently of special interest. Nevertheless, until recently, addressing questions about individual genes or fine regulations in such structures was very difficult due to the close arrangement of the genome, almost preventing the production of targeted simple or multiple mutants. The CRISPR/Cas9 new tool offers new opportunities as the setting of gene deletion strategies in various genetic backgrounds. Here, we used this technology on the cold acclimation CBF pathway in different accessions of Arabidopsis thaliana with the same set of guide RNAs. Deleted lines free of T-DNA have been produced for simple or multiple copies of CBF genes and evaluated for cold tolerance after acclimation. Expression levels of CBF genes and five COR genes have also been analyzed. Our data suggest first that two or three missing CBF genes are necessary to induce a strong reduction in cold tolerance and secondly that most deletions have a low impact on the expression of remaining CBF copies which contradicts the previous hypothesis in the literature. Our results thus show that the CRISPR/Cas9 deletion strategy is a useful performance tool to investigate how tandem gene arrays work.