Cold acclimation is an important adaptive response of plants from temperate regions to increase their freezing tolerance after being exposed to low nonfreezing temperatures. The three CBF genes are well known to be involved in cold acclimation. As the 3 CBF genes are linked tandemly in the Arabidopsis genome, it is almost impossible to obtain cbf triple mutants using traditional genetic methods. Recently, using the CRISPR/Cas9 technology, we generated cbf single, double, and triple mutants. Our results showed that the cbf triple mutants are extremely sensitive to freezing stress. In addition, the cbf triple mutants are defective in early development and salt tolerance. Interestingly, the cbf1 cbf3 double mutants show increased expression of the CBF2 gene and some downstream cold-responsive genes and display increased freezing tolerance, compared to the wild type, revealing that CBF1 and CBF3 negatively regulate CBF2 expression.
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