Identification and Characterization of () and Genes by CRISPR/Cas9-Targeted Mutagenesis in Chinese Cabbage ( L.).

The CRISPR/Cas9 site-directed gene-editing system offers great advantages for identifying gene function and crop improvement. The circadian clock measures and conveys day length information to control rhythmic hypocotyl growth in photoperiodic conditions, to achieve optimal fitness, but operates through largely unknown mechanisms. Here, we generated core circadian clock evening components, () , , and (both and double knockout) mutants, using CRISPR/Cas9 genome editing in Chinese cabbage, where 9-16 genetic edited lines of each mutant were obtained. The targeted deep sequencing showed that each mutant had 2-4 different mutation types at the target sites in the and genes. To identify the functions of and genes, hypocotyl length, and mRNA and protein levels of core circadian clock morning components, () and () and were examined under light/dark cycles and continuous light conditions. The and double mutants showed longer hypocotyls, lower core circadian clock morning component mRNA and protein levels, and a shorter circadian rhythm than wildtype (WT). On the other hand, the mutant was not significantly different from WT. These results suggested that two paralogous genes may not be associated with the same regulatory function in Chinese cabbage. Taken together, our results demonstrated that CRISPR/Cas9 is an efficient tool for achieving targeted genome modifications and elucidating the biological functions of circadian clock genes in , for both breeding and improvement.