Multiplex CRISPR/Cas9 Mutagenesis of BrVRN1 Delays Flowering Time in Chinese Cabbage ( Brassica rapa L. ssp. pekinensis )

The VERNALIZATION1 ( VRN1 ) gene is a crucial transcriptional repressor involved in triggering the transition to flowering in response to prolonged cold. To develop Chinese cabbage ( Brassica rapa L. ssp. pekinensis ) plants with delayed flowering time, we designed a multiplex CRISPR/Cas9 platform that allows the co-expression of four sgRNAs targeting different regions of the endogenous BrVRN1 gene delivered via a single binary vector built using the Golden Gate cloning system. DNA sequencing analysis revealed site-directed mutations at two target sites: gRNA1 and gRNA2. T 1 mutant plants with a 1-bp insertion in BrVRN1 exhibited late flowering after the vernalization. Additionally, we identified ‘transgene-free’ BrVRN1 mutant plants without any transgenic elements from the GE 1 (gene-editing 1) and GE 2 generations. All GE 2 mutant plants contained successful edits in two out of three BrVRN1 orthologs and displayed delayed flowering time. In GE 2 mutant plants, the floral repressor gene FLC1 was expressed during vernalization; but the floral integrator gene FT was not expressed after vernalization. Taken together, our data indicate that the BrVRN1 genes act as negative regulators of FLC1 expression during vernalization in Chinese cabbage, raising the possibility that the ‘transgene-free’ mutants of BrVRN1 developed in this study may serve as useful genetic resources for crop improvement with respect to flowering time regulation.