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

Abstract

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. T1 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 GE1 (gene-editing 1) and GE2 generations. All GE2 mutant plants contained successful edits in two out of three BrVRN1 orthologs and displayed delayed flowering time. In GE2 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.

Multiplex CRISPR/Cas9‐mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality

“…In this study, mutation of MsFTa1 via CRISPR/Cas9 gene editing resulted in tetra‐allelic homozygous mutant plants with delayed flowering and increased plant height and fresh and dry biomass compared to the control plants (Figures 2 , 4 ). The results of this study are consistent with prior findings on the manipulation of plant genes known to promote flowering in improved agronomic traits, yield and quality in soybean (Cai et al ., 2019 ; Xu et al ., 2021 ), M. truncatula (Tadege et al ., 2015 ), alfalfa (Lorenzo et al ., 2020 ), rice (Cui et al ., 2021 ; Liu et al ., 2021 ; Wu et al ., 2020 ; Zhang et al ., 2019 ), switchgrass (Gou et al ., 2019 ), Chinese cabbage (Hong et al ., 2021 ) and tomato (Rajendran et al ., 2021 ; Shalit et al ., 2009 ). Recently, the role of the FTa1 gene in alfalfa was confirmed by down‐regulating its expression through an artificial microRNA (amiRNA) (Lorenzo et al ., 2020 ).…”

“…For instance, the heading date of rice was delayed by fine‐tuning flowering time genes ( Ehd1 and Hd2 ) using CRISPR/Cas9, which resulted in prolonged vegetative growth period with improved yield potential for flexible cropping systems in diverse environments (Liu et al ., 2021 ; Wu et al ., 2020 ; Zhang et al ., 2019 ). Flowering time in Chinese cabbage ( Brassica rapa L.) was also delayed by targeting BrVRN1 using multiple guide RNAs (gRNAs) in the multiplex CRIPSPR/Cas9 construct to generate lines with different flowering times for crop improvement (Hong et al ., 2021 ). Similarly, CRISPR/Cas9 mutagenesis in soybean targeting two flowering genes ( GmFT2a and GmFT5a ) improved adaptability of soybean to different environments with desirable transgene‐free mutant lines (Cai et al ., 2018 , 2019 ).…”

CRISPR/Cas9-mediated gene editing to confer turnip mosaic virus (TuMV) resistance in Chinese cabbage (Brassica rapa)

“…Recently, genome editing in Chinese cabbage has been performed for different traits, such as FLOWERING LOCUS C (FLC) for early flowering, BrVRN1 gene for delayed flowering, and PR55/B gene for self-incompatibility [ 41–43 ]. Moreover, DNA-free (transgene-free) genome editing by targeting the vernalization determinant FRIGIDA and phytoene desaturase gene (FRI and PDS) genes has also been done to pave toward minimizing GMO legislation-related issues [ 34 ].…”

Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage

“…Only a few studies have reported modifying genes using CRISPR/Cas9 in B. rapa . These studies focused on genes involved in flowering time [ 12 ], leaf color transition [ 13 ], methylation of pectin [ 14 ], self-incompatibility [ 15 ], and circadian rhythms [ 16 ]. However, considering that the end goal of CRISPR/Cas9 GE is to obtain a transgene-free homozygous plant harboring a precise modification of a specific target gene, and the altered sequence and the resulting traits should be stably inherited, there is still a need for efficiency improvement and diversification of objectives in the CRISPR/Cas9 application for B. rapa .…”