We demonstrate the functionality of these vectors through biotinylation assays in tobacco ( Nicotiana benthamiana ) plants. To allow for the use of the constructs in a range of experiments we have designed assembly modules that encode the biotin ligases fused to different linkers as well as different commonly used subcellular localization sequences. To facilitate the use of proximity-labelling in plants, we have generated a collection of constructs that can be used for the rapid cloning of TurboID and MiniTurbo fusion proteins using the Golden Gate cloning method. These biotinylated proteins can then be isolated by affinity purification using streptavidin-coated beads and identified by mass spectrometry. Expression of BioID (or of its derivates TurboID and MiniTurbo) fused to a bait protein results in the biotinylation of proximal proteins. One proximity labelling approach makes use of a promiscuous bacterial biotin ligase, termed BioID. Proximity-labelling has emerged as a powerful tool for the detection of weak and transient interactions between proteins as well as the characterization of subcellular proteomes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the FAD2 and FATB multigenes. These three vectors were transformed into soybeans using the Agrobacterium-mediated method.
In practice, we constructed multiple sgRNAs targeting multiple genes of FAD2 and FATB in soybean using this protocol. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. 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.ĬRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. In GE2 mutant plants, the floral repressor gene FLC1 was expressed during vernalization but the floral integrator gene FT was not expressed after vernalization. All GE2 mutant plants contained successful edits in two out of three BrVRN1 orthologs and displayed delayed flowering time. Additionally, we identified ‘transgene-free’ BrVRN1 mutant plants without any transgenic elements from the GE1 (gene-editing 1) and GE2 generations. T1 mutant plants with a 1-bp insertion in BrVRN1 exhibited late flowering after the vernalization. DNA sequencing analysis revealed site-directed mutations at two target sites: gRNA1 and gRNA2. 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. To develop Chinese cabbage (Brassica rapa L. The VERNALIZATION1 (VRN1) gene is a crucial transcriptional repressor involved in triggering the transition to flowering in response to prolonged cold.
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