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. 2022 Dec 29;24(1):541.
doi: 10.3390/ijms24010541.

CRISPR/Cas9-Mediated Mutagenesis of BrLEAFY Delays the Bolting Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Yun-Hee Shin  1 , Young-Doo Park  1
Affiliations

CRISPR/Cas9-Mediated Mutagenesis of BrLEAFY Delays the Bolting Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Yun-Hee Shin et al. Int J Mol Sci. .

Abstract

Chinese cabbage has unintended bolting in early spring due to sudden climate change. In this study, late-bolting Chinese cabbage lines were developed via mutagenesis of the BrLEAFY (BrLFY) gene, a transcription factor that determines floral identity, using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system. Double-strand break of the target region via gene editing based on nonhomologous end joining (NHEJ) was applied to acquire useful traits in plants. Based on the 'CT001' pseudomolecule, a single guide RNA (sgRNA) was designed and the gene-editing vector was constructed. Agrobacterium-mediated transformation was used to generate a Chinese cabbage line in which the sequence of the BrLFY paralogs was edited. In particular, single base inserted mutations occurred in the BrLFY paralogs of the LFY-7 and LFY-13 lines, and one copy of T-DNA was inserted into the intergenic region. The selected LFY-edited lines displayed continuous vegetative growth and late bolting compared to the control inbred line, 'CT001'. Further, some LFY-edited lines showing late bolting were advanced to the next generation. The T-DNA-free E1LFY-edited lines bolted later than the inbred line, 'CT001'. Overall, CRISPR/Cas9-mediated mutagenesis of the BrLFY gene was found to delay the bolting time. Accordingly, CRISPR/Cas9 is considered an available method for the molecular breeding of crops.

Keywords: Brassica rapa; CRISPR/Cas9; LEAFY gene; floral identity; late bolting.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
CRISPR/Cas9 gene editing vector construction targeting BrLFY paralogs. (A) Gene structure of the BrLFY paralogs. Black box, exon regions; Black line, intron regions; Blue arrow, target region of single guide RNA (sgRNA); and Red line, conserved domain. (B) Structure of CRISPR/Cas9 gene editing vector with sgRNA targeting the BrLFY paralogs based on pHAtC. LB, left border; T1, NOS terminator; HygR, hygromycin resistance gene; P1, NOS promoter; P2, Arabidopsis U6 promoter; P3, 35S promoter; Cas9hc:NLS:HA, human-codon-optimized Cas9 with the nuclear localization signal and an HA epitope; T2, 35S terminator; RB, right border; and Blue box, sgRNA, and sgRNA scaffold. (C) Chromatograms of single guide RNA (sgRNA) sequence in the BrLFY paralogs of inbred line, ‘CT001’. sgRNA sequence is underlined in black and blue rectangles indicating PAM sequence.
Figure 2
Figure 2
Identification of the tentative E0 LFY-edited lines by polymersase chain reaction (PCR) analysis using the HygR and Cas9hc primer sets. The 709 bp and 654 bp PCR amplicons are indicated by an arrow, respectively. P, positive control; M, 100 bp DNA ladder; N, negative control; Numbering lane, tentative LFY-edited lines.
Figure 3
Figure 3
Sequence-based detection of mutations induced by CRISPR/Cas9 system in the BrLFY paralogs of the E0 LFY-edited lines. (A) PCR analysis results based on the target gene-specific amplifying primer sets of the E0 LFY-edited lines. M, 100 bp DNA ladder; Numbering lane, LFY-edited lines. (B) Mutagenesis of CT001_A02071870 at the genomic DNA and amino acid levels in the LFY-7 and LFY-13 edited lines. (C) Mutagenesis of CT001_A06214910 at the genomic DNA and amino acid levels in the LFY-7 and LFY-13 edited lines. sgRNA sequence is underlined in black and PAM sequence is shown in blue font. Nucleotide and amino acid mutations are indicated by a red font and base insertions are highlighted in yellow boxes. Termination codon is indicated in red asterisk.
Figure 4
Figure 4
Confirmation of the bolting time and flower bud phenotype for ‘CT001’ and the E0 LFY-edited lines. Red arrow, emergence of the first flower bud.
Figure 5
Figure 5
Analysis of T-DNA copy number and insertion site in the E0 LFY-edited lines. (A) Southern hybridization analysis results confirming the number of T-DNA copies inserted into the E0 LFY-edited line genome. Forty micrograms of genomic DNA were digested with EcoRI for 4 h and separated overnight on a 1.0% agarose gel at 20 V. Digested DNA was blotted onto a Hybond N+ nylon membrane. Probe DNA with α-32P-labeled dCTP was obtained, and hybridization was carried out at 65 °C in a shaking incubator. M, λ HindIII molecular ladder; N, negative control; Lane, E0 LFY-edited lines showing late-bolting. (B) Results of the junction region sequences of T-DNA in the E0 LFY-edited lines genome using a modified variable argument thermal asymmetric interlaced PCR (VA-TAIL PCR) analysis. T-DNA was inserted into the intergenic region of the genome of the E0 LFY-edited lines.
Figure 6
Figure 6
Identification of the generated mutagenesis of the BrLFY paralogs in the T-DNA-free E1 LFY-edited lines. (A) Mutagenesis of CT001_A02071870 at the genomic DNA level in the E1 LFY-edited lines. (B) Mutagenesis of CT001_A06214910 at the genomic DNA level in the E1 LFY-edited lines. sgRNA sequence is underlined in black and blue rectangles indicating PAM sequence. Insertion mutations are indicated using red font.
Figure 7
Figure 7
Confirmation of bolting time and normal growth of ‘CT001’ and E1 T-DNA-free LFY-edited lines. (A) Early stage of bolting. Bolting of the inbred line ‘CT001’ (left); (B) Middle stage of bolting. Bolting of the E1 T-DNA-free LFY-edited lines (middle and right); (C) End stage of bolting. Left, inbred line ‘CT001’; Middle and right, E1 T-DNA-free LFY-edited lines. All lines showed normal growth.
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References

    1. Chowdhury Z., Mohanty D., Giri M.K., Venables B.J., Chaturvedi R., Chao A., Petros R.A., Shah J. Dehydroabietinal promotes flowering time and plant defense in Arabidopsis via the autonomous pathway genes FLOWERING LOCUS D, FVE, and RELATIVE OF EARLY FLOWERING 6. J. Exp. Bot. 2020;71:4903–4913. doi: 10.1093/jxb/eraa232. - DOI - PubMed
    1. Wu Z., Fang X., Zhu D., Dean C. Autonomous pathway: FLOWERING LOCUS C repression through an antisense-mediated chromatin-silencing mechanism. Plant Physiol. 2020;182:27–37. doi: 10.1104/pp.19.01009. - DOI - PMC - PubMed
    1. Singh G.L., Singh V. Systems scale characterization of circadian rhythm pathway in Camellia sinensis. Comput. Struct. Biotechnol. J. 2022;20:598–607. doi: 10.1016/j.csbj.2021.12.026. - DOI - PMC - PubMed
    1. Preston J.C. Evolutionary genetics of core eudicot inflorescence and flower development. Int. J. Plant Dev. Biol. 2010;4:17–29.
    1. Goslin K., Zheng B., Serrano-Mislata A., Rae L., Ryan P.T., Kwaśniewska K., Thomson B., Ó’Maoiléidigh D.S., Madueño F., Wellmer F., et al. Transcription factor interplay between LEAFY and APETALA1/CAULIFLOWER during floral initiation. Plant Physiol. 2017;174:1097–1109. doi: 10.1104/pp.17.00098. - DOI - PMC - PubMed

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