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Article

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

by
Joon Ki Hong
,
Eun Jung Suh
,
Sang Ryeol Park
,
Jihee Park
and
Yeon-Hee Lee
*
Agricultural Biotechnology Department, National Institute of Agricultural Sciences, Rural Development Administration, 370 Nongsaengmyeong-ro, Jeonju 54874, Korea
*
Author to whom correspondence should be addressed.
Agriculture 2021, 11(12), 1286; https://0-doi-org.brum.beds.ac.uk/10.3390/agriculture11121286
Submission received: 11 November 2021 / Revised: 3 December 2021 / Accepted: 13 December 2021 / Published: 17 December 2021
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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.

1. Introduction

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is an important vegetable crop that contributes to human health as a source of beneficial dietary fiber, vitamins, and minerals [1]. Because it is responsive to seed vernalization, Chinese cabbage is prone to premature bolting, which affects crop yield and quality. This is particularly true in spring varieties that are often exposed to extended periods of cold during winter [2,3,4].
A properly timed transition from vegetative to reproductive development is the most important step in the life cycle of a flowering plant. Flowering time plasticity has evolved to integrate plants’ perceptions of both seasonal and developmental changes to maximize reproductive success in various environments. Flowering time has also emerged as a key trait affecting yield in commercial agronomic and horticultural crops because certain biotic and abiotic stresses can be avoided altogether by inducing or delaying the transition to flowering. Multiple pathways, such as photoperiod sensation, vernalization, gibberellin signaling, and responses to autonomous signals, are involved in the timing of floral transition during specific seasons, and altered flowering time phenotypes have been generated in multiple cultivated species through targeted genetic modification [5,6,7,8,9]. Vernalization, the induction of flowering by prolonged cold, ensures that plants flower in favorable spring conditions, and is usually required for flowering in Brassicaceae. Because Chinese cabbage can sense low temperatures during seed germination, it sometimes undergoes early flowering (bolting) due to low temperatures in the fall [10,11]. Premature bolting leads to a decline in commercial quality and a loss of market value, so a high bolting resistance is preferred when breeding cultivars of this species [10].
To facilitate these breeding efforts, extensive genetic analysis in Arabidopsis thaliana has identified several genes, including FRIGIDA (FRI) and FLOWERING LOCUS C (FLC), that play vital roles in the flowering process. FRI encodes a plant-specific nuclear protein that promotes high expression of FLC [12]. FLC encodes a MADS-box transcription factor that prevents flowering by suppressing the expression of the floral integrator genes FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) [13,14,15,16]. The FLC expression level negatively correlates with flowering time after vernalization, and is suppressed by exposure to a long period of cold temperatures [17]. The duration of vernalization, therefore, represses FLC expression in order to promote flowering in vernalization-responsive late-flowering plants. Thus, FLC is thought to be a major determinant of the vernalization response. In Arabidopsis, FLC expression is decreased through a vernalization mechanism involving VERNALIZATION INSENSITIVE 3 (VIN3), and this repression is epigenetically maintained by a Polycomb-like chromatin regulation involving VERNALIZATION 2 (VRN2), a Su(z)12 homolog, VERNALIZATION 1 (VRN1), and LIKE-HETEROCHROMATIN PROTEIN 1 [18,19,20,21,22,23].
VRN1 encodes a DNA-binding protein containing two plant-specific B3 domains that is required for stable repression of the major target of the vernalization pathway, the floral repressor FLC, during prolonged cold treatment [23,24]. In Arabidopsis, expression levels of AtVRN1 and AtVRN2 are not affected by vernalization [22,23]. However, vrn1–2 mutant plants grown in extended short-day (ESD) conditions showed reduced FT but wild-type FLC expression, suggesting that VRN1 regulates FLC and FT [23]. In Chinese cabbage, the expression of BrVRN1 genes is altered by cold treatment, according to patterns that vary among the three paralogous genes: two BrVRN1 genes are downregulated during cold treatment, and one is upregulated. It is therefore possible that the suppression of BrFLC expression is dependent on the upregulation of BrVRN1 by vernalization in B. rapa [25].
Despite genetic analysis of floral transition regulation in Arabidopsis and the identification of floral regulator genes in Chinese cabbage, there have been few reports on the mechanism of floral transition in Chinese cabbage specifically [26,27]. Recently, several studies have set out to directly control flowering time via CRISPR/Cas9-mediated genome editing [28,29]. In this study, we used the CRISPR/Cas9 system for targeted mutagenesis of BrVRN1 homologs (BrVRN1s) to introduce the late-flowering trait into B. rapa [25]. CRISPR/Cas9-mediated multi-target mutagenesis of BrVRN1s gave new insights into flowering time regulation and demonstrated that genome editing can be used to improve agricultural traits in B. rapa.

2. Material and Methods

2.1. sgRNA Target Site Design and Vector Construction

sgRNAs were designed using the web tool Cas-Designer (http://www.rgenome.net/cas-designer/, accessed on 8 July 2019) [30], which displayed all optional sgRNA sequences (20 bp) immediately followed by a 5′-NGG protospacer-adjacent motif (PAM) sequence in the forward or reverse strand. Sequences of the Chinese cabbage VRN1 genes (BrVRN1a, Bra022375, BrVRN1b, Bra037544, and BrVRN1c, Bra001729) were compared to find a region with matching sequences (Figure 1a, Figure S1 and Table S1). Then, four sgRNAs were designed in the corresponding region to target the BrVRN1a (Bra022375) gene [25]. To construct a plasmid vector carrying four sgRNAs and the Cas9 cassette, we designed a multiplex CRISPR/Cas9 platform that allows the co-expression of four sgRNA modules targeting different sites of BrVRN1 in one binary vector (pAGM4723) using Golden Gate assembly (Figure 1b) [31]. The sequence of Cas9 was assembled downstream of the Cauliflower mosaic virus (CaMV) 2× 35S promoter along with four custom sgRNAs driven by four Arabidopsis U6-26 promoters. The hygromycin-resistance gene (hpt) driven by a nos promoter was used as a selectable marker. The final vector was introduced into Agrobacterium tumefaciens GV3101 and then used for the transformation of Chinese cabbage.

2.2. Generation of Transgenic Chinese Cabbage Plants

The late-flowering Chinese cabbage cultivar ‘inbred line DA001’ was used for transformation according to previously published methods with minor modifications [32,33]. Surface-sterilized seeds were placed on Murashige and Skoog (MS) medium (MS basal medium, 3% sucrose, and 0.8% Phytagar) and germinated in a culture room under long-day conditions (16 h light/8 h dark) at 25 °C for 7 days. Hypocotyls were cut into 0.5 cm segments and placed on pre-culture medium (MS basal medium, 1 mg/L NAA, 3 mg/L BA, 2 mg/L AgNO3, 3% sucrose, and 0.8% Phytagar) for 2 days. The pre-cultured hypocotyls were inoculated with Agrobacterium suspension (OD600 = 0.3) in MS liquid medium for 15–20 min with gentle shaking. The inoculated hypocotyls were blotted with sterilized filter paper to remove excess bacterial suspension and co-cultivated on co-cultivation medium (MS basal medium, 1 mg/L NAA, 3 mg/L BA, 2 mg/L AgNO3, 100 μM acetosyringone, 3% sucrose, and 0.8% Phytagar) for 3 days in darkness at 25%. Hypocotyls were then washed with sterilized water containing 100 mg/L carbenicillin and 250 mg/L cefotaxime and transferred to selective medium (MS basal medium, 1 mg/L NAA, 3 mg/L BA, 2 mg/L AgNO3, 3% sucrose, 100 mg/L carbenicillin, 250 mg/L cefotaxime, and 0.8% Phytagar) supplemented with 10 mg/L hygromycin as a selective agent. Callus that formed on hypocotyls was sub-cultured on fresh selective medium every 2–3 weeks. Healthy and elongated shoots were excised and transferred to a phytohormone-free MS medium to induce rooting. The transformed plantlets were grown in a greenhouse after acclimatization.

2.3. Confirmation of the Mutations of Transgenic Plants

To confirm the stable integration of transgenes in Chinese cabbage, the dual-genomic DNA-PCR was performed. Genomic DNA PCR was carried out using a pair of hpt-specific primers (forward, 5′-AGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTC-3′; reverse, 5′-TCTACACAGCCATCGGTCCAGACGGCCGCGCTTCTG-3′; 961 bp PCR product) and two pairs of Cas9-specific primers (SP1-forward, 5′-TCCTCCCGAAAAGGAACAGCGAC-3′; SP1-reverse, 5′-CATTCGTTTCCGGCCGTTTTCAAG-3′; 311 bp PCR product, SP2-forward, 5′-CAACTGGTTCAGACTTACAATCAGC-3′; SP2-reverse, 5′-TTCCAAGATGGGCTTAATAAATTTG-3′; 1031 bp PCR product). The PCR reactions were performed using 35 cycles of 30 s at 95 °C, 30 s at 55 °C, and 40 s at 72 °C, followed by a final extension at 72 °C for 10 min. The resulting PCR products were analyzed using electrophoresis and ethidium bromide staining.
To verify BrVRN1 mutations in gene-edited plants, genomic DNA was isolated from transgenic leaf samples using the CTAB method, and then PCR, Sanger sequencing, and deep sequencing were performed. To confirm BrVRN1 mutations in T0 and T1 generation plants, as well as in gene-edited plants without transgenes (GE1 plants, sequences adjacent to the four sgRNA target positions were amplified with primer sets (target1 and target2, 5′-AGGACGAGCTATCCGTAGCA-3′, 5′-GGCAGAGTTGCCTTCGTATC-3′; target3 and target4, 5′-TACCATTCCTCCGCTCTCAT-3′, 5′-TAGGAGTTGGTGTCGCCTCT-3′). PCR products were subjected to deep sequencing performed at ToolGen Inc. (ToolGen, Seoul, Korea) to detect mutations [34]. Also, to verify the mutations in the three BrVRN1 genes in the GE1 and GE2 plants, Sanger sequencing was performed using three gene-specific amplicons generated using intron-specific primer sets (BrVRN1a, 5′-TATACAATGTTCCAGGAATAGTAATGGAC-3′, 5′-ACCAATCATCACAATCAATCCAAGAATG-3′; BrVRN1b, 5′-GAACCATTTGGATTGTGATTTTAAATTAA-3′, 5′-AATTTTCGGTTCAGTTCAGCCGGTTTGG-3′; BrVRN1c, 5′-CCTAAAGGTTACAAGCA0GTCTCATTTAT-3′, 5′-TCACTACTAAACAATAGTCTGAAGAATC-3′). PCR amplicons from GE1 and GE2 plants were cloned into the pGEM-T vector (Promega, Madison, WI, USA), and 100 plasmids were Sanger-sequenced. For verification of on- and off-target editing events, as well as homozygosity, in the GE2 generation, SNP and insertion/deletion (indel) analyses were performed using next-generation sequencing (NGS) at SEEDERS Inc. (SEEDERS, Daejeon, Korea). Raw sequencing reads of individual paralogs were used as queries, which were then compared to the provided non-edited reference genome sequence (Table S2) [35,36,37,38,39,40].

2.4. RNA Extraction and Real-Time Reverse Transcription Quantitative PCR (qPCR) Analysis

To analyze the expression of floral-related genes in transgenic plants, qPCR was performed using primer sets specific to flowering genes (BrFLC1, 5′-GTGCGGCTGCTTGAACAT-3′, 5′-CCTGACCAGGCTGGAGAAA-3′; BrFT, 5′-ACTTTGGTTATGGTGGATCCAGATGT-3′, and 5′-GGAAGGCCGAGATTGTATAGGGAA-3′) [41,42]. Samples were collected from the leaf tissue of GE2 plants at different growth stages. Total RNA was extracted from 100 mg of leaf tissue using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and treated with DNase I (New England Biolabs, Ipswich, MA, USA) to remove genomic DNA. First-strand cDNA was synthesized from 2 μg of total RNA using MMLV reverse transcriptase (RNaseH-free; Toyobo, Osaka, Japan) according to the manufacturer’s instructions. The cDNA was resuspended in nuclease-free water (Promega, Madison, WI, USA) and used for qPCR analysis with a CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). BrActin was used as an internal control for normalizing mRNA levels [41,42]. Three biological replicates were analyzed for each qRT-PCR assay, and Student’s t-test was used for statistical analyses.

2.5. Flowering Time Analysis

Transgenic Chinese cabbage plants with 4–5 leaves were transferred to a 4 °C growth chamber (for vernalization) under long-day conditions (16 h light/8 h dark) for 40–60 days. After vernalization, plants were transplanted into 25 cm diameter pots and transferred to a greenhouse to observe flowering time phenotypes in March and September [32]. The flowering time of GE1 and GE2 plants was recorded as the number of days until the appearance of a bolt with a floral bud. Progeny seeds were obtained using bud-pollination.

3. Results

3.1. Identification and CRISPR-Cas9 Mutagenesis of BrVRN1 Genes

To identify orthologs of the AtVRN1 gene in Chinese cabbage, we used an AtVRN1 (At3g18990) sequence for a BLAST query in the B. rapa database (BRAD, http://brassicadb.org, accessed on 15 March 2019), which revealed three paralogs (Table S1), which we refer to as BrVRN1a, BrVRNb, and BrVRN1c. The sequence similarity between the paralogs ranged between 89.1% and 90.7%. A previous study showed that under cold treatment, BrVRN1a (Bra022376) is upregulated, while its paralogs BrVRN1b (Bra037544) and BrVRN1c (Bra001729) are downregulated [25,41]. We expected that knocking out any one paralog in Chinese cabbage would give comparable phenotypic results and therefore focused on BrVRN1a. We selected four target sites (gRNA1-gRNA4) in the second exon of BrVRN1a (Figure 1a, Figure S1 and Table S3) and transformed the corresponding multiplex plasmid vector carrying four sgRNAs and a Cas9 cassette into Chinese cabbage (B. rapa L. ssp. pekinensis) via Agrobacterium-mediated transformation (Figure 1b).
Next, we performed hypocotyl transformation to introduce the CRISPR/Cas9 construct into the late-flowering Chinese cabbage cultivar ‘inbred line DA001’. The efficiency of callus formation from hypocotyls infected with Agrobacterium on selection medium supplemented with hygromycin was 36% (data not shown), and we obtained nineteen independent T0 transformants (Table S4). Before transferring the transgenic plants to the soil, we used genomic DNA extracted from T0 leaf tissue to examine the CRISPR/Cas9-induced mutations at the target sites using deep-sequencing analysis (data not shown). We identified mutations in 10 independent T0 lines regenerated into whole plants, demonstrating the efficacy of the selected sgRNAs in Chinese cabbage. Deep-sequencing of these lines revealed an overlay of more than two different sequences (Figure S2), suggesting that these were chimeric plants containing different mutations and that some mutations might have occurred somatically. Among these 10 mutants, we were able to harvest seeds from three lines (#1, #2, and #7), which we planted under vernalization conditions in Jeonju, Korea, and used for further studies.

3.2. Generation of ‘Transgene-Free’ Mutant Lines

We expected that chimeric T0 plants would give rise to complex segregation patterns in the T1 generation and that homozygous mutants could only be selected from later generations. We tested thirty-seven T1 plants (19 from line #1, 13 from line #2, and 5 from line #7) for the presence of transgenes. Also, to obtain BrVRN1a mutants without any transgenic elements of the sgRNAs/Cas9 vector, we screened ‘transgene-free’ plants through a dual-PCR strategy that used two sets of primers flanking the Cas9 and hph (hygromycin B phosphotransferase) genes (Figure S3). This showed that two (#2-2 and #2-8) of the 13 #2 T1 mutants were ‘transgene-free’, and their 20 offspring plants were all ‘transgene-free’ BrVRN1 mutants (Figure S4). We then conducted deep-sequencing of two selected ‘transgene-free’ GE1 (gene-editing 1) lines, which confirmed BrVRN1 gene editing based on the proportion of indels. The 1-bp insertion type was most frequently identified in the BrVRN1-gRNA1 and BrVRN1-gRNA2 target regions but was not found in the BrVRN1-gRNA3 and BrVRN1-gRNA4 target regions (Figure S5).
Next, we sequenced the PCR products from the genomic DNA of each mutant to search for mutations within the targeted regions in three paralogs (BrVRN1a-c). Cloning the amplicons containing the target regions into plasmids and subsequent sequencing confirming the chimeric nature of the T1 plants. Interestingly, five (#2-1, -2, -3, -8, and -13) mutant plants showed three types of mutations at two target sites in two genes (BrVRN1a, b) (Figure 2). Two types of mutations were found at target sites BrVRN1a-gRNA1 and -gRNA2 (A-insertion and C-insertion), and the 1-bp insertion type was most frequently identified. A type of mutation was also found at target site BrVRN1b-gRNA2 (A-insertion), and the 1-bp insertion type was most frequently identified. However, no mutations at the target sites BrVRN1c-gRNA1 and BrVRN1c-gRNA2 were generated in T1 plants (Figure 2). All types of frameshift mutations induced by CRISPR/Cas9 at target sites of BrVRN1a and BrVRN1b generated premature translation termination codons (Figure 3). Unfortunately, no mutations at target sites gRNA3 and gRNA4 in BrVRN1a were identified in T1 plants. This suggests that the mutations at these two target sites were not inherited from the T0 generation to the T1 generation.

3.3. Stable Inheritance of Induced Mutations and Phenotypes of the Mutants

To validate whether CRISPR/Cas9-mediated mutagenesis of BrVRN1s was linked to a late-bolting phenotype, we compared the flowering times of the three T1 mutant lines (#1, #2, and #7) with that of wild-type plants under vernalization conditions. All three mutant lines had delayed flowering time (Figure S6 and Table 1). After a 60-day vernalization period, the average time to bolting after the end of the vernalization period was approximately nine days in wild-type plants, but 19–20 days in the CRISPR/Cas9-mutagenized #1 and #2 T1 plants. In addition, after 40 days of vernalization, wild-type plants bolted 15–16 days after the end of vernalization, while the T1 mutant #7 plants were delayed by 8–11 days, bolting 23–27 days after the end of vernalization (Table 1). Thus, bolting time was delayed in all mutants regardless of cold treatment season and duration (Table 1), suggesting that the targeted gene-edits of BrVRN1s are sufficient to induce late-bolting phenotypes in Chinese cabbage. In order to use the mutants showing delayed bolting as breeding material, we attempted to select mutants in which the transgenes had been removed and the mutations were homogeneous. We then analyzed GE2 generations of the two GE1 lines that had the Cas9 gene removed (Figure S3) and selected two ‘transgene-free’ mutants (#2-2 and #2-8) for phenotypic analysis. We self-crossed the #2-2 and #2-8 GE1 plants to obtain GE2 seeds and analyzed 10 plants (20 total) from each line for their genotypes and bolting characteristics.
To determine whether the BrVRN1 mutants could transmit the induced mutations and phenotypes to their progenies, we grew the GE2 progeny of the GE1 #2-2 and #2-8 lines under vernalization conditions. We examined Sanger sequencing results of three gene-specific amplicons from the 20 GE2 plants and found that targeted mutagenesis of BrVRN1s induced by CRISPR/Cas9 was stably inherited and maintained consistent mutation types from the GE1 generation to the GE2 generation (Figure S7). The average time to bolting after 40-day vernalization was approximately 16 days in wild-type plants, but ranged from 37 to 59 days in the #2-2 and #2-8 GE2 plants, meaning that bolting was delayed by 21–43 days (Table 2). These results confirmed that the BrVRN1 mutant GE2 plants showed a delayed flowering phenotype under vernalization conditions, suggesting that the BrVRN1a and BrVRN1b knockout carried the controlled late-flowering trait in Chinese cabbage (Figure 4).

3.4. Expression Patterns of Floral Regulators in GE2 Mutant Plants

Because the BrVRN1 GE2 mutants displayed late-flowering phenotypes, we considered the possibility that BrVRN1s may regulate the expression of other floral genes, such as FLC1 and FT [23]. Therefore, we analyzed the expression patterns of BrFLC1 and BrFT in the leaves of vernalized and unvernalized wild-type and ‘transgene-free’ GE2 mutant plants with different flowering times using qPCR (Figure 5). Unvernalized wild-type Chinese cabbage showed high BrFLC1 expression, whereas BrFT expression was undetectable, probably because of the high level of BrFLC1 expression. BrFLC1 expression was reduced in the wild-type plant during a 40-day cold treatment. BrFT expression was undetectable before vernalization, but began to increase towards the end of vernalization; in general, the BrFT expression level was inversely proportional to that of BrFLC [32]. In GE2 mutant plants, the expression of BrFLC1 decreased more slowly than in wild-type plants and remained detectable after vernalization. In addition, FT expression was never observed in the mutants regardless of vernalization status. However, BrVRN1a expression in mutants at the RNA level was similar to that of the wild type, occurring only after vernalization (Figure S8). These results revealed that the altered patterns of BrFLC1 and BrFT expression induced by BrVRN1 mutations could result in the late flowering time phenotypes of mutant plants. These data indicate that the BrVRN1s are important determinants of vernalization-responsive flowering time through their regulation of BrFLC1 and BrFT expression in Chinese cabbage.

3.5. Analysis of Potential Off-Target Changes

To confirm the specificity of BrVRN1 gene editing via CRISPR/Cas9 in Chinese cabbage, and to avoid affecting the phenotypic statistics by including off-target mutations when identifying homologous mutants, we analyzed the potential off-target effects based on SNP and indel proportions in the 10 GE2 mutant plants (five plants each of the #2-2 and #2-8 lines) using NGS (Tables S3 and S5). All of the examined potential off-target sites possessed mismatches of only 1–3 bp compared with the on-target guide sequences. We did not observe any mutations in the potential off-target sites we examined in GE2 plants (Table 3). Also, the mutation types (BrVRN1a; A-insertion and C-insertion, BrVRN1b; A-insertion) in GE1 mutants (#2-2 and #2-8) were inherited without any transgenic elements in the GE2 generation (Table 4 and Table 5). In this experiment, we identified homozygous double mutations (BrVRN1a and BrVRN1b), but no heterozygous mutations in the 10 GE2 mutant (Table 5).

4. Discussion

The transition from vegetative to reproductive growth is a key developmental switch in the life cycle of flowering plants. Flowering is regulated by a complex regulatory network that modulates flowering time in response to various environmental stimuli and developmental statuses [32,43]. Chinese cabbage is a typical vernalization-sensitive plant. Genetic analyses have identified several genes, including VERNALIZATION1 (VRN1), involved in the control of flowering time in Chinese cabbage during vernalization [23,25,41,44].
VRN1 encodes a DNA binding protein containing two plant-specific B3 domains that is required for the increase in H3K27me3 (trimethylation of lysine 27 in histone H3) epigenetic marks that causes transcriptional repression of FLC during prolonged cold treatment [23,26]. In previous studies, expression levels of AtVRN1 and AtVRN2 were not affected by vernalization in Arabidopsis [22,23]. In contrast to Arabidopsis, in Chinese cabbage one of the three BrVRN1 paralogs (Bra022376) was upregulated by cold treatment, whereas the other two (Bra037544 and Bra001729) were downregulated [25,41]. These results suggest that the flowering pathway in Chinese cabbage is somehow different and more complex than that of Arabidopsis [25,41]. Despite the importance of VRN1 genes in controlling vernalization-responsive flowering time, phenotypic effects of the BrVRN1 genes in Chinese cabbage have not been fully investigated.
The CRISPR/Cas9 system has been reliable means to perform functional studies on multi-gene clusters of unknown function and is becoming one of the most effective tools for creating desirable phenotypes in crop plants. In this study, we assessed the molecular variation in the flowering time of Chinese cabbage via CRISPR/Cas9-mediated mutagenesis of BrVRN1 genes. Among the three BrVRN1 genes, we chose four target sites in the second exon of BrVRN1a (Figure 1, Figure S1 and Table S1) [25,41]. This multiplex CRISPR/Cas9 system-generated target gene mutations with a highly diverse pattern of indel mutations in the T0 generation. CRISPR/Cas9 editing using constitutively strong promoters such as CaMV 35S may generate unwanted somatic mutations in various tissues and growth periods. The various mutations induced through CRISPR/Cas9 in the T0 generations may then be unstably inherited, and it is, therefore, difficult to maintain consistent mutation types between the T0 and T1 generations. These undesired and unstable mutations in Chinese cabbage can be segregated out of the genome via self-pollination [45]. Notably, not all T0 plants were capable of generating seeds, with only three out of ten edited lines (#1, #2, #7 lines) forming seeds (Figure S2 and Table S4). By deep sequencing of T1 plants from the #2 line without transgenes, we verified the presence of A and C base insertions at the gRNA1 and gRNA2 target sites (Figure 2 and Figure S5), confirming that the CRISPR/Cas9-induced mutations were passed down from the T0 to the T1 generation. In addition, targeted mutations in two BrVRN1 orthologs (BrVRN1a and BrVRN1b) in the T1 heterozygous mutant lines were stably transmitted to the GE2 generation (Figure 2, Figure S7 and Table 4). These mutations, caused by base insertions in the coding regions, were expected to inactivate the BrVRN1a and BrVRN1b proteins by causing frameshifts (Figure 3). In contrast, no mutations at target sites BrVRN1a gRNA3 or gRNA4 or BrVRN1c gRNA1 or gRNA2 were identified in the T1 and GE2 mutant plants (Figure 2, Figure S5 and Table 5). This suggests that the gene targeting efficiency of sgRNAs may be affected by their relative position in the expression cassette and the chromosome structure of targeted genes, as has also been observed in other plant species [31,46]. Taken together, our results demonstrate the feasibility of introducing transmissible mutations induced by CRISPR/Cas9 through the propagation of stable transgenic plants, which could be widely used for reverse genetics in Chinese cabbage.
In applying CRISPR/Cas9 gene editing, it is important to consider off-target sites, and the homology between sgRNAs and candidate off-target sites has been reported to be the main factor influencing off-target mutations [47,48]. To examine the specificity of CRISPR/Cas9 in Chinese cabbage and avoid confounding our phenotype statistics by including off-target sites, we investigated the off-target regions of four target sites of BrVRN1 through NGS analysis. We observed no mutations at any of the off-target sites (Table 3), most likely as a result of careful target selection. The risk of off-target edits by CRISPR/Cas9 in Chinese cabbage may also be low in general on account of the low frequency of somatic mutations in tissue-culture-based transformation [48]. In addition, undesired off-target mutations in plants could be eliminated by hybridization [45]. Therefore, we predicted that the complex mutation patterns of the BrVRN1 in T0 plants could be eliminated by hybridization (Figure S2 and Figure 2).
Chinese cabbage possesses three BrVRN1 genes, but it is not known whether all three are involved in controlling flowering time [25,41]. However, the BrVRN1a and BrVRN1b double-mutant plants did display a delayed flowering phenotype (Figure 4 and Table 2). Although our data do not indicate whether the BrVRN1b gene directly or indirectly influenced late-flowering phenotypes, they do suggest that the induced late-flowering phenotypes by the double-mutations of BrVRN1 genes attests to the complexity underlying multiple signalling networks. Therefore, further experiments are required to define how much the BrVRN1 genes contribute to the regulation of flowering time by vernalization in comparison with BrFLC genes. However, the frameshift mutations induced by CRISPR/Cas9 at two target sites of BrVRN1 generated premature translation termination codons through amino acid substitution in the corresponding protein (Figure 3). This result suggests that the mutated genes were not translated as complete proteins containing two B3 domains, resulting in a loss of protein function. Therefore, their inability to suppress FLC expression in warm conditions following prolonged cold exposure resulted in a late-flowering phenotype [23,44]. In this study, the mutants for which BrVRN1 gene editing was confirmed showed a delayed flowering time in each generation (from the T0 to the T2 generation), regardless of the season or the low-temperature treatment period (Table 1 and Table 2). In addition, the bolting time of GE2 mutant plants ranged from 37 to 59 days, whereas the GE1 mutant plants required from 23 to 27 days, which may have been caused by converting heterozygous mutations in the targeted genes to homozygous using selfing-crossing (Table 5). Our results suggest that BrVRN1 genes play an important role in the regulation of flowering time in Chinese cabbage by regulating the expression of the floral repressor FLC and floral integrator FT (Figure 5). In addition, these results suggest the possibility of developing breeding material with a delayed flowering time in Brassicaceae with a similar flowering pathway mechanism using VRN1 gene knockouts.
Recently, genome-editing technology has provided a new approach for developing crops that can replace genetically modified organisms (GMOs), whose safety has been debated [45]. The CRISPR/Cas9 system can accurately generate site-directed mutations in specific genes. The exogenous transgenes (Cas9, sgRNA, promoters, and selectable markers) inserted into a plant genome by Agrobacterium-mediated transformation can be removed in later generations via genetic segregation through selfing-crossing or hybridization approaches. These ‘transgene-free’ mutants have been generated by CRISPR/Cas9 editing in many plant species, such as Arabidopsis [49,50], rice [51,52], wheat [53], tomato [54], and soya bean [45]. In the present study, we obtained ‘transgene-free’ targeted genome-modified Chinese cabbage that was homozygous for the null alleles of endogenous BrVRN1a and BrVRN1b without any transgenic elements in the GE1 and GE2 generations (Figures S3 and S4, Table 4 and Table 5). Therefore, ‘transgene-free’ BrVRN1 mutants can provide breeding materials for developing Brassica crops with delayed flowering time. Chinese cabbage is very sensitive to low temperatures, which limits its geographical range of cultivation, and our study may contribute to expanded regional introduction and domestication of this species.

5. Conclusions

In conclusion, this is the first report of the development of a Chinese cabbage strain with a delayed flowering time trait through BrVRN1 gene editing using a multiplex CRISPR/Cas9 system. BrVRN1-specific gene editing notably delayed flowering times and induced the production of heritable mutations. Taken together, our data indicate that BrVRN1 acts as a positive regulator of the induction of flowering in Chinese cabbage, thus raising the possibility that this may serve as a useful genetic source for crop improvement with respect to local environments and climate change.

Supplementary Materials

The following are available online at https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/agriculture11121286/s1, Figure S1. Comparison of the nucleotide sequences of three BrVRN1 genes with target sites of CRISPR/Cas9. Figure S2. Genome-editing analysis of T0 plants by deep-sequencing. Figure S3. The confirmation of the presence of hygromycin resistant gene (hph) and Cas9 transgenes using genomic PCR analysis in T1 transgenic lines. Figure S4. The confirmation of the presence of Cas9 transgenes using genomic PCR analysis in ‘transgene-free’ BrVRN1 mutant GE2 lines. Figure S5. Genome-editing analysis of T0 lines by deep-sequencing. Figure S6. Flowering-time phenotype in BrVRN1 mutant GE1 plants. Figure S7. Target gene mutations by the multiplex CRISPR/Cas9 system in the GE2 generation. Figure S8. Expression patterns of BrVRN1a in ‘transgene-free’ BrVRN1 mutant GE2 lines. Table S1. Features of BrVRN1 genes used in this study. Table S2. Summary of Brassica rapa L. (v3.0) reference genome. Table S3. Statistics of target four-gRNA position on the reference v3 genome. Table S4. CRISPR/Cas9-mediated targeted mutagenesis of BrVRN1 in the T0 generation. Table S5. Statistics of final trimmed whole genome resequencing data from ‘transgene-free’ BrVRN1 mutant GE2 lines.

Author Contributions

Y.-H.L. conceived and designed the research. J.K.H. and J.P. performed experiments. J.K.H., E.J.S. and S.R.P. collected and analyzed the data. J.K.H. and Y.-H.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was carried out with the support of “Research Program for Agricultural Biotechnology (PJ014174)”, National Institute of Agricultural Science, Rural Development Administration, Republic of Korea. This study was supported by 2021 the RDA Fellowship Program of National Institute of Agricultural Science, Rural Development Administration, Republic of Korea.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Multiplex gene editing construction of BrVRN1 family gene. (a) Gene structure of BrVRN1a with target sites of CRISPR/Cas9 designed in the second exon. Blue stripe, exon; black line, intron; grey stripe, UTR (untranslated regions). The underlined nucleotides indicate the target sites (named gRNA1 to gRNA4). Nucleotides in red represent PAM sequences. PAM, protospacer adjacent motif. (b) Structure of CRISPR/Cas9 platform with BrVRN1a four sgRNAs vector based on pAGM4723. AtU6P, Arabidopsis U6 promoter; Pnos, nopaline synthase promoter; Tnos, nopaline synthase terminator; LB, left border; RB, right border; HygR+, hygromycin-resistance gene; OCST, octopine synthase terminator; L3E, end linker 3; CaMV35P, 35S promoter.
Figure 1. Multiplex gene editing construction of BrVRN1 family gene. (a) Gene structure of BrVRN1a with target sites of CRISPR/Cas9 designed in the second exon. Blue stripe, exon; black line, intron; grey stripe, UTR (untranslated regions). The underlined nucleotides indicate the target sites (named gRNA1 to gRNA4). Nucleotides in red represent PAM sequences. PAM, protospacer adjacent motif. (b) Structure of CRISPR/Cas9 platform with BrVRN1a four sgRNAs vector based on pAGM4723. AtU6P, Arabidopsis U6 promoter; Pnos, nopaline synthase promoter; Tnos, nopaline synthase terminator; LB, left border; RB, right border; HygR+, hygromycin-resistance gene; OCST, octopine synthase terminator; L3E, end linker 3; CaMV35P, 35S promoter.
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Figure 2. Target gene mutations by the multiplex CRISPR/Cas9 system in the T1 generation. The mutated DNA sequences of target regions are provided. Gene mutations are in red. Wt, untransformed wild-type plants; lanes #2-2 to -8, selected GE1 lines, respectively.
Figure 2. Target gene mutations by the multiplex CRISPR/Cas9 system in the T1 generation. The mutated DNA sequences of target regions are provided. Gene mutations are in red. Wt, untransformed wild-type plants; lanes #2-2 to -8, selected GE1 lines, respectively.
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Figure 3. Nucleotide and deduced amino acid sequences of insertion variants of BrVRN1 homologs in #2 GE1 mutant lines. Nucleotide residues are numbered starting with the methionine initiation codon (ATG). The deduced amino acid sequences are presented by a single letter code above the nucleotide sequence. The original amino acid sequences and changed codes are colored in green and red, respectively. The stop codon is denoted by an asterisk.
Figure 3. Nucleotide and deduced amino acid sequences of insertion variants of BrVRN1 homologs in #2 GE1 mutant lines. Nucleotide residues are numbered starting with the methionine initiation codon (ATG). The deduced amino acid sequences are presented by a single letter code above the nucleotide sequence. The original amino acid sequences and changed codes are colored in green and red, respectively. The stop codon is denoted by an asterisk.
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Figure 4. Flowering-time phenotype in ‘transgene-free’ BrVRN1 GE2 mutant lines. Pictures were taken 31 days at 25 °C after a 40-day exposure to 4 °C. Wt, untransformed wild-type plants; #2-2 and #2-8, selected ‘transgene-free’ GE2 mutant lines, respectively.
Figure 4. Flowering-time phenotype in ‘transgene-free’ BrVRN1 GE2 mutant lines. Pictures were taken 31 days at 25 °C after a 40-day exposure to 4 °C. Wt, untransformed wild-type plants; #2-2 and #2-8, selected ‘transgene-free’ GE2 mutant lines, respectively.
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Figure 5. Expression patterns of floral regulator genes in ‘transgene-free’ BrVRN1 mutant GE2 lines. BrFLC, B. rapa FLOWERING LOCUS C; FT, FLOWERING LOCUS T. 0dV, 20 day-old plants before cold treatment (4 °C); 20dV, 20-day exposure to 4 °C; 40dV, 40-day exposure to 4 °C; 40dV10 and 40dV15, 10 days and 15 days at 25 °C after a 40-day exposure to 4 °C, respectively. Wt, untransformed wild-type plants; lanes #2-2-1 to #2-8-10, selected ‘transgene-free’ GE2 mutant lines, respectively.
Figure 5. Expression patterns of floral regulator genes in ‘transgene-free’ BrVRN1 mutant GE2 lines. BrFLC, B. rapa FLOWERING LOCUS C; FT, FLOWERING LOCUS T. 0dV, 20 day-old plants before cold treatment (4 °C); 20dV, 20-day exposure to 4 °C; 40dV, 40-day exposure to 4 °C; 40dV10 and 40dV15, 10 days and 15 days at 25 °C after a 40-day exposure to 4 °C, respectively. Wt, untransformed wild-type plants; lanes #2-2-1 to #2-8-10, selected ‘transgene-free’ GE2 mutant lines, respectively.
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Table
Table 1. Phenotypes of flowering time in Chinese cabbage GE1 (T1) mutant plants after vernalization. GE1 mutant plants were exposed to low temperature (4 ℃) for 40 or 60 days.
Table 1. Phenotypes of flowering time in Chinese cabbage GE1 (T1) mutant plants after vernalization. GE1 mutant plants were exposed to low temperature (4 ℃) for 40 or 60 days.
Vernalization PeriodTarget GeneGE1 LineDays to Visible Flowering Bud in Plants
12 June–12 October 2020 (60 days)No gene targetedWt-1~109 (9 ± 0) a
BrVRN1 (multi-target)#1-1~1919-20 (19.4 ± 0.116 **)
BrVRN1 (multi-target)#2-1~1319-20 (19.2 ± 0.104 **)
28 September–10 November 2020 (40 days)No gene targetedWt-1~515-16 (15.6 ± 0.245)
BrVRN1 (multi-target)#7-1~523-27 (25.4 ± 0.748 **)
a (parentheses): The range of values obtained by mean ± SE with 95% confidence limits (**; p-value < 0.001, Student’s t-test).
Table
Table 2. Phenotypes of flowering time in Chinese cabbage GE2 mutant plants after vernalization. ‘Transgene-free’ GE2 plants were exposed to low temperature (4 ℃) for 40 days.
Table 2. Phenotypes of flowering time in Chinese cabbage GE2 mutant plants after vernalization. ‘Transgene-free’ GE2 plants were exposed to low temperature (4 ℃) for 40 days.
Vernalization PeriodTarget GeneGE2 LineDays to Visible Flowering Bud in Plants
21 January–2 March 2021 (40 days)No gene targetedWt-1~1016 (16 ± 0) a
BrVRN1
(multi-target, transgene-free)		#2-2-1~1037–59 (52.7 ± 2.395 **)
BrVRN1
(multi-target, transgene-free)		#2-8-1~1038–56 (48.3 ± 2.408 **)
a (parentheses): The range of values obtained by mean ± SD with 95% confidence limits (**; p-value < 0.001, Student’s t-test).
Table
Table 3. Effect of chromatin structure on Cas9 activity at off-target sites of the ‘transgene-free’ GE2 mutant plants.
Table 3. Effect of chromatin structure on Cas9 activity at off-target sites of the ‘transgene-free’ GE2 mutant plants.
TargetTarget SequenceMismatchNumber of Found TargetsNumber of SNP PositionsNumber of In/Del Positions
gRNA1ACACGCCAAACGTGACCATCNGG1000
2000
3100
gRNA2GACCGTTACTCAATCCGCATNGG1000
2000
3000
gRNA3AGAAGAAGGATAAACAACCTNGG1000
2000
3610
gRNA4ATAGCGGCCACAGTAACTTCNGG1000
2000
3000
total 710
Excluded the on-target sites searched of three BrVRN1 genes. Nucleotides in red represent PAM sequence.
Table
Table 4. Statistics of alignment to BrVRN1a-4gRNAs vector.
Table 4. Statistics of alignment to BrVRN1a-4gRNAs vector.
LineNo. of Total Trimmed Reads *1No. of Mapped Reads *2Mapped Region *3 (%)
WT143,832,42424 (0.00%)374 (2.81%)
#2-2-1110,834,7981 (0.00%)0 (0.00%)
#2-2-2144,407,53813 (0.00%)0 (0.00%)
#2-2-3129,575,6546 (0.00%)0 (0.00%)
#2-2-4160,433,7268 (0.00%)0 (0.00%)
#2-2-5120,246,0264 (0.00%)0 (0.00%)
#2-8-1130,463,1464 (0.00%)0 (0.00%)
#2-8-2153,956,5467 (0.00%)0 (0.00%)
#2-8-3151,410,9502 (0.00%)0 (0.00%)
#2-8-4160,952,6266 (0.00%)0 (0.00%)
#2-8-5126,426,79012 (0.00%)0 (0.00%)
*1 No. of Total trimmed reads: Total number of clean reads used for read alignment through pre-processing. *2 No. of Mapped reads: When clean reads are aligned, the number of reads mapped to the vector sequence. *3 Mapped region: Compared to the vector 13,301 bp sequence, the base pair of the region covered by read alignment.
Table
Table 5. Summary of zygosity and genotype of BrVRN1 mutations in the ‘transgene-free’ GE2 mutant plants.
Table 5. Summary of zygosity and genotype of BrVRN1 mutations in the ‘transgene-free’ GE2 mutant plants.
LineTargetType of MutationBrVRN1aBrVRN1bBrVRN1c
ReadsRead Rate (%)ZygosityReadsRead Rate (%)ZygosityReadsRead Rate (%)Zygosity
#2-2-1~5gRNA1Wt10 Homozygous115 Wt1710Wt
+A10391.15-0-
Total113 115 171
gRNA2Wt11 Homozygous4 Homozygous1650Wt
+C/+A11090.909395.87-
Total121-97 165
gRNA3Wt147 Wt- ----
SNP/indel-0---
Total147 - -
gRNA4Wt124 Wt- -- -
SNP/indel-0----
Total124 - -
#2-8-1~5gRNA1Wt5 Homozygous108 Wt1710Wt
+A11595.83-0-
Total120 108 171
gRNA2Wt11 Homozygous8 Homozygous1550Wt
+C/+A13592.4612193.80-
Total146 129 155
gRNA3Wt151 Wt- ----
SNP/indel-0---
Total151 - -
gRNA4Wt149 Wt- ----
SNP/indel-0---
Total149 - -
* Read rate indicates the percentage of the number of mutations in the total mapped read count. Plus symbol denotes insertion. Homologous: read rate ≥ 90% and heterozygous: 40% ≤ read rate < 90%.
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Hong, J.K.; Suh, E.J.; Park, S.R.; Park, J.; Lee, Y.-H. Multiplex CRISPR/Cas9 Mutagenesis of BrVRN1 Delays Flowering Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis). Agriculture 2021, 11, 1286. https://0-doi-org.brum.beds.ac.uk/10.3390/agriculture11121286

AMA Style

Hong JK, Suh EJ, Park SR, Park J, Lee Y-H. Multiplex CRISPR/Cas9 Mutagenesis of BrVRN1 Delays Flowering Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis). Agriculture. 2021; 11(12):1286. https://0-doi-org.brum.beds.ac.uk/10.3390/agriculture11121286

Chicago/Turabian Style

Hong, Joon Ki, Eun Jung Suh, Sang Ryeol Park, Jihee Park, and Yeon-Hee Lee. 2021. "Multiplex CRISPR/Cas9 Mutagenesis of BrVRN1 Delays Flowering Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)" Agriculture 11, no. 12: 1286. https://0-doi-org.brum.beds.ac.uk/10.3390/agriculture11121286

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