Developing a UV-visible reporter-assisted CRISPR/Cas9 gene editing system to alter flowering time in Chrysanthemum indicum.

Chrysanthemum (Chrysanthemum morifolium Ramat.) is an economically important ornamental crop worldwide. This typical obligate short-day (SD) herbaceous perennial in the Asteraceae (Compositae) family is a useful model for studying the photoperiodic control of flowering. However, the complex, heterozygous chrysanthemum genome and the self-incompatibility of this species have hindered basic and applied research on its horticultural and physiological properties. Isolating and characterizing mutants in specific genes is critical to dissecting gene function for both basic and applied research. In the first report of genome editing in chrysanthemum, six CmDMC1 (DISRUPTION OF MEIOTIC CONTROL 1) genes were simultaneously targeted by TALENs (Shinoyama et al., 2020). However, the design of sequence-specific TALENs is cumbersome and the needed recombinant plasmids are large. In this regard, CRISPR/Cas9-mediated genome editing holds advantages in vector design and assembly, especially when targeting multiple genes, and has been widely used in many organisms. Nevertheless, the high frequency of chimeric events and low editing efficiency has hindered the application of the CRISPR/Cas9 system in chrysanthemum (Kishi-Kaboshi et al., 2017). Only a single study has reported a successful knockout for a TCP transcription factor gene in chrysanthemum (C. morifolium) using a conventional CRISPR/Cas9-mediated system (Li et al., 2022). Chrysanthemum indicum L. is often used as a model for cultivated chrysanthemum since it is a progenitor of cultivated chrysanthemum, and as a health food and anti-inflammatory herb in traditional Chinese medicine for over 2000 years. In this study, we chose a diploid C. indicum as material (2n = 2x = 18) (Figure S1) and first targeted its single-copy Phytoene desaturase (CiPDS) gene. We cloned four single-guide RNAs (sgRNAs) based on the CiPDS sequence (Figure 1a,b) into pDIRECT-22C, which can simultaneously express multiple sgRNAs using the Csy-type ribonuclease 4 (Csy4) enzyme (Čermák et al., 2017). We measured the editing efficiency of each sgRNA in C. indicum protoplasts by high-throughput sequencing (Data S1). The editing efficiencies of sgRNA1-4 were 8.22 ± 1.2%, 7.82 ± 0.3%, 4.02 ± 0.4%, and 9.14 ± 1.0%, respectively (Figure 1b). We chose sgRNA1 and sgRNA2 to knockout CiPDS because they had the highest editing efficiencies and target the first CiPDS exon (Figure 1a). Since the selection of optimal promoters is important for high-efficiency genome editing, we tested five different promoters to drive Cas9 and the sgRNAs (Figure 1c): the cauliflower mosaic virus (CaMV) 35S, Cestrum yellow leaf curling virus (CmYLCV), parsley (Petroselinum crispum) Ubiquitin (PcUbi), Arabidopsis thaliana YAOZHE (YAO), and C. indicum Ubiquitin (CiUbi) promoters. We also codon-optimized Cas9 based on the codon usage of A. thaliana (AtCas9) and C. indicum (CiCas9). We tested the editing efficiencies of the resulting nine constructs in at least two independent experiments each (Table S1). In total, we transformed 7786 C. indicum leaf discs with the nine constructs via Agrobacterium tumefaciens-mediated transformation (Figure 1c). The construct harbouring 35S:AtCas9 and PcUbi:sgRNA showed the highest editing efficiency among all experiments and was designated pDIRECT-Ci-opti (Construct 5 in Figure 1c and Table S1). We obtained eight albino plants by transformation using pDIRECT-Ci-opti, with a biallelic editing efficiency of 0.74% (Figure 1c). One albino plant showed a bushy phenotype (Figure 1d1), whereas most plants developed normal roots and leaves (Figure 1e). Sanger sequencing revealed that all mutations in the albino plants occurred solely at the sgRNA1 target site and consisted of deletions and insertions (Figure 1e and Figure S2). Among the 14 albino plants generated (Figure 1c), four showed variegated phenotypes (28.6%), suggesting a high level of chimerism (Figure 1d2,d3). When CiPDS is used as a visual marker to validate genome editing, chimerism is easily scorable, but scoring is time-consuming and laborious for most target genes without a visual mutant phenotype. To further improve the screening efficiency and solve the chimerism issue, we generated transgenic C. indicum expressing eYGFPuv (35S:eYGFPuv), encoding a protein with bright fluorescence under ultraviolet (UV) light that is visible to the naked eye (Chin et al., 2018) and has been used as a visible reporter for gene expression and stable transformation in plants (Yuan et al., 2021). All organs of 35S:eYGFPuv C. indicum plants exhibited fluorescence (Figure 1f). We then targeted eYGFPuv with specific sgRNAs for exploitation as a visible marker to efficiently screen gene-edited plants while eliminating chimeric plants. We cloned both CiPDS sgRNA1 and sgRNA2 and eYGFPuv sgRNA1 and sgRNA2 into pDIRECT-Ci-opti (pDIRECT-Ci-opti-CiPDS-eYGFPuv, Figure 1g) and transformed this construct into 35S:eYGFPuv leaf discs. While leaf discs from 35S:eYGFPuv C. indicum exhibited bright fluorescence, those from the wild type (WT) did not exhibit fluorescence under UV light (Figure 1h1). Following transformation with pDIRECT-Ci-opti-CiPDS-eYGFPuv, some 35S:eYGFPuv-derived calli lacked eYGFPuv fluorescence, suggesting that they harboured eYGFPuv mutation(s) (Figure 1h2). In addition, some regenerated non-fluorescent plantlets were not albino (Figure 1h3), suggesting that these plantlets were eygfpuv biallelic mutants but not Cipds biallelic mutants. Other regenerated plantlets without fluorescence were albino (Figure 1h4), indicating that they were eygfpuv Cipds biallelic mutants. We also identified variegated plantlets based on eYGFPuv fluorescence (Figure 1h5). Using traditional antibiotic selection, we obtained 50 hygromycin-resistant transgenic plantlets. Among them, we identified eight Cipds biallelic mutants, representing a biallelic mutant screening efficiency of 16.0% (Figure 1i). We also selected 11 plants without eYGFPuv fluorescence that were eygfpuv biallelic mutants. Among them, seven were albino, with a biallelic mutant screening efficiency for eygfpuv of 63.6% (Figure 1i). Therefore, using mutated eYGFPuv as a visible marker improved screening efficiency and largely eliminated chimeric plants. The production of flowering in chrysanthemum usually requires the artificial regulation of day length. One major goal of chrysanthemum breeding is to create photoperiod-insensitive or early-flowering chrysanthemum cultivars. TERMINAL FLOWER 1 (TFL1) inhibits flowering in chrysanthemum. Overexpressing TFL1 delays flowering time in chrysanthemum (Gao et al., 2019; Higuchi and Hisamatsu, 2015). Here, we identified two TFL1 homologues in C. indicum: CiTFL1a and CiTFL1b. We generated three Citfl1a biallelic mutants and one Citfl1b homozygous mutant using our CRISPR/Cas9 platform. The three Citfl1a biallelic mutants contained small deletions and insertions in the first CiTFL1a exon. The Citfl1b mutant contained the same 1-bp insertion in the third CiTFL1b exon in both gene copies (Figure 1j and Figure S3). The edited plants showed different degrees of early flowering, with the Citfl1a mutants exhibiting the earliest flowering (Figure 1k). Under SD conditions, flower buds emerged at 41.3 ± 1.2 days in WT plants but at 16.7 ± 0.6 and 32.7 ± 1.2 days in Citfl1a and Citfl1b plants, respectively (Figure 1l). The visible reporter-assisted CRISPR/Cas9 system developed in this study should facilitate research and breeding of chrysanthemum. pDIRECT-22C was a gift from Dr. Daniel Voytas (Addgene plasmid # 91135). This work was supported by the National Key Research and Development Program of China (2022YFF1003104, 2019YFD1001500) and the National Natural Science Foundation of China (grants 31822045, 32072611). The authors declare no competing interests. C.M. and L.L. conceived and designed the experiments; Y.X., J.L., M.H., X.L., and T.J. performed the experiments; C.M., L.L., and Y.Z. analysed the data; C.M. and L.L. wrote the article. Data S1 High-throughput sequencing of four CiPDS sgRNA editing targets in C. indicum protoplasts. Table S1 Editing efficiency of engineered vectors constructed using different promoters and codon-optimized Cas9s. Figure S1 Ploidy of C. indicum used in this study, as determined by flow cytometry. Figure S2 Sequencing analysis of Cipds mutants by Sanger sequencing. Figure S3 Sequencing analysis of Citfl1a and Citfl1b mutants by Sanger sequencing. 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