Establishment of a CRISPR/Cas9 gene-editing system for Chrysanthemum morifolium
2024
Qi Chen | Xin Zhang | Ruibing Jin | Hongyu Mao
Chrysanthemum morifolium ranks among the four highest-selling cut flowers worldwide. Gene editing is an important tool to research gene function, but efficient and precise genome-editing tools are not available for chrysanthemum. Here, we established a CRISPR/Cas9-mediated gene-editing system to explore gene functions and enhance the breeding of chrysanthemum. We used the Golden Gate Assembly system to construct CRISPR/Cas9 vectors for dual targeting of the Phytoene Dehydro (PDS) gene. To test the accuracy of sgRNA design, we initially used the transient CRISPR/Cas9 editing in plants (TCEP) method. Target gene expression in nine plants subjected to transient transfection was 19.1%–52% of the normal level, confirming the feasibility of target gene knockout. We carried out stable transformation; PCR and sequencing of the target sites showed that four of eight albino plants obtained had been stably edited at the target sites. We further assessed the editing efficiency of the system by targeting another gene, CmTGA1, chosen because of its potential importance in Chrysanthemum White Rust (CWR) disease progression. Our data indicates that combining transient and stable transformation improves the efficiency and success rate of genome fixed-point editing. The effective, heritable CRISPR/Cas9-mediated genome-editing system we have established here lays the foundation for functional gene studies and genetic improvement of C. morifolium.
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