Simultaneous Knockout of <i>Tk1‐SST</i> and <i>Tk1‐FFT</i> via <scp>CRISPR</scp> /Cas9 Enhances the Natural Rubber Accumulation in <scp> <i>Taraxacum kok‐saghyz</i> </scp>

Taraxacum kok-saghyz (TKS) synthesises natural rubber (NR) and inulin using sucrose as a carbon source. However, molecular mechanisms regulating inulin and NR accumulation remain largely unclear. Here, we report the generation of double-gene homozygous mutants, 1-sst1-fft, by simultaneously knocking out two key genes responsible for inulin biosynthesis (Tk1-SST and Tk1-FFT) using CRISPR/Cas9 technology. The 1-sst1-fft mutants exhibited significant increases in rosette leaf number, flower number, leaf area, whole-plant biomass and seed set. Moreover, inulin biosynthesis was abolished in 1-sst1-fft, leading to significant changes in sugar composition, particularly a marked increase in sucrose levels. Notably, NR accumulation more than doubled, with no significant change in molecular weight and most terpenoid accumulation also increased in 1-sst1-fft, both being positively correlated with sucrose levels. For the first time, this study reports the generation of an inulin synthesis-deficient mutant in plants, emphasising the essential roles of 1-SST and 1-FFT in regulating carbon partitioning and, consequently, modulating important traits and metabolite accumulation. Transcriptomic analysis revealed fundamental genes involved in sucrose metabolism, sugar signalling and transport and NR elongation were significantly upregulated in 1-sst1-fft; accompanied by enhanced enzymatic activities of sucrose-phosphate synthase and invertase. These findings demonstrate that blocking inulin biosynthesis in 1-sst1-fft redirects sucrose towards NR biosynthesis, highlighting the dual role of sucrose as both a carbon source and signalling molecule in modulating plant growth and development and metabolite synthesis. Our study provides a successful approach to enhancing NR accumulation by modifying carbon allocation in TKS, offering novel insights into high-yield breeding strategies.