Rubber particles (RPs) are specialized organelles for the biosynthesis and storage of natural rubber in rubber-producing plants. However, the mechanisms underlying the biogenesis and development of RPs remain unclear. In this study, two latex-specific cis-prenyltransferases (CPTs), TkCPT1 and TkCPT2, were identified in Taraxacum kok-saghyz, with almost identical orthologues retained across other Taraxacum species. For the first time, Tkcpt1 single and Tkcpt1/2 double mutants were successfully generated using the CRISPR/Cas9 system. Rubber biosynthesis was significantly depressed in Tkcpt1 mutants and completely blocked in Tkcpt1/2 mutants. The absence of RPs in the Tkcpt1/2 was confirmed using oil red O and Nile red staining, high-speed centrifugal stratification, cryo-SEM and TEM on fresh latex or laticifer cells. Transcriptomic and proteomic analyses revealed that, in the latex of Tkcpt1/2, rubber biosynthesis was blocked at the protein level, while metabolomic profiling indicated an enrichment of lipids and terpenoids. Furthermore, knockout of TkCPTL1, a latex-specific CPT-like gene that encodes a rubber transferase activator, resulted in outright disruption of rubber biosynthesis and RP ontogeny, a phenotype similar to that of Tkcpt1/2 mutants. These findings indicate that rubber biosynthesis is a driving force for the biogenesis and development of RPs, providing new insights into rubber production mechanisms.