Natural rubber (NR) is an industrially essential biopolymer due to its irreplicable properties. To date the only necessary components identified for NR biosynthesis are two membrane-bound proteins, cis-prenyltransferase (CPT) and CPT binding protein (CBP) that form a complex in which CPT catalyzes the addition of isopentyl pyrophosphate (IPP) subunits to an allylic primer molecule. Reconstitution of CPT/CBP protein complexes in vitro is insufficient for NR production. Therefore, plant systems are essential for elucidating the factors that influence NR production.Lettuce is an ideal model to study NR as it produces high quality, long NR polymers (Mw ? 1 million Da) and is amenable to molecular genetic studies. In this work a CRISPR-Cas9 gene editing system was used to produce lscpt3 mutants. These mutants were NR deficient, suggesting that LsCPT3 is the sole lettuce CPT paralog responsible for NR biosynthesis. Using this mutant background, NR produced by heterologous CPTs in lettuce laticifers was evaluated. Interestingly, CPTs from guayule and goldenrod produced NR with an average Mw that was 1.8-fold and 14-fold greater than in the native plant. This finding demonstrates that CPT is insufficient in determining NR Mw and suggests that the unique cellular context of lettuce laticifers is an important variable in determining NR Mw.Two paralogs of CBP, LsCBP1 and LsCBP2, have been identified in lettuce. CRISPR-Cas9 was implemented to generate NR deficient lscbp2 mutants with no obvious developmental or physiological phenotypes. This data suggests gene duplication and neofunctionalization of LsCBP2 has resulted in a CBP specific for NR biosynthesis in lettuce.Lettuce also accumulates sesquiterpene lactones (STLs) in its laticifers that pull from the same IPP pool as NR. To understand the coordination of these two metabolites, lettuce lines expressing GUS-promoter constructs for STL biosynthetic genes were produced. These lines revealed that STL genes are localized to the parenchyma cells neighbouring laticifers, despite downstream STLs accumulating in the laticifer. These results contribute to a greater understanding of NR biosynthesis in the model plant lettuce. The unique cellular context of laticifers and evolution of a NR specific CBP provide additional insight for the development of biotech enabled NR production.