Tobacco has been cultivated for hundreds of years and a source of income for many around the world. It’s not only found in the modern day commercially sold cigarette that includes several other ingredients besides tobacco, but is also consumed in the form of cigars and hookahs. And, some even roll their own tobacco which eliminates unwanted ingredients. Moreover, tobacco is used as an insecticide that can be boiled and seeped in water then applied to gardens to reduce harmful insects.
Nicotine’s Role In Cancer
As the active and addictive component of cigarettes, nicotine is the most well known of the four major alkaloids in the tobacco plant. Alkaloids are basic compounds with pharmacological properties found not only in nicotine and coffee giving that bitter taste when consumed, but also in pain-reducing agents such as morphine as well as quinine; the antimalarial drug.
While many people may believe it’s the nicotine that causes cancer when smoked, it’s not the carcinogen itself. However, nicotine starts the reaction that does in fact lead to cancer. When dried, a chemical reaction known as nitrosation occurs in tobacco producing tobacco-specific-nitrosamines (TSNAs) which are labeled as carcinogens.
Manipulating TSNA N-Nitrosonornicotine (NNN) With Gene Editing
Scientists have found genes linking to the formation of one particular TSNA abundant in tobacco plants; N-nitrosonornicotine (NNN). Figure 1 shows the conversion of nicotine to NNN. Identification of the genes involved in any chemical process allows for the possibility to modify the amount. Therefore, it makes sense that researchers used a gene editing technique called RNA interference (RNAi) to silence the CYP82E4 gene involved in the conversion of nicotine to the cancer causing nornicotine. 1
In addition to being a carcinogen, nornicotine has also been linked to potentially altering both the efficacy and toxicity of commonly used steroid drugs giving yet another reason to reduce the amount in tobacco plants. 1
The research implicates the CYP82E2 gene family, and specifically the CYP82E4 gene as playing a major role in the metabolic conversion of nicotine to nornicotine in tobacco. 1 CYP82E4 is a gene within the Cytochrome P450 family of enzymes that in humans, is involved in several metabolic reactions and in plants, is involved in many biosynthetic reactions.
Newer Gene Editing Tools Like CRISPR And Cas-CLOVER More Effective Than RNAi
Novel gene editing proteins such as, Cas9, transcription activator-like effector nucleases (TALEN), and zinc-finger nucleases (ZFN) target specific genes in a deliberate manner. This is compared to previous tools that randomly insert, delete, or cause a frameshift in the sequence being edited; dramatically altering gene function.
Previous research in fruit ripening enzymes used gene technology such as RNAi which reduces gene expression at the mRNA level while newer technologies such as CRISPR and Cas-CLOVER completely and permanently silences the gene at the DNA level.
Besides the temporary reduction vs. a permanent solution, RNAi silencing method suffers limitations due to high off-target effects and GMO transgenesis.
Demeetra’s Cas-CLOVER Gene Editing System Validated In Tobacco
Cas-CLOVER was first introduced and validated to have little to no off-targets due to its dimeric nature in cell lines such as human T-cells.
As shown in Figure 2, the Cas-CLOVER gene editing system utilizes a catalytically inactive Cas9 protein fused to the Clo51 nucleus domain which works as monomers recruited by a pair of guide RNAs (gRNA) to introduce targeted mutations. And when both subunits are properly recruited to the target-site, it leads to dimerization and activation of the Clo51 nucleus domain, leading to targeted gene disruptions.
Figure 1: Cas-CLOVER gene editing system
Shown in a previous publication2, we were able to validated the activity of Cas-CLOVER in plants by targeted inactivation of the RNA-dependent RNA polymerase 6 (RDR6) gene in tobacco achieving an efficiency of at least 18% in the first target which is significant for tobacco.
Advanced gene editing technologies enable plant biotechnologists with the ability to make targeted knockouts and insertions. This scarless gene editing means no heterologous genes are left in the plant genome, producing a non-GMO crop and simplifying the process. We recognize the impact that gene editing tools have on the future of science, agriculture, and pharmaceuticals. In particular, Demeetra offers licenses for their novel gene editing Cas-CLOVER system.
Enhancing tobacco production is just one example that can benefit from gene editing technology. Our rapidly growing population with threats of climate change and global pandemics continue to be factors that pressure our food and medicine security, increasing the need for more productive agriculture traits and biomanufacturing systems is essential.
If you are interested in finding ways to minimize TSNAs in tobacco plants or and/or other gene modifications, contact Demeetra for support in your tobacco gene editing research.
References
- Siminszky, B., Gavilano, L., Bowen, S. W., & Dewey, R. E. (2005). Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase. Proceedings of the National Academy of Sciences of the United States of America, 102(41), 14919–14924. https://doi.org/10.1073/pnas.0506581102
- Norman D., Rector, K., Tateo, M., Crawford, J. (2020). Targeted editing of Tobacco with Cas-CLOVER™: the clean alternative to CRISPR/Cas9 for plant genome editing. American Society of Plant Biology Poster Presentation. 10.46678/PB.20.1053452