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The applications of CRISPR mediated genome editing are continuing to grow. The number of individuals addicted to nicotine is also continuing to grow. The solution? CRISPR-edited nicotine-free tobacco plants, say researchers Felix Stehle and Julia Schachtsiek from the Technical University of Dortmund in Germany.



In a study published in Plant Biotechnology, the researchers adopted CRISPR technology to disable six enzymes that are involved in the production of nicotine in the tobacco plant. Nicotine is a highly addictive substance.



Statistics suggest that there are approximately 50 million people in the United States that are addicted to some form of tobacco, whether it be cigarettes, cigars, snuff or chewing tobacco. Recent efforts have applied RNA silencing methods for upper pathways genes that encode the putrescine N‐methyltransferase (PMT) or A622, a phosphatidylinositol‐4‐phosphate (PIP)‐family member of NADPH reductases. The RNA silencing approach either resulted in increased biosynthesis of other alkaloids or were limited in success to the hairy root cultures and not whole plants.



"We aimed for a simple CRISPR Cas9‐based knockout strategy and searched for an identical target sequence present in all six published coding sequences (BBLa, BBLc, BBLd.2 originated from Nicotiana sylvestris; BBLb, BBLd.1, BBLe from Nicotiana tomentosiformis)," the authors comment in their publication.



We spoke with Schachtsi to learn more about the research study and the implications that surround growing CRISPR-edited plants.



Molly Campbell (MC): Please can you tell us about the background work leading to this study



Julia Schachtsiek (JS): We started the research three years ago. We just wondered why no real no-nicotine tobacco plant was available and not much was known regarding nicotine reduction. MC: Why did you choose CRISPR genome-editing to modify the tobacco plants, over other gene-editing techniques? JS: Using classical mutagenesis methods it is almost impossible to knock out all six genes responsible for nicotine biosynthesis just by chance. The CRISPR technology opens the possibility to edit the genome without any footprints left behind. Moreover, it is possible to target more than one gene, which makes gene editing much faster. In our study, it was possible to target a complete gene family, responsible for the last step in the nicotine biosynthesis, with only one guide RNA sequence



MC: Please can you briefly describe the methods of the study?



JS: In this study, we used the CRISPR technology to target all genes responsible for the last step in nicotine biosynthesis. Resulting plants were analyzed with respect to their nicotine content and desired knockouts of the six targeted genes.



MC: What were your findings, and which finding excited you the most?



JS: We generated the first nicotine-free, non-transgenic tobacco plant. The most exciting fact was that we were able to target all six genes within one approach and we did not find any negative effects on growth or primary metabolism.



MC: What implications are there for growing genetically edited plants?



JS: From the point of a scientist, the plant we generated is NOT transgenic. It just carries single point mutations in the desired genes. In Europe, plants are evaluated by the process and not by the final result. Thus, it is only by law still transgenic. Almost all fruits and vegetables you can buy are “mutated” with regards to the wild type varieties. This is done by mutagenic chemicals or radioactive radiation. This is still allowed, and the result is the same as with the method we utilized. On the contrary, using CRISPR Cas9 you have precise modifications. Using other methods, you cannot control what is knocked out and the resulting plants will carry more than the desired mutations. Therefore, growing plants of the type as described above represent no bigger risk than the cultivation of any (bred) plants.



MC: What approvals are required?



JS: Cultivation of this plant in Europe is currently not possible. It is necessary to sensitize and to inform the public of facts and differences between the different gene editing methods and their products. The modern gene editing methods allow the fast and easy formation of modified plants without carrying any transgenes. Therefore, these plants are not different to classical bred plants.



MC: Logistically, would it be possible to grow the gene-edited plants on a large scale?

JS: This is possible. Unfortunately, Europe restricted the cultivation of CRIPSR edited plants last year, although no transgenes are present in the plant. However, cultivation of the plant is allowed in the rest of the world.



MC: What are the next steps for this research?



JS: It would be interesting to see if the same results can be obtained with tobacco varieties used commercially in the tobacco industry to get nicotine-free cigarettes on the market. Furthermore, the application of this method to Nicotiana benthamiana, a strain used in industry for the production of proteins, might be useful to expand the production spectrum with this plant.



Julia Schachtsiek was speaking with Molly Campbell, Science Writer, Technology Networks.