Nicotine‐free, nontransgenic tobacco (Nicotiana tabacum l.) edited by CRISPR‐Cas9

Abstract

Worldwide approximately 1.1 billion people are smokers and more than 7 million people die fromthe negative effects of smokingevery year(WHO report, 2017). One of the main natural ingredients causingdependence on tobacco is nicotine. Tobacco with a lowered nicotine content could help people to overcome their nicotine addiction. Nicotine-free (or nicotine reduced) cigarettes may contribute to reduce the number of smokersand nicotine consumption, thus reducing the risk of death from tobacco use. Most genes involved in the nicotine biosynthesis in tobacco are known and well characterized (Dewey and Xie, 2013). This opens the possibility to employ genetic engineering approaches to alter the alkaloid content of the plant, and in particularto reduce the nicotine content. Nicotine itself is composed of a pyrrolidine and a pyridine ring, whichare synthesized in independent pathways (Figure 1 A). Recent approaches dealt with the silencing of upper pathway genes encoding the putrescine N-methyltransferase (PMT) or A622, a phosphatidylinositol-4-phosphate (PIP)-family member of NADPH-reductases. The applied RNA silencing methods resulted either in the increased biosynthesisof other alkaloids like anatabine(i.e. Wang et\xa0al., 2009) or were only successful in hairy root cultures and BY-2 cells, but not in whole plants(Kajikawa et\xa0al., 2009). The final oxidation step in the biosynthesis ofnicotine, as well as anatabine and anabasine, is proposed to be catalyzed by flavoproteins of the berberine bridge enzyme-like (BBL) family(Kajikawa et\xa0al., 2011). The knockdown of the three most highly expressed BBL-genes(BBLa-BBLc) by RNAi or the knockout with EMS-induced mutations resulted in a reduction of the nicotine content without increasing the contentof other alkaloids(Kajikawa et\xa0al., 2011; Lewis et\xa0al., 2015). Recently, the BBL-gene family in tobacco was expanded by the identification of BBLd.2 and BBLe,leading to six known isoforms(Kajikawa et\xa0al., 2017). Thus, the simultaneousknockout of theseBBL-genes is a promising approach to generate a nicotine-free tobacco plant. This article is protected by copyright. All rights reserved.