Impact of CRISPR/Cas9-induced mutations in nicotine biosynthesis core genes A622 and BBL on tobacco: Reduction in nicotine content and developmental abnormalities

Tobacco (Nicotiana tabacum) is known for its psychoactive alkaloid nicotine, which presents significant public health challenges. Recent research has linked the final stages of nicotine biosynthesis with the BBL and A622 genes, yet this part of the biosynthetic pathway remains largely unexplored, representing a 'black box' in our understanding. In our study, we employed a multi-target CRISPR/Cas9 system to target homologous genes of BBL and A622 in commercial tobacco varieties Virginia, creating various mutants. This led to significant variations in plant development and alkaloid content. Notably, mutant lines a622a-38–5 and a622l-3–9 with exon-intron boundary deletions exhibited significantly decreased plant height and leaf number, along with a substantial reduction in alkaloids, including nicotine. Particularly, double mutants in the A622 family displayed more severe effects than sextuple BBL mutants, emphasizing the distinctive role of A622 in nicotine synthesis and plant development. Our findings demonstrate that mutations in A622 and BBL genes can drastically reduce nicotine and anatabine content, with some cases showing reductions up to 99.6%. These results underscore the potential of genome editing in developing tobacco varieties with significantly lower nicotine levels. This study not only enhances our understanding of nicotine biosynthesis but also contributes to public health efforts by providing a pathway to develop less addictive tobacco products.