CRISPRi-Mediated Down-Regulation of the Cinnamate-4-Hydroxylase (C4H) Gene Enhances the Flavonoid Biosynthesis in Nicotiana tabacum

SIMPLE SUMMARY: Flavonoids are natural compounds in plants. They play a critical role in plant growth and pathogen defense. Due to their health benefits, flavonoids have gained much attention as potent therapeutic agents. However, the low abundance of flavonoids in nature has limited their exploitation. Hence, this study aimed to enhance flavonoid production by silencing the cinnamate-4-hydroxylase (C4H) enzyme using the clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology. Our results showed that the C4H-silenced tobacco cells had a lower NtC4H expression level compared to wild-type. This was concurred by the flavonoid analysis, where the accumulation of C4H’s substrate in the C4H-silenced cells was significantly higher than in the wild-type. Our findings provide valuable insight into the future development of CRISPRi to manipulate plant metabolite biosynthesis. ABSTRACT: Flavonoids are an important class of natural compounds present in plants. However, despite various known biological activities and therapeutic potential, the low abundance of flavonoids in nature limits their development for industrial applications. In this study, we aimed to enhance flavonoid production by silencing cinnamate-4-hydroxylase (C4H), an enzyme involved in the branch point of the flavonoid biosynthetic pathway, using the clustered regularly interspaced short palindromic repeats interference (CRISPRi) approach. We designed three sgRNAs targeting the promoter region of NtC4H and cloned them into a CRISPRi construct. After being introduced into Nicotiana tabacum cell suspension culture, the transformed cells were sampled for qPCR and liquid chromatography-mass spectrometry analyses. Sixteen of 21 cell lines showed PCR-positive, confirming the presence of the CRISPRi transgene. The NtC4H transcript in the transgenic cells was 0.44-fold lower than in the wild-type. In contrast, the flavonoid-related genes in the other branching pathways, such as Nt4CL and NtCHS, in the C4H-silenced cells showed higher expression than wild-type. The upregulation of these genes increased their respective products, including pinostrobin, naringenin, and chlorogenic acid. This study provides valuable insight into the future development of CRISPRi-based metabolic engineering to suppress target genes in plants.