CRISPR-Mediated Transcriptional Activation of Squalene Synthase to Increase the Biosynthesis of Anticancer Metabolite Inotodiol in Inonotus Obliquus: A Research Protocol

Abstract

Chaga (Inonotus obliquus) is a parasitic fungus of birch trees used in the traditional medicine of Russia, China, and other Eurasian countries. The secondary metabolites produced by I. obliquus are known to possess anticancer, anti-inflammatory, antiviral, antioxidant, and hypoglycemic activity, while posing no known adverse effects. The therapeutic properties of I. obliquus are thus of particular medical interest. Several studies have demonstrated that the triterpenoids produced by I. obliquus provide the anticancer effects. Specifically, the triterpenoid inotodiol has demonstrated promising antitumor effects in human cervical cancer HeLa cells. Unfortunately, the limited natural abundance of I. obliquus impedes use of this fungus as a source of inotodiol for clinical applications. Furthermore, attempts to culture I. obliquus in a laboratory setting are limited by the low expression of biosynthetic gene clusters. While chemical syntheses of inotodiol avoid these challenges, they are hindered by low yield, cost, and time. To address these challenges, we propose using CRISPR-mediated transcriptional activation (CRISPRa) to boost the expression of the enzyme (squalene synthase (SQS)) that mediates the biosynthesis of inotodiol in I. obliquus, thereby increasing production of the therapeutic metabolite. The level of inotodiol production achieved by endonuclease deficient Cas9s fused to transcriptional activator domains (dCas9-VPR) will be compared using a control group with no single guide RNA (sgRNA) as well as three test groups with sgRNA sequences, varying in their distance upstream from the SQS gene. To evaluate the proposed CRISPRa system, transcriptomic, proteomic, and metabolomic analyses will be implemented. To our knowledge, CRISPRa methodology has not yet been used to improve the yield of I. obliquus metabolites.