Manipulation of Cannabinoid Biosynthesis via Transient RNAi Expression

Area of Science:

• Plant Molecular Biology
• Biotechnology
• Pharmacology

Background:

• Cannabis sativa produces valuable phytocannabinoids with pharmaceutical applications.
• Understanding the function of cannabinoid biosynthesis genes is crucial for harnessing these compounds.
• Previous research lacked in vivo methods to directly investigate these genes' roles.

Purpose of the Study:

• To report the first in vivo modulation of cannabinoid biosynthesis genes in Cannabis sativa using RNA interference (RNAi).
• To elucidate the specific roles of THCAS, CBDAS, and CBCAS genes in cannabinoid synthesis.
• To establish a functional genomics approach for future genetic studies and strain development.

Main Methods:

• Agroinfiltration of RNAi constructs targeting THCAS, CBDAS, and CBCAS genes into Cannabis sativa leaf segments.
• Utilizing vacuum infiltration for efficient delivery of RNAi constructs.
• Quantifying gene expression levels using real-time quantitative PCR to assess downregulation and off-targeting effects.

Main Results:

• Significant downregulation of THCAS (92%), CBDAS (97%), and CBCAS (70%) was achieved using a CBDAS-targeting RNAi construct.
• Evidence of gene cross-regulation was observed, with CBCAS upregulation (76%) when a CBCAS-targeting construct was used.
• RNAi demonstrated significant off-targeting of highly homologous transcripts, indicating complex gene interactions.

Conclusions:

• RNAi via agroinfiltration is an effective method for in vivo modulation of cannabinoid biosynthesis genes in Cannabis sativa.
• This technique provides valuable insights into gene regulation and the functional relationships between cannabinoid synthesis genes.
• The developed RNAi approach facilitates functional genomics screens and the engineering of designer cannabis strains with tailored cannabinoid profiles.