AUTHOR=Matchett-Oates Lennon , Spangenberg German C. , Cogan Noel O. I. 

TITLE=Manipulation of Cannabinoid Biosynthesis via Transient RNAi Expression

JOURNAL=Frontiers in Plant Science

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.773474

DOI=10.3389/fpls.2021.773474

ISSN=1664-462X

ABSTRACT=<p><italic>Cannabis sativa</italic> L. produces unique phytocannabinoids, which are used for their pharmaceutical benefits. To date, there are no reports of <italic>in vivo</italic> engineering targeting the cannabinoid biosynthesis genes to greater elucidate the role each of these genes play in synthesis of these medically important compounds. Reported here is the first modulation of cannabinoid biosynthesis genes using RNAi <italic>via</italic> agroinfiltration. Vacuum infiltrated leaf segments of the Cannbio-2 <italic>C. sativa</italic> strain, transfected with different RNAi constructs corresponding to <italic>THCAS, CBDAS</italic>, and <italic>CBCAS</italic> gene sequences, showed significant downregulation of all cannabinoid biosynthesis genes using real-time quantitative PCR. Using RNAi, significant off-targeting occurs resulting in the downregulation of highly homologous transcripts. Significant (<italic>p</italic> &lt; 0.05) downregulation was observed for <italic>THCAS</italic> (92%), <italic>CBDAS</italic> (97%), and <italic>CBCAS</italic> (70%) using pRNAi-GG-<italic>CBDAS-UNIVERSAL</italic>. Significant (<italic>p</italic> &lt; 0.05) upregulation of <italic>CBCAS</italic> (76%) and non-significant upregulation of <italic>THCAS</italic> (13%) were observed when transfected with pRNAi-GG-<italic>CBCAS</italic>, suggesting the related gene’s ability to synthesize multiple cannabinoids. Using this approach, increased understanding of the relationship between cannabinoid biosynthesis genes can be further elucidated. This RNAi approach enables functional genomics screens for further reverse genetic studies as well as the development of designer cannabis strains with over-expression and/or downregulation of targeted cannabinoid biosynthesis genes. Functional genomics screens, such as these, will further provide insights into gene regulation of cannabinoid biosynthesis in <italic>Cannabis</italic>.</p>