Manipulating the Biosynthesis of Bioactive Compound Alkaloids for Next-Generation Metabolic Engineering in Opium Poppy Using CRISPR-Cas 9 Genome Editing Technology.

Yagiz Alagoz,Tugba Gurkok,Baohong Zhang,Turgay Unver

doi:10.1038/srep30910

Yagiz Alagoz, Tugba Gurkok + Show 2 more

Open Access

https://doi.org/10.1038/srep30910

Copy DOI

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) endonuclease system is a powerful RNA-guided genome editing tool. CRISPR/Cas9 has been well studied in model plant species for targeted genome editing. However, few studies have been reported on plant species without whole genome sequence information. Currently, no study has been performed to manipulate metabolic pathways using CRISPR/Cas9. In this study, the type II CRISPR/SpCas9 system was used to knock out, via nonhomologous end-joining genome repair, the 4′OMT2 in opium poppy (Papaver somniferum L.), a gene which regulates the biosythesis of benzylisoquinoline alkaloids (BIAs). For sgRNA transcription, viral-based TRV and synthetic binary plasmids were designed and delivered into plant cells with a Cas9 encoding-synthetic vector by Agrobacterium-mediated transformation. InDels formed by CRISPR/Cas9 were detected by sequence analysis. Our results showed that the biosynthesis of BIAs (e.g. morphine, thebaine) was significantly reduced in the transgenic plants suggesting that 4′OMT2 was efficiently knocked-out by our CRISPR-Cas9 genome editing approach. In addition, a novel uncharacterized alkaloid was observed only in CRISPR/Cas9 edited plants. Thus, the applicabilitiy of the CRISPR/Cas9 system was demonstrated for the first time for medicinal aromatic plants by sgRNAs transcribed from both synthetic and viral vectors to regulate BIA metabolism and biosynthesis.

Highlights

P. somniferum (2n = 22), from the family of Papaveraceae, is one of the two opium poppy species known to biosynthesize morphine[30,31]
It has been reported that benzylisoquinoline alkaloids (BIAs) production in opium poppy can be manipulated by altering the expression of some particular genes in the BIA pathway
We targeted the poppy 4′OMT2 gene by introducing double strand brakes (DSBs) and observed insertion and deletions (InDels) mutations directed by Non-Homologous End Joining (NHEJ) repair mechanism

Summary

Introduction

This step is catalysed by 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT)[35] (Fig. 1) Several end products such as morphine, noscapine and papaverine are derived from S-reticuline via different BIA pathways. Over-expression and TRV-mediated gene silencing studies in opium poppy revealed that the amount of alkaloid biosynthesis could be manipulated in a tissue-specific manner[38,39,40,41] Both the over-expression and the silencing of (R, S)-reticuline 7-O-methyltransferase (7OMT) and 3′-hydroxyl-N-methylcoclaurine 4′-O-methyltransferase (4′OMT2) genes revealed their regulatory roles in BIA production in different tissues[42]. We used the CRISPR/Cas[9] system to knock out 4′OMT (isoform-2) which is a critical regulatory gene involved in the biosynthesis of some bioactive compounds produced in the medicinal aromatic plant, opium poppy (Papaver somniferum L.) using both synthetic and viral-based delivery systems

Methods

Results

Conclusion