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

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.

Figure 1. Morphine, thebaine and codeine biosynthesis in opium poppy is catalyzed by several essential enzymes including 4′OMT2.

By delivering CRISPR/Cas9 system components of sgRNA and Cas9 inside plant tissues with agroinoculation, it’s possible to knock-out the desired gene segment to manipulate BIA production flux for engineered biosynthesis. This figure shows the target for CRISPR/Cas9.

Figure 2. After induction of mutagenesis in the Ps4′OMT gene region by CRISPR/Cas9 system, targeted sites were amplified from outside by PCR and InDels were identified by Sanger dideoxy sequencing.

Both results show that genome knockout lines were generated and the majority of gene knockout deleted 1 to 4 nt from the genome sequences.

Figure 3. Alkaloid levels were signifncatly reduced in CRISPR/Cas9 knockout opium poppy leaves measured by HPLC-ToF/MS.

The amounts were ×10⁻³ mg/g. Error bars represent SD. *P < 0.005, using one-way ANOVA Tukey’s test. SB: Synthetic Backbone; VB: Viral Backbone.

Figure 4. The chromatogram of the measured alkaloids in genome knockout leaves as well the control leaves.

(A) known alkaloids; M: morphine isomers; C: codeine; S: S-reticuline; T: thebaine L: laudanosine; N: noscapine; P: papaverine, (B) unidentified peak only represented in CRISPRed plants.