title: 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.
authors.affiliation: Department of Biology, Faculty of Science, Çankırı Karatekin University, Çankırı18100, Turkey.|Hawkesbury Institute for the Environment, Western Sydney University, Richmond, New South Wales 2753, Australia.
authors.forename: Yagiz
authors.initials: Y
authors.lastname: Alagoz
authors.affiliation: Department of Biology, Faculty of Science, Çankırı Karatekin University, Çankırı18100, Turkey.
authors.forename: Tugba
authors.initials: T
authors.lastname: Gurkok
authors.affiliation: Department of Biology, East Carolina University, Greenville, NC 27858, USA.
authors.forename: Baohong
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Department of Biology, Faculty of Science, Çankırı Karatekin University, Çankırı18100, Turkey.|Izmir International Biomedicine and Genome Institute (iBG-izmir), Dokuz Eylul University, Balcova 35340 Izmir, Turkey.
authors.forename: Turgay
authors.initials: T
authors.lastname: Unver
mesh_terms: D044182:Benzylisoquinolines / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D000072669:Gene Editing / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors; D018745:Genome, Plant; D060847:Metabolic Engineering; D010207:Papaver / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010940:Plant Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
chemical_list: D044182:Benzylisoquinolines; D010940:Plant Proteins