Metabolic engeneering in chicory by CRISPR/Cas9 editing

Abstract

Chicory (Cichorium intybus L.) is a valuable crop grown mostly in northern France, Belgium and the Netherlands, whose roots, leaves and etiolated shoots are used as vegetable, coffee replacement and remedy for several conditions including inflammation. Roots of chicory are an important source of inulin, a prebiotic fiber and a sweetener. Chicory also contains bioactive terpene compounds that protect the plant against herbivores, but are also responsible for medicinal properties of the plant, as they have anti-microbial, anti-cancer and anti-inflammatory activity. Biosynthesis of plant bioactive metabolites can be modified using new plant breeding techniques, especially new gene editing techniques like CRISPR/Cas9 to improve their health-promoting qualities. Chicory was transformed with Agrobacterium tumefaciens carrying CRISPR/Cas9 constructs to knock-out expression of key genes in the sesquiterpene biosynthesis pathway. Regenerated plants were selected on media with kanamycin and further tested by PCR for the presence of transgenes. Obtained plants were also tested for mutations in target genes, by PCR with primers surrounding the mutation site detecting larger indels and by Sanger sequencing detecting smaller indels. Further analysis was performed detecting heteroduplex DNA fragments as a result of either hetero- or homozygous mutation events.