Unraveling the sesquiterpene lacton pathway using CRISPR/Cas9 genome editing in chicory and witloof

Cichorium intybus var. sativum (chicory) and var. foliosum (witloof) are economically important crops with a high nutritional value due to many specialized metabolites, including sesquiterpene lactones (SLs). However, sesquiterpene lactones are responsible for a bitter taste, limiting the use of Cichorium for industrial purposes. Editing specific genes from the SL pathway in Cichorium would lead to changes in the SL metabolite pathway and thus result in altered bitterness.
The genes germacrene A synthase (GAS), germacrene A oxidase (GAO), costunolide synthase (COS) and kauniolide synthase (KLS) are already known to control production of SLs. To identify new genes in the SL biosynthetic pathway in C. intybus, a comprehensive genome-wide screen was executed. By this, paralogs of the known members (GAS, GAO, COS and KLS) were identified and gene families were annotated. Previous genome-wide transcriptome profiling studies demonstrated that a Methyl Jasmonate (MeJA) treatment may trigger the upregulation of genes encoding enzymes involved secondary metabolic pathways, including the SL biosynthetic pathway. Therefore, we performed a comprehensive transcriptome analysis of MeJA treated samples in three species (C. intybus var. sativum, C. intybus var. foliosum and Lactuca sativa), to identify MeJA-inducible members of gene families putatively involved in the SL biosynthetic pathway. Combining the genome-wide gene family annotation with their MeJA-inducibility allowed to identify ten CiGAS, six CiGAO, four CiCOS and ten CiKLS candidate genes putatively related to the SL biosynthetic pathway. These candidate genes were used in co-expression assays in the heterologous host tobacco (Nicotiana benthamiana), to confirm their involvement in the SL biosynthetic pathway. Catalytic activity of two CiGAO paralogs and four CiCOS paralogs is already shown. The catalytic activity of other CiGAO and CiKLS candidate genes are analyzed at the moment.
Inducing CRISPR/Cas9 knock-outs of these selected SL candidate genes in Cichorium and analyzing the SL metabolite production of obtained mutated plants could validate their gene function in planta and alter bitterness production. Therefore a CRISPR/Cas9 protoplast transfection method was developed to create mutated Cichorium genotypes (De Bruyn et al., 2020). Targeting one paralog of CiGAS, CiGAO and CiCOS revealed a range of mutated genotypes. Using ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) it was shown that mutant containing a mutation in both CiGAO alleles have a lower amount of some SL metabolites, compared to plants with no mutation or plants with only one mutated allele. Now protoplasts transfected with multiple CRISPR/Cas9 sgRNAs targeting multiple paralogous SL candidate genes are regenerating into plants. Regenerants will be screened by HiPlex amplicon sequencing to verify CRISPR/Cas9 mutation efficiency. Analyses on the mutants by UHPLC-MS/MS will provide insights in the function of the SL candidate genes, the formation of the SL compounds and hence the bitterness in Cichorium.