Inactivation of the germacrene A synthase genes by CRISPR/Cas9 eliminates the biosynthesis of sesquiterpene lactones in Cichorium intybus L.

Abstract

Chicory (Cichorium intybus var. sativum) is an industrial crop species cultivated for the production of a fructose polymer inulin, which is used as a low-calorie sweetener and prebiotic. Besides, inulin chicory taproots also accumulate sesquiterpene lactones (STLs). These are bitter tasting compounds, which need to be removed during inulin extraction, resulting in additional costs. In this work, we describe chicory lines where STL accumulation is almost completely eliminated. Genome editing using the CRISPR/Cas9 system was used to inactivate four genes that encode the enzyme that performs the first dedicated step in STL synthesis, germacrene A synthase (CiGAS). Chicory lines were obtained that carried null mutations in all four CiGAS genes. Lines lacking functional CiGAS alleles showed a normal phenotype upon greenhouse cultivation and show nearly complete elimination of the STL synthesis in the roots. It was shown that the reduction in STLs could be attributed to mutations in genetically linked copies of the CiGAS-short gene and not the CiGAS-long gene, which is relevant for breeding the trait into other cultivars. The inactivation of the STL biosynthesis pathway led to increase in phenolic compounds as well as accumulation of squalene in the chicory taproot, presumably due to increased availability of farnesyl pyrophosphate (FFP). These results demonstrate that STLs are not essential for chicory growth and that the inhibition of the STL biosynthesis pathway reduced the STL levels chicory which will facilitate inulin extraction.

CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots

“…CRISPR/Cas9 mutagenesis is shown as an efficient strategy to inactivate genes in chicory. In this work we transiently expressed CRISPR/Cas9 reagents encoded on plasmids in protoplasts and as observed previously, transient plasmid protocols can result in Cas9 integration in the chicory genome despite the absence of selection ( Bernard et al, 2019 ; Cankar et al, 2021 ). Through the transient transfection of a ribonucleoprotein complex we were able to identify CiKLS mutants that lack additional insertions.…”

“…The integration of Cas9 in the genome was observed in lines A1, A34, and A153 but not in the other five analyzed lines. The integration of Cas9 has been previously observed when using plasmid-based transfection of CRISPR/Cas9 reagents for chicory genome editing ( Bernard et al, 2019 ; Cankar et al, 2021 ).…”

“…For each transfection a mix of 90 μg of the Cas9 expression plasmid and 30 μg of a plasmid carrying sgRNAs was prepared in a 50 μl final volume. For the RNP approach the protocol has been previously described ( Cankar et al, 2021 ). The plasmid or RNPs were added to 300 μl of protoplast solution and mixed gently.…”

“…Efficient genome editing approaches using CRISPR/Cas9 have been developed for chicory and efficient protocols for subsequent plant regeneration have been described ( Bernard et al, 2019 ; De Bruyn et al, 2020 ). Transient transfection of either plasmid encoded CRISPR/Cas9 reagents or ribonucleoprotein complexes was used successfully to modulate terpene biosynthesis in chicory ( Cankar et al, 2021 ). Upon inactivation of the germacrene A synthases that catalyze the first dedicated step in the biosynthesis of chicory sesquiterpenes, nearly complete elimination of STLs in chicory was demonstrated ( Cankar et al, 2021 ).…”

“…Transient transfection of either plasmid encoded CRISPR/Cas9 reagents or ribonucleoprotein complexes was used successfully to modulate terpene biosynthesis in chicory ( Cankar et al, 2021 ). Upon inactivation of the germacrene A synthases that catalyze the first dedicated step in the biosynthesis of chicory sesquiterpenes, nearly complete elimination of STLs in chicory was demonstrated ( Cankar et al, 2021 ).…”