CRISPR/Cas9-Targeted Mutagenesis of CiGAS and CiGAO to Reduce Bitterness in Chicory (Cichorium intybus L.)

Abstract

Background: Chicory (Cichorium intybus L.), a member of the Asteraceae family, is known for its numerous health benefits, including its prebiotic, digestive, antioxidant or anti-inflammatory effects. Used as a coffee substitute, chicory roots is also appreciated for its bitterness, which can prove to be a disadvantage for other uses in food. The bitterness of chicory is largely linked to the presence of sesquiterpene lactones (STLs) in the roots.

Methods: In order to create less bitter industrial chicory varieties, CRISPR/Cas9 technology was used to inhibit the first two genes of the STL biosynthetic pathway: germacrene A synthase (CiGAS), short form, and germacrene A oxidase (CiGAO). To determine the impact of these reductions on the perception of bitterness, a sensory analysis of 13 field-grown chicories genotypes, contrasting for their STL composition, allowed the construction of obtain a bitterness scale by correlating STL content with perceived bitterness. The edited chicories were positioned on this scale according to their STL content.

Results: Biallelic mutations in two of the copies of CiGAS-short form or in the CiGAO gene led to a reduction in STL content of edited chicories and a reduction in bitterness, or even an absence of perception, was obtained for some mutants.

Conclusions: The use of the CRISPR/Cas9 tool as well as the choice of targets therefore makes it possible to modulate the bitterness of chicory.

Keywords: CRISPR/Cas9; Cichorium intybus L.; bitterness; germacrene A oxidase; germacrene A synthase; hairy roots; sesquiterpene lactones.