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

Abstract

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