Chicory is a relatively little-studied crop and gene editing protocols had to be developed almost from scratch. Variants of CRISPR methods were developed that differ in the extent, and the way DNA is used to deliver the CRISPR system generating the genome edits. These methods were systematically evaluated for their efficiency and safety, studying potential off-target editing in each case. Off-target mutations caused by the CRISPR system were not detected in any of the methods used or any of the lines analyzed. When DNA was used to deliver the CRISPR system via stable transformation, in some plants different edits within the same plant were observed. Apparently, the stably integrated CRISPR complex continues to make edits at the remaining target sites while the plant is developing. When DNA with the CRISPR system was delivered transiently, in addition to the intended edits, in some plants also CRISPR DNA fragments were found unintentionally integrated in the genome. In both cases, these plants can be recognized and discarded. When the CRISPR-system was delivered as a protein complex instead of via DNA, only edits at the expected target sites occurred. This makes this method as highly interesting for commercial applications.
The methods were implemented to generate a multitude of chicory varieties: varieties with a higher quality of healthy inulin because inulin degradation in autumn is prevented, as well as varieties that are reduced in bitter terpenes which greatly facilitates the extraction of inulin. In addition, varieties were created that accumulate specific terpenes of potential interest for medicinal use. The latter was guided by bioassays that have identified terpenes from chicory roots with very interesting anti-inflammatory and antimicrobial activity. Finally, the self-incompatibility of chicory was disrupted, simplifying crossing and allowing for more efficient breeding. Through these experiments scientific knowledge was generated on chicory biology and in particularly on its bioactive compounds, their biosynthesis and storage.
A Stakeholder Advisory Group with representatives of industry, academia, agriculture and end-users interacted with the CHIC consortium during the entire project period. In addition, consultations with a broader range of stakeholders covering the entire agriculture and food value chain, policy makers and regulators helped to clarify hindering and facilitating factors for genome-edited plants in general and for root chicory innovation in particular. Scenarios differing in aspects such as whether CRISPR edited chicory is regulated as GMO or not, and what type of products are isolated from them, were evaluated for their socio-economic and environmental impacts over the whole value chain. They show that inulin and/or terpene production based on the new chicory variants create more jobs, generate higher value added, and reduce greenhouse gas emissions and primary energy demand compared to the current process.
A lot of emphasis was put on communication about the CHIC project and its aims. A website from which flyers, newsletters and explanatory movies can be accessed and social media channels, were set up. Artists visited laboratories and created art works allowing for another type of interaction with the general publics. An educational game (MyChicFarm) was developed and CHIC partners visited schools and organized panel discussions targeting in particular young citizens.
CHIC also monitored regulations related to genome editing worldwide. For the EU, the European Court of Justice ruled that all genome edited plants are regulated as GMOs. The regulatory field is at the moment changing fast with big differences appearing between continents and countries. The project clearly shows that the promising food related applications will only be further pursued by business actors in the EU if the regulatory status for certain gene edited plants is changed.
