Developing CRISPR activation/CRISPR interference toolset for fine-tuning production of metabolites in plants

Informations projet

METABOTUNE

N° de convention de subvention: 101151286

DOI

10.3030/101151286

Projet clôturé

Date de signature de la CE 8 Mars 2024

Date de début 1 Mai 2024

Date de fin 30 Avril 2026

Financé au titre de

• Marie Skłodowska-Curie Actions (MSCA)

Coût total

Aucune donnée

Contribution de l’UE

€ 175 920,00

Coordonné par

VIB VZW
Belgium

Periodic Reporting for period 1 - METABOTUNE (Developing CRISPR activation/CRISPR interference toolset for fine-tuning production of metabolites in plants)

Période du rapport: 2024-05-01 au 2026-04-30

Résumé du contexte et des objectifs généraux du projet

Specialised metabolites are plant compounds that are not involved in central metabolic processes, but have specialised roles, such as defences against pathogens and herbivores. Their bioactivity often makes them valuable to us humans, and many are used as medicines. However, due to their specialised roles, they are usually absent or present only in small amounts, and their production requires induction by some form of stress. The plant systems most amenable to manipulation at an industrial scale are cultures of suspension cells and hairy roots. They can be grown in liquid cultures, upscaled to large volumes, and treated with stress inducers that elicit production of specialised metabolites. But, these elicitors are non-selective, inducing undesired metabolic pathways as well as the desired ones, and usually prevent cells from further growing, reducing their biomass.

The main objective of METABOTUNE is to develop a CRISPR-based toolset for fine-tuning metabolite production in plant cultures. By targeting specific genes for activation using CRISPR activation (CRISPRa) only the desired pathways may be activated, and through inactivation using CRISPR interference (CRISPRi) undesired branches of the target pathways can be blocked, resulting in increased production of target metabolites, while avoiding the growth inhibition caused by elicitors.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

We tested RNA-targeting CRISPRi tool for use in plant metabolic engineering. In this system, the Cas enzyme cuts RNA molecules instead of DNA molecules, and is targeted to specific RNAs using complementary guide RNAs (gRNAs), small RNA molecules bound by Cas enzymes. This tool was first adapted for cloning using an efficient molecular cloning system, simplifying the creation of vectors used for plant transformation. We then demonstrated that this tool can work with single guide RNAs (gRNAs), and that the efficiency of cutting target RNAs likely depends on their secondary structures. CRISPRi could silence genes when transiently expressed in Nicotiana benthamiana tobacco plants, both exogenous gene and one endogenous metabolic gene. However, the silencing of additional three endogenous metabolic genes was not successful, potentially due to lower expression levels of the genes. The tool is now being tested in cell suspension liquid cultures of the Arabidopsis thaliana plant on the phenylpropanoid metabolic pathway.

Simultaneously, CRISPRa tools were tested for use in Arabidopsis cell suspension cultures. We tested two different CRISPRa systems, which were shown to work either in whole plants or transiently in tobacco. The results were negative for both. We investigated why these tools might not work in cell cultures, and demonstrated that it is most likely due to epigenetic repression of target genes, preventing typical gene activators from working in cell cultures. Two epigenetic CRISPRa tools proved unsuccessful for use in cell cultures. Further tests and optimisation are ongoing.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

The main objective was not achieved, but it was shown that RNA-targeting CRISPRi may be used in metabolic engineering to reduce the expression of highly expressed genes. This is beneficial in pathways where there is high expression of undesired side-branches that might be necessary for plant function, and complete blockage of them would have deleterious effects. The tests of CRISPRi in cell cultures is ongoing, and if proven successful will have potential for exploitation.

CRISPRa tools may not be applicable to plant cell cultures due to their strong metabolic gene expression. Further research on epigenetic CRISPRa tools is needed and it is ongoing in the host laboratory. If both tools prove successful and can be combined, the potential for exploitation of the outcomes will be even greater.