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

Project Information

METABOTUNE

Grant agreement ID: 101151286

DOI

10.3030/101151286

Project closed

EC signature date 8 March 2024

Start date 1 May 2024

End date 30 April 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 175 920,00

Coordinated by

VIB VZW
Belgium

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

Reporting period: 2024-05-01 to 2026-04-30

Summary of the context and overall objectives of the project

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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

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.