Project description
A new technology for editing large DNA segments
The recent advent of the CRISPR-Cas9 technology has revolutionised targeted genome editing by generating small insertions and deletions at the target site. The EU-funded GenDels project will work on an alternative CRISPR-based technology that employs the Cas3 enzyme which combines helicase and nuclease activity. The aim is to achieve high-throughput gene-editing of large DNA segments in human cells for health-related applications or to decipher the role of non-coding DNA variants in disease. Moreover, this technology could be exploited for the editing of bacterial cells for synthetic biological and metabolic purposes.
Objective
The advent of the revolutionary genome editing technique CRISPR-Cas9 has enabled targeted gene mutation, repression, and activation, facilitating impactful biological findings. However, Cas9 as an unbiased DNA deletion tool is limited in its ability to interrogate large regions of DNA of unknown function, because it predominantly generates very small (<20 bp) insertions and deletions at its target site. The capacity to rapidly and efficiently generate large genomic deletions does not currently exist and would be an extremely useful tool for research, allowing for rapid strain engineering of bacterial cells for synthetic biological and metabolic engineering purposes. Additionally, this technology would allow for the interrogation of large segments of non-coding DNA in human cells, much of which has unknown function, but whose variants are often associated with human disease. In this proposal, I aim to develop a Type I-C CRISPR-Cas system employing the Cas3 enzyme (completely distinct from Cas9), which naturally possess coupled nuclease and helicase activity, for high-throughput gene-editing purposes in various prokaryotic, as well as human cells. My preliminary results have shown that this is a credible approach, as I have been able to generate individually, as well as in combination, multiple deletions in bacterial organisms exceeding 60 kb in size. A focal point of the proposal is to adapt this system for use in human cells, which would provide a novel basic research tool with unprecedented capabilities and also could be utilized in human health-related applications. The proposal aims to address this later possibility by utilizing the developed system to treat human cell lines infected with difficult-to-treat pathogenic viruses.
Fields of science
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Programme(s)
Funding Scheme
MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)Coordinator
69117 Heidelberg
Germany