Why did we fund this project?
This award aims to replace the use of rodents in genotoxicity risk assessment studies by optimising a method using next generation sequencing to detect double stranded breaks (DSBs).
Candidate drugs are tested for risk of genotoxicity by analysing DNA damage and cellular responses after treatment. The main regulatory assay involves dosing rodents, typically mice, with the chemical to be analysed. Animals are then sacrificed to remove the bone marrow allowing analysis of the blood cells for markers of DNA damage such as the presence of micronuclei. Professor Simon Reed has developed a method to detect DSBs in human cells using next generation DNA sequencing technologies. The technique can also detect rare DSBs caused by endogenous processes such as DNA replication.
The student will now further develop the next generation sequencing technique to measure chemical induced DSBs. The student will develop skills in library preparation, adaptor design, and next generation sequencing techniques.
This award is jointly funded by Unilever.
Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage, especially the DNA double strand break (DSB). Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Current assays involve in vivo cell-based analysis of surrogate markers for breaks, such as the gamma H2AX focus assay, or the tail moment of DNA from the Comet assay. The main regulatory assay for the detection of DNA damage caused by clastogens and aneugens is the animal-based rodent in vivo micronucleus assay.
Novel next generation DNA sequencing technologies now make it possible to revolutionise the way we test for factors affecting the stability of the genome, and this could also include the testing of chemicals and compounds generated by humans, and those found in the natural environment. Furthermore, novel genome editing technologies offer the possibility of novel therapeutic modalities, which also need to be safety-tested for their potential genotoxic effects due to the established problem of 'off-target' editing. In this regard, we recently developed a novel method to capture DSBs induced by CRISPR-Cas9 editing of the genome, with a view to measuring such off-target editing. Remarkably, we have determined that the method, which we call INDUCE-Seq, is exquisitely sensitive to detecting DSBs in the genome over a very broad dynamic range. Current assays for detecting genetic damage (eg gamma H2AX and Comet assays) are far less sensitive. DSBs can be caused by both endogenous processes, such as DNA replication and gene transcription, but also by induced events such as restriction enzyme digestion, or nuclease-dependent genome editing. We have shown that INDUCE-seq is capable of simultaneously detecting both rare endogenous breaks in the genome, as well as highly abundant targeted breaks over a vast dynamic range.
We seek to train a student in the principles of 3Rs research with a view to training them to help build the future tools that will replace the current use of animals in genotoxicity testing of novel chemicals and compounds. This will enable the safe development of new materials in the future. We plan to further develop INDUCE-seq to test for the measurement of chemical induced DSBs, to provide additional genomic DNA damage data to add to the battery of genomic and proteomic analyses currently being developed in the drive to establish the necessary tools for next generation risk assessment (NGRA). The aim of such initiatives, which include the EPAs Toxcast programme and the related Tox21 consortium, is the successful introduction of NGRA, with a view to the eventual elimination of animal testing of chemicals.