Periodic Reporting for period 2 - CIRCULAR VISION (Circular DNA in diagnosis and disease models) Reporting period: 2021-10-01 to 2022-09-30 Summary of the context and overall objectives of the project Circular DNA is emerging as a novel biomarker for many types of cancer and inflammatory disorders. However, we lack the technology for detecting circular DNA and for generating appropriate disease models. Many human diseases, including cancers and inflammatory disorders, are associated with the production of ‘circular DNA’ from chromosomes. These molecules have potential as biomarkers, and in the case of cancers are potential drivers of disease progression. However, the technology for detecting circular DNA and for creating disease models is lacking, meaning we cannot explore their potential use in diagnostics. CIRCULAR VISIONs goal is to explore the new opportunities circular DNA creates in early diagnosis and monitoring of disease, particularly screening, and to create an experimental system to understand the link between disease and circular DNA.CIRCULAR VISION explores this brand new territory by creating highly sensitive whole genome screens for circular DNA that correlate with disease, using lung cancer (LC) and inflammatory bowel disease (IBD) as model examples. We develop and combine novel methods in molecular biology, microfluidics, DNA sequencing and bioinformatics in order to identify new diagnostic markers. Such markers for IBD and LC will then be adapted to clinical diagnosis and prognosis using advanced image analysis and cytometry methods. Finally, CIRCULAR VISION will show the causal link between circular DNA and cancer, by producing disease models with oncogenes on circular DNA.CIRCULAR VISION assembles key pioneers in the emerging field of circular DNA with leading clinical experts and key commercial players in cytometry and genomics. We are convinced that our technology has broad applications in early noninvasive diagnosis of cancer and monitoring of inflammatory diseases. We believe our technology will lay the foundation for future research into circular DNA biology and spur future drug development. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The consortium finalized an efficient protocol for the purification of circular DNA and tested the newly developed protocol on plasma in a study of patients with lung cancer and another study of patients with inflammatory bowels disease. A computational biology study of the inter-laboratory analysis of the samples was performed and an array of tools for mining of circular-DNA data was also developed. The project further developed algorithms for identification of circular DNA, especially those that have an impact on phenotype. We have also implemented and developed new methods for enumeration of nucleic acids, e.g. circular DNA, in cells and in cell-free samples. Protocols and proper workflows for fabricating microfluidic chips were established and optimised.The purification of circular DNA from pilot plasma was carried out and we also purified, sequenced and mapped circular DNA from plasma of hundreds of lung cancer patients and patients with other cancers that serve as controls. We further enrolled a hundred IBD patients in a prospective study where we followed them over a year. We sampled biopsies and plasma from hundreds of samples and we started the sequencing and analysis of circular DNA from the first hundred samples, to identify circular elements that can be used to mark different stages of the disease.Several strains with reporter genes on circular DNA were generated in fission yeast, and we observed that endogenous circular DNA behaves differently from circular DNA coming from the environment around the yeast. Lastly we developed an optimized CRISPR-C method for generating circular DNA in human cells. We finally published our findings in 9 publications, 7 of which were in high impact journals. We reached the European citizens through podcasts, videos and a newsletter with stories and documentaries about our research and what it means to be a patient with IBD or cancer. 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) We hope to revolutionize the detection of circular DNA by producing efficient and accurate technologies demonstrated in relevant environments for circular DNA visualization, purification, sequencing and computation processing methods.We will exploit the potential of circular DNA as an early biomarker for blood-based diagnosis and efficient prognosis of IBD and cancer.By establishing model cell lines where circular DNA can be removed, inserted and monitored at will we want to find out how circular DNAs form in vivo, how some of them contribute to oncogenesis in human cells and how most cells eliminate these molecules.