In the Crosetto lab, we are interested to understand what makes the genome of eukaryotic cells prone to break, and what the consequences of this fragility are in the context of healthy or diseased cells. We have pioneered the first method for direct labeling of DNA double-strand breaks (DSB) and their genome-wide localization using massively parallel sequencing (BLESS, Crosetto et al., Nature Methods 2013). Recently, we developed BLISS, a quantitative and versatile method for genome-wide DSB localization that can quantify and map endogenous DSBs and breaks induced by nucleases like CRISPR-Cas, even in precious samples of few thousands of cells and in tissue sections (Yan et al., Nature Communications 2017). We are now applying BLISS together with other ‘omic’ methods and high-resolution microscopy techniques, to investigate genome fragility in a variety of projects outlined below.
Research Area I: Genome fragility and cancer
The ultimate goal in this project is to construct a Cancer Breakome Atlas of the genome-wide pattern of DSBs (the ‘Breakome’) in different cancer types. Such atlas is expected to reveal different mechanisms of ongoing genome instability, and to identify patterns of DSBs that are predictive of a better or worse clinical outcome. Initially, we aim to profile a broad spectrum of established cancer cell lines, starting from cell lines that have been thoroughly characterized in the Encyclopedia of DNA Elements (ENCODE) project. We will then extend our efforts to patient-derived tumor samples, aiming to profile the Breakome at the time of diagnosis and, when possible, in biopsies obtained at relapse.
Research Area II: Genome fragility and neurological disorders
The goal of this project is to investigate the connection between DSBs and copy number variants (CNVs) in the context of the nervous system. Germline and somatic CNVs have been implicated in the genesis of several neurological diseases, including autism spectrum and neurodegenerative disorders. In this project, we apply our BLISS method to map DSBs in human stem/progenitor cells (NSPCs) and patient-derived induced pluripotent stem cells (iPSCs) undergoing different types of perturbations (replication stress, transcription stress, gene-specific mutations). We then perform BLISS and other genome-wide measurements in order to correlate the formation of CNVs with the genomic landscape of DSBs and chromatin features.
Research Area III: Genome fragility and aging
The goal of this project is to investigate the role of DSBs and DSB-related mutations in physiological aging. In particular, we are interested in exploring whether programmed DSBs that recurrently form during transcription lead, over the course of aging, to mutations in regulatory regions that in turn re-wire global transcription. In this project, we use primary cells from individuals of different ages, as well as from the same individual at different ages, and perform BLISS and deep sequencing to identify DSB hotspots that over the course of time give rise to mutations in gene promoters and enhancers. We then assess the effects of selected mutations on transcription in vitro using CRISPR-Cas technology.