The nucleus, far from solely being the repository of genomic information, is increasingly viewed as a complex physical entity, mechanically and chemically coupled to its cellular environment. It is now clear that the three-dimensional architecture and the physical properties of the genome play a central role in its genomic and non-genomic functions. In recent years, novel approaches based on chromatin conformation capture techniques, complementing genome-wide assays and fixed-cell imaging tools have revealed multiple layers of conformational organization closely linked to genome function, including gene regulation, DNA replication, chromosome segregation, differentiation and others. Yet, despite the rapid evolution in our understanding of genome architecture in the past years, several roadblocks remain, that critically hold up progress in genome biology. First, experiments are predominantly performed on population of cells or in fixed conditions and there is a clear need to carry out measurements at the level of single living cells in order to address the conformational dynamics of chromatin at all scales, from the formation of loops to that of domains, compartments and territories. Second, most experiments (notably imaging measurements) are based on the passive observation of chromatin, possibly combined with global alterations such as gene knock-down. As a result, it is often difficult to distinguish between correlation and causality. Third, the dynamics and conformation of chromatin is governed by many energy-driven processes, and understanding the properties of chromatin as active matter is essential. Finally, chromosomes are mechanical objects with a conformation that can be dynamically modified as a function of the mechanical stresses exerted on the nucleus. Overall there is a clear need for a comprehensive description of the genome that account for the physical properties of chromosomes, notably including the out-of-equilibrium and mechanical processes that contribute to their organisation and dynamics in the nucleus. To address this challenge, we propose a novel approach that consists in mechanically perturbing the genome architecture, at the single chromosome level and with genomic specificity, and directly assessing the associated effects on nuclear organization and genomic functions. To do so, we will leverage our consortium’s unique expertise in cell biophysics, genome editing and nanoprobe chemistry to design an innovative nanotoolbox for chromosome imaging and manipulation. Specifically, we will develop a highly versatile and scalable strategy to decorate genomic loci in live cells with fluorescent quantum dots or magnetic nanoparticles via specific targeting using recombinant catalytically inactive CRISPR/Cas9 proteins, complexed with synthetic gRNAs. These novel functionalized nanoprobes will allow us to image and physically manipulate individual chromosomes at specific genomic sites in mammalian cells and to record the effect of these mechanical perturbations (in the pN range) on chromosomal conformations and functions. By doing so, we will directly assess: (i) the rheological and elastic properties of individual chromosomes in interphase and the role of out-of-equilibrium processes in their multiscale conformations, (ii) how mechanical forces affect transcriptional activity. Our approach, which goes well beyond the state-of-the art in genome research, will contribute to bridge the gap between population biochemical assays and the biophysical models of chromatin. Thereby, we will achieve a more integrative understanding of nuclear organization and dynamics and we will open many prospects for the study of a variety of scientific questions on different genome functions (transcription, repair, replication) and on the alteration of genome organization during processes as diverse as development, differentiation, disease or ageing.

Genome editing mediated by CRISPR-Cas9 has shown great promise for the treatment of retinal dystrophies (RD). Currently, adeno-associated viruses are the most widely used vectors for retinal gene therapies but their small packaging capacity and permanent transgene expression makes them suboptimal for CRISPR-Cas delivery. Transient delivery of Cas9 protein and its guide RNA as ribonucleoprotein (RNP) complexes have been reported in the retinal pigment epithelium (RPE) and into the inner ear cells in vivo. Members of our consortium investigated transient delivery of either Cas9 mRNA or Cas9 RNP into the retinal pigment epithelium (RPE) and photoreceptors as these are the target cells for most prevalent inherited retinal degenerations. Cas9 mRNA complexed with different lipid or peptide vectors led to low rates of indels at the target sequence in vivo and mostly in the RPE. Major changes to the delivery system are needed to increase the efficiency of gene editing, and finally, safety and cost of the therapeutic approach need to be taken into account when designing such vector systems. Our objectives are to address some of these challenges in this project by developing a novel polymer based non-viral carrier for in vivo targeted delivery of CRISPR-based RNP complexes; and to assess the efficacy and safety of our novel delivery approach in a mouse model of retinal dystrophy.

Complex gene regulatory networks govern organism development and homeostasis. Dysfunction of these networks impacts fundamental cellular functions, leading to many human diseases. The characterization of complexes involved in these fundamental cellular pathways, the study of their cellular dynamics and localisation and the way they evolve in a pathological context become major challenges of modern biology. Among the fundamental cellular complexes, TFIIH is at the cross roads of genome expression and repair. Mutations in three genes coding for TFIIH subunits give rise to three genetic disorders harbouring complex phenotypes. Therefore, a wealth of genetic information on TFIIH in humans can be exploited to understand the way this protein complex is participating in the maintenance of cell homeostasis. A consortium of four teams in strong synergy will implement a project with the aim to provide a new level of insight into structure and function of TFIIH. To this end, the consortium undertakes to characterize macromolecular assemblies of complexes involving TFIIH and its partners. We will focus on the dynamic of the complex at work in cells, the localisation of its different subunits and main partners at high resolution in normal or pathological context. In vitro, we will analyse the fine regulation of TFIIH enzymatic activities inside the complex and in relation with its partners. We will dissect the dysfunction implemented by disease-causing mutations on the complex and on its interaction with its partners. To successfully implement this project and achieve our goal, the consortium relies on the high scientific quality of the associated teams and will leverage its leading-edge expertise in genome editing, structural biology, biochemistry, and functional genomics. The project will also benefit from strong scientific platforms and services and will be facilitated by the unique multi-disciplinary expertise of the host institutions. Finally, this project, based on strong preliminary data, will also provide valuable tools to the scientific community. The social and scientific impact of this multi-scale study on TFIIH is high not only for patients with genetic disease-causing mutations in TFIIH but also for the healthy population since more and more acquired disorders are characterized as transcription and/or DNA repair syndromes

For several millennia, the grains of fruit kefir (or water kefir) have been exchanged from hand to hand from one grower/drinker to another. These exchanges contribute to the domestication and dissemination of bacterial communities with pre and probiotic properties and appreciated by many users for their effect on digestive well-being and general anti-inflammatory properties. A key feature of kefir ferment is that the grain cannot be produced de novo. There is therefore a connection between all the kefir grains in circulation on the planet today. The data available today, obtained from sequencing work and microbiological characterizations from several laboratories, including the MNHN, show a significant biodiversity of kefir grains, with the frequent presence of a few species and a significant potential for the discovery of new species of bacteria and yeast. The origin of this biodiversity, the dynamics of the stability of a given consortium during its transmission and circulation, are however not known today. Understanding the rules for the evolution of kefir is an issue that interests scientists, artisanal kefir producers as much as the hundreds of thousands of kefir users around the world who want to maintain certain strains and develop recipes and know-how allowing the maintenance of taste and well-being properties of a drink from a specific consortium. The challenge of this proposal is to co-construct with a civil society actor, a research laboratory and kefir-drinking consumer participants, a participatory research experience testing the resilience and evolution capacities of a grain of kefir, at the microbiological, visual and organoleptic level, during its circulation on French territory (possibly French-speaking). This research will be accompanied by a bi-monthly, interactive and participatory animation via a webinar animated alternately by scientists and civil society actors, in order to exchange and co-construct the participatory studies of tomorrow around this family of ferments whose the current boom responds to societal demands for sustainable, healthy, environmentally friendly and energy-responsible food.

Telomeres are short tandemly repeated DNA sequences at the end of chromosomes that preserve genome stability. Abnormal telomere shortening causes telomere syndrome, a rare monogenic disease characterized by bone marrow failure Dyskeratosis Congenita, aplastic anemia and idiopathic pulmonary fibrosis (IPF). Strikingly, rare heterozygous variations predicted to be deleterious in genes encoding for the subunits of RPA genes were identified in patients with short telomeres developing IPF. RPA is a heterotrimeric ssDNA binding protein involved in DNA replication, recombination and repair. Our preliminary results show that RPA patient variations provoke telomeric defects in cells harboring these mutations. These data strengthen our hypothesis that RPA is required to maintain repeated telomeric sequences in human. Our objective is to investigate the role of RPA at telomeres through the characterization of several RPA mutations and to validate RPA genes as genetic factor for telomere diseases.