The 4D Nucleome Project [preprint]

The spatial organization of the genome and its dynamics contribute to gene expression and cellular function in normal development as well as in disease. Although we are increasingly well equipped to determine a genome\u27s sequence and linear chromatin composition, studying the three-dimensional organization of the genome with high spatial and temporal resolution remains challenging. The 4D Nucleome Network aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the long term goal of gaining deeper mechanistic understanding of how the nucleus is organized. The project will develop and benchmark experimental and computational approaches for measuring genome conformation and nuclear organization, and investigate how these contribute to gene regulation and other genome functions. Further efforts will be directed at applying validated experimental approaches combined with biophysical modeling to generate integrated maps and quantitative models of spatial genome organization in different biological states, both in cell populations and in single cells

Application of Aptamers Improves CRISPR-Based Live Imaging of Plant Telomeres

Development of live imaging techniques for providing information how chromatin is organized in living cells is pivotal to decipher the regulation of biological processes. Here, we demonstrate the improvement of a live imaging technique based on CRISPR/Cas9. In this approach, the sgRNA scaffold is fused to RNA aptamers including MS2 and PP7. When the dead Cas9 (dCas9) is co-expressed with chimeric sgRNA, the fluorescent coat protein-tagged for MS2 and PP7 aptamers (tdMCP-FP and tdPCP-FP) are recruited to the targeted sequence. Compared to previous work with dCas9:GFP, we show that the quality of telomere labeling was improved in transiently transformed Nicotiana benthamiana using aptamer-based CRISPR-imaging constructs. Labeling is influenced by the copy number of aptamers and less by the promoter types. The same constructs were not applicable for labeling of repeats in stably transformed plants and roots. The constant interaction of the RNP complex with its target DNA might interfere with cellular processes

The road ahead in genetics and genomics

In celebration of the 20th anniversary of Nature Reviews Genetics, we asked 12 leading researchers to reflect on the key challenges and opportunities faced by the field of genetics and genomics. Keeping their particular research area in mind, they take stock of the current state of play and emphasize the work that remains to be done over the next few years so that, ultimately, the benefits of genetic and genomic research can be felt by everyone

Characterization of chromatin mobility upon DNA damage in Arabidopsis thaliana

Plant cells are subject to high levels of DNA damage from dependence on sunlight for energy and the associated exposure to biotic and abiotic stresses. Double-strand breaks (DSBs) are a particularly deleterious type of DNA damage, potentially leading to chromosome rearrangements or loss of entire chromosome arms. The presence of efficient and accurate repair mechanisms may be particularly important for sedentary organisms with late separation of the germline, such as plants. DSB repair is accomplished by two main pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ is achieved by stabilization and re-ligation of broken DNA ends, often with a loss or mutation of bases. HR is a more complex and conservative mechanism in which intact homologous regions are used as a template for repair. The molecular mechanisms that control DSB signaling and repair have been characterized extensively. Nonetheless, little is known about how the homology search happens in the crowded space of the cell nucleus. This thesis reveals the methodology to capture chromatin motion to investigate nuclear dynamics in different developmental and cellular contexts. Using live imaging approaches, we measured chromosome mobility by tracking the motion of specific loci using the lacO/LacI and ParB/parS tagging systems in Arabidopsis thaliana.Our results have shown that chromatin mobility is affected by cell differentiation level, cell cycle phase, or genomic position, and that chromatin mobility increases when DNA damage is induced. Moreover, we observed an increase in chromatin mobility upon the induction of DNA damage, specifically at the S/G2 phases of the cell cycle. Importantly, this increase in mobility in S/G2 was lost on sog1-1 mutant, a central transcription factor of the DNA damage response (DDR), indicating that repair mechanisms actively regulate chromatin mobility upon DNA damage. Studies have shown that HR is the predominant DSB repair pathway occurring during S/G2 phase. Therefore, we investigated the mobility of two GFP-tagged HR regulators, RAD51 and RAD54, corresponding to early and late HR. DSB sites show remarkably high mobility levels at the early HR stage. Subsequently, a drastic decrease in DSB mobility is observed, which seems to be associated with the relocation of DSBs to the nucleus periphery.Altogether, our study suggests chromatin mobility as a non-negligible factor for DNA repair in plants, which may facilitate physical searching in the nuclear space thereby helping to locate a homologous template during homology-directed DNA repair

Induced Pluripotent Stem Cells Meet Genome Editing

It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments - the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology - have fundamentally reshaped our approach to biomedical research, stem cell biology, and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided and set the stage for their success.National Institutes of Health (U.S.) (Grant 1R01NS088538-01)National Institutes of Health (U.S.) (Grant 2R01MH104610-15

Modeling genetic epilepsies in a dish

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, provide a powerful platform for mechanistic studies of disorders of neurodevelopment and neural networks. hPSC models of autism, epilepsy, and other neurological disorders are also advancing the path toward designing and testing precision therapies. The field is evolving rapidly with the addition of genome‐editing approaches, expanding protocols for the two‐dimensional (2D) differentiation of different neuronal subtypes, and three‐dimensional (3D) human brain organoid cultures. However, the application of these techniques to study complex neurological disorders, including the epilepsies, remains a challenge. Here, we review previous work using both 2D and 3D hPSC models of genetic epilepsies, as well as recent advances in the field. We also describe new strategies for applying these technologies to disease modeling of genetic epilepsies, and discuss current challenges and future directions.Key FindingsZebrafish post‐embryonic intestinal development is slow during the first two weeks due to proliferation pattern.Transformation to the juvenile intestine is preceded by increased proliferation and changes in mitotic pattern.cells integrate between proliferating fold base epithelial cells and may regulate proliferation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153080/1/dvdy79.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/153080/2/dvdy79_am.pd

Combining CRISPR-Cas9 and Proximity Labeling to Illuminate Chromatin Composition, Organization, and Regulation

A bacterial and archaeal adaptive immune system, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), has recently been engineered for genome editing. This RNA-guided platform has simplified genetic manipulation and holds promise for therapeutic applications. However, off-target editing has been one of the major concerns of the commonly used Streptococcus pyogenes Cas9 (SpyCas9). Despite extensive enzyme engineering to reduce off-target editing of SpyCas9, we have turned to nature and uncovered a Cas9 ortholog from Neisseria meningitidis (Nme) with high fidelity. In the first part of my thesis, we have systematically characterized Nme1Cas9 for engineering mammalian genomes and demonstrated its high specificity by genome-wide off-targeting detection methods in vitro and in cellulo, and thus provided a new platform for accurate genome editing. Due to its flexibility, CRISPR is becoming a versatile tool not only for genome editing, but also for chromatin manipulation. These alternative applications are possible because of the programmable targeting capacity of catalytically dead Cas9 (dCas9). In the second part of my thesis, we have combined dCas9 with the engineered plant enzyme ascorbate peroxidase (APEX2) to develop a proteomic method called dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging (C-BERST). Relying on the spatially restricted, fast biotin labeling of proteins near defined genomic loci, C-BERST enables the high-throughput identification of known telomere- and centromere- associated proteomes and novel factors. Furthermore, we have extended C-BERST to map the c-fos promoter and gained new insights regarding the dynamic transcriptional regulation process. Taken together, C-BERST can advance our understanding of chromatin regulators and their roles in nuclear and chromosome biology