Inter-homologue repair in fertilized human eggs?, bioRxiv, 2017-08-29
Many human diseases have an underlying genetic component. The development and application of methods to prevent the inheritance of damaging mutations through the human germline could have significant health benefits, and currently include preimplantation genetic diagnosis and carrier screening. Ma et al. take this a step further by attempting to remove a disease mutation from the human germline through gene editing1. They assert the following advances (i) the correction of a pathogenic gene mutation responsible for hypertrophic cardiomyopathy in human embryos using CRISPR-Cas9 and (ii) the avoidance of mosaicism in edited embryos. In the case of correction, the authors conclude that repair using the homologous chromosome was as or more frequent than mutagenic nonhomologous end-joining (NHEJ). Their conclusion is significant, if validated, because such a “self-repair” mechanism would allow gene correction without the introduction of a repair template. While the authors’ analyses relied on the failure to detect mutant alleles, here we suggest approaches to provide direct evidence for inter-homologue recombination and discuss other events consistent with the data. We also review the biological constraints on inter-homologue recombination in the early embryo.
biorxiv cell-biology 100-200-users 2017Robust and Bright Genetically Encoded Fluorescent Markers for Highlighting Structures and Compartments in Mammalian Cells, bioRxiv, 2017-07-07
To increase our understanding of cells, there is a need for specific markers to identify biomolecules, cellular structures and compartments. One type of markers comprises genetically encoded fluorescent probes that are linked with protein domains, peptides andor signal sequences. These markers are encoded on a plasmid and they allow straightforward, convenient labeling of cultured mammalian cells by introducing the plasmid into the cells. Ideally, the fluorescent marker combines favorable spectroscopic properties (brightness, photostability) with specific labeling of the structure or compartment of interest. Here, we report on our ongoing efforts to generate robust and bright genetically encoded fluorescent markers for highlighting structures and compartments in living cells.
biorxiv cell-biology 200-500-users 2017CRISPR-Cas9 genome editing in human cells works via the Fanconi Anemia pathway, bioRxiv, 2017-05-12
AbstractCRISPR-Cas9 genome editing creates targeted double strand breaks (DSBs) in eukaryotic cells that are processed by cellular DNA repair pathways. Co-administration of single stranded oligonucleotide donor DNA (ssODN) during editing can result in high-efficiency (>20%) incorporation of ssODN sequences into the break site. This process is commonly referred to as homology directed repair (HDR) and here referred to as single stranded template repair (SSTR) to distinguish it from repair using a double stranded DNA donor (dsDonor). The high efficacy of SSTR makes it a promising avenue for the treatment of genetic diseases1,2, but the genetic basis of SSTR editing is still unclear, leaving its use a mostly empiric process. To determine the pathways underlying SSTR in human cells, we developed a coupled knockdown-editing screening system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. Unexpectedly, we found that SSTR requires multiple components of the Fanconi Anemia (FA) repair pathway, but does not require Rad51-mediated homologous recombination, distinguishing SSTR from repair using dsDonors. Knockdown of FA genes impacts SSTR without altering break repair by non-homologous end joining (NHEJ) in multiple human cell lines and in neonatal dermal fibroblasts. Our results establish an unanticipated and central role for the FA pathway in templated repair from single stranded DNA by human cells. Therapeutic genome editing has been proposed to treat genetic disorders caused by deficiencies in DNA repair, including Fanconi Anemia. Our data imply that patient genotype andor transcriptome profoundly impact the effectiveness of gene editing treatments and that adjuvant treatments to bias cells towards FA repair pathways could have considerable therapeutic value.
biorxiv cell-biology 100-200-users 2017The Human Cell Atlas, bioRxiv, 2017-05-09
AbstractThe recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body, by undertaking a Human Cell Atlas Project as an international collaborative effort. The aim would be to define all human cell types in terms of distinctive molecular profiles (e.g., gene expression) and connect this information with classical cellular descriptions (e.g., location and morphology). A comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, as well as provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas.
biorxiv cell-biology 500+-users 2017Mitochondria are physiologically maintained at close to 50 °C, bioRxiv, 2017-05-03
AbstractIn endothermic species, heat released as a product of metabolism ensures stable internal temperature throughout the organism, despite varying environmental conditions. Mitochondria are major actors in this thermogenic process. Part of the energy released by the oxidation of respiratory substrates drives ATP synthesis and metabolite transport, while a noticeable proportion is released as heat. Using a temperature-sensitive fluorescent probe targeted to mitochondria, we measured mitochondrial temperature in situ under different physiological conditions. At a constant external temperature of 38 °C, mitochondria were more than 10 °C warmer when the respiratory chain was fully functional, both in HEK293cells and primary skin fibroblasts. This differential was abolished in cells lacking mitochondrial DNA or by respiratory inhibitors, but preserved or enhanced by expressing thermogenic enzymes such as the alternative oxidase or the uncoupling protein 1. The activity of various RC enzymes was maximal at, or slightly above, 50 °C. Our study prompts a re-examination of the literature on mitochondria, taking account of the inferred high temperature.
biorxiv cell-biology 200-500-users 2017Investigation of the cellular reprogramming phenomenon referred to as stimulus-triggered acquisition of pluripotency (STAP), bioRxiv, 2015-09-29
In January 2014, it was reported that strong external stimuli, such as a transient low-pH stressor, was capable of inducing the reprogramming of mammalian somatic cells, resulting in the generation of pluripotent cells (Obokata et al. 2014a, b). This cellular reprograming event was designated 'stimulus-triggered acquisition of pluripotency' (STAP) by the authors of these reports. However, after multiple instances of scientific misconduct in the handling and presentation of the data were brought to light, both reports were retracted. To investigate the actual scientific significance of the purported STAP phenomenon, we sought to repeat the original experiments based on the methods presented in the retracted manuscripts and other relevant information. As a result, we have concluded that the STAP phenomenon as described in the original studies is not reproducible.
biorxiv cell-biology 0-100-users 2015