Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
We report a genome-wide association scan in over 6,000 Latin Americans for features of scalp hair (shape, colour, greying, balding) and facial hair (beard thickness, monobrow, eyebrow thickness). We found 18 signals of association reaching genome-wide significance (P values 5 × 10(-8) to 3 × 10(-119)), including 10 novel associations. These include novel loci for scalp hair shape and balding, and the first reported loci for hair greying, monobrow, eyebrow and beard thickness. A newly identified locus influencing hair shape includes a Q30R substitution in the Protease Serine S1 family member 53 (PRSS53). We demonstrate that this enzyme is highly expressed in the hair follicle, especially the inner root sheath, and that the Q30R substitution affects enzyme processing and secretion. The genome regions associated with hair features are enriched for signals of selection, consistent with proposals regarding the evolution of human hair.
DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20-30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling.
Systems analysis of sex differences reveals an immunosuppressive role for testosterone in the response to influenza vaccination
- Proceedings of the National Academy of Sciences of the United States of America
- Published about 4 years ago
Females have generally more robust immune responses than males for reasons that are not well-understood. Here we used a systems analysis to investigate these differences by analyzing the neutralizing antibody response to a trivalent inactivated seasonal influenza vaccine (TIV) and a large number of immune system components, including serum cytokines and chemokines, blood cell subset frequencies, genome-wide gene expression, and cellular responses to diverse in vitro stimuli, in 53 females and 34 males of different ages. We found elevated antibody responses to TIV and expression of inflammatory cytokines in the serum of females compared with males regardless of age. This inflammatory profile correlated with the levels of phosphorylated STAT3 proteins in monocytes but not with the serological response to the vaccine. In contrast, using a machine learning approach, we identified a cluster of genes involved in lipid biosynthesis and previously shown to be up-regulated by testosterone that correlated with poor virus-neutralizing activity in men. Moreover, men with elevated serum testosterone levels and associated gene signatures exhibited the lowest antibody responses to TIV. These results demonstrate a strong association between androgens and genes involved in lipid metabolism, suggesting that these could be important drivers of the differences in immune responses between males and females.
Exercise induces beneficial responses in the brain, which is accompanied by an increase in BDNF, a trophic factor associated with cognitive improvement and the alleviation of depression and anxiety. However, the exact mechanisms whereby physical exercise produces an induction in brain Bdnf gene expression are not well understood. While pharmacological doses of HDAC inhibitors exert positive effects on Bdnf gene transcription, the inhibitors represent small molecules that do not occur in vivo. Here, we report that an endogenous molecule released after exercise is capable of inducing key promoters of the Mus musculus Bdnf gene. The metabolite β-hydroxybutyrate, which increases after prolonged exercise, induces the activities of Bdnf promoters, particularly promoter I, which is activity-dependent. We have discovered that the action of β-hydroxybutyrate is specifically upon HDAC2 and HDAC3, which act upon selective Bdnf promoters. Moreover, the effects upon hippocampal Bdnf expression were observed after direct ventricular application of β-hydroxybutyrate. Electrophysiological measurements indicate that β-hydroxybutyrate causes an increase in neurotransmitter release, which is dependent upon the TrkB receptor. These results reveal an endogenous mechanism to explain how physical exercise leads to the induction of BDNF.
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 4 years ago
To identify molecular mechanisms underlying the prospective health advantages associated with psychological well-being, we analyzed leukocyte basal gene expression profiles in 80 healthy adults who were assessed for hedonic and eudaimonic well-being, as well as potentially confounded negative psychological and behavioral factors. Hedonic and eudaimonic well-being showed similar affective correlates but highly divergent transcriptome profiles. Peripheral blood mononuclear cells from people with high levels of hedonic well-being showed up-regulated expression of a stress-related conserved transcriptional response to adversity (CTRA) involving increased expression of proinflammatory genes and decreased expression of genes involved in antibody synthesis and type I IFN response. In contrast, high levels of eudaimonic well-being were associated with CTRA down-regulation. Promoter-based bioinformatics implicated distinct patterns of transcription factor activity in structuring the observed differences in gene expression associated with eudaimonic well-being (reduced NF-κB and AP-1 signaling and increased IRF and STAT signaling). Transcript origin analysis identified monocytes, plasmacytoid dendritic cells, and B lymphocytes as primary cellular mediators of these dynamics. The finding that hedonic and eudaimonic well-being engage distinct gene regulatory programs despite their similar effects on total well-being and depressive symptoms implies that the human genome may be more sensitive to qualitative variations in well-being than are our conscious affective experiences.
Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries.
Hair differentiates from follicle stem cells through progenitor cells in the matrix. In contrast to stem cells in the bulge, the identities of the progenitors and the mechanisms by which they regulate hair shaft components are poorly understood. Hair is also pigmented by melanocytes in the follicle. However, the niche that regulates follicular melanocytes is not well characterized. Here, we report the identification of hair shaft progenitors in the matrix that are differentiated from follicular epithelial cells expressing transcription factor KROX20. Depletion of Krox20 lineage cells results in arrest of hair growth, confirming the critical role of KROX20(+) cells as antecedents of structural cells found in hair. Expression of stem cell factor (SCF) by these cells is necessary for the maintenance of differentiated melanocytes and for hair pigmentation. Our findings reveal the identities of hair matrix progenitors that regulate hair growth and pigmentation, partly by creating an SCF-dependent niche for follicular melanocytes.
Modern whole-organism genome analysis, in combination with biomass estimates, allows us to estimate a lower bound on the total information content in the biosphere: 5.3 × 1031 (±3.6 × 1031) megabases (Mb) of DNA. Given conservative estimates regarding DNA transcription rates, this information content suggests biosphere processing speeds exceeding yottaNOPS values (1024 Nucleotide Operations Per Second). Although prokaryotes evolved at least 3 billion years before plants and animals, we find that the information content of prokaryotes is similar to plants and animals at the present day. This information-based approach offers a new way to quantify anthropogenic and natural processes in the biosphere and its information diversity over time.
COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. In a previous paper (Dodonova et al., 2017), we determined a complete structural model of the in vitro reconstituted COPI coat. Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β'-COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.