Molecular recording of mammalian embryogenesis.

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes

Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation

While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%–99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways

N plus 2 Supersonic Concept Development and Systems Integration

Supersonic airplanes for two generations into the future (N+2, 2020-2025 EIS) were designed: the 100 passenger 765-072B, and the 30 passenger 765-076E. Both achieve a trans-Atlantic range of about 4000nm. The larger 765-072B meets fuel burn and emissions goals forecast for the 2025 time-frame, and the smaller 765-076E improves the boom and confidence in utilization that accompanies lower seat count. The boom level of both airplanes was reduced until balanced with performance. The final configuration product is two "realistic", non-proprietary future airplane designs, described in sufficient detail for subsequent multi-disciplinary design and optimization, with emphasis on the smaller 765-076E because of its lower boom characteristics. In addition IGES CAD files of the OML lofts of the two example configurations, a non-proprietary parametric engine model, and a first-cycle Finite Element Model are also provided for use in future multi-disciplinary analysis, optimization, and technology evaluation studies

Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens

Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes—such as transcriptional profiles—at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays. Keywords: single-cell RNA-seq; pooled screen; CRISPR; epistasis; genetic interaction

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.National Human Genome Research Institute (U.S.) (Grant P50HG006193

Beliefs and experiences of fear of hypoglycemiaand use of uncooked cornstarch before bedtime in persons with type 1-diabetes

Introduction: Among persons living with type 1-diabetes hypoglycemia and fear of hypoglycemia remain limiting barriers for achieving optimal glucose control and a good quality of life. Fear of hypoglycemia has been found stable over time if not treated. Uncooked cornstarch has been found to reduce the risk of hypoglycemia but has not been studied in relation to fear of hypogly-cemia. The aims of this study were to through clinical data, self-reported measures and clinical interviews explore subjects’ experience of using un-cooked cornstarch before bedtime and their beliefs and experiences of fear of hypoglycemia. Methods: Mixed methods with both quantitative and qualita-tive data were used. Self-reported measures of hypoglycemia and fear of hy-poglycemia were compared to subjects’ responses during a clinical interview. The interviews were analyzed with a functional behavior analytical approach. Results: A total of five subjects took part in the study. One subject perceived the uncooked cornstarch helpful in reducing hypoglycemia. Several subjects could recall frightening hypoglycemic episodes triggering their fear. Three out of the five subjects reported avoidance behaviors such as excessive self-monitoring of blood glucose or overeating related to fear of hypoglyce-mia. Conclusions: The uncooked cornstarch was found appetizing but was not perceived as having an effect on BG or hypoglycemia frequency. The clinical interviews confirmed previous research regarding experience of hy-poglycemia and fear of hypoglycemia

A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response

Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR