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

Understanding the errors of SHAPE-directed RNA structure modeling

Single-nucleotide-resolution chemical mapping for structured RNA is being rapidly advanced by new chemistries, faster readouts, and coupling to computational algorithms. Recent tests have shown that selective 2'-hydroxyl acylation by primer extension (SHAPE) can give near-zero error rates (0-2%) in modeling the helices of RNA secondary structure. Here, we benchmark the method using six molecules for which crystallographic data are available: tRNA(phe) and 5S rRNA from Escherichia coli, the P4-P6 domain of the Tetrahymena group I ribozyme, and ligand-bound domains from riboswitches for adenine, cyclic di-GMP, and glycine. SHAPE-directed modeling of these highly structured RNAs gave an overall false negative rate (FNR) of 17% and a false discovery rate (FDR) of 21%, with at least one helix prediction error in five of the six cases. Extensive variations of data processing, normalization, and modeling parameters did not significantly mitigate modeling errors. Only one varation, filtering out data collected with deoxyinosine triphosphate during primer extension, gave a modest improvement (FNR = 12%, and FDR = 14%). The residual structure modeling errors are explained by the insufficient information content of these RNAs' SHAPE data, as evaluated by a nonparametric bootstrapping analysis. Beyond these benchmark cases, bootstrapping suggests a low level of confidence (<50%) in the majority of helices in a previously proposed SHAPE-directed model for the HIV-1 RNA genome. Thus, SHAPE-directed RNA modeling is not always unambiguous, and helix-by-helix confidence estimates, as described herein, may be critical for interpreting results from this powerful methodology.Comment: Biochemistry, Article ASAP (Aug. 15, 2011

Case series: convalescent plasma therapy for patients with COVID-19 and primary antibody deficiency

Patients with primary antibody deficiency are at risk for severe and in many cases for prolonged COVID-19. Convalescent plasma treatment of immunocompromised individuals could be an option especially in countries with limited access to monoclonal antibody therapies. While studies in immunocompetent COVID19 patients have demonstrated only a limited benefit, evidence for the safety, timing, and effectiveness of this treatment in antibody-deficient patients is lacking. Here, we describe 16 cases with primary antibody deficiency treated with convalescent plasma in four medical centers. In our cohort, treatment was associated with a reduction in viral load and improvement of clinical symptoms, even when applied over a week after onset of infection. There were no relevant side effects besides a short-term fever reaction in one patient. Longitudinal full-genome sequencing revealed the emergence of mutations in the viral genome, potentially conferring an antibody escape in one patient with persistent viral RNA shedding upon plasma treatment. However, he resolved the infection after a second course of plasma treatment. Thus, our data suggest a therapeutic benefit of convalescent plasma treatment in patients with primary antibody deficiency even months after infection. While it appears to be safe, PCR follow-up for SARS-CoV-2 is advisable and early re-treatment might be considered in patients with persistent viral shedding

SEIS: Insight's Seismic Experiment for Internal Structure of Mars.

By the end of 2018, 42 years after the landing of the two Viking seismometers on Mars, InSight will deploy onto Mars' surface the SEIS (Seismic Experiment for Internal Structure) instrument; a six-axes seismometer equipped with both a long-period three-axes Very Broad Band (VBB) instrument and a three-axes short-period (SP) instrument. These six sensors will cover a broad range of the seismic bandwidth, from 0.01&nbsp;Hz to 50&nbsp;Hz, with possible extension to longer periods. Data will be transmitted in the form of three continuous VBB components at 2 sample per second (sps), an estimation of the short period energy content from the SP at 1&nbsp;sps and a continuous compound VBB/SP vertical axis at 10&nbsp;sps. The continuous streams will be augmented by requested event data with sample rates from 20 to 100 sps. SEIS will improve upon the existing resolution of Viking's Mars seismic monitoring by a factor of ∼ 2500 at 1&nbsp;Hz and ∼ 200 000 at 0.1&nbsp;Hz. An additional major improvement is that, contrary to Viking, the seismometers will be deployed via a robotic arm directly onto Mars' surface and will be protected against temperature and wind by highly efficient thermal and wind shielding. Based on existing knowledge of Mars, it is reasonable to infer a moment magnitude detection threshold of M w ∼ 3 at 40 ∘ epicentral distance and a potential to detect several tens of quakes and about five impacts per year. In this paper, we first describe the science goals of the experiment and the rationale used to define its requirements. We then provide a detailed description of the hardware, from the sensors to the deployment system and associated performance, including transfer functions of the seismic sensors and temperature sensors. We conclude by describing the experiment ground segment, including data processing services, outreach and education networks and provide a description of the format to be used for future data distribution.Electronic supplementary materialThe online version of this article (10.1007/s11214-018-0574-6) contains supplementary material, which is available to authorized users

Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest

Anthrax is a globally important animal disease and zoonosis. Despite this, our current knowledge of anthrax ecology is largely limited to arid ecosystems, where outbreaks are most commonly reported. Here we show that the dynamics of an anthrax-causing agent, Bacillus cereus biovar anthracis, in a tropical rainforest have severe consequences for local wildlife communities. Using data and samples collected over three decades, we show that rainforest anthrax is a persistent and widespread cause of death for a broad range of mammalian hosts. We predict that this pathogen will accelerate the decline and possibly result in the extirpation of local chimpanzee (Pan troglodytes verus) populations. We present the epidemiology of a cryptic pathogen and show that its presence has important implications for conservation

The Far Side of Mars: Two Distant Marsquakes Detected by InSight

For over three Earth years the Marsquake Service has been analyzing the data sent back from the Seismic Experiment for Interior Structure¿the seismometer placed on the surface of Mars by NASA¿s InSight lander. Although by October 2021, the Mars seismic catalog included 951 events, until recently all these events have been assessed as lying within a radius of 100° of InSight. Here we report two distant events that occurred within days of each other, located on the far side of Mars, giving us our first glimpse into Mars¿ core shadow zone. The first event, recorded on 25 August 2021 (InSight sol 976), shows clear polarized arrivals that we interpret to be PP and SS phases at low frequencies and locates to Valles Marineris, 146° ± 7° from InSight. The second event, occurring on 18 September 2021 (sol 1000), has significantly more broadband energy with emergent PP and SS arrivals, and a weak phase arriving before PP that we interpret as Pdiff¿. Considering uncertain pick times and poorly constrained travel times for Pdiff¿, we estimate this event is at a distance between 107° and 147° from InSight. With magnitudes of MMaw 4.2 and 4.1, respectively, these are the largest seismic events recorded so far on Mars.Anna C. Horleston, Jessica C. E. Irving,and Nicholas A. Teanby are funded by the UKSA under Grant Numbers ST/R002096/1, ST/W002523/1, and ST/W002515/1.Nikolaj L. Dahmen, Cecilia Duran, Géraldine Zenhäusern, andSimon C. Stähler would like to acknowledge support from Eidgenössische Technische Hochschule (ETH) through the ETH+ funding scheme (ETH+02 19-1: “Planet Mars”). The French coauthors acknowledge the funding support provided by CNES and the Agence Nationale de la Recherche (ANR-19-CE31-0008-08 MAGIS) for SEIS operation and SEIS Science analysis. Alexander E. Stott acknowledges the French Space Agency CNES and ANR (ANR-19-CE31-0008-08). Caroline Beghein and Jiaqi Li were supported by NASA InSight Participating Scientist Program (PSP) Grant Number 80NSSC18K1679. This article is InSight Contribution Number 236

Active Chromatin Marks Are Retained on X Chromosomes Lacking Gene or Repeat Silencing Despite XIST/Xist Expression in Somatic Cell Hybrids

X-chromosome inactivation occurs early in mammalian development and results in the inactive X chromosome acquiring numerous hallmarks of heterochromatin. While XIST is a key player in the inactivation process, the method of action of this ncRNA is yet to be determined.To assess which features of heterochromatin may be directly recruited by the expression and localization of the XIST RNA we have analyzed a mouse/human somatic cell hybrid in which expression of human and mouse XIST/Xist has been induced from the active X by demethylation. Such hybrids had previously been demonstrated to disconnect XIST/Xist expression from gene silencing and we confirm maintenance of X-linked gene expression, even close to the Xist locus, despite the localized expression of mouse Xist.Loss of the active chromatin marks H3 acetylation and H3 lysine 4 methylation was not observed upon XIST/Xist expression, nor was there a gain of DNA methylation; thus these marks of facultative heterochromatin are not solely dependent upon Xist expression. Cot-1 holes, regions of depleted RNA hybridization with a Cot-1 probe, were observed upon Xist expression; however, these were at reduced frequency and intensity in these somatic cells. Domains of human Cot-1 transcription were observed corresponding to the human chromosomes in the somatic cell hybrids. The Cot-1 domain of the X was not reduced with the expression of XIST, which fails to localize to the human X chromosome in a mouse somatic cell background. The human inactive X in a mouse/human hybrid cell also shows delocalized XIST expression and an ongoing Cot-1 domain, despite X-linked gene silencing. These results are consistent with recent reports separating Cot-1 silencing from genic silencing, but also demonstrate repetitive element expression from an otherwise silent X chromosome in these hybrid cells

Surface waves and crustal structure on Mars

We detected surface waves from two meteorite impacts on Mars. By measuring group velocity dispersion along the impact-lander path, we obtained a direct constraint on crustal structure away from the InSight lander. The crust north of the equatorial dichotomy had a shear wave velocity of approximately 3.2 kilometers per second in the 5- to 30-kilometer depth range, with little depth variation. This implies a higher crustal density than inferred beneath the lander, suggesting either compositional differences or reduced porosity in the volcanic areas traversed by the surface waves. The lower velocities and the crustal layering observed beneath the landing site down to a 10-kilometer depth are not a global feature. Structural variations revealed by surface waves hold implications for models of the formation and thickness of the martian crust.D.K., S.C., D.G., J.C., C.D., A. K., S.C.S., N.D., and G.Z. were supported by the ETH+ funding scheme (ETH+02 19-1: “Planet Mars”). Marsquake Service operations at ETH Zürich were supported by ETH Research grant ETH-06 17-02. N.C.S. and V.L. were supported by NASA PSP grant no. 80NSSC18K1628. Q.H. and E.B. are funded by NASA grant 80NSSC18K1680. C.B. and J.L. were supported by NASA InSight PSP grant no. 80NSSC18K1679. S.D.K. was supported by NASA InSight PSP grant no. 80NSSC18K1623. P.L., E.B., M.D., H.S., E.S., M.W., Z.X., T.W., M.P., R.F.G. were supported by CNES and the Agence Nationale de la Recherche (ANR-19-CE31-0008-08 MAGIS) for SEIS operation and SEIS Science analysis. A.H., C.C. and W.T.P. were supported by the UKSA under grant nos. ST/R002096/1, ST/ W002523/1 and ST/V00638X/1. Numerical computations of McMC Approach 2 were performed on the S-CAPAD/DANTE platform (IPGP, France) and using the HPC resources of IDRIS under the allocation A0110413017 made by GENCI. A.H. was supported by the UKSA under grant nos. ST/R002096/1 and ST/W002523/1. F.N. was supported by InSight PSP 80NSSC18K1627. I.J.D. was supported by NASA InSight PSP grant no. 80NSSC20K0971. L.V.P. was funded by NASANNN12AA01C with subcontract JPL-1515835. The research was carried out in part by W.B.B., M.G. and M.P.P. at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration (80NM0018D0004)Peer reviewe

Seismic detection of the martian core

Clues to a planet's geologic history are contained in its interior structure, particularly its core. We detected reflections of seismic waves from the core-mantle boundary of Mars using InSight seismic data and inverted these together with geodetic data to constrain the radius of the liquid metal core to 1830 +/- 40 kilometers. The large core implies a martian mantle mineralogically similar to the terrestrial upper mantle and transition zone but differing from Earth by not having a bridgmanite-dominated lower mantle. We inferred a mean core density of 5.7 to 6.3 grams per cubic centimeter, which requires a substantial complement of light elements dissolved in the iron-nickel core. The seismic core shadow as seen from InSight's location covers half the surface of Mars, including the majority of potentially active regions-e.g., Tharsis-possibly limiting the number of detectable marsquakes.This is InSight contribution 200. We acknowledge NASA, CNES, and partner agencies and institutions (UKSA, SSO, ESA-PRODEX, DLR, JPL, IPGP-CNRS, ETHZ, IC, and MPS-MPG) for the development of SEIS. Numerical simulations were supported by a grant from the Swiss National Supercomputing Centre (CSCS) under project ID s922 as well as HPC resources of CINES under the allocation A0090407341, made by GENCI. We thank B. Dintrans, director of CINES, for his efficient handling of our request for computational time. Figures were created using matplotlib (83), seismic data processing was done in ObsPy (84), and numerical evaluation was done in NumPy and SciPy (85, 86). Funding: S.C.S., A.K., D.G., J.C., A.C.D., G.Z., and N.D. acknowledge support from ETHZ through the ETH+ funding scheme (ETH+2 19-1: “Planet MARS”). S.C.S. acknowledges funding from ETH research grant ETH-10 17-3. W.B.B., A.G.M., M.P.P., and S.E.S. were supported by the NASA InSight mission and funds from the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration (80NM0018D0004). D.A. has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement 724690). The French teams acknowledge support from CNES as well as Agence Nationale de la Recherche (ANR-14-CE36-0012-02 and ANR-19-CE31-0008-08). A.R. was financially supported by the Belgian PRODEX program managed by the European Space Agency in collaboration with the Belgian Federal Science Policy Office. M.S. wishes to thank SANIMS (RTI2018-095594-B-I00). M.v.D. received support from the ERC under the European Union’s Horizon 2020 program (grant no. 714069). D.S. and C.S. acknowledge funding from ETH research grant ETH-06 17-02. J.C.E.I. acknowledges support from NASA grant 80NSSC18K1633. N.S., D.K., Q.H., R.M., V.L., and A.G.M. acknowledge NASA grant 80NSSC18K1628 for support. V.L. acknowledges support from the Packard Foundation. W.T.P. and C.C. received funding from the UK Space Agency, grant ST/S001239/1. A.H. was funded by the UK Space Agency (grant ST/R002096/1). A.-C.P. acknowledges the financial support and endorsement from the DLR Management Board Young Research Group Leader Program and the Executive Board Member for Space Research and Technology. Author contributions: S.C.S., D.G., S.C., R.F.G., Q.H., D.K., V.L., M.S., N.S., D.S., É.S., C.S., and G.Z. analyzed the seismic data and made ScS arrival time picks. S.C.S., P.L., D.G., Z.X., C.C., and W.T.P. performed the statistical analysis of the observed signals. S.C.S., Q.H., N.S., R.M., and A.G.M. identified the arrivals as ScS waves based on interior models from A.K., H.S., and A.R. A.K., M.D., A.C.D., and H.S. performed the inversions. S.C.S., A.K., P.L., D.G., D.A., J.C.E.I., M.K., C.P., A.-C.P., A.R., T.G., and S.E.S. participated and contributed to the interpretation of the results. Review of the continuous data and detection of marsquakes was done by S.C.S., S.C., G.Z., C.C., N.D., J.C., M.v.D., T.K., M.P., and A.H. with operational support by É.B., C.P., and P.M.D. S.C.S. and A.K. wrote the central part of the paper with contributions from H.S., N.S., D.A., J.C.E.I., A.G.M., A.-C.P., A.R., J.C., and M.v.D. J.C.E.I., R.M., M.K., and V.L. reviewed the contributions to the supplementary materials. The InSight mission is managed by W.B.B., M.P.P., and S.E.S. The SEIS instrument development was led by P.L., D.G., W.T.P., and W.B.B. Supplementary section 1 was written by M.S., D.S., and É.S. with contributions from S.C.S., C.S., and Z.X. Supplementary section 2 was written by D.K. and V.L. with contributions from J.C.E.I. and N.S. Supplementary section 3 was written by M.S. and É.S. Supplementary section 4 was written by R.F.G. with contributions from M.D. Supplementary section 5 was written by Q.H. with contributions from N.S. Supplementary section 6 was written by S.C.S. with contributions from the authors of the other supplements. Supplementary section 7 was written by Z.X. and C.C. with contributions from P.L. and W.T.P. Supplementary section 8 was written by A.K., M.D., A.C.D., and H.S. Supplementary section 9 was written by M.D. Supplementary section 10 was written by A.C.D., A.K., and M.D. Supplementary section 11 was written by D.A. and A.R. with contributions from A.K. Competing interests: The authors declare that they have no competing interests. Data and materials availability: We thank the operators of JPL, SISMOC, MSDS, IRIS-DMC, and PDS for providing SEED SEIS data (87). Three hundred interior models derived in this study are available from MSDS (88)

Seismic Constraints on the Thickness and Structure of the Martian Crust from InSight

NASA¿s InSight mission [1] has for the first time placed a very broad-band seismometer on the surface of Mars. The Seismic Experiment for Interior Structure (SEIS) [2] has been collecting continuous data since early February 2019. The main focus of InSight is to enhance our understanding of the internal structure and dynamics of Mars, which includes the goal to better constrain the crustal thickness of the planet [3]. Knowing the present-day crustal thickness of Mars has important implications for its thermal evolution [4] as well as for the partitioning of silicates and heat-producing elements between the different layers of Mars. Current estimates for the crustal thickness of Mars are based on modeling the relationship between topography and gravity [5,6], but these studies rely on different assumptions, e.g. on the density of the crust and upper mantle, or the bulk silicate composition of the planet and the crust. The resulting values for the average crustal thickness differ by more than 100%, from 30 km to more than 100 km [7]. New independent constraints from InSight will be based on seismically determining the crustal thickness at the landing site. This single firm measurement of crustal thickness at one point on the planet will allow to constrain both the average crustal thickness of Mars as well as thickness variations across the planet when combined with constraints from gravity and topography [8]. Here we describe the determination of the crustal structure and thickness at the InSight landing site based on seismic receiver functions for three marsquakes compared with autocorrelations of InSight data [9].We acknowledge NASA, CNES, partner agencies and institutions (UKSA, SSO,DLR, JPL, IPGP-CNRS, ETHZ, IC, MPS-MPG) and the operators of JPL, SISMOC, MSDS, IRIS-DMC and PDS for providing SEED SEIS data. InSight data is archived in the PDS, and a full list of archives in the Geosciences, Atmospheres, and Imaging nodes is at https://pds-geosciences.wustl.edu/missions/insight/. This work was partially carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration. ©2021, California Institute of Technology. Government sponsorship acknowledge