Crystal structure of Cas9 in complex with guide RNA and target DNA.

The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.

Conserved cell types with divergent features in human versus mouse cortex.

Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease. Here we used single-nucleus RNA-sequencing analysis to perform a comprehensive study of cell types in the middle temporal gyrus of human cortex. We identified a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse, with excitatory types being less layer-restricted than expected. Comparison to similar mouse cortex single-cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of properties of human cell types. Despite this general conservation, we also found extensive differences between homologous human and mouse cell types, including marked alterations in proportions, laminar distributions, gene expression and morphology. These species-specific features emphasize the importance of directly studying human brain.

Mechanisms and consequences of mRNA destabilization during viral infections.

During viral infection there is dynamic interplay between the virus and the host to regulate gene expression. In many cases, the host induces the expression of antiviral genes to combat infection, while the virus uses "host shut-off" systems to better compete for cellular resources and to limit the induction of the host antiviral response. Viral mechanisms for host shut-off involve targeting translation, altering host RNA processing, and/or inducing the degradation of host mRNAs. In this review, we discuss the diverse mechanisms viruses use to degrade host mRNAs. In addition, the widespread degradation of host mRNAs can have common consequences including the accumulation of RNA binding proteins in the nucleus, which leads to altered RNA processing, mRNA export, and changes to transcription.

SARS-CoV-2 Nsp1 mediated mRNA degradation requires mRNA interaction with the ribosome.

Nsp1 is a SARS-CoV-2 host shutoff factor that both represses cellular translation and promotes host RNA decay. However, it is unclear how these two activities are connected and interact with normal translation processes. Here, we performed mutational analyses of Nsp1, and these revealed that both the N and C terminal domains of Nsp1 are important for translational repression. Furthermore, we demonstrate that specific residues in the N terminal domain are required for cellular RNA degradation but not bulk translation shutoff of host mRNAs, thereby separating RNA degradation from translation repression. We also present evidence that Nsp1 mediated RNA degradation requires engagement of the ribosome with mRNA. First, we observe that cytosolic lncRNAs, which are not translated, escape Nsp1 mediated degradation. Second, inhibition of translation elongation with emetine does not prevent Nsp1 mediated degradation, while blocking translation initiation before 48S ribosome loading reduces mRNA degradation. Taken together, we suggest that Nsp1 represses translation and promotes mRNA degradation only after ribosome engagement with the mRNA. This raises the possibility that Nsp1 may trigger RNA degradation through pathways that recognize stalled ribosomes.

Fixed single-cell transcriptomic characterization of human radial glial diversity.

The diverse progenitors that give rise to the human neocortex have been difficult to characterize because progenitors, particularly radial glia (RG), are rare and are defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed Fixed and Recovered Intact Single-cell RNA (FRISCR), a method for profiling the transcriptomes of individual fixed, stained and sorted cells. Using FRISCR, we profiled primary human RG that constitute only 1% of the midgestation cortex and classified them as ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identified vRG and oRG markers and molecular profiles, an essential step for understanding human neocortical progenitor development. FRISCR allows targeted single-cell profiling of any tissues that lack live-cell markers.

Transcriptomic cytoarchitecture reveals principles of human neocortex organization.

Variation in cytoarchitecture is the basis for the histological definition of cortical areas. We used single cell transcriptomics and performed cellular characterization of the human cortex to better understand cortical areal specialization. Single-nucleus RNA-sequencing of 8 areas spanning cortical structural variation showed a highly consistent cellular makeup for 24 cell subclasses. However, proportions of excitatory neuron subclasses varied substantially, likely reflecting differences in connectivity across primary sensorimotor and association cortices. Laminar organization of astrocytes and oligodendrocytes also differed across areas. Primary visual cortex showed characteristic organization with major changes in the excitatory to inhibitory neuron ratio, expansion of layer 4 excitatory neurons, and specialized inhibitory neurons. These results lay the groundwork for a refined cellular and molecular characterization of human cortical cytoarchitecture and areal specialization.

Test sensitivity is secondary to frequency and turnaround time for COVID-19 screening.

The COVID-19 pandemic has created a public health crisis. Because SARS-CoV-2 can spread from individuals with presymptomatic, symptomatic, and asymptomatic infections, the reopening of societies and the control of virus spread will be facilitated by robust population screening, for which virus testing will often be central. After infection, individuals undergo a period of incubation during which viral titers are too low to detect, followed by exponential viral growth, leading to peak viral load and infectiousness and ending with declining titers and clearance. Given the pattern of viral load kinetics, we model the effectiveness of repeated population screening considering test sensitivities, frequency, and sample-to-answer reporting time. These results demonstrate that effective screening depends largely on frequency of testing and speed of reporting and is only marginally improved by high test sensitivity. We therefore conclude that screening should prioritize accessibility, frequency, and sample-to-answer time; analytical limits of detection should be secondary.

HIV and COVID-19: review of clinical course and outcomes.

Background:Understanding the relationship between HIV and SARS-CoV-2 has important public health implications.Objective:To summarize current research on COVID-19 among people with HIV (PWH) as published through 15 July 2021.Methods: We conducted a search of PubMed, Scopus, preprint databases (medRxiv, bioRxiv), and the references of publications found using key terms relevant to COVID-19 ('COVID-19' OR 'SARS-CoV-2' OR 'coronavirus') AND to HIV ('HIV' OR 'Human Immunodeficiency Virus' OR 'AIDS' OR 'Acquired Immunodeficiency Syndrome'). We summarized all articles that reported data or opinions on SARS-CoV-2 and HIV coinfection.Conclusions: Although many initial case series and cohort studies found no increased risk for SARS-CoV-2 infection or severe COVID-19 outcomes among PWH, recent studies have signaled an increased risk for severe COVID-19 disease progression even in the setting of well-controlled HIV. Whether this is due to the increased prevalence of comorbidities in PWH and other social determinants of health is unknown. These conflicting findings highlight the continued need for COVID-19 related research among PWH that addresses COVID-19 disease course as well as exacerbation of existing comorbidities already disproportionately represented among PWH.

Genome-wide binding analysis of 195 DNA binding proteins reveals "reservoir" promoters and human specific SVA-repeat family regulation.

A key aspect in defining cell state is the complex choreography of DNA binding events in a given cell type, which in turn establishes a cell-specific gene-expression program. Here we wanted to take a deep analysis of DNA binding events and transcriptional output of a single cell state (K562 cells). To this end we re-analyzed 195 DNA binding proteins contained in ENCODE data. We used standardized analysis pipelines, containerization, and literate programming with R Markdown for reproducibility and rigor. Our approach validated many findings from previous independent studies, underscoring the importance of ENCODE's goals in providing these reproducible data resources. We also had several new findings including: (i) 1,362 promoters, which we refer to as 'reservoirs,' that are defined by having up to 111 different DNA binding-proteins localized on one promoter, yet do not have any expression of steady-state RNA (ii) Reservoirs do not overlap super-enhancer annotations and distinct have distinct properties from super-enhancers. (iii) The human specific SVA repeat element may have been co-opted for enhancer regulation and is highly transcribed in PRO-seq and RNA-seq. Collectively, this study performed by the students of a CU Boulder computational biology class (BCHM 5631 -Spring 2020) demonstrates the value of reproducible findings and how resources like ENCODE that prioritize data standards can foster new findings with existing data in a didactic environment.

Single-cell and single-nucleus RNA-seq uncovers shared and distinct axes of variation in dorsal LGN neurons in mice, non-human primates, and humans.

Abundant evidence supports the presence of at least three distinct types of thalamocortical (TC) neurons in the primate dorsal lateral geniculate nucleus (dLGN) of the thalamus, the brain region that conveys visual information from the retina to the primary visual cortex (V1). Different types of TC neurons in mice, humans, and macaques have distinct morphologies, distinct connectivity patterns, and convey different aspects of visual information to the cortex. To investigate the molecular underpinnings of these cell types, and how these relate to differences in dLGN between human, macaque, and mice, we profiled gene expression in single nuclei and cells using RNA-sequencing. These efforts identified four distinct types of TC neurons in the primate dLGN: magnocellular (M) neurons, parvocellular (P) neurons, and two types of koniocellular (K) neurons. Despite extensively documented morphological and physiological differences between M and P neurons, we identified few genes with significant differential expression between transcriptomic cell types corresponding to these two neuronal populations. Likewise, the dominant feature of TC neurons of the adult mouse dLGN is high transcriptomic similarity, with an axis of heterogeneity that aligns with core vs. shell portions of mouse dLGN. Together, these data show that transcriptomic differences between principal cell types in the mature mammalian dLGN are subtle relative to the observed differences in morphology and cortical projection targets. Finally, alignment of transcriptome profiles across species highlights expanded diversity of GABAergic neurons in primate versus mouse dLGN and homologous types of TC neurons in primates that are distinct from TC neurons in mouse.

Test sensitivity is secondary to frequency and turnaround time for COVID-19 surveillance.

The COVID-19 pandemic has created a public health crisis. Because SARS-CoV-2 can spread from individuals with pre-symptomatic, symptomatic, and asymptomatic infections, the re-opening of societies and the control of virus spread will be facilitated by robust surveillance, for which virus testing will often be central. After infection, individuals undergo a period of incubation during which viral titers are usually too low to detect, followed by an exponential viral growth, leading to a peak viral load and infectiousness, and ending with declining viral levels and clearance. Given the pattern of viral load kinetics, we model surveillance effectiveness considering test sensitivities, frequency, and sample-to-answer reporting time. These results demonstrate that effective surveillance depends largely on frequency of testing and the speed of reporting, and is only marginally improved by high test sensitivity. We therefore conclude that surveillance should prioritize accessibility, frequency, and sample-to-answer time; analytical limits of detection should be secondary.

Transcriptomic evidence that von Economo neurons are regionally specialized extratelencephalic-projecting excitatory neurons.

von Economo neurons (VENs) are bipolar, spindle-shaped neurons restricted to layer 5 of human frontoinsula and anterior cingulate cortex that appear to be selectively vulnerable to neuropsychiatric and neurodegenerative diseases, although little is known about other VEN cellular phenotypes. Single nucleus RNA-sequencing of frontoinsula layer 5 identifies a transcriptomically-defined cell cluster that contained VENs, but also fork cells and a subset of pyramidal neurons. Cross-species alignment of this cell cluster with a well-annotated mouse classification shows strong homology to extratelencephalic (ET) excitatory neurons that project to subcerebral targets. This cluster also shows strong homology to a putative ET cluster in human temporal cortex, but with a strikingly specific regional signature. Together these results suggest that VENs are a regionally distinctive type of ET neuron. Additionally, we describe the first patch clamp recordings of VENs from neurosurgically-resected tissue that show distinctive intrinsic membrane properties relative to neighboring pyramidal neurons.

Single-nucleus and single-cell transcriptomes compared in matched cortical cell types.

Transcriptomic profiling of complex tissues by single-nucleus RNA-sequencing (snRNA-seq) affords some advantages over single-cell RNA-sequencing (scRNA-seq). snRNA-seq provides less biased cellular coverage, does not appear to suffer cell isolation-based transcriptional artifacts, and can be applied to archived frozen specimens. We used well-matched snRNA-seq and scRNA-seq datasets from mouse visual cortex to compare cell type detection. Although more transcripts are detected in individual whole cells (~11,000 genes) than nuclei (~7,000 genes), we demonstrate that closely related neuronal cell types can be similarly discriminated with both methods if intronic sequences are included in snRNA-seq analysis. We estimate that the nuclear proportion of total cellular mRNA varies from 20% to over 50% for large and small pyramidal neurons, respectively. Together, these results illustrate the high information content of nuclear RNA for characterization of cellular diversity in brain tissues.

Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type.

We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large 'rosehip'-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1+CCK+, CNR1-SST-CALB2-PVALB-) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.

Single-Cell Profiling of an In Vitro Model of Human Interneuron Development Reveals Temporal Dynamics of Cell Type Production and Maturation.

GABAergic interneurons are essential for neural circuit function, and their loss or dysfunction is implicated in human neuropsychiatric disease. In vitro methods for interneuron generation hold promise for studying human cellular and functional properties and, ultimately, for therapeutic cell replacement. Here we describe a protocol for generating cortical interneurons from hESCs and analyze the properties and maturation time course of cell types using single-cell RNA-seq. We find that the cell types produced mimic in vivo temporal patterns of neuron and glial production, with immature progenitors and neurons observed early and mature cortical neurons and glial cell types produced late. By comparing the transcriptomes of immature interneurons to those of more mature neurons, we identified genes important for human interneuron differentiation. Many of these genes were previously implicated in neurodevelopmental and neuropsychiatric disorders.

A Single-Cell Roadmap of Lineage Bifurcation in Human ESC Models of Embryonic Brain Development.

During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/ß-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders.