A Broad-Spectrum Inhibitor of CRISPR-Cas9.

CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9.

A scoutRNA Is Required for Some Type V CRISPR-Cas Systems.

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.

Interferon-γ Represses M2 Gene Expression in Human Macrophages by Disassembling Enhancers Bound by the Transcription Factor MAF.

Mechanisms by which interferon (IFN)-γ activates genes to promote macrophage activation are well studied, but little is known about mechanisms and functions of IFN-γ-mediated gene repression. We used an integrated transcriptomic and epigenomic approach to analyze chromatin accessibility, histone modifications, transcription-factor binding, and gene expression in IFN-γ-primed human macrophages. IFN-γ suppressed basal expression of genes corresponding to an "M2"-like homeostatic and reparative phenotype. IFN-γ repressed genes by suppressing the function of enhancers enriched for binding by transcription factor MAF. Mechanistically, IFN-γ disassembled a subset of enhancers by inducing coordinate suppression of binding by MAF, lineage-determining transcription factors, and chromatin accessibility. Genes associated with MAF-binding enhancers were suppressed in macrophages isolated from rheumatoid-arthritis patients, revealing a disease-associated signature of IFN-γ-mediated repression. These results identify enhancer inactivation and disassembly as a mechanism of IFN-γ-mediated gene repression and reveal that MAF regulates the macrophage enhancer landscape and is suppressed by IFN-γ to augment macrophage activation.

Prevention of antimicrobial prescribing among infants following maternal vaccination against respiratory syncytial virus.

SignificanceStrategies to reduce consumption of antimicrobial drugs are needed to contain the growing burden of antimicrobial resistance. Respiratory syncytial virus (RSV) is a prominent cause of upper and lower respiratory tract infections, as a single agent and in conjunction with bacterial pathogens, and may thus contribute to the burden of both inappropriately treated viral infections and appropriately treated polymicrobial infections involving bacteria. In a double-blind, randomized, placebo-controlled trial, administering an RSV vaccine to pregnant mothers reduced antimicrobial prescribing among their infants by 12.9% over the first 3 mo of life. Our findings implicate RSV as an important contributor to antimicrobial exposure among infants and demonstrate that this exposure is preventable by use of effective maternal vaccines against RSV.

The Relationship between Event Boundary Strength and Pattern Shifts across the Cortical Hierarchy During Naturalistic Movie-viewing.

Our continuous experience is spontaneously segmented by the brain into discrete events. However, the beginning of a new event (an event boundary) is not always sharply identifiable: Phenomenologically, event boundaries vary in salience. How are the response profiles of cortical areas at event boundaries modulated by boundary strength during complex, naturalistic movie-viewing? Do cortical responses scale in a graded manner with boundary strength, or do they merely detect boundaries in a binary fashion? We measured "cortical boundary shifts" as transient changes in multivoxel patterns at event boundaries with different strengths (weak, moderate, and strong), determined by across-participant agreement. Cortical regions with different processing timescales were examined. In auditory areas, which have short timescales, cortical boundary shifts exhibited a clearly graded profile in both group-level and individual-level analyses. In cortical areas with long timescales, including the default mode network, boundary strength modulated pattern shift magnitude at the individual participant level. We also observed a positive relationship between boundary strength and the extent of temporal alignment of boundary shifts across different levels of the cortical hierarchy. In addition, hippocampal activity was highest at event boundaries for which cortical boundary shifts were most aligned across hierarchical levels. Overall, we found that event boundary strength modulated cortical pattern shifts strongly in sensory areas and more weakly in higher-level areas and that stronger boundaries were associated with greater alignment of these shifts across the cortical hierarchy.

Causal and Chronological Relationships Predict Memory Organization for Nonlinear Narratives.

While recounting an experience, one could employ multiple strategies to transition from one part to the next. For instance, if the event was learned out of linear order, one could recall events according to the time they were learned (temporal), similar events (semantic), events occurring nearby in time (chronological), or events produced by the current event (causal). To disentangle the importance of these factors, we had participants watch the nonlinear narrative, "Memento," under different task instructions and presentation orders. For each scene of the film, we also separately computed semantic and causal networks. From these derivations, we contrasted the evidence for temporal, semantic, chronological, or causal strategies during recall. Critically, there was stronger evidence for the causal and chronological strategies than semantic or temporal strategies. Moreover, the causal and chronological strategies outperformed the temporal one even after asking participants to recall the film in the presented order, underscoring the fundamental nature of causal structure in scaffolding understanding and organizing recall. Nevertheless, time still marginally predicted recall transitions, suggesting it still operates as a weak signal in the presence of more salient forms of structure. In addition, semantic and causal network properties predicted scene memorability, including showing a stronger role for causes of an event than its outgoing effects. In summary, these effects highlight the importance of accounting for complex, causal networks in knowledge building and memory.

Accelerating attribute-focused process and product development through the development and deployment of autonomous process analytical technology platform system.

Enabling real-time monitoring and control of the biomanufacturing processes through product quality insights continues to be an area of focus in the biopharmaceutical industry. The goal is to manufacture products with the desired quality attributes. To realize this rigorous attribute-focused Quality by Design approach, it is critical to support the development of processes that consistently deliver high-quality products and facilitate product commercialization. Time delays associated with offline analytical testing can limit the speed of process development. Thus, developing and deploying analytical technology is necessary to accelerate process development. In this study, we have developed the micro sequential injection process analyzer and the automatic assay preparation platform system. These innovations address the unmet need for an automatic, online, real-time sample acquisition and preparation platform system for in-process monitoring, control, and release of biopharmaceuticals. These systems can also be deployed in laboratory areas as an offline analytical system and on the manufacturing floor to enable rapid testing and release of products manufactured in a good manufacturing practice environment.

Respiratory Syncytial Virus Vaccination during Pregnancy and Effects in Infants.

BACKGROUND: Respiratory syncytial virus (RSV) is the dominant cause of severe lower respiratory tract infection in infants, with the most severe cases concentrated among younger infants. METHODS: Healthy pregnant women, at 28 weeks 0 days through 36 weeks 0 days of gestation, with an expected delivery date near the start of the RSV season, were randomly assigned in an overall ratio of approximately 2:1 to receive a single intramuscular dose of RSV fusion (F) protein nanoparticle vaccine or placebo. Infants were followed for 180 days to assess outcomes related to lower respiratory tract infection and for 364 days to assess safety. The primary end point was RSV-associated, medically significant lower respiratory tract infection up to 90 days of life, and the primary analysis of vaccine efficacy against the primary end point was performed in the per-protocol population of infants (prespecified criterion for success, lower bound of the 97.52% confidence interval [CI] of ≥30%). RESULTS: A total of 4636 women underwent randomization, and there were 4579 live births. During the first 90 days of life, the percentage of infants with RSV-associated, medically significant lower respiratory tract infection was 1.5% in the vaccine group and 2.4% in the placebo group (vaccine efficacy, 39.4%; 97.52% CI, -1.0 to 63.7; 95% CI, 5.3 to 61.2). The corresponding percentages for RSV-associated lower respiratory tract infection with severe hypoxemia were 0.5% and 1.0% (vaccine efficacy, 48.3%; 95% CI, -8.2 to 75.3), and the percentages for hospitalization for RSV-associated lower respiratory tract infection were 2.1% and 3.7% (vaccine efficacy, 44.4%; 95% CI, 19.6 to 61.5). Local injection-site reactions among the women were more common with vaccine than with placebo (40.7% vs. 9.9%), but the percentages of participants who had other adverse events were similar in the two groups. CONCLUSIONS: RSV F protein nanoparticle vaccination in pregnant women did not meet the prespecified success criterion for efficacy against RSV-associated, medically significant lower respiratory tract infection in infants up to 90 days of life. The suggestion of a possible benefit with respect to other end-point events involving RSV-associated respiratory disease in infants warrants further study. (Funded by Novavax and the Bill and Melinda Gates Foundation; ClinicalTrials.gov NCT02624947.).

Tumor necrosis factor induces GSK3 kinase-mediated cross-tolerance to endotoxin in macrophages.

Endotoxin tolerance, a key mechanism for suppressing excessive inflammatory cytokine production, is induced by prior exposure of macrophages to Toll-like receptor (TLR) ligands. Induction of cross-tolerance to endotoxin by endogenous cytokines has not been investigated. Here we show that prior exposure to tumor necrosis factor (TNF) induced a tolerant state in macrophages, with less cytokine production after challenge with lipopolysaccharide (LPS) and protection from LPS-induced death. TNF-induced cross-tolerization was mediated by suppression of LPS-induced signaling and chromatin remodeling. TNF-induced cross-tolerance was dependent on the kinase GSK3, which suppressed chromatin accessibility and promoted rapid termination of signaling via the transcription factor NF-κB by augmenting negative feedback by the signaling inhibitors A20 and IκBα. Our results demonstrate an unexpected homeostatic function for TNF and a GSK3-mediated mechanism for the prevention of prolonged and excessive inflammation.

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we design a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

Naturalistic Audio-Movies reveal common spatial organization across "visual" cortices of different blind individuals.

Occipital cortices of different sighted people contain analogous maps of visual information (e.g. foveal vs. peripheral). In congenital blindness, "visual" cortices respond to nonvisual stimuli. Do visual cortices of different blind people represent common informational maps? We leverage naturalistic stimuli and inter-subject pattern similarity analysis to address this question. Blindfolded sighted (n = 22) and congenitally blind (n = 22) participants listened to 6 sound clips (5-7 min each): 3 auditory excerpts from movies; a naturalistic spoken narrative; and matched degraded auditory stimuli (Backwards Speech, scrambled sentences), during functional magnetic resonance imaging scanning. We compared the spatial activity patterns evoked by each unique 10-s segment of the different auditory excerpts across blind and sighted people. Segments of meaningful naturalistic stimuli produced distinctive activity patterns in frontotemporal networks that were shared across blind and across sighted individuals. In the blind group only, segment-specific, cross-subject patterns emerged in visual cortex, but only for meaningful naturalistic stimuli and not Backwards Speech. Spatial patterns of activity within visual cortices are sensitive to time-varying information in meaningful naturalistic auditory stimuli in a broadly similar manner across blind individuals.

Conservative and liberal attitudes drive polarized neural responses to political content.

People tend to interpret political information in a manner that confirms their prior beliefs, a cognitive bias that contributes to rising political polarization. In this study, we combined functional magnetic resonance imaging with semantic content analyses to investigate the neural mechanisms that underlie the biased processing of real-world political content. We scanned American participants with conservative-leaning or liberal-leaning immigration attitudes while they watched news clips, campaign ads, and public speeches related to immigration policy. We searched for evidence of "neural polarization": activity in the brain that diverges between people who hold liberal versus conservative political attitudes. Neural polarization was observed in the dorsomedial prefrontal cortex (DMPFC), a brain region associated with the interpretation of narrative content. Neural polarization in the DMPFC intensified during moments in the videos that included risk-related and moral-emotional language, highlighting content features most likely to drive divergent interpretations between conservatives and liberals. Finally, participants whose DMPFC activity closely matched that of the average conservative or the average liberal participant were more likely to change their attitudes in the direction of that group's position. Our work introduces a multimethod approach to study the neural basis of political cognition in naturalistic settings. Using this approach, we characterize how political attitudes biased information processing in the brain, the language most likely to drive polarized neural responses, and the consequences of biased processing for attitude change. Together, these results shed light on the psychological and neural underpinnings of how identical information is interpreted differently by conservatives and liberals.

High-Order Areas and Auditory Cortex Both Represent the High-Level Event Structure of Music.

Recent fMRI studies of event segmentation have found that default mode regions represent high-level event structure during movie watching. In these regions, neural patterns are relatively stable during events and shift at event boundaries. Music, like narratives, contains hierarchical event structure (e.g., sections are composed of phrases). Here, we tested the hypothesis that brain activity patterns in default mode regions reflect the high-level event structure of music. We used fMRI to record brain activity from 25 participants (male and female) as they listened to a continuous playlist of 16 musical excerpts and additionally collected annotations for these excerpts by asking a separate group of participants to mark when meaningful changes occurred in each one. We then identified temporal boundaries between stable patterns of brain activity using a hidden Markov model and compared the location of the model boundaries to the location of the human annotations. We identified multiple brain regions with significant matches to the observer-identified boundaries, including auditory cortex, medial prefrontal cortex, parietal cortex, and angular gyrus. From these results, we conclude that both higher-order and sensory areas contain information relating to the high-level event structure of music. Moreover, the higher-order areas in this study overlap with areas found in previous studies of event perception in movies and audio narratives, including regions in the default mode network.

COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme.

Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K/Q/A, and N501Y mutations, at least one of which is found in nearly all major variants. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all targeted SNP mutations. An analysis pipeline for CRISPR-based SNP identification from 261 clinical samples yielded a SNP concordance of 97.3% and agreement of 98.9% (258 of 261) for SARS-CoV-2 lineage classification, using SARS-CoV-2 whole-genome sequencing and/or real-time RT-PCR as test comparators. We also showed that detection of the single E484A mutation was necessary and sufficient to accurately identify Omicron from other major circulating variants in patient samples. These findings demonstrate the utility of CRISPR-based DETECTR as a faster and simpler diagnostic method compared with sequencing for SARS-CoV-2 variant identification in clinical and public health laboratories.

Synergistic activation of inflammatory cytokine genes by interferon-γ-induced chromatin remodeling and toll-like receptor signaling.

Synergistic activation of inflammatory cytokine genes by interferon-γ (IFN-γ) and Toll-like receptor (TLR) signaling is important for innate immunity and inflammatory disease pathogenesis. Enhancement of TLR signaling, a previously proposed mechanism, is insufficient to explain strong synergistic activation of cytokine production in human macrophages. Rather, we found that IFN-γ induced sustained occupancy of transcription factors STAT1, IRF-1, and associated histone acetylation at promoters and enhancers at the TNF, IL6, and IL12B loci. This priming of chromatin did not activate transcription but greatly increased and prolonged recruitment of TLR4-induced transcription factors and RNA polymerase II to gene promoters and enhancers. Priming sensitized cytokine transcription to suppression by Jak inhibitors. Genome-wide analysis revealed pervasive priming of regulatory elements by IFN-γ and linked coordinate priming of promoters and enhancers with synergistic induction of transcription. Our results provide a synergy mechanism whereby IFN-γ creates a primed chromatin environment to augment TLR-induced gene transcription.

Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain.

Understanding the conformational dynamics of CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is of the utmost importance for improving its genome editing capability. Here, molecular dynamics simulations performed using Anton-2 - a specialized supercomputer capturing micro-to-millisecond biophysical events in real time and at atomic-level resolution - reveal the activation process of the endonuclease Cas9 toward DNA cleavage. Over the unbiased simulation, we observe that the spontaneous approach of the catalytic domain HNH to the DNA cleavage site is accompanied by a remarkable structural remodeling of the recognition (REC) lobe, which exerts a key role for DNA cleavage. Specifically, the significant conformational changes and the collective conformational dynamics of the REC lobe indicate a mechanism by which the REC1-3 regions 'sense' nucleic acids, 'regulate' the HNH conformational transition, and ultimately 'lock' the HNH domain at the cleavage site, contributing to its catalytic competence. By integrating additional independent simulations and existing experimental data, we provide a solid validation of the activated HNH conformation, which had been so far poorly characterized, and we deliver a comprehensive understanding of the role of REC1-3 in the activation process. Considering the importance of the REC lobe in the specificity of Cas9, this study poses the basis for fully understanding how the REC components control the cleavage of off-target sequences, laying the foundation for future engineering efforts toward improved genome editing.

Temporal integration of narrative information in a hippocampal amnesic patient.

Default network regions appear to integrate information over time windows of 30 â€‹s or more during narrative listening. Does this long-timescale capability require the hippocampus? Amnesic behavior suggests that regions other than the hippocampus can independently support some online processing when input is continuous and semantically rich: amnesics can participate in conversations and tell stories spanning minutes, and when tested immediately on recently heard prose they are able to retain some information. We hypothesized that default network regions can integrate the semantically coherent information of a narrative across long time windows, even in the absence of an intact hippocampus. To test this prediction, we measured BOLD activity in the brain of a hippocampal amnesic patient (D.A.) and healthy control participants while they listened to a 7 min narrative. The narrative was played either in its intact form, or as a paragraph-scrambled version, which has been previously shown to interfere with the long-range temporal dependencies in default network activity. In the intact story condition, D.A.'s moment-by-moment BOLD activity spatial patterns were similar to those of controls in low-level auditory cortex as well as in some high-level default network regions (including lateral and medial posterior parietal cortex). Moreover, as in controls, D.A.'s response patterns in medial and lateral posterior parietal cortex were disrupted when paragraphs of the story were presented in a shuffled order, suggesting that activity in these areas did depend on information from 30 â€‹s or more in the past. Together, these results suggest that some default network cortical areas can integrate information across long timescales, even when the hippocampus is severely damaged.

Propagation of Information Along the Cortical Hierarchy as a Function of Attention While Reading and Listening to Stories.

How does attention route information from sensory to high-order areas as a function of task, within the relatively fixed topology of the brain? In this study, participants were simultaneously presented with 2 unrelated stories-one spoken and one written-and asked to attend one while ignoring the other. We used fMRI and a novel intersubject correlation analysis to track the spread of information along the processing hierarchy as a function of task. Processing the unattended spoken (written) information was confined to auditory (visual) cortices. In contrast, attending to the spoken (written) story enhanced the stimulus-selective responses in sensory regions and allowed it to spread into higher-order areas. Surprisingly, we found that the story-specific spoken (written) responses for the attended story also reached secondary visual (auditory) regions of the unattended sensory modality. These results demonstrate how attention enhances the processing of attended input and allows it to propagate across brain areas.

Overlap between hippocampal pre-encoding and encoding patterns supports episodic memory.

It is well-established that whether the information will be remembered or not depends on the extent to which the learning context is reinstated during post-encoding rest and/or at retrieval. It has yet to be determined, however, if the fundamental importance of contextual reinstatement to memory extends to periods of spontaneous neurocognitive activity prior to learning. We thus asked whether memory performance can be predicted by the extent to which spontaneous pre-encoding neural patterns resemble patterns elicited during encoding. Individuals studied and retrieved lists of words while undergoing fMRI-scanning. Multivoxel hippocampal patterns during resting periods prior to encoding resembled hippocampal patterns at encoding most strongly for items that were subsequently remembered. Furthermore, across subjects, the magnitude of similarity correlated with a behavioral measure of episodic recall. The results indicate that the neural context before learning is an important determinant of memory.

Learning Naturalistic Temporal Structure in the Posterior Medial Network.

The posterior medial network is at the apex of a temporal integration hierarchy in the brain, integrating information over many seconds of viewing intact, but not scrambled, movies. This has been interpreted as an effect of temporal structure. Such structure in movies depends on preexisting event schemas, but temporal structure can also arise de novo from learning. Here, we examined the relative role of schema-consistent temporal structure and arbitrary but consistent temporal structure on the human posterior medial network. We tested whether, with repeated viewing, the network becomes engaged by scrambled movies with temporal structure. Replicating prior studies, activity in posterior medial regions was immediately locked to stimulus structure upon exposure to intact, but not scrambled, movies. However, for temporally structured scrambled movies, functional coupling within the network increased across stimulus repetitions, rising to the level of intact movies. Thus, temporal structure is a key determinant of network dynamics and function in the posterior medial network.