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Hitching a ride with a retroelement Retroviruses and retroelements have inserted their genetic code into mammalian genomes throughout evolution. Although many of these integrated virus-like sequences pose a threat to genomic integrity, some have been retooled by mammalian cells to perform essential roles in development. Segel et al . found that one of these retroviral-like proteins, PEG10, directly binds to and secretes its own mRNA in extracellular virus–like capsids. These virus-like particles were then pseudotyped with fusogens to deliver functional mRNA cargos to mammalian cells. This potentially provides an endogenous vector for RNA-based gene therapy. —DJ

Understanding genome organization requires integration of DNA sequence and three-dimensional spatial context; however, existing genome-wide methods lack either base pair sequence resolution or direct spatial localization. Here, we describe in situ genome sequencing (IGS), a method for simultaneously sequencing and imaging genomes within intact biological samples. We applied IGS to human fibroblasts and early mouse embryos, spatially localizing thousands of genomic loci in individual nuclei. Using these data, we characterized parent-specific changes in genome structure across embryonic stages, revealed single-cell chromatin domains in zygotes, and uncovered epigenetic memory of global chromosome positioning within individual embryos. These results demonstrate how IGS can directly connect sequence and structure across length scales from single base pairs to whole organisms.

IscB proteins are putative nucleases encoded in a distinct family of IS200/IS605 transposons and are likely ancestors of the RNA-guided endonuclease Cas9, but the functions of IscB and its interactions with any RNA remain uncharacterized. Using evolutionary analysis, RNA sequencing, and biochemical experiments, we reconstructed the evolution of CRISPR-Cas9 systems from IS200/IS605 transposons. We found that IscB uses a single noncoding RNA for RNA-guided cleavage of double-stranded DNA and can be harnessed for genome editing in human cells. We also demonstrate the RNA-guided nuclease activity of TnpB, another IS200/IS605 transposon-encoded protein and the likely ancestor of Cas12 endonucleases. This work reveals a widespread class of transposon-encoded RNA-guided nucleases, which we name OMEGA (obligate mobile element–guided activity), with strong potential for developing as biotechnologies.

© 2020 American Association for the Advancement of Science. All rights reserved. The number of disease risk genes and loci identified through human genetic studies far outstrips the capacity to systematically study their functions. We applied a scalable genetic screening approach, in vivo Perturb-Seq, to functionally evaluate 35 autism spectrum disorder/neurodevelopmental delay (ASD/ND) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing mouse brain in utero, followed by single-cell RNAsequencing of perturbed cells in the postnatal brain. We identified cell type-specific and evolutionarily conserved gene modules from both neuronal and glial cell classes. Recurrent gene modules and cell types are affected across this cohort of perturbations, representing key cellular effects across sets of ASD/ND risk genes. In vivo Perturb-Seq allows us to investigate how diverse mutations affect cell types and states in the developing organism.

© 2019 American Association for the Advancement of Science. All Rights Reserved. Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.

Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.

Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses.

Synthetic tissue-hydrogel methods have enabled superresolution investigation of biological systems using diffraction-limited microscopy. However, chemical modification by fixatives can cause loss of antigenicity, limiting molecular interrogation of the tissue gel. Here, we present epitope-preserving magnified analysis of proteome (eMAP) that uses purely physical tissue-gel hybridization to minimize the loss of antigenicity while allowing permanent anchoring of biomolecules. We achieved success rates of 96% and 94% with synaptic antibodies for mouse and marmoset brains, respectively. Maximal preservation of antigenicity allows imaging of nanoscopic architectures in 1000-fold expanded tissues without additional signal amplification. eMAP-processed tissue gel can endure repeated staining and destaining without epitope loss or structural damage, enabling highly multiplexed proteomic analysis. We demonstrated the utility of eMAP as a nanoscopic proteomic interrogation tool by investigating molecular heterogeneity in inhibitory synapses in the mouse brain neocortex and characterizing the spatial distributions of synaptic proteins within synapses in mouse and marmoset brains.

Copyright © 2020 The Authors, some rights reserved. Parkinson's disease is characterized by decreased dopamine and increased beta-band oscillatory activity accompanying debilitating motor and mood impairments. Coordinate dopamine-beta opposition is considered a normative rule for basal ganglia function. We report a breakdown of this rule. We developed multimodal systems allowing the first simultaneous, chronic recordings of dopamine release and beta-band activity in the striatum of nonhuman primates during behavioral performance. Dopamine and beta signals were anticorrelated over secondslong time frames, in agreement with the posited rule, but at finer time scales, we identified conditions in which these signals were modulated with the same polarity. These measurements demonstrated that task-elicited beta suppressions preceded dopamine peaks and that relative dopamine-beta timing and polarity depended on reward value, performance history, movement, and striatal domain. These findings establish a new view of coordinate dopamine and beta signaling operations, critical to guide novel strategies for diagnosing and treating Parkinson's disease and related neurodegenerative disorders.

Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in <2 hours. We further demonstrate that SHERLOCK can distinguish the four DENV serotypes, as well as region-specific strains of ZIKV from the 2015–2016 pandemic. Finally, we report the rapid (<1 week) design and testing of instrument-free assays to detect clinically relevant viral single-nucleotide polymorphisms., NIH (Grants AI-100190, 1R01-HG009761, 1R01-MH110049, and 1DP1-HL141201)

Programmable RNA editing enables reversible recoding of RNA information for research and disease treatment. Previously, we developed a programmable adenosine-to-inosine (A-to-I) RNA editing approach by fusing catalytically inactivate RNA-targeting CRISPR-Cas13 (dCas13) with the adenine deaminase domain of ADAR2. Here, we report a cytidine-to-uridine (C-to-U) RNA editor, referred to as RNA Editing for Specific C-to-U Exchange (RESCUE), by directly evolving ADAR2 into a cytidine deaminase. RESCUE doubles the number of mutations targetable by RNA editing and enables modulation of phosphosignaling-relevant residues. We apply RESCUE to drive b-catenin activation and cellular growth. Furthermore, RESCUE retains A-to-I editing activity, enabling multiplexed C-to-U and A-to-I editing through the use of tailored guide RNAs., National Institutes of Health (Grants 1F30-CA210382, 1R01-HG009761, 1R01-MH110049, and 1DP1-HL141201)

Particular deep artificial neural networks (ANNs) are today’s most accurate models of the primate brain’s ventral visual stream. Using an ANN-driven image synthesis method, we found that luminous power patterns (i.e., images) can be applied to primate retinae to predictably push the spiking activity of targeted V4 neural sites beyond naturally occurring levels. This method, although not yet perfect, achieves unprecedented independent control of the activity state of entire populations of V4 neural sites, even those with overlapping receptive fields. These results show how the knowledge embedded in today’s ANN models might be used to noninvasively set desired internal brain states at neuron-level resolution, and suggest that more accurate ANN models would produce even more accurate control., National Eye Institute (Grant R01-EY014970), Office of Naval Research (Grant MURI-114407)

The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third G is compensated by newly introduced non-base-specific interactions, enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells., NICHD (Grants P01HD087157, R01HD088412), NIMH (Grants 5DP1-MH100706,1R01-MH110049), NIDDK (Grant 5R01DK097768-03)

The field of bioelectronic medicines seeks to modulate electrical signaling within peripheral organs, providing temporally precise control of physiological functions. This is usually accomplished with implantable devices, which are often unsuitable for interfacing with soft and highly vascularized organs. Here, we demonstrate an alternative strategy for modulating peripheral organ function, which relies on the endogenous expression of a heat-sensitive cation channel, transient receptor potential vanilloid family member 1 (TRPV1), and heat dissipation by magnetic nanoparticles (MNPs) in remotely applied alternating magnetic fields. We use this approach to wirelessly control adrenal hormone secretion in genetically intact rats. TRPV1-dependent calcium influx into the cells of adrenal cortex and medulla is sufficient to drive rapid release of corticosterone and (nor)epinephrine. As altered levels of these hormones have been correlated with mental conditions such as posttraumatic stress disorder and major depression, our approach may facilitate the investigation of physiological and psychological impacts of stress., National Institutes of Health BRAIN Initiative (Grant 1R01MH111872)

Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology., National Institute of Allergy and Infectious Diseases (U.S.) (Grant R01AI117043), United States. Air Force. Office of Scientific Research (Grant FA9550-14-1-0060), National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706), National Institute of Mental Health (U.S.) (Grant 1R01-MH110049)

CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion., National Institutes of Health (Grant 1R01-HG009761), National Institutes of Health (Grant 1R01-MH110049), National Institutes of Health (Grant 1DP1-HL141201)

Humans process information hierarchically. In the presence of hierarchies, sources of failures are ambiguous. Humans resolve this ambiguity by assessing their confidence after one or more attempts. To understand the neural basis of this reasoning strategy, we recorded from dorsomedial frontal cortex (DMFC) and anterior cingulate cortex (ACC) of monkeys in a task in which negative outcomes were caused either by misjudging the stimulus or by a covert switch between two stimulus-response contingency rules. We found that both areas harbored a representation of evidence supporting a rule switch. Additional perturbation experiments revealed that ACC functioned downstream of DMFC and was directly and specifically involved in inferring covert rule switches. These results‏ reveal the computational principles of hierarchical reasoning, as implemented by cortical circuits.

Cancer genomics consortia have charted the landscapes of numerous human cancers. Whereas somemutations were found in classical oncogenes and tumor suppressors, others have not yet been functionally studied in vivo. To date, a comprehensive assessment of how these genes influence oncogenesis is lacking. We performed direct highthroughput in vivo mapping of functional variants in an autochthonous mouse model of cancer. Using adenoassociated viruses (AAVs) carrying a single-guide RNA (sgRNA) library targeting putative tumor suppressor genes significantly mutated in human cancers, we directly pool-mutagenized the livers of Cre-inducible CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) mice. All mice that received the AAV-mTSG library developed liver cancer and diedwithin 4 months.We usedmolecular inversion probe sequencing of the sgRNA target sites to chart the mutational landscape of these tumors, revealing the functional consequence of multiple variants in driving liver tumorigenesis in immunocompetent mice. AAV-mediated autochthonous CRISPR screens provide a powerful means for mapping a provisional functional cancer genome atlas of tumor suppressors in vivo., Damon Runyon Cancer Research Foundation (DRG-2117-12; DFS-13-15), Melanoma Research Foundation (412806, 16-003524), St. Baldrick’s Foundation (426685), American Cancer Society (IRG 58-012-54), Cancer Research Institute (New York, N.Y.), Breast Cancer Alliance, Cancer Research Institute (New York, N.Y.) (Clinic and Laboratory Integration Program), American Association for Cancer Research (499395), United States. Department of Defense (W81XWH-17-1-0235), National Cancer Institute (U.S.) (1U54CA209992), National Cancer Institute (U.S.) (5P50CA196530-A10805), National Cancer Institute (U.S.) (4P50CA121974-A08306), National Institutes of Health (U.S.) (R01-CA133404), National Institutes of Health (U.S.) (R01-GM034277), National Institutes of Health (U.S.) (CCNE), Skolkovo Foundation, Casimir-Lambert Fund, National Institutes of Health (U.S.) (grant 1R01-HG009761), National Institutes of Health (U.S.) (grant R01-MH110049), National Institutes of Health (U.S.) (grant DP1-HL141201), Howard Hughes Medical Institute, New York Stem Cell Foundation, Simons, Paul G. Allen Family, Vallee Foundations, Poitras Center for Affective Disorders Research at MIT, Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT

Infants understand that people pursue goals, but how do they learn which goals people prefer? We tested whether infants solve this problem by inverting a mental model of action planning, trading off the costs of acting against the rewards actions bring. After seeing an agent attain two goals equally often at varying costs, infants expected the agent to prefer the goal it attained through costlier actions. These expectations held across three experiments that conveyed cost through different physical path features (height, width, and incline angle), suggesting that an abstract variable—such as “force,” “work,” or “effort”—supported infants’ inferences. We modeled infants’ expectations as Bayesian inferences over utility-theoretic calculations, providing a bridge to recent quantitative accounts of action understanding in older children and adults.

Single-cell RNA sequencing (RNA-Seq) provides rich information about cell types and states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation of rare neurons from adult tissue is challenging and markers for each phase are limited. Here, we develop Div-Seq, which combines scalable single-nucleus RNA-Seq (sNuc-Seq) with pulse labeling of proliferating cells by 5-ethynyl-2′-deoxyuridine (EdU) to profile individual dividing cells. sNuc-Seq and Div-Seq can sensitively identify closely related hippocampal cell types and track transcriptional dynamics of newborn neurons within the adult hippocampal neurogenic niche, respectively. We also apply Div-Seq to identify and profile rare newborn neurons in the adult spinal cord, a noncanonical neurogenic region. sNuc-Seq and Div-Seq open the way for unbiased analysis of diverse complex tissues., National Institute of Mental Health (U.S.) (Grant U01MH105960), National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706), National Institute of Mental Health (U.S.) (Grant 1R01-MH110049)

The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9., National Institutes of Health (U.S.) (Award 1DP1-MH100706), National Institutes of Health (U.S.) (1R01-DK097768)

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications., United States. Air Force Office of Scientific Research (Grant FA9550-14-1-0060), Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-14-1-0006), National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706), National Institutes of Health (U.S.) (Grant 1R01-MH110049)

Defects in the mitochondrial respiratory chain (RC) underlie a spectrum of human conditions, ranging from devastating inborn errors of metabolism to aging. We performed a genome-wide Cas9-mediated screen to identify factors that are protective during RC inhibition. Our results highlight the hypoxia response, an endogenous program evolved to adapt to limited oxygen availability. Genetic or small-molecule activation of the hypoxia response is protective against mitochondrial toxicity in cultured cells and zebrafish models. Chronic hypoxia leads to a marked improvement in survival, body weight, body temperature, behavior, neuropathology, and disease biomarkers in a genetic mouse model of Leigh syndrome, the most common pediatric manifestation of mitochondrial disease. Further preclinical studies are required to assess whether hypoxic exposure can be developed into a safe and effective treatment for human diseases associated with mitochondrial dysfunction., National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706), National Institute of Mental Health (U.S.) (Grant 1R01-MH110049), National Institute of Neurological Diseases and Stroke (U.S.) (Grant 5R01DK097768-03)

A large body of previous neuroimaging studies suggests that multiple languages are processed and organized in a single neuroanatomical system in the bilingual brain, although differential activation may be seen in some studies because of different proficiency levels and/or age of acquisition of the two languages. However, one important possibility is that the two languages may involve interleaved but functionally independent neural populations within a given cortical region, and thus, distinct patterns of neural computations may be pivotal for the processing of the two languages. Using functional magnetic resonance imaging (fMRI) and multivariate pattern analyses, we tested this possibility in Chinese-English bilinguals when they performed an implicit reading task. We found a broad network of regions wherein the two languages evoked different patterns of activity, with only partially overlapping patterns of voxels in a given region. These regions, including the middle occipital cortices, fusiform gyri, and lateral temporal, temporoparietal, and prefrontal cortices, are associated with multiple aspects of language processing. The results suggest the functional independence of neural computations underlying the representations of different languages in bilinguals.

Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to the mdx mousemodel of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force.This work establishes CRISPR-Cas9-based genome editing as a potential therapy to treat DMD., Muscular Dystrophy Association (Award MDA277360), National Institutes of Health (U.S.) (Grant 5DP1-MH100706), National Institutes of Health (U.S.) (Grant R01DK097768)

The noncoding genome affects gene regulation and disease, yet we lack tools for rapid identification and manipulation of noncoding elements. We developed a CRISPR screen using ∼18,000 single guide RNAs targeting > 700 kilobases surrounding the genes NF1, NF2, and CUL3, which are involved in BRAF inhibitor resistance in melanoma. We find that noncoding locations that modulate drug resistance also harbor predictive hallmarks of noncoding function. With a subset of regions at the CUL3 locus, we demonstrate that engineered mutations alter transcription factor occupancy and long-range and local epigenetic environments, implicating these sites in gene regulation and chemotherapeutic resistance. Through our expansion of the potential of pooled CRISPR screens, we provide tools for genomic discovery and for elucidating biologically relevant mechanisms of gene regulation., National Institutes of Health (U.S.) (Award F32-DK096822), National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706), National Institute of Mental Health (U.S.) (Grant 1R01-MH110049)

Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle., National Institute of General Medical Sciences (U.S.) (Grant T2GM007753), National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706), National Institutes of Health (U.S.) (Grant 5R01DK097768-03)

Emotional expressions of others are salient biological stimuli that automatically capture attention and prepare us for action. We investigated the early cortical dynamics of automatic visual discrimination of fearful body expressions by monitoring cortical activity using magnetoencephalography. We show that right parietal cortex distinguishes between fearful and neutral bodies as early as 80-ms after stimulus onset, providing the first evidence for a fast emotion-attention-action link through human dorsal visual stream., National Institutes of Health (U.S.) (NIH grant RO1NS44824), Netherlands Organization for Scientific Research (NWO grant R 95-403), Netherlands Organization for Scientific Research (NWO grant NWO grant 451-05-014), Mind Research Institute, Massachusetts General Hospital. Center for Functional Neuroimaging Technologies (NIH grant number P41 RR14075), National Institutes of Health (U.S.) (NIH grant 5R01EB009048), National Institutes of Health (U.S.) (NIH grant 5R01NS037462), National Institutes of Health (U.S.) (NIH grant R01NS57500), Swiss National Science Foundation (PPOOP3_130191), Human Frontier Science Program (Strasbourg, France) (grant number RGP0054/ 2004-C), European Commission (EU project COBOL (FDP6-NEST-043403)), European Commission (EU project TANGO (FP7 FET-Open))

The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that “enhanced specificity” SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity., Massachusetts Institute of Technology. Simons Center for the Social Brain, National Institute of General Medical Sciences (U.S.) (T32GM007753), Paul & Daisy Soros Fellowships for New Americans (New York, N.Y.), National Institute of Mental Health (U.S.) (5DP1-MH100706), National Institute of Mental Health (U.S.) (1R01MH110049), National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (5R01DK097768-03), National Science Foundation (U.S.) (Waterman Award), W. M. Keck Foundation, New York Stem Cell Foundation, Damon Runyon Cancer Research Foundation, Kinship Foundation. Searle Scholars Program, Merkin Foundation, Vallee Foundation

The ability to recognize people by their voice is an important social behavior. Individuals differ in how they pronounce words, and listeners may take advantage of language-specific knowledge of speech phonology to facilitate recognizing voices. Impaired phonological processing is characteristic of dyslexia and thought to be a basis for difficulty in learning to read. We tested voice-recognition abilities of dyslexic and control listeners for voices speaking listeners’ native language or an unfamiliar language. Individuals with dyslexia exhibited impaired voice-recognition abilities compared with controls only for voices speaking their native language. These results demonstrate the importance of linguistic representations for voice recognition. Humans appear to identify voices by making comparisons between talkers’ pronunciations of words and listeners’ stored abstract representations of the sounds in those words., Ellison Medical Foundation, National Institutes of Health (U.S.) (Grant UL1RR025758), National Science Foundation (U.S.). Graduate Research Fellowship

Available in PMC 2015 July 30., In optical microscopy, fine structural details are resolved by using refraction to magnify images of a specimen. We discovered that by synthesizing a swellable polymer network within a specimen, it can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved, a process we call expansion microscopy (ExM). Thus, this process can be used to perform scalable superresolution microscopy with diffraction-limited microscopes. We demonstrate ExM with apparent ~70-nanometer lateral resolution in both cultured cells and brain tissue, performing three-color superresolution imaging of ~107 cubic micrometers of the mouse hippocampus with a conventional confocal microscope., National Institutes of Health (U.S.) (NIH Director’s Pioneer Award 1DP1NS087724), National Institutes of Health (U.S.) (NIH Transformative Research Award 1R01MH103910-01), New York Stem Cell Foundation (Robertson Investigator Award), National Science Foundation (U.S.). Center for Brains, Minds and Machines (CBMM) (NSF CCF-1231216), National Science Foundation (U.S.) (NSF CAREER Award CBET 1053233)

The clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated genes (Cas) adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the class 2 type VI CRISPR-Cas effector C2c2 and demonstrate its RNA-guided ribonuclease function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis shows that C2c2 is guided by a single CRISPR RNA and can be programmed to cleave single-stranded RNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, mutations of which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools., United States. Dept. of Energy (Computational Science Graduate Fellowship), Massachusetts Institute of Technology. Simons Center for the Social Brain, National Institute of General Medical Sciences (U.S.) (Award T32GM007753), National Institutes of Health (U.S.) (National Institute of Mental Health (U.S.) Grants 5DP1-MH100706 and 1R01-MH110049), National Science Foundation (U.S.), New York Stem Cell Foundation, Simons Foundation, Paul G. Allen Family Foundation, Vallee Foundation, Poitras Foundation, Robert Metcalfe, David R. Cheng

Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology., National Institutes of Health (U.S.) (NIH grant R01-GM34277), National Institutes of Health (U.S.) (NIH grant R01-CA133404), National Institutes of Health (U.S.) (NIH Director's New Innovator Award (DP2AI104556)), National Institutes of Health (U.S.) (NIH Director's Pioneer Award (DP1MH100706))

Oxygen deprivation followed by reoxygenation causes pathological responses in many disorders, including ischemic stroke, heart attacks, and reperfusion injury. Key aspects of ischemia-reperfusion can be modeled by a Caenorhabditis elegans behavior, the O2-ON response, which is suppressed by hypoxic preconditioning or inactivation of the O[subscript 2]-sensing HIF (hypoxia-inducible factor) hydroxylase EGL-9. From a genetic screen, we found that the cytochrome P450 oxygenase CYP-13A12 acts in response to the EGL-9–HIF-1 pathway to facilitate the O2-ON response. CYP-13A12 promotes oxidation of polyunsaturated fatty acids into eicosanoids, signaling molecules that can strongly affect inflammatory pain and ischemia-reperfusion injury responses in mammals. We propose that roles of the EGL-9–HIF-1 pathway and cytochrome P450 in controlling responses to reoxygenation after anoxia are evolutionarily conserved., National Institutes of Health (U.S.) (Grant GM24663), National Science Foundation (U.S.). Graduate Research Fellowship Program, Massachusetts Institute of Technology. Undergraduate Research Opportunities Program, Helen Hay Whitney Foundation (Postdoctoral Fellowship)

Dysfunctions in frontostriatal brain circuits have been implicated in neuropsychiatric disorders, including those characterized by the presence of repetitive behaviors. We developed an optogenetic approach to block repetitive, compulsive behavior in a mouse model in which deletion of the synaptic scaffolding gene, Sapap3, results in excessive grooming. With a delay-conditioning task, we identified in the mutants a selective deficit in behavioral response inhibition and found this to be associated with defective down-regulation of striatal projection neuron activity. Focused optogenetic stimulation of the lateral orbitofrontal cortex and its terminals in the striatum restored the behavioral response inhibition, restored the defective down-regulation, and compensated for impaired fast-spiking neuron striatal microcircuits. These findings raise promising potential for the design of targeted therapy for disorders involving excessive repetitive behavior., Massachusetts Institute of Technology. Simons Center for the Social Brain, Eunice Kennedy Shriver National Institute of Child Health and Human Development (U.S.) (NIH R37 HD028341), United States. Defense Advanced Research Projects Agency (W911NF1010059), National Institute of Mental Health (U.S.) (NIH R01 MH081201)

Biogenic amines such as serotonin and dopamine are intercellular signaling molecules that function widely as neurotransmitters and neuromodulators. We have identified in the nematode Caenorhabditis elegans three ligand-gated chloride channels that are receptors for biogenic amines: LGC-53 is a high-affinity dopamine receptor, LGC-55 is a high-affinity tyramine receptor, and LGC-40 is a low-affinity serotonin receptor that is also gated by choline and acetylcholine. lgc-55 mutants are defective in a behavior that requires endogenous tyramine, which indicates that this ionotropic tyramine receptor functions in tyramine signaling in vivo. Our studies suggest that direct activation of membrane chloride conductances is a general mechanism of action for biogenic amines in the modulation of C. elegans behavior., National Institutes of Health (U.S.) (Grant GM24663), Howard Hughes Medical Institute, Life Sciences Research Foundation, Medical Foundation, Inc.

available in PMC 2014 May 29, The rapid encoding of contextual memory requires the CA3 region of the hippocampus, but the necessary genetic pathways remain unclear. We found that the activity-dependent transcription factor Npas4 regulates a transcriptional program in CA3 that is required for contextual memory formation. Npas4 was specifically expressed in CA3 after contextual learning. Global knockout or selective deletion of Npas4 in CA3 both resulted in impaired contextual memory, and restoration of Npas4 in CA3 was sufficient to reverse the deficit in global knockout mice. By recruiting RNA polymerase II to promoters and enhancers of target genes, Npas4 regulates a learning-specific transcriptional program in CA3 that includes many well-known activity-regulated genes, which suggests that Npas4 is a master regulator of activity-regulated gene programs and is central to memory formation., McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology (MIT Presidential Marcus Fellowship to Honor Norman B. Leventhal), Massachusetts Institute of Technology. Simons Center for the Social Brain, National Science Foundation (U.S.) (NSF grant IOS 0919159), Whitehall Foundation (research grant), Anonymous Foundation, John Merck Fund (John Merck Scholars Program), National Institutes of Health (U.S.) (NIH grant MH091220-01)

Electrical recordings in humans and monkeys show attentional enhancement of evoked responses and gamma synchrony in ventral stream cortical areas. Does this synchrony result from intrinsic activity in visual cortex or from inputs from other structures? Using paired recordings in the frontal eye field (FEF) and area V4, we found that attention to a stimulus in their joint receptive field leads to enhanced oscillatory coupling between the two areas, particularly at gamma frequencies. This coupling appeared to be initiated by FEF and was time-shifted by about 8 to 13 milliseconds across a range of frequencies. Considering the expected conduction and synaptic delays between the areas, this time-shifted coupling at gamma frequencies may optimize the postsynaptic impact of spikes from one area upon the other, improving cross-area communication with attention., Grant EY017292, Grant EY17921

Optogenetic tools are DNA-encoded molecules that, when genetically targeted to cells, enable the control of specific physiological processes within those cells through exposure to light. These tools can pinpoint how these specific processes affect the emergent properties of a complex biological system, such as a mammalian organ or even an entire animal. They can also allow control of a biological system for therapeutic or bioengineering purposes. Many of the optical control tools explored to date are single-component reagents containing a photoactive signaling domain. An interesting question is raised by comparing optogenetics to synthetic biology. In the latter, interchangeable and modular DNA-encoded parts are assembled into complex biological circuits, thus enabling sophisticated logic and computation as well as the production of biologics and reagents (1, 2). Is it possible to devise strategies for the temporally precise cell-targeted optical control of complex engineered biological computational or chemical-synthetic pathways? Such a marriage of optogenetics and synthetic biology—which one might call synthetic physiology—would open up the ability to use optogenetics to trigger and regulate engineered synthetic biology systems, which in turn could execute computational and biological programs of great complexity (3). On page 1565 of this issue, Ye et al. (4) explore such a hybrid approach to controlling a biological system, as well as the bioengineering and preclinical capabilities opened up by such an approach.