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      In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9.

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          Abstract

          Probing gene function in the mammalian brain can be greatly assisted with methods to manipulate the genome of neurons in vivo. The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) can be used to edit single or multiple genes in replicating eukaryotic cells, resulting in frame-shifting insertion/deletion (indel) mutations and subsequent protein depletion. Here, we delivered SpCas9 and guide RNAs using adeno-associated viral (AAV) vectors to target single (Mecp2) as well as multiple genes (Dnmt1, Dnmt3a and Dnmt3b) in the adult mouse brain in vivo. We characterized the effects of genome modifications in postmitotic neurons using biochemical, genetic, electrophysiological and behavioral readouts. Our results demonstrate that AAV-mediated SpCas9 genome editing can enable reverse genetic studies of gene function in the brain.

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          Most cited references23

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          Receptive fields of single neurones in the cat's striate cortex

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            Is Open Access

            The UCSC Genome Browser database: update 2011

            The University of California, Santa Cruz Genome Browser (http://genome.ucsc.edu) offers online access to a database of genomic sequence and annotation data for a wide variety of organisms. The Browser also has many tools for visualizing, comparing and analyzing both publicly available and user-generated genomic data sets, aligning sequences and uploading user data. Among the features released this year are a gene search tool and annotation track drag-reorder functionality as well as support for BAM and BigWig/BigBed file formats. New display enhancements include overlay of multiple wiggle tracks through use of transparent coloring, options for displaying transformed wiggle data, a ‘mean+whiskers’ windowing function for display of wiggle data at high zoom levels, and more color schemes for microarray data. New data highlights include seven new genome assemblies, a Neandertal genome data portal, phenotype and disease association data, a human RNA editing track, and a zebrafish Conservation track. We also describe updates to existing tracks.
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              The story of Rett syndrome: from clinic to neurobiology.

              The postnatal neurodevelopmental disorder Rett syndrome (RTT) is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2), a transcriptional repressor involved in chromatin remodeling and the modulation of RNA splicing. MECP2 aberrations result in a constellation of neuropsychiatric abnormalities, whereby both loss of function and gain in MECP2 dosage lead to similar neurological phenotypes. Recent studies demonstrate disease reversibility in RTT mouse models, suggesting that the neurological defects in MECP2 disorders are not permanent. To investigate the potential for restoring neuronal function in RTT patients, it is essential to identify MeCP2 targets or modifiers of the phenotype that can be therapeutically modulated. Moreover, deciphering the molecular underpinnings of RTT is likely to contribute to the understanding of the pathogenesis of a broader class of neuropsychiatric disorders.
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                Author and article information

                Journal
                Nat. Biotechnol.
                Nature biotechnology
                1546-1696
                1087-0156
                Jan 2015
                : 33
                : 1
                Affiliations
                [1 ] 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] McGovern Institute for Brain Research, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
                [2 ] Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
                [3 ] 1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
                [4 ] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
                Article
                nbt.3055 NIHMS701422
                10.1038/nbt.3055
                25326897
                4be4972f-c074-4f5a-ae84-a22846dc1615
                History

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