Blog
About

17
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor (dSaCas9 KRAB) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9 KRAB efficacy for gene silencing in vivo, we silenced transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9 KRAB and a Pcsk9-targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels. Despite a moderate host response to dSaCas9 KRAB expression, Pcsk9 repression is maintained for 24 weeks after a single treatment, demonstrating the potential for long-term gene silencing in post-mitotic tissues with dSaCas9 KRAB. In vivo programmable gene silencing enables studies that link gene regulation to complex phenotypes and expands the CRISPR-Cas9 perturbation toolbox for basic research and gene therapy applications.

          Abstract

          Repression of gene transcription using CRISPR-Cas9 has been achieved in vitro but not for delivery into adult animal models. Here, the authors use AAV8 to deliver the transcriptional repressor dSaCas9 KRAB to the cholesterol regulator Pcsk9, and show repression up to 24 weeks and reduced cholesterol levels in mice.

          Related collections

          Most cited references 16

          • Record: found
          • Abstract: found
          • Article: not found

          CRISPR RNA-guided activation of endogenous human genes

          Catalytically inactive CRISPR-associated 9 nuclease (dCas9) can be directed by short guide RNAs (gRNAs) to repress endogenous genes in bacteria and human cells. Here we show that a dCas9-VP64 transcriptional activation domain fusion protein can be directed by single or multiple gRNAs to increase expression of specific endogenous human genes. These results provide an important proof-of-principle that CRISPR-Cas systems can be used to target heterologous effector domains in human cells.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

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

            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.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Functional annotation of native enhancers with a Cas9 -histone demethylase fusion

              Understanding of mammalian enhancer function is limited by the lack of a technology to rapidly and thoroughly test their cell type-specific function. Here, we use a nuclease-deficient (d)Cas9 histone demethylase fusion to functionally characterize previously described and novel enhancer elements for their roles in the embryonic stem cell state. Further, we distinguish the mechanism of action of dCas9-LSD1 at enhancers from previous dCas9-effectors.
                Bookmark

                Author and article information

                Contributors
                charles.gersbach@duke.edu
                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group UK (London )
                2041-1723
                26 April 2018
                26 April 2018
                2018
                : 9
                Affiliations
                [1 ]ISNI 0000 0004 1936 7961, GRID grid.26009.3d, Department of Biomedical Engineering, , Duke University, ; Durham, 27708 NC USA
                [2 ]ISNI 0000 0004 1936 7961, GRID grid.26009.3d, Center for Genomic and Computational Biology, , Duke University, ; Durham, 27708 NC USA
                [3 ]ISNI 0000000100241216, GRID grid.189509.c, University Program in Genetics and Genomics, , Duke University Medical Center, ; Durham, 27710 NC USA
                [4 ]ISNI 0000 0004 1936 7961, GRID grid.26009.3d, Division of Laboratory Animal Resources, , Duke University School of Medicine, ; Durham, 27710 NC USA
                [5 ]ISNI 0000000100241216, GRID grid.189509.c, Department of Orthopaedic Surgery, , Duke University Medical Center, ; Durham, 27710 NC USA
                Article
                4048
                10.1038/s41467-018-04048-4
                5920046
                29700298
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                Categories
                Article
                Custom metadata
                © The Author(s) 2018

                Uncategorized

                Comments

                Comment on this article