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      Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing

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          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.

          Summary

          Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses’ silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.

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          Highlights

          • Repurposing the ERV silencing machinery for targeted epigenetic gene silencing
          • Hit-and-run delivery of combinations of engineered transcriptional repressors (ETRs)
          • Inheritable and stimulation-resistant silencing of endogenous genes by DNA methylation
          • Portable to different cell types and DNA binding platforms and amenable to multiplexing

          Abstract

          Transient co-expression of engineered transcriptional repressors (ETRs) allows for stable and highly specific epigenetic silencing of endogenous genes, which is amenable to multiplexing and can be reverted by targeted DNA demethylation.

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          Most cited references 25

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          Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

          TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.
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            Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers

            Technologies that facilitate the targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we have generated a programmable acetyltransferase based on the CRISPR/Cas9 gene regulation system, consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. This fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, corresponding with robust transcriptional activation of target genes from promoters, proximal enhancers, and distal enhancers. Gene activation by the targeted acetyltransferase is highly specific across the genome. In contrast to conventional dCas9-based activators, the acetyltransferase effectively activates genes from enhancer regions and with individual guide RNAs. The core p300 domain is also portable to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a new robust tool for manipulating gene regulation.
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              BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis

              Summary Enhancers, critical determinants of cellular identity, are commonly identified by correlative chromatin marks and gain-of-function potential, though only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously we identified an erythroid enhancer of BCL11A, subject to common genetic variation associated with fetal hemoglobin (HbF) level, whose mouse ortholog is necessary for erythroid BCL11A expression. Here we develop pooled CRISPR-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for HbF reinduction. The detailed enhancer map will inform therapeutic genome editing. The screening approach described here is generally applicable to functional interrogation of noncoding genomic elements.
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                Author and article information

                Contributors
                Journal
                Cell
                Cell
                Cell
                Cell Press
                0092-8674
                1097-4172
                22 September 2016
                22 September 2016
                : 167
                : 1
                : 219-232.e14
                Affiliations
                [1 ]San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy
                [2 ]Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milan, Italy
                [3 ]Center for Translational Genomics and Bioinformatics, IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy
                Author notes
                []Corresponding author naldini.luigi@ 123456hsr.it
                [∗∗ ]Corresponding author lombardo.angelo@ 123456hsr.it
                [4]

                Co-first authors

                [5]

                Lead Contact

                Article
                S0092-8674(16)31236-3
                10.1016/j.cell.2016.09.006
                5039111
                27662090
                © 2016 The Author(s)

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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