RNA Modification & Editing

Continuous genetic recording with self-targeting CRISPR-Cas in human cells

Wed, 12/09/2015 - 00:00
Perli SD1, Cui CH2, Lu TK3.
Science. 2016 Sep 9;353(6304). pii: aag0511. doi: 10.1126/science.aag0511. Epub 2016 Aug 18.
Malin Akerblom
Time
12:00pm
The ability to record molecular events in vivo would enable monitoring of signaling dynamics within cellular niches and critical factors that orchestrate cellular behavior. We present a self-contained analog memory device for longitudinal recording of molecular stimuli into DNA mutations in human cells. This device consists of a self-targeting guide RNA (stgRNA) that repeatedly directs Streptococcus pyogenes Cas9 nuclease activity toward the DNA that encodes the stgRNA, thereby enabling localized, continuous DNA mutagenesis as a function of stgRNA expression. We demonstrate programmable and multiplexed memory storage in human cells triggered by exogenous inducers or inflammation, both in vitro and in vivo. This tool, Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE), provides a unique strategy for investigating cell biology in vivo and enables continuous evolution of targeted DNA sequences.
RNA Modification & Editing
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malinakerblom
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Whole organism lineage tracing by combinatorial and cumulative genome editing

Thu, 05/26/2016 - 00:00
McKenna A1, Findlay GM1, Gagnon JA2, Horwitz MS3, Schier AF4, Shendure J5.
Science.
Michael Boettcher
Time
12:00pm
Multicellular systems develop from single cells through distinct lineages. However, current lineage tracing approaches scale poorly to whole, complex organisms. Here, we use genome editing to progressively introduce and accumulate diverse mutations in a DNA barcode over multiple rounds of cell division. The barcode, an array of CRISPR/Cas9 target sites, marks cells and enables the elucidation of lineage relationships via the patterns of mutations shared between cells. In cell culture and zebrafish, we show that rates and patterns of editing are tunable and that thousands of lineage-informative barcode alleles can be generated. By sampling hundreds of thousands of cells from individual zebrafish, we find that most cells in adult organs derive from relatively few embryonic progenitors. In future analyses, genome editing of synthetic target arrays for lineage tracing (GESTALT) can be used to generate large-scale maps of cell lineage in multicellular systems for normal development and disease.
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micboe00
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Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements

Tue, 12/01/2015 - 00:00
Thakore PI1,2, D'Ippolito AM2,3, Song L2,4, Safi A2,4, Shivakumar NK1, Kabadi AM1,2, Reddy TE2,5, Crawford GE2,4, Gersbach CA1,2,6.
Nat Methods.
Estefania Tarifeno
Time
12:00pm
Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.
RNA Modification & Editing
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etarisal
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RNA Directed Non-Mendelian Inheritance

Mon, 11/16/2015 - 00:00
James E DiCarlo, Alejandro Chavez, Sven L Dietz, Kevin M Esvelt & George M Church
Nature Biotechnology
Michael McManus
Time
12:00pm
RNA-guided gene drives capable of spreading genomic alterations made in laboratory organisms through wild populations could be used to address environmental and public health problems. However, the possibility of unintended genome editing occurring through the escape of strains from laboratories, coupled with the prospect of unanticipated ecological change, demands caution. We report the efficacy of CRISPR-Cas9 gene drive systems in wild and laboratory strains of the yeast Saccharomyces cerevisiae. Furthermore, we address concerns surrounding accidental genome editing by developing and validating methods of molecular confinement that minimize the risk of unwanted genome editing. We also present a drive system capable of overwriting the changes introduced by an earlier gene drive. These molecular safeguards should enable the development of safe CRISPR gene drives for diverse organisms.
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mmcmanus
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Cas9 gRNA engineering for genome editing activation and repression

Mon, 09/07/2015 - 00:00
Samira Kiani1,2,11, Alejandro Chavez3–5,11,
Marcelle Tuttle3, Richard N Hall1,2, Raj Chari5,
Dmitry Ter-Ovanesyan3,5, Jason Qian3,5,
Benjamin W Pruitt3, Jacob Beal6, Suhani Vora1,3,5, Joanna Buchthal3, Emma J K Kowal3,
Mohammad R Ebrahimkhani1,7, James J Collins1–3,8–10, Ron Weiss1,2,7 & George Church3,5
Nature Methods
Ryan Hromyak
Time
12:00pm
We demonstrate that by altering the length of Cas9- associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.
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Ryan Hromyak
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Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells

Thu, 06/11/2015 - 00:00
Grow EJ1, Flynn RA2, Chavez SL3, Bayless NL4, Wossidlo M5, Wesche DJ6, Martin L2, Ware CB7, Blish CA8, Chang HY2, Pera RA9, Wysocka J10.
Nature
Boris Reznik
Time
12:00pm
Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome1 . The most recently acquired human ERV is HERVK(HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor2,3. Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins4 . However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection5–7. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development.
RNA Modification & Editing
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breznik25
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Engineered CRISPR-Cas9 nucleases with altered PAM specificities

Thu, 07/23/2015 - 00:00
Kleinstiver BP1, Prew MS2, Tsai SQ1, Topkar VV2, Nguyen NT2, Zheng Z3, Gonzales AP4, Li Z5, Peterson RT4, Yeh JR6, Aryee MJ7, Joung JK1
Nature
Scott Hickey
Time
12:00pm
Although CRISPR-Cas9 nucleases are widely used for genome editing1,2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3–6. As a result, it can often be difficult to target doublestranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis7 . In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.
RNA Modification & Editing
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shickey
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APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages

Tue, 04/21/2015 - 00:00
Sharma S1, Patnaik SK2, Taggart RT1, Kannisto ED2, Enriquez SM3, Gollnick P3, Baysal BE1.
Nat Commun
Tristan Eifler
Time
12:00pm
The extent, regulation and enzymatic basis of RNA editing by cytidine deamination are incompletely understood. Here we show that transcripts of hundreds of genes undergo sitespecific C4U RNA editing in macrophages during M1 polarization and in monocytes in response to hypoxia and interferons. This editing alters the amino acid sequences for scores of proteins, including many that are involved in pathogenesis of viral diseases. APOBEC3A, which is known to deaminate cytidines of single-stranded DNA and to inhibit viruses and retrotransposons, mediates this RNA editing. Amino acid residues of APOBEC3A that are known to be required for its DNA deamination and anti-retrotransposition activities were also found to affect its RNA deamination activity. Our study demonstrates the cellular RNA editing activity of a member of the APOBEC3 family of innate restriction factors and expands the understanding of C4U RNA editing in mammals.
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Tristan
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Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers

Fri, 05/01/2015 - 00:00
Isaac B Hilton, Anthony M D’Ippolito, Christopher M Vockley, Pratiksha I Thakore, Gregory E Crawford,
Timothy E Reddy & Charles A Gersbach
Nature Biotechnology
Michael Boettcher
Time
12:00pm
Technologies that enable 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 describe a programmable, CRISPR-Cas9-based acetyltransferase consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. The fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, leading to robust transcriptional activation of target genes from promoters and both proximal and distal enhancers. Gene activation by the targeted acetyltransferase was highly specific across the genome. In contrast to previous dCas9-based activators, the acetyltransferase activates genes from enhancer regions and with an individual guide RNA. We also show that the core p300 domain can be fused to other programmable DNA-binding proteins. These results support ta
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micboe00
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