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Unknown,Transcriptomics,Genomics,Proteomics

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Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system

ABSTRACT: Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3 to 4 sgRNAs binding to the proximal promoters suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system. We used microarray to assay the gene expression changes after transfection of dCas9VP160 with the different sgRNAs

ORGANISM(S): Homo sapiens

SUBMITTER: Albert Cheng

PROVIDER: E-GEOD-49701 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system

Project description:Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3 to 4 sgRNAs binding to the proximal promoters suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.

2013-08-10 | GSE49701 | GEO

Potent and targeted activation of HIV-1 using the CRISPR/Cas9 activator Complex

Project description:Integration of the HIV-1 provirus in the host genome ensures a persistent supply of latently infected cells. This latent reservoir is recalcitrant to antiretroviral therapy (ART) making lifelong treatment the only option for patients. The â??shock and killâ?? strategy aims to eradicate latent HIV by reactivating proviral gene expression followed by ART treatment. Gene specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising small guide RNAs (sgRNAs) with a nuclease deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). Â We engineered this system to target 23 sites within the LTR promoter of HIV-1 and identified a â??hotspotâ?? for activation. We studied the functionality of activating sgRNAs to transcriptionally modulate the latent proviral genome across multiple differentÂ in vitroÂ latency cell models including several J-Lat, ACH2 J1.1 and the CEM T cell model comprising a single clonal population of integrated mCherry-IRES-Tat from a full-length HIV LTR (LChIT).Â While we observed variable responses of latent cell models to well-characterized chemical stimuli, we detected consistent efficient activation of latent virus mediated by activator sgRNAs.Â In addition, transcriptome analysis of chemically treated cells revealed massive non-specific gene dysregulation whereas by comparison, dCas9-VP64/sgRNAs induced specific activation of the integrated provirus.Â In conclusion, we show the potential for CRISPR-mediated gene activation systems to provide enhanced efficiency and specificity in a targeted latency reactivation strategy. This represents a promising approach to a â??functional cureâ?? of HIV/AIDS. Three experimental conditions (sgRNA control, TNF treated and sgRNA against the LTR of HIV-1) were analyzed in triplicate using two sequencing lanes

2016-08-20 | E-GEOD-72259 | biostudies-arrayexpress

3prime RNA-seq of murine SCLC cell line modified with CRISPR-activation of Myc, Mycl or Mycn

Project description:In cells derived from a genetically engineered Rb1 and Trp53 loss mouse model of SCLC (RP) expression of one of the three MYC family members induced from the endogenous locus using CRISPR activation. Cells were first stably transfected with the lenti-MS2-p65-HSF1 activator plasmid. Respective sgRNAs targeting either Myc, Mycl or MYCN were then cloned into the lentiSAMv2 system and transfected separately into the MS2-p65-HSF1 cells using lentiviral delivery.

2019-07-09 | E-MTAB-7411 | biostudies-arrayexpress

CETCh-Seq of Mammalian Transcription Factors

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CRISPR/Cas9/AAV9-sgRNA Mediated In Vivo Genome Editing Reveals the Indispensability of Myc During Muscle Stem Cells Activation by Remodeling the 3D Chromatin [RNA-seq(FISC-DSC)]

Project description:Skeletal muscle satellite cells (SCs) are muscle stem cells responsible for muscle development and injury induced muscle regeneration. The pace of SC related study, however, is constrained partially by the technological limitations in generating genetically modified mice. Although the ease of use of CRISPR-Cas9 in genome manipulation has been documented in many cell lines and various species, its application in endogenous SCs remains elusive. In this study, we generated muscle-specific Cas9-expressing mice and achieved robust in vivo genome editing in juvenile SCs at the postnatal stage through AAV9 mediated short guide RNAs (sgRNAs) delivery. We also found adult quiescent SCs are reluctant to CRISPR/Cas9 editing despite efficient AAV9 transduction. To edit juvenile SCs in vivo, as a proof-of-concept, we delivered sgRNAs targeting MyoD, a key gene critical for muscle physiology and showed an efficient editing at MyoD locus, resulting in accumulation of SCs and defects in SCs differentiation which resembled the phenotypes reported in MyoD knockout mice. Further application of this system on potential key transcription factors (TFs) involved in SC fate transition, Myc, Bcl6 and Pknox2, unveiled their distinct functions in the early stage of SC activation and injury induced muscle regeneration. In addition, we revealed that Myc orchestrated SCs activation through impinging on 3D chromatin architecture. Altogether we established a robust muscle restricted CRISPR/Cas9-based gene editing platform in endogenous SCs and elucidated the functionality of key factors governing SC activities.

2021-10-27 | GSE133955 | GEO

CRISPR/Cas9/AAV9-sgRNA Mediated In Vivo Genome Editing Reveals the Indispensability of Myc During Muscle Stem Cells Activation by Remodeling the 3D Chromatin[RNA-seq(QSC-Ctrl/sgMyc)]

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