Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells

Kearns, Nicola A.; Genga, Ryan M.; Enuameh, Metewo Selase; Garber, Manuel; Wolfe, Scot A.; Maehr, René

doi:10.1242/dev.103341

Cited by 266 publications

(232 citation statements)

References 28 publications

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“…Human codon-optimized dCas9 (nuclease-dead) from S. pyogenes (23), N. meningitidis, and S. thermophilus (18) were fused to 1XGFP, 2XGFP, 3XGFP, 3Xcherry, or 3XBFP and then subcloned into pHAGE-DEST lentiviral vectors. To optimize the promoters for U2OS and RPE-1 cells, the EF1α promoter in the pHAGE-EF1α-DEST vector was replaced by EFS, SFFV, and CMV-TetO promoters, respectively, resulting in pHAGE-EFS-DEST, pHAGE-SFFV-DEST, and pHAGE-TO-DEST.…”

Section: Methodsmentioning

confidence: 99%

Multicolor CRISPR labeling of chromosomal loci in human cells

Naseri

Reyes-Gutierrez

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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377

350

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The intranuclear location of genomic loci and the dynamics of these loci are important parameters for understanding the spatial and temporal regulation of gene expression. Recently it has proven possible to visualize endogenous genomic loci in live cells by the use of transcription activator-like effectors (TALEs), as well as modified versions of the bacterial immunity clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Here we report the design of multicolor versions of CRISPR using catalytically inactive Cas9 endonuclease (dCas9) from three bacterial orthologs. Each pair of dCas9-fluorescent proteins and cognate single-guide RNAs (sgRNAs) efficiently labeled several target loci in live human cells. Using pairs of differently colored dCas9-sgRNAs, it was possible to determine the intranuclear distance between loci on different chromosomes. In addition, the fluorescence spatial resolution between two loci on the same chromosome could be determined and related to the linear distance between them on the chromosome's physical map, thereby permitting assessment of the DNA compaction of such regions in a live cell.4D nucleome | telomeres | pericentromeric DNA | chromosomes

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Section: Methodsmentioning

confidence: 99%

Multicolor CRISPR labeling of chromosomal loci in human cells

Naseri

Reyes-Gutierrez

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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377

350

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“…Both effectors exhibit complex mechanisms of transactivation via recruitment of secondary transcription factors (Mittler et al, 2003; (Groner et al, 2010). TEMs are designed based Gao et al, 2014;Gilbert et al, 2013;Hu et al, 2014;Kearns et al, 2014Kearns et al, , 2015Lawhorn et al, 2014;Thakore et al, 2015KRAB Cheng et al, 2016Zalatan et al, 2015SID4x Konermann et al, 2013Mxi1 Gilbert et al, 2013 Targeted epigenetic modifiers (TEMs) (McDonald et al, 2016;Vojta et al, 2016), TET1 5mC-hydroxylase (Maeder et al, 2013a;Xu et al, 2016), LSD1 histone demethylase (Mendenhall et al, 2013;Kearns et al, 2015) and the p300 and CBP histone acetyltransferases (Hilton et al, 2015;Cheng et al, 2016). Below, we discuss applications of epigenetic editing by TEMs or indirect modulation by TTFs using dCas9 as DNA-binding platform.…”

Section: Applications Of the Dcas9 Toolmentioning

confidence: 99%

“…Taking that notion into account target sites can be selected based on information regarding genome-bound proteins (e.g. using DNase-seq) (Maeder et al, 2013b;Kearns et al, 2014;Thakore et al, 2015). Alternatively, DNA accessibility and off-target binding are determined ad hoc for every application.…”

Section: Applications Of the Dcas9 Toolmentioning

confidence: 99%

dCas9: A Versatile Tool for Epigenome Editing

Brocken¹,

Tark-Dame²,

Dame³

2018

Current Issues in Molecular Biology

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The epigenome is a heritable layer of information not encoded in the DNA sequence of the genome, but in chemical modifications of DNA or histones. These chemical modifications, together with transcription factors, operate as spatiotemporal regulators of genome activity. Dissecting epigenome function requires controlled site-specific alteration of epigenetic information. Such control can be obtained using designed DNA-binding platforms associated with effector domains to function as targeted transcription factors or epigenetic modifiers. Here, we review the use of dCas9 as a novel and versatile tool for fundamental studies on epigenetic landscapes, chromatin structure and transcription regulation, and the potential of this approach in basic research in these fields.

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“…79 Similarly, the catalytically inactive Cas9 was fused to known transcriptional activator domains and targeted to specific promoter regions by corresponding gRNAs, upregulating the target gene expression. 80 The ability to artificially control the expression of specific target genes not only enables us to better understand gene functions but also to manipulate cell fate through controlled expression of desired sets of pathway genes.…”

Section: Talenmentioning

confidence: 99%

A practical guide to induced pluripotent stem cell research using patient samples

Santostefano

Hamazaki

Biel

et al. 2015

Laboratory Investigation

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Approximately 3 years ago, we assessed how patient induced pluripotent stem cell (iPSC) research could potentially impact human pathobiology studies in the future. Since then, the field has grown considerably with numerous technical developments, and the idea of modeling diseases 'in a dish' is becoming increasingly popular in biomedical research. Likely, it is even acceptable to include patient iPSCs as one of the standard research tools for disease mechanism studies, just like knockout mice. However, as the field matures, we acknowledge there remain many practical limitations and obstacles for their genuine application to understand diseases, and accept that it has not been as straightforward to model disorders as initially proposed. A major practical challenge has been efficient direction of iPSC differentiation into desired lineages and preparation of the large numbers of specific cell types required for study. Another even larger obstacle is the limited value of in vitro outcomes, which often do not closely represent disease conditions. To overcome the latter issue, many new approaches are underway, including three-dimensional organoid cultures from iPSCs, xenotransplantation of human cells to animal models and in vitro interaction of multiple cell types derived from isogenic iPSCs. Here we summarize the areas where patient iPSC studies have provided truly valuable information beyond existing skepticism, discuss the desired technologies to overcome current limitations and include practical guidance for how to utilize the resources. Undoubtedly, these human patient cells are an asset for experimental pathology studies. The future rests on how wisely we use them.

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Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells

Cited by 266 publications

References 28 publications

Multicolor CRISPR labeling of chromosomal loci in human cells

Multicolor CRISPR labeling of chromosomal loci in human cells

dCas9: A Versatile Tool for Epigenome Editing

A practical guide to induced pluripotent stem cell research using patient samples

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