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Citation
Lin, S., Ewen-Campen, B., Ni, X., Housden, B.E., Perrimon, N. (2015). In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila.  Genetics 201(2): 433--442.
FlyBase ID
FBrf0229805
Publication Type
Research paper
Abstract

A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be overexpressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore the most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. In this study, we characterize the dCas9-VPR system in Drosophila cells and in vivo. We show that this system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and that a single guide RNA upstream of the transcription start site can activate high levels of target transcription. We observe marked heterogeneity in guide RNA efficacy for any given gene, and we confirm that transcription is inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, we used dCas9-VPR to identify target genes for Twist and Snail, two highly conserved transcription factors that cooperate during Drosophila mesoderm development. In addition, we simultaneously activated both Twist and Snail to identify synergistic responses to this physiologically relevant combination. Finally, we show that dCas9-VPR can activate target genes and cause dominant phenotypes in vivo, providing the first demonstration of dCas9 activation in a multicellular animal. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of overexpression studies.

PubMed ID
PubMed Central ID
PMC4596659 (PMC) (EuropePMC)
Related Publication(s)
Personal communication to FlyBase

P{UAS-dCas9.VPR} insertions.
Ewen-Campen and Perrimon, 2016.9.29, P{UAS-dCas9.VPR} insertions. [FBrf0233686]

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Secondary IDs
    Language of Publication
    English
    Additional Languages of Abstract
    Parent Publication
    Publication Type
    Journal
    Abbreviation
    Genetics
    Title
    Genetics
    Publication Year
    1916-
    ISBN/ISSN
    0016-6731
    Data From Reference
    Alleles (5)
    Genes (14)
    Cell Lines (1)
    Natural transposons (1)
    Insertions (4)
    Experimental Tools (4)
    Transgenic Constructs (5)