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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.
KEYWORDS CRISPR-Cas9; gene activation; overexpression, gain-of-function
IThasrecently become possibleto activatetranscriptionof target genes from their native genomic locususing nucleasedead Cas9 (dCas9) fused to transcriptional activator domains (Mali et al. 2013; Gilbert et al. 2014; Tanenbaum et al. 2014; Zalatan et al. 2014; Chavez et al. 2015; Konermann et al. 2015). Activating genes from their endogenous transcription start site (TSS) offers several benefits that are complementary to traditional overexpression studies based on cloned cDNAs. For example, the dCas9-mediation activation technique is preferable for genes that are difficult to clone, e.g., if they occur in multiple splice isoforms and/or are very large. In addition, there is evidence that dCas9-mediated activation leads to target gene activation at physiologically relevant levels, as...