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Contributed by Norbert Perrimon, March 23, 2018 (sent for review January 12, 2018; reviewed by Joseph B. Duffy and Stéphane Noselli)
Significance
We present flySAM, a potent system for Cas9-based transcriptional activation (CRISPRa) in Drosophila. flySAM greatly improves on existing in vivo CRISPRa techniques in terms of potency, scalability, and ease of use, and provides a simple and general method for conducting overexpression experiments and screens. flySAM will now serve as the basis for our growing collection of publicly available CRISPRa transgenic fly lines.
Abstract
CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster. We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.
Footnotes
↵1Y.J., R.-G.X., X.R., and B.E.-C. contributed equally to this work.
- ↵2To whom correspondence may be addressed. Email: sun-j12{at}mails.tsinghua.edu.cn; perrimon{at}receptor.med.harvard.edu; or nijq{at}mail.tsinghua.edu.cn.
Author contributions: X.R., B.E.-C., N.P., and J.-Q.N. designed research; R.-G.X., X.R., B.E.-C., R.R., and D.Y.-Z. performed research; Y.J., X.R., R.Z., F.W., D.M., P.P., H.-H.Q., X.W., L.-P.L., B.X., J.-Y.J., Q.L., J.S., and J.-Q.N. contributed new reagents/analytic tools; R.-G.X., X.R., B.E.-C., R.R., and J.Z. analyzed data; and B.E.-C. and J.-Q.N. wrote the paper.
Reviewers: J.B.D., Worcester Polytechnic Institute; and S.N., University of Nice, CNRS.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1800677115/-/DCSupplemental.
Published under the PNAS license.
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Next-generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM
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