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Next-generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM

Yu Jia, Rong-Gang Xu, Xingjie Ren, Ben Ewen-Campen, Rajendhran Rajakumar, Jonathan Zirin, Donghui Yang-Zhou, Ruibao Zhu, Fang Wang, Decai Mao, Ping Peng, Huan-Huan Qiao, Xia Wang, Lu-Ping Liu, Bowen Xu, Jun-Yuan Ji, Qingfei Liu, Jin Sun, Norbert Perrimon and Jian-Quan Ni
PNAS April 16, 2018. 201800677; published ahead of print April 16, 2018. https://doi.org/10.1073/pnas.1800677115
Yu Jia
aGene Regulatory Laboratory, School of Medicine, Tsinghua University, 100084 Beijing, China;
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Rong-Gang Xu
aGene Regulatory Laboratory, School of Medicine, Tsinghua University, 100084 Beijing, China;
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Xingjie Ren
bInstitute for Human Genetics and Department of Neurology, University of California, San Francisco, CA 94143;
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Ben Ewen-Campen
cDepartment of Genetics, Harvard Medical School, Boston, MA 02115;
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Rajendhran Rajakumar
cDepartment of Genetics, Harvard Medical School, Boston, MA 02115;
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Jonathan Zirin
dDrosophila RNAi Screening Center, Department of Genetics, Harvard Medical School, Boston, MA 02115;
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Donghui Yang-Zhou
dDrosophila RNAi Screening Center, Department of Genetics, Harvard Medical School, Boston, MA 02115;
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Ruibao Zhu
aGene Regulatory Laboratory, School of Medicine, Tsinghua University, 100084 Beijing, China;
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Fang Wang
aGene Regulatory Laboratory, School of Medicine, Tsinghua University, 100084 Beijing, China;
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Decai Mao
aGene Regulatory Laboratory, School of Medicine, Tsinghua University, 100084 Beijing, China;eSichuan Academy of Grassland Science, 611731 Chengdu, China;
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Ping Peng
aGene Regulatory Laboratory, School of Medicine, Tsinghua University, 100084 Beijing, China;
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Huan-Huan Qiao
aGene Regulatory Laboratory, School of Medicine, Tsinghua University, 100084 Beijing, China;
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Xia Wang
aGene Regulatory Laboratory, School of Medicine, Tsinghua University, 100084 Beijing, China;
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Lu-Ping Liu
aGene Regulatory Laboratory, School of Medicine, Tsinghua University, 100084 Beijing, China;fTsinghua Fly Center, Tsinghua University, 100084 Beijing, China;gHoward Hughes Medical Institute, Boston, MA 02115;
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Bowen Xu
aGene Regulatory Laboratory, School of Medicine, Tsinghua University, 100084 Beijing, China;
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Jun-Yuan Ji
hDepartment of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843;
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  • ORCID record for Jun-Yuan Ji
Qingfei Liu
iSchool of Pharmaceutical Sciences, Tsinghua University, 100084 Beijing, China;
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Jin Sun
aGene Regulatory Laboratory, School of Medicine, Tsinghua University, 100084 Beijing, China;jTsinghua University–Peking University Joint Center for Life Sciences, 100084 Beijing, China
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  • For correspondence: sun-j12@mails.tsinghua.edu.cnperrimon@receptor.med.harvard.edunijq@mail.tsinghua.edu.cn
Norbert Perrimon
cDepartment of Genetics, Harvard Medical School, Boston, MA 02115;gHoward Hughes Medical Institute, Boston, MA 02115;
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  • For correspondence: sun-j12@mails.tsinghua.edu.cnperrimon@receptor.med.harvard.edunijq@mail.tsinghua.edu.cn
Jian-Quan Ni
aGene Regulatory Laboratory, School of Medicine, Tsinghua University, 100084 Beijing, China;
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  • For correspondence: sun-j12@mails.tsinghua.edu.cnperrimon@receptor.med.harvard.edunijq@mail.tsinghua.edu.cn
  1. Contributed by Norbert Perrimon, March 23, 2018 (sent for review January 12, 2018; reviewed by Joseph B. Duffy and Stéphane Noselli)

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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.

  • CRISPR/Cas9
  • CRISPRa
  • Cas9 activators
  • gain of function

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
Yu Jia, Rong-Gang Xu, Xingjie Ren, Ben Ewen-Campen, Rajendhran Rajakumar, Jonathan Zirin, Donghui Yang-Zhou, Ruibao Zhu, Fang Wang, Decai Mao, Ping Peng, Huan-Huan Qiao, Xia Wang, Lu-Ping Liu, Bowen Xu, Jun-Yuan Ji, Qingfei Liu, Jin Sun, Norbert Perrimon, Jian-Quan Ni
Proceedings of the National Academy of Sciences Apr 2018, 201800677; DOI: 10.1073/pnas.1800677115

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Next-generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM
Yu Jia, Rong-Gang Xu, Xingjie Ren, Ben Ewen-Campen, Rajendhran Rajakumar, Jonathan Zirin, Donghui Yang-Zhou, Ruibao Zhu, Fang Wang, Decai Mao, Ping Peng, Huan-Huan Qiao, Xia Wang, Lu-Ping Liu, Bowen Xu, Jun-Yuan Ji, Qingfei Liu, Jin Sun, Norbert Perrimon, Jian-Quan Ni
Proceedings of the National Academy of Sciences Apr 2018, 201800677; DOI: 10.1073/pnas.1800677115
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