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Author Notes:

Correspondence to: michael.mcmanus@ucsf.edu

The project was conceived and directed by M.B. and M.T.M.

Screen optimisation was performed by M.B. and D.W.

Libraries were designed by J.A.B. with guidance from M.B. and cloned by M.B.

Orthogonal vectors and cell lines were created by M.B.

All screens were performed by M.B., with A.B. assisting in CRISPRa screen analysis.

R.T. developed the computational pipelines and the statistical framework for data analysis for screens with guidance from M.K. R.T. also selected the best-performing sgRNAs for arrayed validation.

M.B. and R.T.W. conducted and analysed arrayed validation experiments.

E.M. and R.T.W. performed western blot analyses.

X.M. and H.F. conducted and analysed TR-FRET experiments.

M.B. and M.T.M. wrote the manuscript with critical input from R.T., M.K., N.Z. and F.M.

All authors read and approved the final manuscript.

Special thanks go to members of the McManus Lab who provided critical feedback during the course of this project and Elizabeth Cahill for excellent technical support.

We also thank Luke A. Gilbert and Marvin E. Tanenbaum for sharing the CRISPRa cell line ahead of its publication.

The authors declare no competing financial interests.

Subjects:

Research Funding:

M.T.M. was supported by NIH/CTD2 (U01CA168370) and IDG (1U01MH105028).

M.K. was supported by NIH/NIGMS New Innovator Award DP2 GM119139, NIH/NCI K99/R00 CA181494, a Stand Up to Cancer Innovative Research Grant and the Chan Zuckerberg Biohub.

J.B. was supported by NIH Training grant T32 GM00715 and an AFPE Predoctoral Fellowship.

H.F. was supported by NIH/CTD2 (U01CA168449).

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Biotechnology & Applied Microbiology
  • PROTEIN-TYROSINE-PHOSPHATASE
  • CHRONIC MYELOID-LEUKEMIA
  • MAMMALIAN-CELLS
  • INTERACTION MAP
  • BCR-ABL
  • IN-VIVO
  • CANCER
  • CRISPR
  • IMATINIB
  • KINASE

Dual gene activation and knockout screen reveals directional dependencies in genetic networks

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Journal Title:

Nature Biotechnology

Volume:

Volume 36, Number 2

Publisher:

, Pages 170-+

Type of Work:

Article | Post-print: After Peer Review

Abstract:

Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair. Based on the results from over 100,000 perturbed gene pairs, we reconstruct a directional dependency network for human K562 leukemia cells and demonstrate how our approach allows the determination of directionality in activating genetic interactions. Our interaction network connects previously uncharacterized genes to well-studied pathways and identifies targets relevant for therapeutic intervention.

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved.

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