Article

AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma

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Abstract

A causative understanding of genetic factors that regulate glioblastoma pathogenesis is of central importance. Here we developed an adeno-associated virus–mediated, autochthonous genetic CRISPR screen in glioblastoma. Stereotaxic delivery of a virus library targeting genes commonly mutated in human cancers into the brains of conditional-Cas9 mice resulted in tumors that recapitulate human glioblastoma. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlated with those in two independent patient cohorts. Co-mutation analysis identified co-occurring driver combinations such as B2mNf1, Mll3Nf1 and Zc3h13Rb1, which were subsequently validated using AAV minipools. Distinct from Nf1-mutant tumors, Rb1-mutant tumors are undifferentiated and aberrantly express homeobox gene clusters. The addition of Zc3h13 or Pten mutations altered the gene expression profiles of Rb1 mutants, rendering them more resistant to temozolomide. Our study provides a functional landscape of gliomagenesis suppressors in vivo.

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Change history

  • Corrected online 21 August 2017

    In the PDF version of this article initially published online, one of the Online Methods headings read “vRNA-seq differential expression analysis”; this has been changed to “RNA-seq differential expression analysis.” The error has been corrected in the print and PDF versions of this article.

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Acknowledgements

We thank all members of the Chen, Sharp, Zhang and Platt laboratories, as well as our colleagues in the Yale Department of Genetics, Systems Biology Institute, Yale Cancer Center and Stem Cell Center, Koch Institute and Broad Institute at MIT for assistance and/or discussions. We thank the Center for Genome Analysis, Center for Molecular Discovery, High Performance Computing Center, West Campus Analytical Chemistry Core and West Campus Imaging Core and Keck Biotechnology Resource Laboratory at Yale, as well as Swanson Biotechnology Center at MIT, for technical support. S.C. is supported by Yale SBI/Genetics Startup Fund, Damon Runyon (DRG-2117-12; DFS-13-15), Melanoma Research Alliance (412806, 16-003524), St. Baldrick's Foundation (426685), American Cancer Society (IRG 58-012-54), Breast Cancer Alliance, Cancer Research Institute (CLIP), AACR (499395), DoD (W81XWH-17-1-0235) and NIH/NCI (1U54CA209992, 5P50CA196530-A10805, 4P50CA121974-A08306). R.J.P. is supported by NCCRMSE and ETH Zurich, the McGovern Institute and NSF (1122374). P.A.S. is supported by NIH (R01-CA133404, R01-GM034277, CCNE), Skoltech Center and the Casimir-Lambert Fund. F.Z. is supported by the NIH/NIMH (5DP1-MH100706 and 1R01-MH110049), NSF, NY Stem Cell Foundation, HHMI, Poitras, Simons, Paul G. Allen Family, Vallee Foundations, D.R. Cheng and B. Metcalfe. C.D.G. and P.R. are supported by an NIH Graduate Training Grant (T32GM007499). R.D.C., M.B.D. and M.W.Y. are supported by an NIH MSTP training grant (T32GM007205). F.S. is supported by NCCRMSE and ETH Zurich. G.W. is supported by RJ Anderson and CRI Irvington Postdoctoral Fellowships.

Author information

Author notes

    • Ryan D Chow
    • , Christopher D Guzman
    • , Guangchuan Wang
    •  & Florian Schmidt

    These authors contributed equally to this work.

    • Mark W Youngblood
    •  & Lupeng Ye

    These authors contributed equally to this work.

Affiliations

  1. Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Ryan D Chow
    • , Christopher D Guzman
    • , Guangchuan Wang
    • , Mark W Youngblood
    • , Lupeng Ye
    • , Youssef Errami
    • , Matthew B Dong
    • , Michael A Martinez
    • , Sensen Zhang
    • , Paul Renauer
    • , Kaya Bilguvar
    • , Murat Gunel
    •  & Sidi Chen
  2. Systems Biology Institute, Yale University School of Medicine, West Haven, Connecticut, USA.

    • Ryan D Chow
    • , Christopher D Guzman
    • , Guangchuan Wang
    • , Lupeng Ye
    • , Youssef Errami
    • , Matthew B Dong
    • , Michael A Martinez
    • , Sensen Zhang
    • , Paul Renauer
    •  & Sidi Chen
  3. Medical Scientist Training Program, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Ryan D Chow
    • , Mark W Youngblood
    • , Matthew B Dong
    • , Murat Gunel
    •  & Sidi Chen
  4. Biological and Biomedical Sciences Program, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Christopher D Guzman
    • , Paul Renauer
    •  & Sidi Chen
  5. Immunobiology Program, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Christopher D Guzman
    •  & Sidi Chen
  6. Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Christopher D Guzman
  7. Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.

    • Florian Schmidt
    •  & Randall J Platt
  8. Department of Chemistry, University of Basel, Basel, Switzerland.

    • Florian Schmidt
    •  & Randall J Platt
  9. Department of Neurosurgery, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Mark W Youngblood
    •  & Murat Gunel
  10. Yale Center for Genome Analysis, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Kaya Bilguvar
    •  & Murat Gunel
  11. Koch Institute for Integrative Cancer Research, MIT, Cambridge, Massachusetts, USA.

    • Phillip A Sharp
  12. Department of Biology, MIT, Cambridge, Massachusetts, USA.

    • Phillip A Sharp
  13. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

    • Feng Zhang
  14. Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA.

    • Feng Zhang
  15. Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Sidi Chen
  16. Stem Cell Center, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Sidi Chen

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Contributions

S.C. and R.J.P. conceived the study, designed the study and performed the initial set of experiments. R.D.C. developed the algorithms and performed integrative analyses of all the data. C.D.G. performed validation, performed histology and established primary cell lines. G.W. performed exome-capture, mutant cell line generation, drug treatment and RNA-seq. F.S. performed AAV production. S.C. performed MRI. M.W.Y. contributed to data analysis. L.Y., Y.E., M.B.D., M.A.M., S.Z. and P.R. contributed to experiments including mouse breeding, genotyping, cloning, cell culture, virus prep, injection, necropsy and sample prep. K.B. assisted in captured and exome sequencing. M.G. provided clinical insights. P.A.S., F.Z., R.J.P. and S.C. jointly supervised the work. R.D.C. and S.C. wrote the manuscript with inputs from all authors.

Competing interests

F.Z. is a cofounder of Editas Medicine and a scientific advisor for Editas Medicine and Horizon Discovery. A patent application has been filed on the methods pertaining to this work.

Corresponding authors

Correspondence to Randall J Platt or Sidi Chen.

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