Article

Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex

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Abstract

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

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BioProject

Data deposits

All reagents described in this manuscript have been deposited with Addgene (plasmid IDs 61422-61427 for SAM component plasmid and 61597 for the human SAM guide RNA library). RNA-seq data are available at BioProject under accession number PRJNA269048.

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Acknowledgements

We would like to thank S. Shehata, K. Zheng, C. Johannessen, L. Garraway, O. Shalem and members of the Zhang laboratory for assistance and helpful discussions. O.O.A. is supported by a NSF Graduate Research Fellowship, J.S.G. is supported by a D.O.E. Computational Science Graduate Fellowship, H.N. is supported by PRESTO from JST and Grant-in-Aid for Scientific Research (B) from JSPS, O.N. is supported by the CREST program and JST, and F.Z. is supported by the NIMH (DP1-MH100706), the NINDS (R01-NS07312401), NSF, the Keck, Searle Scholars, Klingenstein, Vallee, and Simons Foundations, and Bob Metcalfe. CRISPR reagents are available to the academic community through Addgene, and associated protocols, support forum and computational tools are available via the Zhang laboratory website (http://www.genome-engineering.org).

Author information

Author notes

    • Silvana Konermann
    •  & Mark D. Brigham

    These authors contributed equally to this work.

Affiliations

  1. Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, Massachusetts 02142, USA

    • Silvana Konermann
    • , Mark D. Brigham
    • , Alexandro E. Trevino
    • , Julia Joung
    • , Omar O. Abudayyeh
    • , Clea Barcena
    • , Patrick D. Hsu
    • , Naomi Habib
    • , Jonathan S. Gootenberg
    •  & Feng Zhang
  2. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Silvana Konermann
    • , Mark D. Brigham
    • , Alexandro E. Trevino
    • , Omar O. Abudayyeh
    • , Clea Barcena
    • , Patrick D. Hsu
    • , Jonathan S. Gootenberg
    •  & Feng Zhang
  3. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Silvana Konermann
    • , Mark D. Brigham
    • , Alexandro E. Trevino
    • , Omar O. Abudayyeh
    • , Clea Barcena
    • , Patrick D. Hsu
    • , Jonathan S. Gootenberg
    •  & Feng Zhang
  4. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Silvana Konermann
    • , Mark D. Brigham
    • , Alexandro E. Trevino
    • , Julia Joung
    • , Omar O. Abudayyeh
    • , Clea Barcena
    • , Patrick D. Hsu
    • , Jonathan S. Gootenberg
    •  & Feng Zhang
  5. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Jonathan S. Gootenberg
  6. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi Bunkyo, Tokyo 113-0032, Japan

    • Hiroshi Nishimasu
    •  & Osamu Nureki
  7. JST, PRESTO 2-11-16 Yayoi Bunkyo, Tokyo 113-0032, Japan

    • Hiroshi Nishimasu

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Contributions

S.K. and F.Z. conceived the project. S.K., M.D.B., A.E.T. and F.Z. designed the experiments. S.K., M.D.B., A.E.T., C.B., P.D.H. and J.J. performed experiments and analysed data. H.N. and O.N. helped with structural interpretation. N.H. performed the RNA-seq analysis. J.S.G. performed the depletion guide efficacy analysis. O.O.A. performed the analysis of clinical data sets. S.K., A.E.T., P.D.H. and F.Z. wrote the paper with help from all authors.

Competing interests

The authors have filed a patent application related to this work

Corresponding author

Correspondence to Feng Zhang.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Sequences and Supplementary Tables 1-5 (see separate files for Supplementary Tables 6 and 7).

CSV files

  1. 1.

    Supplementary Table

    This file contains Supplementary Table 6, which is a list of sgRNA target sequences for the human genome.

  2. 2.

    Supplementary Table

    This file contains Supplementary Table 7, which shows the normalized raw counts of sgRNAs from all of the screens conducted in this study.