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Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease

Nature Biotechnology volume 33, pages 1159–1161 (2015)Cite this article

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A Corrigendum to this article was published on 07 April 2016

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Abstract

We have developed a CRISPR-based method that uses catalytically active Cas9 and distinct single guide (sgRNA) constructs to knock out and activate different genes in the same cell. These sgRNAs, with 14- to 15-bp target sequences and MS2 binding loops, can activate gene expression using an active Streptococcus pyogenes Cas9 nuclease, without inducing double-stranded breaks. We use these 'dead RNAs' to perform orthogonal gene knockout and transcriptional activation in human cells.

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Figure 1: dRNAs can mediate robust gene activation using an active Cas9.
Figure 2: dRNAs can specifically upregulate gene expression, have a specificity profile similar to 20bp sgRNA activators, and can be used for orthogonal gene control in human cells.

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Change history

  • 04 February 2016

    In the version of this article initially published, when discussing the data in Figure 2b, on p. 1160, we wrote, “...targeting the same HBG1/2 promoter and found they had 32 and 55 perturbed transcripts....” This should have been “31 and 55 perturbed transcripts” as in the sentence in the figure legend discussing the same data. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

The authors thank K. Zheng and I. Slaymaker for their support and input. J.E.D. is supported by a Life Science Research Foundation post-doctoral fellowship of the Cystic Fibrosis Foundation. O.O.A. is supported by a Friends of the McGovern Institute Fellowship. J.S.G. is supported by a Department of Energy (DOE) Computational Science Graduate Fellowship. F.Z. is supported by the National Institute of Mental Health (NIMH) (1DP1-MH100706), the Poitras, Vallee, Simons, Paul G. Allen and New York Stem Cell Foundations, and David R. Cheng and Bob Metcalfe. The authors plan to make the reagents widely available to the academic community through Addgene and to provide software tools via the Zhang laboratory website (http://www.genome-engineering.org/).

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Author notes

  1. James E Dahlman and Omar O Abudayyeh: These authors contributed equally to this work.

Authors and Affiliations

  1. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    James E Dahlman, Omar O Abudayyeh, Julia Joung, Jonathan S Gootenberg, Feng Zhang & Silvana Konermann

  2. Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA

    James E Dahlman

  3. McGovern Institute for Brain Research, MIT, Cambridge, Massachusetts, USA

    Omar O Abudayyeh, Julia Joung, Jonathan S Gootenberg, Feng Zhang & Silvana Konermann

  4. Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA

    Omar O Abudayyeh, Julia Joung, Jonathan S Gootenberg, Feng Zhang & Silvana Konermann

  5. Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA

    Omar O Abudayyeh, Julia Joung, Jonathan S Gootenberg, Feng Zhang & Silvana Konermann

  6. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

    Jonathan S Gootenberg

Authors
  1. James E Dahlman
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  5. Feng Zhang
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Contributions

J.E.D., O.O.A., F.Z. and S.K. conceived this study and designed the experiments. J.E.D., O.O.A., S.K., J.J. and J.S.G. performed experiments. J.E.D., O.O.A., F.Z. and S.K. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to James E Dahlman or Silvana Konermann.

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Competing interests

J.E.D., O.O.A., F.Z. and S.K. have filed for intellectual property rights related to material described in this publication.

Integrated supplementary information

Supplementary Figure 1 dRNAs can mediate robust gene activation using an active Cas9.

Three different dRNAs targeting the HBG1 promoter region were designed. The length of the RNA targeting sequence was varied from 11nt to 20nt. HBG1 mRNA (normalized to GAPDH, and compared to cells transfected with GFP plasmid) was quantified, as well HBG1 indel frequency. In all cases, guides were designed with MS2 binding loops in the tetraloops and stem loop two11, and were co-transfected with active Cas9 and the MPH transcriptional activation complex. Average +/− SEM is plotted, N=2-3 replicates group.

Supplementary Figure 2 20bp dRNAs with mismatches at the 5′ end can activate transcription.

Four dRNAs were designed to target the HBG1 promoter region with a series of 5′ end mismatches (red). Target indel formation occurred consistently when sixteen or more nucleotides were matched to target DNA. Gene activation was observed with as few as 11 bp of homology to the target DNA. Average +/− SEM is plotted, N=2-3 replicates/ group.

Supplementary Figure 3 Transcriptome-wide mRNA profiles for ten different sgRNAs targeting HBG1/2

20nt sgRNAs with MS2 binding loops were co-transfected with dCas9 and the MPH complex. A previously published nuclease OT score13 did not significantly correlate with guide specificity. (In all cases, N=3 replicates / group).

Supplementary Figure 4 dRNAs activate target gene expression with active Cas9

A375 cells were transduced with lentivirus containing a dRNA. (a) Indel formation was measured at 0.6% and 0.05% for DNA sites targeted by ITGA9 and EGFR dRNAs, respectively. (b) ITGA9 and EGFR mRNA levels (normalized to GAPDH) were quantified. In all cases, average + / − SEM is plotted, and N=3 replicates / group.

Supplementary information

Supplementary Text

Supplementary Figures 1-4 and Supplementary Notes (PDF 2748 kb)

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Dahlman, J., Abudayyeh, O., Joung, J. et al. Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat Biotechnol 33, 1159–1161 (2015). https://doi.org/10.1038/nbt.3390

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