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Transgenic mice for in vivo epigenome editing with CRISPR-based systems

Nature Methods volume 18, pages 965–974 (2021)Cite this article

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Abstract

CRISPR-Cas9 technologies have dramatically increased the ease of targeting DNA sequences in the genomes of living systems. The fusion of chromatin-modifying domains to nuclease-deactivated Cas9 (dCas9) has enabled targeted epigenome editing in both cultured cells and animal models. However, delivering large dCas9 fusion proteins to target cells and tissues is an obstacle to the widespread adoption of these tools for in vivo studies. Here, we describe the generation and characterization of two conditional transgenic mouse lines for epigenome editing, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. By targeting the guide RNAs to transcriptional start sites or distal enhancer elements, we demonstrate regulation of target genes and corresponding changes to epigenetic states and downstream phenotypes in the brain and liver in vivo, and in T cells and fibroblasts ex vivo. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.

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Fig. 1: AAV-based gRNA and Cre recombinase delivery to Rosa26:LSL-dCas9p300 mice activates Pdx1 gene expression and catalyzes targeted histone acetylation.
Fig. 2: Epigenomic enhancement of Fos in vivo increases excitability in CA1 neurons.
Fig. 3: AAV-based gRNA and Cre recombinase delivery to Rosa26:LSL-dCas9KRAB mice represses Pcsk9 and catalyzes targeted histone methylation.
Fig. 4: Epigenome editing in T cells for activation and repression of Foxp3.

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Data availability

Raw sequencing files are available from the NCBI Gene Expression Omnibus via SuperSeries accession GSE146848. Source data are provided with this paper.

Code availability

Data processing and analysis code is made available through Zenodo80 and on GitHub (https://github.com/ReddyLab/gemberling-et-al-NMETH-A42509C).

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Acknowledgements

We thank J. Deng and B. Ryu for assistance with viral injections, and K. Franks for his support in performing the electrophysiology recordings. This work has been supported by the Allen Distinguished Investigator Program through The Paul G. Allen Frontiers Group to C.A.G., Translating Duke Health Initiative to K.D.P., Open Philanthropy to C.A.G., National Institutes of Health (NIH) grants R33DA041878 to A.E.W. and C.A.G., R01DA036865 to C.A.G., U01AI146356 to C.A.G., UM1HG009428 to T.E.R., M.C. and C.A.G., UG3AR075336 to A.A., and R01GM115474 to M.C., National Science Foundation (NSF) grant EFMA-1830957 to C.A.G., Defense Advanced Research Projects Agency (DARPA) grant HR0011-19-2-0008 to C.A.G., a Pfizer-NCBiotech Distinguished Postdoctoral Fellowship in Gene Therapy to J.C.B., and a Swiss National Science Foundation Postdoctoral Fellowship to V.C.

Author information

Author notes

  1. Isaac B. Hilton

    Present address: Department of Bioengineering, Rice University, Houston, TX, USA

  2. Isaac B. Hilton

    Present address: Department of Biosciences, Rice University, Houston, TX, USA

  3. These authors contributed equally: Matthew P. Gemberling, Keith Siklenka.

Authors and Affiliations

  1. Department of Biomedical Engineering, Duke University, Durham, NC, USA

    Matthew P. Gemberling, Ariel Kantor, Josephine C. Bodle, Heather Daniels, Isaac B. Hilton, Aravind Asokan & Charles A. Gersbach

  2. Center for Advanced Genomic Technologies, Duke University, Durham, NC, USA

    Matthew P. Gemberling, Keith Siklenka, Alejandro Barrera, Ariel Kantor, Luke C. Bartelt, Josephine C. Bodle, Heather Daniels, Aravind Asokan, Maria Ciofani, Kenneth D. Poss, Timothy E. Reddy, Anne E. West & Charles A. Gersbach

  3. Department of Biostatistics and Bioinformatics, Duke University School of Medicine, Durham, NC, USA

    Keith Siklenka, Alejandro Barrera, Liqing Li, Courtney A. Williams, Luke C. Bartelt & Timothy E. Reddy

  4. Department of Neurobiology, Duke University School of Medicine, Durham, NC, USA

    Erica Rodriguez, Katherine R. Tonn-Eisinger, Fang Liu, Mariah F. Hazlett & Anne E. West

  5. Department of Cell Biology, Duke University Medical Center, Durham, NC, USA

    Valentina Cigliola, Kenneth D. Poss & Charles A. Gersbach

  6. Regeneration Next Initiative, Duke University, Durham, NC, USA

    Valentina Cigliola, Aravind Asokan, Kenneth D. Poss & Charles A. Gersbach

  7. Department of Surgery, Duke University Medical Center, Durham, NC, USA

    Victoria J. Madigan, Aravind Asokan & Charles A. Gersbach

  8. Division of Laboratory Animal Resources, Duke University School of Medicine, Durham, NC, USA

    Douglas C. Rouse

  9. Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA

    Aravind Asokan

  10. Department of Immunology, Duke University Medical Center, Durham, NC, USA

    Maria Ciofani

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  1. Matthew P. Gemberling
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Contributions

M.P.G., K.S., E.R., K.R.T.-E., F.L., A.K., V.C., M.F.H., L.C.B., C.A.W. and J.C.B. conducted experiments and analyzed data. H.D., D.C.R. and L.L. assisted with the mouse experiments. A.B. and K.S. performed ChIP-Seq and RNA-seq analysis. M.P.G., K.S., A.E.W. and C.A.G. wrote portions of the paper. I.B.H. provided critical reagents. V.J.M. and A.A. produced AAV9 for the mouse experiments. M.C., K.D.P., T.E.R., A.E.W. and C.A.G. provided guidance on the experimental design and interpretation of results. All authors edited the text.

Corresponding author

Correspondence to Charles A. Gersbach.

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Competing interests

C.A.G., I.B.H. and T.E.R. have filed patent applications related to CRISPR technologies for genome engineering. C.A.G. is an advisor to Tune Therapeutics, Sarepta Therapeutics, Levo Therapeutics and Iveric Bio, and a co-founder of Tune Therapeutics, Element Genomics and Locus Biosciences. A.A. is a co-founder of and advisor to StrideBio and TorqueBio. T.E.R. is a co-founder of Element Genomics. M.P.G. is a co-founder and employee of Tune Therapeutics. All other authors have no competing interests.

Additional information

Peer review information Nature Methods thanks Randall J. Platt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Madhura Mukhopadhyay was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–13.

Reporting Summary

Supplementary Table 1

Sequences for CRISPR protospacers, RT–qPCR primers and ChIP-qPCR primers

Supplementary Data 1

Statistical source data for Supplementary Fig. 2

Supplementary Data 2

Statistical source data for Supplementary Fig. 7

Supplementary Data 3

Statistical source data for Supplementary Fig. 8

Supplementary Data 4

Statistical source data for Supplementary Fig. 12

Supplementary Data 5

Unprocessed western blot for Supplementary Fig. 1A

Supplementary Data 6

Unprocessed western blot for Supplementary Fig. 1B

Supplementary Data 7

Unprocessed western blot for Supplementary Fig. 7D

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Gemberling, M.P., Siklenka, K., Rodriguez, E. et al. Transgenic mice for in vivo epigenome editing with CRISPR-based systems. Nat Methods 18, 965–974 (2021). https://doi.org/10.1038/s41592-021-01207-2

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