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

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.

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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.

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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.

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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.

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Supplementary information

Supplementary Information

Supplementary Figures 1–13.

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Supplementary Table 1

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

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Statistical source data for Supplementary Fig. 2

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Statistical source data for Supplementary Fig. 7

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Statistical source data for Supplementary Fig. 12

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Unprocessed western blot for Supplementary Fig. 1A

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Unprocessed western blot for Supplementary Fig. 1B

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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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