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Inducible in vivo genome editing with CRISPR-Cas9

Nature Biotechnology volume 33, pages 390–394 (2015)Cite this article

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Abstract

CRISPR-Cas9-based genome editing enables the rapid genetic manipulation of any genomic locus without the need for gene targeting by homologous recombination. Here we describe a conditional transgenic approach that allows temporal control of CRISPR-Cas9 activity for inducible genome editing in adult mice. We show that doxycycline-regulated Cas9 induction enables widespread gene disruption in multiple tissues and that limiting the duration of Cas9 expression or using a Cas9D10A (Cas9n) variant can regulate the frequency and size of target gene modifications, respectively. Further, we show that this inducible CRISPR (iCRISPR) system can be used effectively to create biallelic mutation in multiple target loci and, thus, provides a flexible and fast platform to study loss-of-function phenotypes in vivo.

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Figure 1: Inducible genome editing in mouse ESCs.
Figure 2: Inducible genome editing in adult mice.
Figure 3: Frequency and type of gene modifications following Cas9 or Cas9n induction in mice.

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References

  1. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  2. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  Google Scholar 

  3. Ran, F.A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    Article  CAS  Google Scholar 

  4. Li, D. et al. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat. Biotechnol. 31, 681–683 (2013).

    Article  CAS  Google Scholar 

  5. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    Article  CAS  Google Scholar 

  6. Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).

    Article  CAS  Google Scholar 

  7. Hochedlinger, K., Yamada, Y., Beard, C. & Jaenisch, R. Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell 121, 465–477 (2005).

    Article  CAS  Google Scholar 

  8. Premsrirut, P.K. et al. A rapid and scalable system for studying gene function in mice using conditional RNA interference. Cell 145, 145–158 (2011).

    Article  CAS  Google Scholar 

  9. Moser, A.R. et al. Homozygosity for the Min allele of Apc results in disruption of mouse development prior to gastrulation. Dev. Dyn. 203, 422–433 (1995).

    Article  CAS  Google Scholar 

  10. Su, L.K. et al. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science 256, 668–670 (1992).

    Article  CAS  Google Scholar 

  11. González, F. et al. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell 15, 215–226 (2014).

    Article  Google Scholar 

  12. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).

    Article  CAS  Google Scholar 

  13. Sansom, O.J. et al. Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration. Genes Dev. 18, 1385–1390 (2004).

    Article  CAS  Google Scholar 

  14. Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762–1772 (2011).

    Article  CAS  Google Scholar 

  15. Reed, K.R. et al. A limited role for p53 in modulating the immediate phenotype of Apc loss in the intestine. BMC Cancer 8, 162 (2008).

    Article  Google Scholar 

  16. Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014).

    Article  CAS  Google Scholar 

  17. Dow, L.E. et al. Conditional reverse tet-transactivator mouse strains for the efficient induction of TRE-regulated transgenes in mice. PLoS ONE 9, e95236 (2014).

    Article  Google Scholar 

  18. Maddalo, D. et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516, 423–427 (2014).

    Article  CAS  Google Scholar 

  19. Platt, R.J. et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).

    Article  CAS  Google Scholar 

  20. Sánchez-Rivera, F.J. et al. Rapid modelling of cooperating genetic events in cancer through somatic genome editing. Nature 516, 428–431 (2014).

    Article  Google Scholar 

  21. Xue, W. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380–384 (2014).

    Article  CAS  Google Scholar 

  22. Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    Article  CAS  Google Scholar 

  23. Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  Google Scholar 

  24. Dow, L.E. et al. A pipeline for the generation of shRNA transgenic mice. Nat. Protoc. 7, 374–393 (2012).

    Article  CAS  Google Scholar 

  25. Sanjana, N.E. et al. A transcription activator-like effector toolbox for genome engineering. Nat. Protoc. 7, 171–192 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J.Simon for technical assistance with animal colonies, and other members of the Lowe laboratory for advice and discussions. We thank the MSKCC transgenic core facility for production of transgenic animals by blastocyst injection and the MSKCC Genomics core facility for library preparation and Illumina sequencing. This work was supported by a program project grant number CA-013106 from the National Cancer Institute (NCI). L.E.D. was supported by a K22 Career Development Award from the NCI/ National Institutes of Health (NIH) under award number 1K22CA181280-01. K.P.O'R. was supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the NIH under award number T32GM07739 to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program. G.L. was supported by an American Cancer Society postdoctoral fellowship PF-13-037-01 and NIH F32 grant 1F32CA177072-01. S.W.L. is an investigator of the Howard Hughes Medical Institute and the Geoffrey Beene Chair for Cancer Biology.

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  1. Lukas E Dow and Jonathan Fisher: These authors contributed equally to this work.

Authors and Affiliations

  1. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Lukas E Dow, Jonathan Fisher, Kevin P O'Rourke, Edward R Kastenhuber, Geulah Livshits, Darjus F Tschaharganeh & Scott W Lowe

  2. Hematology/Medical Oncology Division, Department of Medicine, Weill-Cornell Medical College, New York, New York, USA

    Lukas E Dow & Ashlesha Muley

  3. Weill-Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD program, New York, New York, USA

    Kevin P O'Rourke

  4. Gerstner Sloan Kettering Graduate School of Biomedical Sciences, New York, New York, USA

    Edward R Kastenhuber

  5. Bioinformatics Core Facility, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Nicholas D Socci

  6. Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Scott W Lowe

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  1. Lukas E Dow
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Contributions

L.E.D. conceived of the project, performed and analyzed experiments and wrote the paper. J.F. performed and analyzed experiments and wrote the paper. K.P.O'R., A.M., G.L. and D.F.T. performed and analyzed experiments. E.R.K. and N.D.S. developed informatics pipelines and analyzed data. S.W.L. supervised experiments, analyzed data and wrote the paper.

Corresponding authors

Correspondence to Lukas E Dow or Scott W Lowe.

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The authors declare no competing financial interests.

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Supplementary Figures 1–7 and Supplementary Tables 1–3 (PDF 10365 kb)

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Dow, L., Fisher, J., O'Rourke, K. et al. Inducible in vivo genome editing with CRISPR-Cas9. Nat Biotechnol 33, 390–394 (2015). https://doi.org/10.1038/nbt.3155

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