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Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing

Abstract

Genome sequencing studies have shown that human malignancies often bear mutations in four or more driver genes1, but it is difficult to recapitulate this degree of genetic complexity in mouse models using conventional breeding. Here we use the CRISPR-Cas9 system of genome editing2,3,4 to overcome this limitation. By delivering combinations of small guide RNAs (sgRNAs) and Cas9 with a lentiviral vector, we modified up to five genes in a single mouse hematopoietic stem cell (HSC), leading to clonal outgrowth and myeloid malignancy. We thereby generated models of acute myeloid leukemia (AML) with cooperating mutations in genes encoding epigenetic modifiers, transcription factors and mediators of cytokine signaling, recapitulating the combinations of mutations observed in patients. Our results suggest that lentivirus-delivered sgRNA:Cas9 genome editing should be useful to engineer a broad array of in vivo cancer models that better reflect the complexity of human disease.

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Figure 1: Stable modification of hematopoietic stem cells by a lentiviral sgRNA:Cas9 delivery system.
Figure 2: Multiplex gene targeting induces clonal development in vivo.
Figure 3: Myeloid malignancy modeling with multiplex genome editing.

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References

  1. The Cancer Genome Atlas Research Network. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N. Engl. J. Med. 368, 2059–2074 (2013).

  2. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Scuoppo, C. et al. A tumour suppressor network relying on the polyamine-hypusine axis. Nature 487, 244–248 (2012).

    Article  CAS  Google Scholar 

  6. Zender, L. et al. An oncogenomics-based in vivo RNAi screen identifies tumor suppressors in liver cancer. Cell 135, 852–864 (2008).

    Article  CAS  Google Scholar 

  7. Hwang, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227–229 (2013).

    Article  CAS  Google Scholar 

  8. 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 

  9. Friedland, A.E. et al. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat. Methods 10, 741–743 (2013).

    Article  CAS  Google Scholar 

  10. Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31, 686–688 (2013).

    Article  CAS  Google Scholar 

  11. Wood, A.J. et al. Targeted genome editing across species using ZFNs and TALENs. Science 333, 307 (2011).

    Article  CAS  Google Scholar 

  12. Sander, J.D. et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat. Biotechnol. 29, 697–698 (2011).

    Article  CAS  Google Scholar 

  13. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  Google Scholar 

  14. Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

    Article  CAS  Google Scholar 

  15. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nat. Biotechnol. 30, 460–465 (2012).

    Article  CAS  Google Scholar 

  16. Lee, B.H. et al. FLT3 mutations confer enhanced proliferation and survival properties to multipotent progenitors in a murine model of chronic myelomonocytic leukemia. Cancer Cell 12, 367–380 (2007).

    Article  CAS  Google Scholar 

  17. 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 

  18. Kelly, L.M. & Gilliland, D.G. Genetics of myeloid leukemias. Annu. Rev. Genomics Hum. Genet. 3, 179–198 (2002).

    Article  CAS  Google Scholar 

  19. Zhang, Y., Taylor, B.R., Shannon, K. & Clapp, D.W. Quantitative effects of Nf1 inactivation on in vivo hematopoiesis. J. Clin. Invest. 108, 709–715 (2001).

    Article  CAS  Google Scholar 

  20. Tanaka, S. et al. Ezh2 augments leukemogenicity by reinforcing differentiation blockage in acute myeloid leukemia. Blood 120, 1107–1117 (2012).

    Article  CAS  Google Scholar 

  21. Challen, G.A. et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat. Genet. 44, 23–31 (2012).

    Article  CAS  Google Scholar 

  22. Moran-Crusio, K. et al. Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation. Cancer Cell 20, 11–24 (2011).

    Article  CAS  Google Scholar 

  23. Quivoron, C. et al. TET2 inactivation results in pleiotropic hematopoietic abnormalities in mouse and is a recurrent event during human lymphomagenesis. Cancer Cell 20, 25–38 (2011).

    Article  CAS  Google Scholar 

  24. Mead, A.J. et al. FLT3-ITDs instruct a myeloid differentiation and transformation bias in lymphomyeloid multipotent progenitors. Cell Reports 3, 1766–1776 (2013).

    Article  CAS  Google Scholar 

  25. Schambach, A. et al. Equal potency of gammaretroviral and lentiviral SIN vectors for expression of O6-methylguanine-DNA methyltransferase in hematopoietic cells. Mol. Ther. 13, 391–400 (2006).

    Article  CAS  Google Scholar 

  26. Kim, J.H. et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS ONE 6, e18556 (2011).

    Article  CAS  Google Scholar 

  27. Morozova, K.S. et al. Far-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy. Biophys. J. 99, L13–L15 (2010).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank C. Morton and A. Hawkins from the Brigham and Women's Cytogenetics Core and R. Bronson from the DF/HCC Rodent Pathology Core for technical assistance and discussions. The authors thank F. Zhang for providing the CRISPR-Cas components. The authors also thank A. Schambach, E. Charpentier, J. Kroenke and A. Mullally for useful discussions, and thank S. Schwartz and B. Haas for assistance with analysis of sequencing data. C. Baum and A. Schambach of the Hannover Medical School, Hannover, Germany, kindly provided RRL.PPT.SFFV.IRES.eGFP.pre*, and D. Trono of EPFL, Lausanne, Switzerland, kindly provided both pMD2.G (Addgene plasmid 12259) and psPAX2 (Addgene plasmid 12260). This work was supported by funding from the National Institutes of Health (P01 CA108631), a Leukemia and Lymphoma Society Scholar Award, the SPARC consortium, a Center for Excellence in Genome Science grant (5P50HG006193-02 from the National Human Genome Research Institute) (A.R.) and Klarman Family Foundation at The Broad Institute (A.R.). D.H. was funded by the German Cancer Foundation (Mildred-Scheel Fellowship). M.S.K. is an EMBO and European Hematology Association Fellow.

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Authors

Contributions

D.H., M.S.K. and B.L.E. designed experiments. D.H., M.S.K., D.Y., R.B., R.V.P., M.E.C. and A.T. performed the experiments. D.H., M.S.K., D.Y., R.B., J.C.A., A.R. and B.L.E. analyzed and interpreted the data. D.H. and B.L.E. wrote the manuscript.

Corresponding author

Correspondence to Benjamin L Ebert.

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Supplementary Figures 1–18, Supplementary Tables 1–4 and Supplementary Methods and Data (PDF 42106 kb)

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Heckl, D., Kowalczyk, M., Yudovich, D. et al. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nat Biotechnol 32, 941–946 (2014). https://doi.org/10.1038/nbt.2951

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  • DOI: https://doi.org/10.1038/nbt.2951

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