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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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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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.

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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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