Abstract
Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3–5 weeks.
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All data generated or analyzed during this study are included in either this paper or our original research study5,6. Source data are provided with this paper.
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Acknowledgements
D.S. is supported by the European Research Council (Consolidator Grant 648521) and the Deutsche Forschungsgemeinschaft (SA1374/4-2; SFB 1321 Project-ID 329628492, SFB 1371 Project-ID 395357507). R.R. is supported by the European Research Council (Consolidator Grants PACA-MET (819642) and MSCA-ITN-ETN (861196)), the Deutsche Forschungsgemeinschaft (DFG RA1629/2-1; SFB1321), the German Cancer Consortium and the Deutsche Krebshilfe (70114314).
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T.K., J.L., R.M., J.W., S.M., R.O. and R.R. conceptualized, designed or developed workflows, tools or procedures. U.E. provided resources and critical input to HTVI experiments. P.A., D.R., S. Brummer and S.E produced and purified AAV. T.K., J.L., R.M., J.W., S.M., R.O., N.G. and J.G. performed wet-lab experiments. N.A.K. performed computational analysis. A.A., J.M., M.S.-S., M.R., G.S. and D.S. provided biological resources. T.K, J.L. and R.R. wrote the manuscript with input from R.M., J.W., S.A.W., S. Bärthel, C.F., A.P. and C.J.B.
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Maresch, R. et al. Nat. Commun. 7, 10770 (2016): https://doi.org/10.1038/ncomms10770
Weber, J. et al. Proc. Natl Acad. Sci. USA 112, 13982–13987 (2015): https://doi.org/10.1073/pnas.1512392112
Mueller, S. et al. Nature 554, 62–68 (2018): https://doi.org/10.1038/nature25459
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Source Data Fig. 4c
Quantification of transduction efficiencies of scAAV8 in the pancreas and the liver in Rosa26mT/mG reporter mice.
Source Data Fig. 5b
Quantification of CRISPR editing efficiencies in the liver and pancreas upon scAAV8-based sgRNA delivery.
Source Data Fig. 6
Statistical data.
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Kaltenbacher, T., Löprich, J., Maresch, R. et al. CRISPR somatic genome engineering and cancer modeling in the mouse pancreas and liver. Nat Protoc 17, 1142–1188 (2022). https://doi.org/10.1038/s41596-021-00677-0
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DOI: https://doi.org/10.1038/s41596-021-00677-0
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