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CRISPR somatic genome engineering and cancer modeling in the mouse pancreas and liver

Nature Protocols volume 17, pages 1142–1188 (2022)Cite this article

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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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Fig. 1: Overview of workflows for somatic genome editing in the mouse liver and pancreas.
Fig. 2: Overview of CRISPR delivery approaches to the pancreas or liver and schemes of Cas9 GEMMs for somatic CRISPR engineering.
Fig. 3: Overview of CRISPR systems and applications.
Fig. 4: Efficacy of viral and nonviral nucleic acid delivery methods for the pancreas and liver of mice.
Fig. 5: Quantification of CRISPR editing efficiencies in the liver and pancreas upon scAAV8-based sgRNA delivery.
Fig. 6: Induction of pancreatic ductal adenocarcinoma (PDAC) through somatic genome engineering in mice.
Fig. 7: Intrachromosomal rearrangements induced by somatic CRISPR–Cas9 multiplexing in the mouse liver by using HTVI.
Fig. 8: Interchromosomal rearrangements induced by CRISPR–Cas9 multiplexing in the pancreas.
Fig. 9: Mutational analysis of CRISPR-edited tumors and corresponding cell lines.

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Data availability

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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Author notes

  1. These authors contributed equally: Thorsten Kaltenbacher, Jessica Löprich.

Authors and Affiliations

  1. Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany

    Thorsten Kaltenbacher, Jessica Löprich, Roman Maresch, Julia Weber, Sebastian Müller, Rupert Oellinger, Nina Groß, Joscha Griger, Niklas de Andrade Krätzig, Sebastian A. Widholz, Anja Pfaus, Christian J. Braun & Roland Rad

  2. Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany

    Thorsten Kaltenbacher, Jessica Löprich, Roman Maresch, Julia Weber, Sebastian Müller, Rupert Oellinger, Nina Groß, Joscha Griger, Niklas de Andrade Krätzig, Sebastian A. Widholz, Stefanie Bärthel, Chiara Falcomatà, Anja Pfaus, Marc Schmidt-Supprian, Dieter Saur & Roland Rad

  3. Institute of Pharmacology and Toxicology, Technical University of Munich, Munich, Germany

    Petros Avramopoulos, Deepak Ramanujam, Sabine Brummer & Stefan Engelhardt

  4. DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany

    Petros Avramopoulos, Deepak Ramanujam & Stefan Engelhardt

  5. Institute of Experimental Cancer Therapy, Technical University of Munich, Munich, Germany

    Stefanie Bärthel, Chiara Falcomatà & Dieter Saur

  6. Department of Medicine II, University Hospital, LMU Munich, Munich, Germany

    Ahmed Alnatsha & Julia Mayerle

  7. German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany

    Julia Mayerle, Marc Schmidt-Supprian, Dieter Saur & Roland Rad

  8. Institute of Experimental Hematology, School of Medicine, Technical University of Munich, Munich, Germany

    Marc Schmidt-Supprian

  9. Department of Medicine II, Klinikum rechts der Isar, School of Medicine, Technical University of Munich, Munich, Germany

    Maximilian Reichert, Günter Schneider, Ursula Ehmer, Dieter Saur & Roland Rad

  10. Department of Pediatrics, Dr. von Hauner Children’s Hospital, University Hospital, LMU Munich, Munich, Germany

    Christian J. Braun

  11. Hopp Children’s Cancer Center Heidelberg (KiTZ), German Cancer Research Center (DKFZ), Heidelberg, Germany

    Christian J. Braun

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  1. Thorsten Kaltenbacher
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Contributions

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.

Corresponding author

Correspondence to Roland Rad.

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Nature Protocols thanks Takahiro Kodama and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

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

Supplementary information

Source data

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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