title: Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice.
abstract: Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis, in vivo synthetic lethality screening and chromosome engineering. Negative-selection screening in the pancreas using multiplexed-CRISPR/Cas9 confirms the vulnerability of pancreatic cells to Brca2-inactivation in a Kras-mutant context. We also demonstrate modelling of chromosomal deletions and targeted somatic engineering of inter-chromosomal translocations, offering multifaceted opportunities to study complex structural variation, a hallmark of pancreatic cancer. The low-frequency mosaic pattern of transfection-based CRISPR/Cas9 delivery faithfully recapitulates the stochastic nature of human tumorigenesis, supporting wide applicability for biological/preclinical research.
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.|German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
authors.forename: Roman
authors.initials: R
authors.lastname: Maresch
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Mueller
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Veltkamp
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.
authors.forename: Rupert
authors.initials: R
authors.lastname: Öllinger
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Mathias
authors.initials: M
authors.lastname: Friedrich
authors.affiliation: Institute of Radiology, Klinikum rechts der Isar, Technischen Universität München, 81675 Munich, Germany.
authors.forename: Irina
authors.initials: I
authors.lastname: Heid
authors.affiliation: Department of Pathology, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Steiger
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.|German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Weber
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.|German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Engleitner
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.|German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
authors.forename: Maxim
authors.initials: M
authors.lastname: Barenboim
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Klein
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Sandra
authors.initials: S
authors.lastname: Louzada
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Ruby
authors.initials: R
authors.lastname: Banerjee
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Strong
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.|German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
authors.forename: Teresa
authors.initials: T
authors.lastname: Stauber
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.|German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Gross
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.|German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
authors.forename: Ulf
authors.initials: U
authors.lastname: Geumann
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Lange
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.|Institute of Virology, Technische Universität München/Helmholtz Zentrum München, 81675 Munich, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Ringelhan
authors.affiliation: Instituto de Biomedicina y Biotecnología de Cantabria, 39011 Santander, Spain.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Varela
authors.affiliation: Helmholtz Zentrum München, Research Unit Radiation Cytogenetics, 85764 Neuherberg, Germany.
authors.forename: Kristian
authors.initials: K
authors.lastname: Unger
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Fengtang
authors.initials: F
authors.lastname: Yang
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.
authors.forename: Roland M
authors.initials: RM
authors.lastname: Schmid
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: George S
authors.initials: GS
authors.lastname: Vassiliou
authors.affiliation: Institute of Radiology, Klinikum rechts der Isar, Technischen Universität München, 81675 Munich, Germany.
authors.forename: Rickmer
authors.initials: R
authors.lastname: Braren
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.
authors.forename: Günter
authors.initials: G
authors.lastname: Schneider
authors.affiliation: Institute of Virology, Technische Universität München/Helmholtz Zentrum München, 81675 Munich, Germany.|Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
authors.forename: Mathias
authors.initials: M
authors.lastname: Heikenwalder
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Allan
authors.initials: A
authors.lastname: Bradley
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.|German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Saur
authors.affiliation: Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.|German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Rad
mesh_terms: D000818:Animals; D024682:BRCA2 Protein / Q000235:genetics; D064113:CRISPR-Cas Systems; D063646:Carcinogenesis / Q000235:genetics*; D002872:Chromosome Deletion; D018274:Electroporation; D005818:Genetic Engineering / Q000379:methods; D016678:Genome; D059014:High-Throughput Nucleotide Sequencing; D007150:Immunohistochemistry; D008279:Magnetic Resonance Imaging; D051379:Mice; D009154:Mutation; D009374:Neoplasms, Experimental / Q000235:genetics; D010179:Pancreas / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000235:genetics*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D017422:Sequence Analysis, DNA; D014162:Transfection / Q000379:methods; D014178:Translocation, Genetic / Q000235:genetics
chemical_list: D024682:BRCA2 Protein; C106711:BRCA2 protein, mouse; C495971:Hras protein, mouse; D016283:Proto-Oncogene Proteins p21(ras)