Multiplexed pancreatic genome engineering and cancer induction by transfection-based CRISPR/Cas9 delivery in mice

Roman Maresch, Sebastian Mueller, Christian Veltkamp, Rupert Öllinger, Mathias Friedrich, Irina Heid, Katja Steiger, Julia Weber, Thomas Engleitner, Maxim Barenboim, Sabine Klein, Sandra Louzada, Ruby Banerjee, Alexander Strong, Teresa Stauber, Nina Gross, Ulf Geumann, Sebastian Lange, Marc Ringelhan, Ignacio Varela, Kristian Unger, Fengtang Yang, Roland M. Schmid, George S. Vassiliou, Rickmer Braren, Günter Schneider, Mathias Heikenwalder, Allan Bradley, Dieter Saur, Roland Rad

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.