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

• Published in: Nature communications 2016-02, Vol.7 (1), p.10770-13, Article 10770
• Main Authors: Maresch, Roman, Mueller, Sebastian, Veltkamp, Christian, Öllinger, Rupert, Friedrich, Mathias, Heid, Irina, Steiger, Katja, Weber, Julia, Engleitner, Thomas, Barenboim, Maxim, Klein, Sabine, Louzada, Sandra, Banerjee, Ruby, Strong, Alexander, Stauber, Teresa, Gross, Nina, Geumann, Ulf, Lange, Sebastian, Ringelhan, Marc, Varela, Ignacio, Unger, Kristian, Yang, Fengtang, Schmid, Roland M., Vassiliou, George S., Braren, Rickmer, Schneider, Günter, Heikenwalder, Mathias, Bradley, Allan, Saur, Dieter, Rad, Roland
• Format: Article
• Language: English
• Subjects: 42/109; 42/41; 42/70; 59/57; 631/1647/1511; 631/1647/1513/1967/3196; 631/208/68; 631/67/1504/1713; Animals; BRCA2 protein; BRCA2 Protein - genetics; Breast cancer; Cancer; Cancer research; Carcinogenesis - genetics; Chromosome Deletion; Chromosome translocations; Chromosomes; Combinatorial analysis; CRISPR; CRISPR-Cas Systems; Electroporation; Engineering; Genes; Genetic engineering; Genetic Engineering - methods; Genetic screening; Genome; Genome editing; Genomes; High-Throughput Nucleotide Sequencing; Humanities and Social Sciences; Immunohistochemistry; In vivo methods and tests; Inactivation; Lethality; Magnetic Resonance Imaging; Medical research; Metastases; Mice; multidisciplinary; Multiplexing; Mutation; Neoplasms, Experimental - genetics; Network analysis; Pancreas; Pancreas - metabolism; Pancreatic cancer; Pancreatic Neoplasms - genetics; Phylogeny; Polymerase Chain Reaction; Proto-Oncogene Proteins p21(ras) - genetics; Science; Science (multidisciplinary); Sequence Analysis, DNA; Stochasticity; Transfection; Transfection - methods; Translocation, Genetic - genetics; Tumorigenesis
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms10770

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. CRISPR/Cas9 technology has been used for genome engineering in vivo . Here, the authors use a transfection technique to deliver multiple guide RNAs to the pancreas of adult mice, allowing genetic screening and chromosome engineering in pancreatic cancer.