Multiplexed Pancreatic Genome Engineering and Cancer Induction by Transfection-Based CRISPR/Cas9 Delivery in Mice

Nat Commun. 2016 Feb 26;7:10770. doi: 10.1038/ncomms10770.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • BRCA2 Protein / genetics
  • CRISPR-Cas Systems
  • Carcinogenesis / genetics*
  • Chromosome Deletion
  • Electroporation
  • Genetic Engineering / methods
  • Genome
  • High-Throughput Nucleotide Sequencing
  • Immunohistochemistry
  • Magnetic Resonance Imaging
  • Mice
  • Mutation
  • Neoplasms, Experimental / genetics
  • Pancreas / metabolism*
  • Pancreatic Neoplasms / genetics*
  • Phylogeny
  • Polymerase Chain Reaction
  • Proto-Oncogene Proteins p21(ras) / genetics
  • Sequence Analysis, DNA
  • Transfection / methods
  • Translocation, Genetic / genetics

Substances

  • BRCA2 Protein
  • BRCA2 protein, mouse
  • Hras protein, mouse
  • Proto-Oncogene Proteins p21(ras)