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.