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.
Highlights
Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation
Bottlenecks and limitations of classic transgenesis are, (i) the long time frames needed to generate and intercross genetically modified mice, (ii) the difficulties to model some aspects of the human disease, (iii) the lack of high-throughput methods for functional interrogation of complex genetic interactions, (iv) the confounding phenotypes emerging in multiallelic crosses of transgenic mice generated in various genetic backgrounds and (v) the lack of tools for efficient modelling of the complex structural variations defining human cancer
green fluorescent protein (GFP)-positive cells could not be observed upon administration of control vectors (n 1⁄4 3 mice), we found that an average of 120 cells per pancreas exhibited a membrane-targeted tdTomato (mT) to membrane-targeted EGFP (mG) switch in mice receiving phosphoglycerate kinase 1 (PGK)-Cre vector
Summary
Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Bottlenecks and limitations of classic transgenesis are, (i) the long time frames needed to generate and intercross genetically modified mice, (ii) the difficulties to model some aspects of the human disease (for example, the stochastic nature of somatic mutations in adult mice), (iii) the lack of high-throughput methods for functional interrogation of complex genetic interactions, (iv) the confounding phenotypes emerging in multiallelic crosses of transgenic mice generated in various genetic backgrounds and (v) the lack of tools for efficient modelling of the complex structural variations defining human cancer. We and others showed recently CRISPR/Cas9-based somatic genome editing in different organs of mice, including the lung, liver, brain and pancreas[22,23,24,25,26,27,28,29] and direct in vivo forward genetic screening[29]. Most approaches to deliver CRISPR/Cas[9] in vivo have limitations, such as the inability or low efficiency of vector multiplexing for complex combinatorial gene editing or the high risk to induce off-target effects due to continuous activity of the system, for example, by virally delivered stably integrated CRISPR/Cas[9]
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