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The dietary change resulting from the domestication of plant and animal species and development of agriculture at different locations across the world was one of the most significant changes in human evolution. An increase in dietary carbohydrates caused an increase in dental caries following the development of agriculture, mediated by the cariogenic oral bacterium Streptococcus mutans. Salivary agglutinin [SAG, encoded by the deleted in malignant brain tumors 1 (DMBT1) gene] is an innate immune receptor glycoprotein that binds a variety of bacteria and viruses, and mediates attachment of S. mutans to hydroxyapatite on the surface of the tooth. In this study we show that multiallelic copy number variation (CNV) within DMBT1 is extensive across all populations and is predicted to result in between 7-20 scavenger-receptor cysteine-rich (SRCR) domains within each SAG molecule. Direct observation of de novo mutation in multigeneration families suggests these CNVs have a very high mutation rate for a protein-coding locus, with a mutation rate of up to 5% per gamete. Given that the SRCR domains bind S. mutans and hydroxyapatite in the tooth, we investigated the association of sequence diversity at the SAG-binding gene of S. mutans, and DMBT1 CNV. Furthermore, we show that DMBT1 CNV is also associated with a history of agriculture across global populations, suggesting that dietary change as a result of agriculture has shaped the pattern of CNV at DMBT1, and that the DMBT1-S. mutans interaction is a promising model of host-pathogen-culture coevolution in humans.

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.

The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest KrasMUTlevels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUTin driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfβ-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.