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Here, we show CRISPR/Cas9-based targeted somatic multiplexmutagenesis and its application for high-throughput analysis of gene function in mice. Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We observed Darwinian selection of target genes, which suppress tumorigenesis in the respective cellular/tissue context, such asPtenorCdkn2a,and conversely found low frequency ofBrca1/2alterations, explaining mutational spectra in human ICC/HCC. Our studies show that multiplexed CRISPR/Cas9 can be used for recessive genetic screening or high-throughput cancer gene validation in mice. The analysis of CRISPR/Cas9-induced tumors provided support for a major role of chromatin modifiers in hepatobiliary tumorigenesis, including that of ARID family proteins, which have recently been reported to be mutated in ICC/HCC. We have also comprehensively characterized the frequency and size of chromosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISPR/Cas9 multiplexing, as well as opportunities for chromosome engineering in the context of hepatobiliary tumorigenesis. Our study describes novel approaches to model and study cancer in a high-throughput multiplexed format that will facilitate the functional annotation of cancer genomes.

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.

Mouse models of human cancer have transformed our ability to link genetics, molecular mechanisms and phenotypes. Both reverse and forward genetics in mice are currently gaining momentum through advances in next-generation sequencing (NGS). Methodologies to analyze sequencing data were, however, developed for humans and hence do not account for species-specific differences in genome structures and experimental setups. Here, we describe standardized computational pipelines specifically tailored to the analysis of mouse genomic data. We present novel tools and workflows for the detection of different alteration types, including single-nucleotide variants (SNVs), small insertions and deletions (indels), copy-number variations (CNVs), loss of heterozygosity (LOH) and complex rearrangements, such as in chromothripsis. Workflows have been extensively validated and cross-compared using multiple methodologies. We also give step-by-step guidance on the execution of individual analysis types, provide advice on data interpretation and make the complete code available online. The protocol takes 2–7 d, depending on the desired analyses. Here, the authors present standardized computational pipelines tailored specifically to the analysis of cancer genome sequencing data from mice. The protocol enables detection of single-nucleotide variants, indels, copy-number variations, loss of heterozygosity and complex rearrangements such as those of chromothripsis.

CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.

Friedrich et al. describe their toolkit for transposon-based insertional mutagenesis in mice for discovering cancer genes. Genome-wide transposon insertion sites are identified, mapped and quantified using QiSeq. Transposon-mediated forward genetics screening in mice has emerged as a powerful tool for cancer gene discovery. It pinpoints cancer drivers that are difficult to find with other approaches, thus complementing the sequencing-based census of human cancer genes. We describe here a large series of mouse lines for insertional mutagenesis that are compatible with two transposon systems, PiggyBac and Sleeping Beauty , and give guidance on the use of different engineered transposon variants for constitutive or tissue-specific cancer gene discovery screening. We also describe a method for semiquantitative transposon insertion site sequencing (QiSeq). The QiSeq library preparation protocol exploits acoustic DNA fragmentation to reduce bias inherent to widely used restriction–digestion-based approaches for ligation-mediated insertion site amplification. Extensive multiplexing in combination with next-generation sequencing allows affordable ultra-deep transposon insertion site recovery in high-throughput formats within 1 week. Finally, we describe principles of data analysis and interpretation for obtaining insights into cancer gene function and genetic tumor evolution.

Background Glioma is the most common intrinsic brain tumor and also occurs in the spinal cord. Activating EGFR mutations are common in IDH1 wild-type gliomas. However, the cooperative partners of EGFR driving gliomagenesis remain poorly understood. Results We explore EGFR -mutant glioma evolution in conditional mutant mice by whole-exome sequencing, transposon mutagenesis forward genetic screening, and transcriptomics. We show mutant EGFR is sufficient to initiate gliomagenesis in vivo, both in the brain and spinal cord. We identify significantly recurrent somatic alterations in these gliomas including mutant EGFR amplifications and Sub1 , Trp53 , and Tead2 loss-of-function mutations. Comprehensive functional characterization of 96 gliomas by genome-wide piggyBac insertional mutagenesis in vivo identifies 281 known and novel EGFR -cooperating driver genes, including Cdkn2a , Nf1 , Spred1 , and Nav3 . Transcriptomics confirms transposon-mediated effects on expression of these genes. We validate the clinical relevance of new putative tumor suppressors by showing these are frequently altered in patients’ gliomas, with prognostic implications. We discover shared and distinct driver mutations in brain and spinal gliomas and confirm in vivo differential tumor suppressive effects of Pten between these tumors. Functional validation with CRISPR-Cas9-induced mutations in novel genes Tead2 , Spred1 , and Nav3 demonstrates heightened EGFRvIII -glioma cell proliferation. Chemogenomic analysis of mutated glioma genes reveals potential drug targets, with several investigational drugs showing efficacy in vitro. Conclusion Our work elucidates functional driver landscapes of EGFR -mutant gliomas, uncovering potential therapeutic strategies, and provides new tools for functional interrogation of gliomagenesis.

Conditional PiggyBac transposase targeted to Rosa26 (tissue-specific PiggyBac transposase, TSPB), SA = splice acceptor; SD = splice donor; CAG = CAG promoter; SB = Sleeping Beauty; PB = PiggyBac inverted repeats; iPBase = insect version of the PiggyBac transposase. Gene inactivation can occur if the transposon inserts in the body of the gene as a consequence of gene trapping which can occur in either orientation because of the presence of two splice acceptors and bidirectional poly(A) (pA) sites. B. Outline of the experimental design: quadruple transgenic mice conditionally activate EGFRvIII expression and PiggyBac transposition in the central nervous system.

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 Kras MUT levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains ( Myc , Yap1 or Nfkb2 ), which collaborate with heterozygous Kras MUT in 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. Oncogenic dosage variation along distinct evolutionary routes defines fundamental aspects of pancreatic cancer biology and phenotypic diversification. Predicting pancreatic cancer phenotypes Despite the availability of hundreds of pancreatic cancer genomes, it has been difficult to associate specific mutation patterns with distinct biological features. To address this, Roland Rad and colleagues tracked genomic alterations during the development of pancreatic cancer, aiming to link mutations to heterogeneous phenotypes. Human and mouse studies reveal that different gene dosages of an activating KRAS mutation are critical determinants of pancreatic cancer biology, including early progression, metastasis, histopathology, cellular plasticity and clinical aggressiveness. Mutant KRAS is amplified through distinct evolutionary routes during tumorigenesis that are defined by prior alterations of specific tumour suppressors and oncogenes. This study sheds light on the mechanisms underlying the phenotypic heterogeneity of pancreatic cancer and may aid advances in diagnosis, prognosis and therapy.

Biliary tract cancers (BTC) represent a heterogeneous group of malignancies with a poor prognosis and rising incidence. Oncogenic FGFR2 fusions are one of several actionable molecular alterations. In this context, selective FGFR tyrosine kinase inhibitors have demonstrated promising and durable response rates and are now approved and included in clinical guidelines. However, secondary kinase mutations frequently arise over time, leading to resistance against these drugs. We present the case of a 41-year-old male patient with metastatic BTC who underwent molecular analysis after disease progression to various established chemotherapy combinations. Testing identified an oncogenic FGFR2 fusion (FGFR2::BICC1). The patient was treated with pemigatinib for 14 months. Upon disease progression, the resistance-associated FGFR2 p. E565A variant was detected in a follow-up biopsy. Treatment was switched to futibatinib, resulting in rapid disease progression. Lacking other therapeutic options, the patient was treated with lenvatinib, supported by previously published data suggesting a potential benefit in similar settings. The treatment was well tolerated, with only a mild increase in transaminases, and the patient remained on treatment with noteworthy effects for 15 months to date. With a growing incidence of BTC and growing use of targeted therapies for FGFR2 alterations, the emergence of secondary resistance-causing point mutations following treatment with approved inhibitors is becoming increasingly challenging. Beyond selective inhibitors, lenvatinib may represent a viable therapeutic option.

Roland Rad and colleagues report development of a new conditional piggyBac transposition system for performing insertional mutagenesis screens in mice. They apply the system to identify new oncogenic driver pathways for pancreatic cancer. Here we describe a conditional piggyBac transposition system in mice and report the discovery of large sets of new cancer genes through a pancreatic insertional mutagenesis screen. We identify Foxp1 as an oncogenic transcription factor that drives pancreatic cancer invasion and spread in a mouse model and correlates with lymph node metastasis in human patients with pancreatic cancer. The propensity of piggyBac for open chromatin also enabled genome-wide screening for cancer-relevant noncoding DNA, which pinpointed a Cdkn2a cis -regulatory region. Histologically, we observed different tumor subentities and discovered associated genetic events, including Fign insertions in hepatoid pancreatic cancer. Our studies demonstrate the power of genetic screening to discover cancer drivers that are difficult to identify by other approaches to cancer genome analysis, such as downstream targets of commonly mutated human cancer genes. These piggyBac resources are universally applicable in any tissue context and provide unique experimental access to the genetic complexity of cancer.