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We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine. Gao, Nowak-Imialek, Chen et al. generate porcine and human stem cells that possess expanded developmental potency for both embryonic and extra-embryonic cell lineages.

Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM -mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM -mutant backgrounds are different to those in BRCA1 -mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation. Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. Here, the authors provide evidence that these hypersensitivities reflect a crucial role for ATM at damaged replication forks being to prevent toxic DNA end-joining leading to chromosome fusions and cell death.

Many gene fusions are reported in tumours and for most their role remains unknown. As fusions are used for diagnostic and prognostic purposes, and are targets for treatment, it is crucial to assess their function in cancer. To systematically investigate the role of fusions in tumour cell fitness, we utilized RNA-sequencing data from 1011 human cancer cell lines to functionally link 8354 fusion events with genomic data, sensitivity to >350 anti-cancer drugs and CRISPR-Cas9 loss-of-fitness effects. Established clinically-relevant fusions were identified. Overall, detection of functional fusions was rare, including those involving cancer driver genes, suggesting that many fusions are dispensable for tumour fitness. Therapeutically actionable fusions involving RAF1 , BRD4 and ROS1 were verified in new histologies. In addition, recurrent YAP1-MAML2 fusions were identified as activators of Hippo-pathway signaling in multiple cancer types. Our approach discriminates functional fusions, identifying new drivers of carcinogenesis and fusions that could have clinical implications. Gene fusions are observed in many cancers but their link to tumour fitness is largely unknown. Here, transcriptomic analysis combined with pharmacological and CRISPR-Cas9 screening of cancer cell lines was used to evaluate the functional linkage between fusions and tumour fitness.

BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR–Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, ‘Shieldin’ (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance. Through CRISPR–Cas9 screen, Dev et al. identified that SHLD1/2 inhibition contributes to PARP-inhibitor resistance. Mechanistically, SHLDs promote non-homologous end-joining and antagonize homologous recombination.

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.

A high-quality sequence assembly of the zebrafish genome reveals the largest gene set of any vertebrate and provides information on key genomic features, and comparison to the human reference genome shows that approximately 70% of human protein-coding genes have at least one clear zebrafish orthologue. The zebrafish genome The genome of the zebrafish — a key model organism for the study of development and human disease — has now been sequenced and published as a well-annotated reference genome. Zebrafish turns out to have the largest gene set of any vertebrate so far sequenced, and few pseudogenes. Importantly for disease studies, comparison between human and zebrafish sequences reveals that 70% of human genes have at least one obvious zebrafish orthologue. A second paper reports on an ongoing effort to identify and phenotype disruptive mutations in every zebrafish protein-coding gene. Using the reference genome sequence along with high-throughput sequencing and efficient chemical mutagenesis, the project's initial results — covering 38% of all known protein-coding genes — they describe phenotypic consequences of more than 1,000 alleles. The long-term goal is the creation of a knockout allele in every protein-coding gene in the zebrafish genome. All mutant alleles and data are freely available at go.nature.com/en6mos . Zebrafish have become a popular organism for the study of vertebrate gene function 1 , 2 . The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease 3 , 4 , 5 . However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes 6 , the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.

DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases fail to capture common C-to-T mutations or distinguish 5-methylcytosine from 5-hydroxymethylcytosine. We present a single base-resolution sequencing methodology that sequences complete genetics and the two most common cytosine modifications in a single workflow. DNA is copied and bases are enzymatically converted. Coupled decoding of bases across the original and copy strand provides a phased digital readout. Methods are demonstrated on human genomic DNA and cell-free DNA from a blood sample of a patient with cancer. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. Simultaneous, phased reading of genetic and epigenetic bases provides a more complete picture of the information stored in genomes and has applications throughout biomedicine. A six-letter sequencing workflow can simultaneously detect genetic and epigenetic bases.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.

Genetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B , two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells. Systematic screens for synthetic lethal gene pairs represent a powerful approach to define interactions that may be exploited in the clinic. Here, the authors use a dual-guide CRISPR screening approach to screen a curated selection of gene pairs across three cell lines and validate the Fam50a/Fam50b synthetic lethality was in isogenic cell models as well as in an in vivo setting.

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.