A CRISPR Resource for Individual, Combinatorial, or Multiplexed Gene Knockout.

We have combined a machine-learning approach with other strategies to optimize knockout efficiency with the CRISPR/Cas9 system. In addition, we have developed a multiplexed sgRNA expression strategy that promotes the functional ablation of single genes and allows for combinatorial targeting. These strategies have been combined to design and construct a genome-wide, sequence-verified, arrayed CRISPR library. This resource allows single-target or combinatorial genetic screens to be carried out at scale in a multiplexed or arrayed format. By conducting parallel loss-of-function screens, we compare our approach to existing sgRNA design and expression strategies.

Asparagine bioavailability governs metastasis in a model of breast cancer.

Using a functional model of breast cancer heterogeneity, we previously showed that clonal sub-populations proficient at generating circulating tumour cells were not all equally capable of forming metastases at secondary sites. A combination of differential expression and focused in vitro and in vivo RNA interference screens revealed candidate drivers of metastasis that discriminated metastatic clones. Among these, asparagine synthetase expression in a patient's primary tumour was most strongly correlated with later metastatic relapse. Here we show that asparagine bioavailability strongly influences metastatic potential. Limiting asparagine by knockdown of asparagine synthetase, treatment with l-asparaginase, or dietary asparagine restriction reduces metastasis without affecting growth of the primary tumour, whereas increased dietary asparagine or enforced asparagine synthetase expression promotes metastatic progression. Altering asparagine availability in vitro strongly influences invasive potential, which is correlated with an effect on proteins that promote the epithelial-to-mesenchymal transition. This provides at least one potential mechanism for how the bioavailability of a single amino acid could regulate metastatic progression.

Erratum: Asparagine bioavailability governs metastasis in a model of breast cancer.

This corrects the article DOI: 10.1038/nature25465.

A computational algorithm to predict shRNA potency.

The strength of conclusions drawn from RNAi-based studies is heavily influenced by the quality of tools used to elicit knockdown. Prior studies have developed algorithms to design siRNAs. However, to date, no established method has emerged to identify effective shRNAs, which have lower intracellular abundance than transfected siRNAs and undergo additional processing steps. We recently developed a multiplexed assay for identifying potent shRNAs and used this method to generate ∼250,000 shRNA efficacy data points. Using these data, we developed shERWOOD, an algorithm capable of predicting, for any shRNA, the likelihood that it will elicit potent target knockdown. Combined with additional shRNA design strategies, shERWOOD allows the ab initio identification of potent shRNAs that specifically target the majority of each gene's multiple transcripts. We validated the performance of our shRNA designs using several orthogonal strategies and constructed genome-wide collections of shRNAs for humans and mice based on our approach.

A model of breast cancer heterogeneity reveals vascular mimicry as a driver of metastasis.

Cancer metastasis requires that primary tumour cells evolve the capacity to intravasate into the lymphatic system or vasculature, and extravasate into and colonize secondary sites. Others have demonstrated that individual cells within complex populations show heterogeneity in their capacity to form secondary lesions. Here we develop a polyclonal mouse model of breast tumour heterogeneity, and show that distinct clones within a mixed population display specialization, for example, dominating the primary tumour, contributing to metastatic populations, or showing tropism for entering the lymphatic or vasculature systems. We correlate these stable properties to distinct gene expression profiles. Those clones that efficiently enter the vasculature express two secreted proteins, Serpine2 and Slpi, which were necessary and sufficient to program these cells for vascular mimicry. Our data indicate that these proteins not only drive the formation of extravascular networks but also ensure their perfusion by acting as anticoagulants. We propose that vascular mimicry drives the ability of some breast tumour cells to contribute to distant metastases while simultaneously satisfying a critical need of the primary tumour to be fed by the vasculature. Enforced expression of SERPINE2 and SLPI in human breast cancer cell lines also programmed them for vascular mimicry, and SERPINE2 and SLPI were overexpressed preferentially in human patients that had lung-metastatic relapse. Thus, these two secreted proteins, and the phenotype they promote, may be broadly relevant as drivers of metastatic progression in human cancer.

Deletion of Mocos Induces Xanthinuria with Obstructive Nephropathy and Major Metabolic Disorders in Mice.

Background: Xanthinuria type II is a rare autosomal purine disorder. This recessive defect of purine metabolism remains an under-recognized disorder. Methods: Mice with targeted disruption of the molybdenum cofactor sulfurase (Mocos) gene were generated to enable an integrated understanding of purine disorders and evaluate pathophysiologic functions of this gene which is found in a large number of pathways and is known to be associated with autism. Results: Mocos-deficient mice die with 4 weeks of age due to renal failure of distinct obstructive nephropathy with xanthinuria, xanthine deposits, cystic tubular dilation, Tamm-Horsfall (uromodulin) protein (THP) deposits, tubular cell necrosis with neutrophils, and occasionally hydronephrosis with urolithiasis. Obstructive nephropathy is associated with moderate interstitial inflammatory and fibrotic responses, anemia, reduced detoxification systems, and important alterations of the metabolism of purines, amino acids, and phospholipids. Conversely, heterozygous mice expressing reduced MOCOS protein are healthy with no apparent pathology. Conclusions: Mocos-deficient mice develop a lethal obstructive nephropathy associated with profound metabolic changes. Studying MOCOS functions may provide important clues about the underlying pathogenesis of xanthinuria and other diseases requiring early diagnosis.

lncRNA Spehd Regulates Hematopoietic Stem and Progenitor Cells and Is Required for Multilineage Differentiation.

Long non-coding RNAs (lncRNAs) show patterns of tissue- and cell type-specific expression that are very similar to those of protein coding genes and consequently have the potential to control stem and progenitor cell fate decisions along a differentiation trajectory. To understand the roles that lncRNAs may play in hematopoiesis, we selected a subset of mouse lncRNAs with potentially relevant expression patterns and refined our candidate list using evidence of conserved expression in human blood lineages. For each candidate, we assessed its possible role in hematopoietic differentiation in vivo using competitive transplantation. Our studies identified two lncRNAs that were required for hematopoiesis. One of these, Spehd, showed defective multilineage differentiation, and its silencing yielded common myeloid progenitors that are deficient in their oxidative phosphorylation pathway. This effort not only suggests that lncRNAs can contribute to differentiation decisions during hematopoiesis but also provides a path toward the identification of functional lncRNAs in other differentiation hierarchies.

Genetic interactions of G-quadruplexes in humans.

G-quadruplexes (G4) are alternative nucleic acid structures involved in transcription, translation and replication. Aberrant G4 formation and stabilisation is linked to genome instability and cancer. G4 ligand treatment disrupts key biological processes leading to cell death. To discover genes and pathways involved with G4s and gain mechanistic insights into G4 biology, we present the first unbiased genome-wide study to systematically identify human genes that promote cell death when silenced by shRNA in the presence of G4-stabilising small molecules. Many novel genetic vulnerabilities were revealed opening up new therapeutic possibilities in cancer, which we exemplified by an orthogonal pharmacological inhibition approach that phenocopies gene silencing. We find that targeting the WEE1 cell cycle kinase or USP1 deubiquitinase in combination with G4 ligand treatment enhances cell killing. We also identify new genes and pathways regulating or interacting with G4s and demonstrate that the DDX42 DEAD-box helicase is a newly discovered G4-binding protein.

lncRNA requirements for mouse acute myeloid leukemia and normal differentiation.

A substantial fraction of the genome is transcribed in a cell-type-specific manner, producing long non-coding RNAs (lncRNAs), rather than protein-coding transcripts. Here, we systematically characterize transcriptional dynamics during hematopoiesis and in hematological malignancies. Our analysis of annotated and de novo assembled lncRNAs showed many are regulated during differentiation and mis-regulated in disease. We assessed lncRNA function via an in vivo RNAi screen in a model of acute myeloid leukemia. This identified several lncRNAs essential for leukemia maintenance, and found that a number act by promoting leukemia stem cell signatures. Leukemia blasts show a myeloid differentiation phenotype when these lncRNAs were depleted, and our data indicates that this effect is mediated via effects on the MYC oncogene. Bone marrow reconstitutions showed that a lncRNA expressed across all progenitors was required for the myeloid lineage, whereas the other leukemia-induced lncRNAs were dispensable in the normal setting.