Academic Appointments


Professional Education


  • Postdoctoral Fellow, University of California, San Francisco, Cellular and Molecular Pharmacology (2013)
  • Ph.D., Harvard University, Biological and Biomedical Sciences (2005)
  • B.S., University of Wisconsin, Madison, Biochemistry and Molecular Biology (1996)

Current Research and Scholarly Interests


Endocytic Pathogens as Tools and Targets

Endocytic pathogens such as protein aggregates, viruses, protein toxins, and bacteria have evolved remarkable ways to enter the cell, disrupt homeostasis, and cause cell death. We use these agents both as probes to understand normal cellular trafficking and signaling events, and to find key targets for therapy.


Stress Signaling to the Cell Death Machinery

Cells have elaborate mechanisms of sensing diverse stresses (oxidative damage, nutrient deprivation, DNA breaks, etc), and must either repair damage or induce cell death. Misregulation of these pathways results in diseases such as cancer and Alzheimer’s. We would like to understand how these signals connect to the death pathway in health and disease in order to improve therapies.

Technology Development and Genetic Interaction Maps

Customized genome-scale gene perturbation libraries: Much of the work we do utilizes genetic screens enabled by novel high-coverage CRISPR/Cas9 libraries (10 sgRNAs/gene) and shRNA libraries (25 shRNAs/gene) we have developed. The high coverage greatly reduces false positive and false negative results. Our platform allows for easy creation of new library designs, and we use a pooled format that can be used to rapidly screen genome-scale libraries in ~1-2 weeks. Libraries can then be analyzed by deep sequencing to quantify changes in sgRNA/shRNA abundance.

Systematic comparison of gene perturbation technologies: We have systematically compared the ability of genome-wide RNAi and CRISPR/Cas9 deletion screens to identify drug targets and essential genes, and have found important differences in their outputs. For example, while both screens perform well in detecting a gold standard set of essential genes, they can identify distinct essential biological processes. Moreover, each technology exhibits its own set of off-target effects and limitations in on-target efficacy.

Using what we have learned about these technologies, we recently created new genome-wide CRISPR libraries that incorporate a number of improved sgRNA features and controls. With a novel statistical framework (casTLE) we can model on- and off-target effects accurately, markedly improve hit detection, and even combine results from screens to improve performance and further limit false positives and false negatives. These studies have demonstrated the utility of parallel screening approaches using complementary technologies to reveal a more complete biological picture.

Systematic genetic interaction maps: We have also developed strategies to systematically knock down/knock out pairs of genes. This has facilitated some of the first systematic genetic interaction maps in mammalian cells. Using these maps, we can understand coordinated gene functions and predict new functions for uncharacterized genes. They also allow us to quickly identify synergistic interactions under stress conditions that we hope to exploit for combination therapies.

Directed evolution using dCas9-targeted somatic hypermutation (CRISPR-X): More recently, we developed a strategy to re-purpose the somatic hypermutation machinery used in antibody diversification to create targeted populations of point mutations. Using dCas9 to recruit a hyperactive variant of the deaminase AID, we can target diverse point mutations within an ~100bp window centered on the sgRNA PAM site. These mutant populations can then be subjected to selection to evolve proteins with improved function or to map the sites of drug-protein interactions. For example, by tiling mutations across PSMB5, we could map known and novel mutations that affect binding to the chemotherapeutic bortezomib.

2019-20 Courses


Stanford Advisees


  • Doctoral Dissertation Reader (AC)
    Nathan Abell, Roy Ang, Jacob Blum, Tony Boutelle, Gun Woo Byeon, Noori Chai, Shi-An Chen, Ching Pin Cheng, Alex Colville, Robert Coukos, Hannah De Jong, Elbegduuren Erdenee, Zintis Inde, Alyssa Kaiser, Marcus Kelly, Kyle Kovary, Ragini Phansalkar, Sarah Pierce, John Pluvinage, Andrew Spencley, Theo Susanto, Nikki Teran, Ryan Vest, Kristen Wells, Robin Yeo
  • Postdoctoral Faculty Sponsor
    Kyuho Han, Roarke Kamber, Roni Levin Konigsberg
  • Doctoral Dissertation Advisor (AC)
    Josh Tycko, David Yao
  • Doctoral Dissertation Co-Advisor (AC)
    Amita Gupta

Graduate and Fellowship Programs


All Publications


  • Systematic Identification of Regulators of Oxidative Stress Reveals Non-canonical Roles for Peroxisomal Import and the Pentose Phosphate Pathway. Cell reports Dubreuil, M. M., Morgens, D. W., Okumoto, K., Honsho, M., Contrepois, K., Lee-McMullen, B., Traber, G. M., Sood, R. S., Dixon, S. J., Snyder, M. P., Fujiki, Y., Bassik, M. C. 2020; 30 (5): 1417

    Abstract

    Reactive oxygen species (ROS) play critical roles inmetabolism and disease, yet a comprehensive analysis of the cellular response to oxidative stress is lacking. To systematically identify regulators ofoxidative stress, we conducted genome-wide Cas9/CRISPR and shRNA screens. This revealed a detailed picture of diverse pathways that control oxidative stress response, ranging from the TCA cycle and DNA repair machineries to iron transport, trafficking, and metabolism. Paradoxically, disrupting the pentose phosphate pathway (PPP) at the level of phosphogluconate dehydrogenase (PGD) protects cells against ROS. This dramatically alters metabolites in the PPP, consistent with rewiring of upper glycolysis to promote antioxidant production. In addition, disruption of peroxisomal import unexpectedly increases resistance to oxidative stress by altering the localization of catalase. Together, these studies provide insights into the roles of peroxisomal matrix import and the PPP in redox biology and represent a rich resource for understanding the cellular response to oxidative stress.

    View details for DOI 10.1016/j.celrep.2020.01.013

    View details for PubMedID 32023459

  • Lipid-droplet-accumulating microglia represent a dysfunctional and proinflammatory state in the aging brain. Nature neuroscience Marschallinger, J., Iram, T., Zardeneta, M., Lee, S. E., Lehallier, B., Haney, M. S., Pluvinage, J. V., Mathur, V., Hahn, O., Morgens, D. W., Kim, J., Tevini, J., Felder, T. K., Wolinski, H., Bertozzi, C. R., Bassik, M. C., Aigner, L., Wyss-Coray, T. 2020

    Abstract

    Microglia become progressively activated and seemingly dysfunctional with age, and genetic studies have linked these cells to the pathogenesis of a growing number of neurodegenerative diseases. Here we report a striking buildup of lipid droplets in microglia with aging in mouse and human brains. These cells, which we call 'lipid-droplet-accumulating microglia' (LDAM), are defective in phagocytosis, produce high levels of reactive oxygen species and secrete proinflammatory cytokines. RNA-sequencing analysis of LDAM revealed a transcriptional profile driven by innate inflammation that is distinct from previously reported microglial states. An unbiased CRISPR-Cas9 screen identified genetic modifiers of lipid droplet formation; surprisingly, variants of several of these genes, including progranulin (GRN), are causes of autosomal-dominant forms of human neurodegenerative diseases. We therefore propose that LDAM contribute to age-related and genetic forms of neurodegeneration.

    View details for DOI 10.1038/s41593-019-0566-1

    View details for PubMedID 31959936

  • Genome-wide analysis of targets of macrolide antibiotics in mammalian cells. The Journal of biological chemistry Gupta, A., Okesli-Armlovich, A., Morgens, D., Bassik, M. C., Khosla, C. 2020

    Abstract

    Macrolide antibiotics, such as erythromycin and josamycin, are polyketide natural products harboring 14-16-membered macrocyclic lactone rings to which various sugars are attached. These antibiotics are extensively used in the clinic because of their ability to inhibit bacterial protein synthesis. More recently, some macrolides have been shown to also possess anti-inflammatory and other therapeutic activities in mammalian cells. To better understand the targets and effects of this drug class in mammalian cells, we used a genome-wide shRNA screen in K562 cancer cells to identify genes that modulate cellular sensitivity to josamycin. Among the most sensitizing hits were proteins involved in mitochondrial translation and the mitochondrial unfolded protein response, glycolysis and the mitogen-activated protein kinase signaling cascade. Further analysis revealed that cells treated with josamycin or other antibacterials exhibited impaired oxidative phosphorylation and metabolic shifts to glycolysis. Interestingly, we observed that knockdown of the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) gene, which contributes to p38 MAPK signaling, sensitized cells to only josamycin but not to other antibacterials. There is a growing interest in better characterizing the therapeutic effects and toxicities of antibiotics in mammalian cells to guide new applications in both cellular and clinical studies. To our knowledge, this is the first report of an unbiased genome-wide screen to investigate the effects of a clinically used antibiotic on human cells.

    View details for DOI 10.1074/jbc.RA119.010770

    View details for PubMedID 31915244

  • Genome-wide synthetic lethal CRISPR screen identifies FIS1 as a genetic interactor of ALS-linked C9ORF72. Brain research Chai, N., Haney, M. S., Couthouis, J., Morgens, D. W., Benjamin, A., Wu, K., Ousey, J., Fang, S., Finer, S., Bassik, M. C., Gitler, A. D. 2019: 146601

    Abstract

    Mutations in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis (ALS). Both toxic gain of function and loss of function pathogenic mechanisms have been proposed. Accruing evidence from mouse knockout studies point to a role for C9ORF72 as a regulator of immune function. To provide further insight into its cellular function, we performed a genome-wide synthetic lethal CRISPR screen in human myeloid cells lacking C9ORF72. We discovered a strong synthetic lethal genetic interaction between C9ORF72 and FIS1, which encodes a mitochondrial membrane protein involved in mitochondrial fission and mitophagy. Mass spectrometry experiments revealed that in C9ORF72 knockout cells, FIS1 strongly bound to a class of immune regulators that activate the receptor for advanced glycation end (RAGE) products and trigger inflammatory cascades. These findings present a novel genetic interactor for C9ORF72 and suggest a compensatory role for FIS1 in suppressing inflammatory signaling in the absence of C9ORF72.

    View details for DOI 10.1016/j.brainres.2019.146601

    View details for PubMedID 31843624

  • Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins. eLife Morgens, D. W., Chan, C., Kane, A. J., Weir, N. R., Li, A., Dubreuil, M. M., Tsui, C. K., Hess, G. T., Lavertu, A., Han, K., Polyakov, N., Zhou, J., Handy, E. L., Alabi, P., Dombroski, A., Yao, D., Altman, R. B., Sello, J. K., Denic, V., Bassik, M. C. 2019; 8

    Abstract

    The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (MOA), which involves halting retrograde vesicle transport to the endoplasmic reticulum (ER). CRISPRi genetic interaction analysis revealed Retro-2 activity resembles disruption of the transmembrane domain recognition complex (TRC) pathway, which mediates post-translational ER-targeting and insertion of tail-anchored (TA) proteins, including SNAREs required for retrograde transport. Cell-based and in vitro assays show that Retro-2 blocks delivery of newly-synthesized TA-proteins to the ER-targeting factor ASNA1 (TRC40). An ASNA1 point mutant identified using CRISPR-mediated mutagenesis abolishes both the cytoprotective effect of Retro-2 against ricin and its inhibitory effect on ASNA1-mediated ER-targeting. Together, our work explains how Retro-2 prevents retrograde trafficking of toxins by inhibiting TA-protein targeting, describes a general CRISPR strategy for predicting the MOA of small molecules, and paves the way for drugging the TRC pathway to treat broad classes of viruses known to be inhibited by Retro-2.

    View details for DOI 10.7554/eLife.48434

    View details for PubMedID 31674906

  • The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature Bersuker, K., Hendricks, J., Li, Z., Magtanong, L., Ford, B., Tang, P. H., Roberts, M. A., Tong, B., Maimone, T. J., Zoncu, R., Bassik, M. C., Nomura, D. K., Dixon, S. J., Olzmann, J. A. 2019

    Abstract

    Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids1,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols3,4. Ferroptosis has been implicated in the cell death that underlies several degenerative conditions2, and induction of ferroptosis by inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death5. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines6, suggesting that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR-Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q10 (CoQ), generating a lipophilic radical-trapping antioxidant (RTA) that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumor xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a new ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutics.

    View details for DOI 10.1038/s41586-019-1705-2

    View details for PubMedID 31634900

  • A ZDHHC5-GOLGA7 Protein Acyltransferase Complex Promotes Nonapoptotic Cell Death. Cell chemical biology Ko, P., Woodrow, C., Dubreuil, M. M., Martin, B. R., Skouta, R., Bassik, M. C., Dixon, S. J. 2019

    Abstract

    Lethal small molecules are useful probes to discover and characterize novel cell death pathways and biochemical mechanisms. Here we report that the synthetic oxime-containing small molecule caspase-independent lethal 56 (CIL56) induces an unconventional form of nonapoptotic cell death distinct from necroptosis, ferroptosis, and other pathways. CIL56-induced cell death requires a catalytically active protein S-acyltransferase complex comprising the enzyme ZDHHC5 and an accessory subunit GOLGA7. The ZDHHC5-GOLGA7 complex is mutually stabilizing and localizes to the plasma membrane. CIL56 inhibits anterograde protein transport from the Golgi apparatus, which may be lethal in the context of ongoing ZDHHC5-GOLGA7 complex-dependent retrograde protein trafficking from the plasma membrane to internal sites. Other oxime-containing small molecules, structurally distinct from CIL56, may trigger cell death through the same pathway. These results define an unconventional form of nonapoptotic cell death regulated by protein S-acylation.

    View details for DOI 10.1016/j.chembiol.2019.09.014

    View details for PubMedID 31631010

  • Phagolysosome resolution requires contacts with the endoplasmic reticulum and phosphatidylinositol-4-phosphate signalling. Nature cell biology Levin-Konigsberg, R., Montano-Rendon, F., Keren-Kaplan, T., Li, R., Ego, B., Mylvaganam, S., DiCiccio, J. E., Trimble, W. S., Bassik, M. C., Bonifacino, J. S., Fairn, G. D., Grinstein, S. 2019

    Abstract

    Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.

    View details for DOI 10.1038/s41556-019-0394-2

    View details for PubMedID 31570833

  • CRISPR-Cas9 Screens Identify the RNA Helicase DDX3X as a Repressor of C9ORF72 (GGGGCC)n Repeat-Associated Non-AUG Translation. Neuron Cheng, W., Wang, S., Zhang, Z., Morgens, D. W., Hayes, L. R., Lee, S., Portz, B., Xie, Y., Nguyen, B. V., Haney, M. S., Yan, S., Dong, D., Coyne, A. N., Yang, J., Xian, F., Cleveland, D. W., Qiu, Z., Rothstein, J. D., Shorter, J., Gao, F., Bassik, M. C., Sun, S. 2019

    Abstract

    Hexanucleotide GGGGCC repeat expansion in C9ORF72 is the most prevalent genetic cause ofamyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism isthe aberrant accumulation of dipeptide repeat (DPR) proteins produced by the unconventional translation of expanded RNA repeats. Here, we performed genome-wide CRISPR-Cas9 screens for modifiers of DPR protein production in human cells.We found that DDX3X, an RNA helicase, suppresses the repeat-associated non-AUG translation of GGGGCC repeats. DDX3X directly binds to (GGGGCC)n RNAs but not antisense (CCCCGG)n RNAs. Its helicase activity is essential for the translation repression. Reduction of DDX3X increases DPR levels in C9ORF72-ALS/FTD patient cells and enhances (GGGGCC)n-mediated toxicity in Drosophila. Elevating DDX3X expression is sufficient to decrease DPR levels, rescue nucleocytoplasmic transport abnormalities, and improve survival of patient iPSC-differentiated neurons. This work identifies genetic modifiers of DPR protein production and provides potential therapeutic targets for C9ORF72-ALS/FTD.

    View details for DOI 10.1016/j.neuron.2019.09.003

    View details for PubMedID 31587919

  • Systematic Identification of Host Cell Regulators of Legionella pneumophila Pathogenesis Using a Genome-wide CRISPR Screen. Cell host & microbe Jeng, E. E., Bhadkamkar, V., Ibe, N. U., Gause, H., Jiang, L., Chan, J., Jian, R., Jimenez-Morales, D., Stevenson, E., Krogan, N. J., Swaney, D. L., Snyder, M. P., Mukherjee, S., Bassik, M. C. 2019

    Abstract

    During infection, Legionella pneumophila translocates over 300 effector proteins into the host cytosol, allowing the pathogen to establish an endoplasmic reticulum (ER)-like Legionella-containing vacuole (LCV) that supports bacterial replication. Here, we perform a genome-wide CRISPR-Cas9 screen and secondary targeted screens in U937 human monocyte/macrophage-like cells to systematically identify host factorsthat regulate killing by L.pneumophila. The screens reveal known host factors hijacked by L.pneumophila, as well as genes spanning diverse trafficking and signaling pathways previously not linked to L.pneumophila pathogenesis. We further characterize C1orf43 and KIAA1109 as regulators ofphagocytosis and show that RAB10 and its chaperone RABIF are required for optimal L.pneumophila replication and ER recruitment to the LCV. Finally, we show that Rab10 protein is recruited to the LCV and ubiquitinated by the effectors SidC/SdcA. Collectively, our results provide a wealth of previously undescribed insights into L.pneumophila pathogenesis and mammalian cell function.

    View details for DOI 10.1016/j.chom.2019.08.017

    View details for PubMedID 31540829

  • CRISPR-Cas9 screens identify regulators of antibody-drug conjugate toxicity. Nature chemical biology Tsui, C. K., Barfield, R. M., Fischer, C. R., Morgens, D. W., Li, A., Smith, B. A., Gray, M. A., Bertozzi, C. R., Rabuka, D., Bassik, M. C. 2019

    Abstract

    Antibody-drug conjugates (ADCs) selectively deliver chemotherapeutic agents to target cells and are important cancer therapeutics. However, the mechanisms by which ADCs are internalized and activated remain unclear. Using CRISPR-Cas9 screens, we uncover many known and novel endolysosomal regulators as modulators of ADC toxicity. We identify and characterize C18ORF8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of screens with ADCs bearing different linkers, we show that a subset of late endolysosomal regulators selectively influence toxicity of noncleavable linker ADCs. Surprisingly, we find cleavable valine-citrulline linkers can be processed rapidly after internalization without lysosomal delivery. Lastly, we show that sialic acid depletion enhances ADC lysosomal delivery and killing in diverse cancer cell types, including with FDA (US Food and Drug Administration)-approved trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal new regulators of endolysosomal trafficking, provide important insights for ADC design and identify candidate combination therapy targets.

    View details for DOI 10.1038/s41589-019-0342-2

    View details for PubMedID 31451760

  • Discovery of small molecule inhibitors of human uridine-cytidine kinase 2 by high-throughput screening. Bioorganic & medicinal chemistry letters Okesli-Armlovich, A., Gupta, A., Jimenez, M., Auld, D., Liu, Q., Bassik, M. C., Khosla, C. 2019

    Abstract

    Clinically relevant inhibitors of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in mammalian de novo pyrimidine synthesis, have strong antiviral and anticancer activity in vitro. However, they are ineffective in vivo due to efficient uridine salvage by infected or rapidly dividing cells. The pyrimidine salvage enzyme uridine-cytidine kinase 2 (UCK2), a 29 kDa protein that forms a tetramer in its active state, is necessary for uridine salvage. Notwithstanding the pharmacological potential of this target, no medicinally tractable inhibitors of the human enzyme have been reported to date. We therefore established and miniaturized an in vitro assay for UCK2 activity and undertook a high-throughput screen against a 40,000-compound library to generate drug-like leads. The structures, activities, and modes of inhibition of the most promising hits are described. Notably, our screen yielded non-competitive UCK2 inhibitors which were able to suppress nucleoside salvage in cells both in the presence and absence of DHODH inhibitors.

    View details for DOI 10.1016/j.bmcl.2019.08.010

    View details for PubMedID 31420268

  • Astrocyte-to-astrocyte contact and a positive feedback loop of growth factor signaling regulate astrocyte maturation GLIA Li, J., Khankan, R. R., Caneda, C., Godoy, M., Haney, M. S., Krawczyk, M. C., Bassik, M. C., Sloan, S. A., Zhan, Y. 2019; 67 (8): 1571–97

    View details for DOI 10.1002/glia.23630

    View details for Web of Science ID 000470958100011

  • Kinetic analysis identifies determinants of sensitivity to MEK inhibitor-induced cell death Inde, Z., Han, K., Bassik, M. C., Dixon, S. J. AMER ASSOC CANCER RESEARCH. 2019
  • Neuronally Enriched RUFY3 Is Required for Caspase-Mediated Axon Degeneration. Neuron Hertz, N. T., Adams, E. L., Weber, R. A., Shen, R. J., O'Rourke, M. K., Simon, D. J., Zebroski, H., Olsen, O., Morgan, C. W., Mileur, T. R., Hitchcock, A. M., Sinnott Armstrong, N. A., Wainberg, M., Bassik, M. C., Molina, H., Wells, J. A., Tessier-Lavigne, M. 2019

    Abstract

    Selective synaptic and axonal degeneration are critical aspects of both brain development and neurodegenerative disease. Inhibition of caspase signaling in neurons is a potential therapeutic strategy for neurodegenerative disease, but no neuron-specific modulators of caspase signaling have been described. Using a mass spectrometry approach, we discovered that RUFY3, a neuronally enriched protein, is essential for caspase-mediated degeneration of TRKA+ sensory axons invitro and invivo. Deletion of Rufy3 protects axons from degeneration, even in the presence of activated CASP3 that is competent to cleave endogenous substrates. Dephosphorylation of RUFY3 at residue S34 appears required for axon degeneration, providing a potential mechanism for neurons to locally control caspase-driven degeneration. Neuronally enriched RUFY3 thus provides an entry pointfor understanding non-apoptotic functions of CASP3 and a potential target to modulate caspase signaling specifically in neurons for neurodegenerative disease.

    View details for DOI 10.1016/j.neuron.2019.05.030

    View details for PubMedID 31221560

  • SLC19A1 Is an Importer of the Immunotransmitter cGAMP. Molecular cell Ritchie, C., Cordova, A. F., Hess, G. T., Bassik, M. C., Li, L. 2019

    Abstract

    2'3'-cyclic-GMP-AMP (cGAMP) is a second messenger that activates the antiviral stimulator of interferon genes (STING) pathway. We recently identified a novel role for cGAMP as a soluble, extracellular immunotransmitter that is produced and secreted by cancer cells. Secreted cGAMP is then sensed by host cells, eliciting an antitumoral immune response. Due to the antitumoral effects of cGAMP, other CDN-based STING agonists are currently under investigation in clinical trials for metastatic solid tumors. However, it is unknown how cGAMP and other CDNs cross the cell membrane to activateintracellular STING. Using a genome-wide CRISPRscreen, we identified SLC19A1 as the first knownimporter of cGAMP and other CDNs, including theinvestigational new drug 2'3'-bisphosphosphothioate-cyclic-di-AMP (2'3'-CDAS). These discoveries will provide insight into cGAMP's role as an immunotransmitter and aid in the development of more targeted CDN-based cancer therapeutics.

    View details for DOI 10.1016/j.molcel.2019.05.006

    View details for PubMedID 31126740

  • CD22 blockade restores homeostatic microglial phagocytosis in ageing brains NATURE Pluvinage, J. V., Haney, M. S., Smith, B. H., Sun, J., Iram, T., Bonanno, L., Li, L., Lee, D. P., Morgens, D. W., Yang, A. C., Shuken, S. R., Gate, D., Scott, M., Khatri, P., Luo, J., Bertozzi, C. R., Bassik, M. C., Wyss-Coray, T. 2019; 568 (7751): 187-+
  • CD22 blockade restores homeostatic microglial phagocytosis in ageing brains. Nature Pluvinage, J. V., Haney, M. S., Smith, B. A., Sun, J., Iram, T., Bonanno, L., Li, L., Lee, D. P., Morgens, D. W., Yang, A. C., Shuken, S. R., Gate, D., Scott, M., Khatri, P., Luo, J., Bertozzi, C. R., Bassik, M. C., Wyss-Coray, T. 2019

    Abstract

    Microglia maintain homeostasis in the central nervous system through phagocytic clearance of protein aggregates and cellular debris. This function deteriorates during ageing and neurodegenerative disease, concomitant with cognitive decline. However, the mechanisms of impaired microglial homeostatic function and the cognitive effects of restoring this function remain unknown. We combined CRISPR-Cas9 knockout screens with RNAsequencing analysis to discover age-related genetic modifiers of microglial phagocytosis. These screens identified CD22, a canonical Bcell receptor, as a negative regulator of phagocytosis that is upregulated on aged microglia. CD22 mediates the anti-phagocytic effect of alpha2,6-linked sialic acid, and inhibition of CD22 promotes the clearance of myelin debris, amyloid-beta oligomers and alpha-synuclein fibrils in vivo. Long-term central nervous system delivery of an antibody that blocks CD22 function reprograms microglia towards a homeostatic transcriptional state and improves cognitive function in aged mice. These findings elucidate a mechanism of age-related microglial impairment and a strategy to restore homeostasis in the ageing brain.

    View details for PubMedID 30944478

  • Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements. Nature communications Tycko, J., Wainberg, M., Marinov, G. K., Ursu, O., Hess, G. T., Ego, B. K., Li, A., Truong, A., Trevino, A. E., Spees, K., Yao, D., Kaplow, I. M., Greenside, P. G., Morgens, D. W., Phanstiel, D. H., Snyder, M. P., Bintu, L., Greenleaf, W. J., Kundaje, A., Bassik, M. C. 2019; 10 (1): 4063

    Abstract

    Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.

    View details for DOI 10.1038/s41467-019-11955-7

    View details for PubMedID 31492858

  • Targeted genomic CRISPR-Cas9 screen identifies MAP4K4 as essential for glioblastoma invasion. Scientific reports Prolo, L. M., Li, A., Owen, S. F., Parker, J. J., Foshay, K., Nitta, R. T., Morgens, D. W., Bolin, S., Wilson, C. M., Vega L, J. C., Luo, E. J., Nwagbo, G., Waziri, A., Li, G., Reimer, R. J., Bassik, M. C., Grant, G. A. 2019; 9 (1): 14020

    Abstract

    Among high-grade brain tumors, glioblastoma is particularly difficult to treat, in part due to its highly infiltrative nature which contributes to the malignant phenotype and high mortality in patients. In order to better understand the signaling pathways underlying glioblastoma invasion, we performed the first large-scale CRISPR-Cas9 loss of function screen specifically designed to identify genes that facilitate cell invasion. We tested 4,574 genes predicted to be involved in trafficking and motility. Using a transwell invasion assay, we discovered 33 genes essential for invasion. Of the 11 genes we selected for secondary testing using a wound healing assay, 6 demonstrated a significant decrease in migration. The strongest regulator of invasion was mitogen-activated protein kinase 4 (MAP4K4). Targeting of MAP4K4 with single guide RNAs or a MAP4K4 inhibitor reduced migration and invasion in vitro. This effect was consistent across three additional patient derived glioblastoma cell lines. Analysis of epithelial-mesenchymal transition markers in U138 cells with lack or inhibition of MAP4K4 demonstrated protein expression consistent with a non-invasive state. Importantly, MAP4K4 inhibition limited migration in a subset of human glioma organotypic slice cultures. Our results identify MAP4K4 as a novel potential therapeutic target to limit glioblastoma invasion.

    View details for DOI 10.1038/s41598-019-50160-w

    View details for PubMedID 31570734

  • Discovery of common and rare genetic risk variants for colorectal cancer NATURE GENETICS Huyghe, J. R., Bien, S. A., Harrison, T. A., Kang, H., Chen, S., Schmit, S. L., Conti, D. V., Qu, C., Jeon, J., Edlund, C. K., Greenside, P., Wainberg, M., Schumacher, F. R., Smith, J. D., Levine, D. M., Nelson, S. C., Sinnott-Armstrong, N. A., Albanes, D., Alonso, M., Anderson, K., Arnau-Collell, C., Arndt, V., Bamia, C., Banbury, B. L., Baron, J. A., Berndt, S. I., Bezieau, S., Bishop, D., Boehm, J., Boeing, H., Brenner, H., Brezina, S., Buch, S., Buchanan, D. D., Burnett-Hartman, A., Butterbach, K., Caan, B. J., Campbell, P. T., Carlson, C. S., Castellvi-Bel, S., Chan, A. T., Chang-Claude, J., Chanock, S. J., Chirlaque, M., Cho, S., Connolly, C. M., Cross, A. J., Cuk, K., Curtis, K. R., de la Chapelle, A., Doheny, K. F., Duggan, D., Easton, D. F., Elias, S. G., Elliott, F., English, D. R., Feskens, E. M., Figueiredo, J. C., Fischer, R., FitzGerald, L. M., Forman, D., Gala, M., Gallinger, S., Gauderman, W., Giles, G. G., Gillanders, E., Gong, J., Goodman, P. J., Grady, W. M., Grove, J. S., Gsur, A., Gunter, M. J., Haile, R. W., Hampe, J., Hampel, H., Harlid, S., Hayes, R. B., Hofer, P., Hoffmeister, M., Hopper, J. L., Hsu, W., Huang, W., Hudson, T. J., Hunter, D. J., Ibanez-Sanz, G., Idos, G. E., Ingersoll, R., Jackson, R. D., Jacobs, E. J., Jenkins, M. A., Joshi, A. D., Joshu, C. E., Keku, T. O., Key, T. J., Kim, H., Kobayashi, E., Kolonel, L. N., Kooperberg, C., Kuehn, T., Kury, S., Kweon, S., Larsson, S. C., Laurie, C. A., Le Marchand, L., Leal, S. M., Lee, S., Lejbkowicz, F., Lemire, M., Li, C. I., Li, L., Lieb, W., Lin, Y., Lindblom, A., Lindor, N. M., Ling, H., Louie, T. L., Mannisto, S., Markowitz, S. D., Martin, V., Masala, G., McNeil, C. E., Melas, M., Milne, R. L., Moreno, L., Murphy, N., Myte, R., Naccarati, A., Newcomb, P. A., Offit, K., Ogino, S., Onland-Moret, N., Pardini, B., Parfrey, P. S., Pearlman, R., Perduca, V., Pharoah, P. P., Pinchev, M., Platz, E. A., Prentice, R. L., Pugh, E., Raskin, L., Rennert, G., Rennert, H. S., Riboli, E., Rodriguez-Barranco, M., Romm, J., Sakoda, L. C., Schafmayer, C., Schoen, R. E., Seminara, D., Shah, M., Shelford, T., Shin, M., Shulman, K., Sieri, S., Slattery, M. L., Southey, M. C., Stadler, Z. K., Stegmaier, C., Su, Y., Tangen, C. M., Thibodeau, S. N., Thomas, D. C., Thomas, S. S., Toland, A. E., Trichopoulou, A., Ulrich, C. M., Van den Berg, D. J., van Duijnhoven, F. B., Van Guelpen, B., van Kranen, H., Vijai, J., Visvanathan, K., Vodicka, P., Vodickova, L., Vymetalkova, V., Weigl, K., Weinstein, S. J., White, E., Win, A., Wolf, C., Wolk, A., Woods, M. O., Wu, A. H., Zaidi, S. H., Zanke, B. W., Zhang, Q., Zheng, W., Scacheri, P. C., Potter, J. D., Bassik, M. C., Kundaje, A., Casey, G., Moreno, V., Abecasis, G. R., Nickerson, D. A., Gruber, S. B., Hsu, L., Peters, U. 2019; 51 (1): 76-+
  • METTL13 Methylation of eEF1A Increases Translational Output to Promote Tumorigenesis. Cell Liu, S., Hausmann, S., Carlson, S. M., Fuentes, M. E., Francis, J. W., Pillai, R., Lofgren, S. M., Hulea, L., Tandoc, K., Lu, J., Li, A., Nguyen, N. D., Caporicci, M., Kim, M. P., Maitra, A., Wang, H., Wistuba, I. I., Porco, J. A., Bassik, M. C., Elias, J. E., Song, J., Topisirovic, I., Van Rechem, C., Mazur, P. K., Gozani, O. 2018

    Abstract

    Increased protein synthesis plays an etiologic role in diverse cancers. Here, we demonstrate that METTL13 (methyltransferase-like 13) dimethylation of eEF1A (eukaryotic elongation factor 1A) lysine 55 (eEF1AK55me2) is utilized by Ras-driven cancers to increase translational output and promote tumorigenesis invivo. METTL13-catalyzed eEF1A methylation increases eEF1A's intrinsic GTPase activity invitro and protein production in cells. METTL13 and eEF1AK55me2 levels are upregulated in cancer and negatively correlate with pancreatic and lung cancer patient survival. METTL13 deletion and eEF1AK55me2 loss dramatically reduce Ras-driven neoplastic growth in mouse models and in patient-derived xenografts (PDXs) from primary pancreatic and lung tumors. Finally, METTL13 depletion renders PDX tumors hypersensitive to drugs thattarget growth-signaling pathways. Together, our work uncovers a mechanism by which lethal cancers become dependent on the METTL13-eEF1AK55me2 axis to meet their elevated protein synthesis requirement and suggests that METTL13 inhibition may constitute a targetable vulnerability of tumors driven by aberrant Ras signaling.

    View details for PubMedID 30612740

  • Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs. eLife Lu, A., Wawro, P., Morgens, D. W., Portela, F., Bassik, M. C., Pfeffer, S. R. 2018; 7

    Abstract

    Extracellular vesicles mediate transfer of biologically active molecules between neighboring or distant cells, and these vesicles may play important roles in normal physiology and the pathogenesis of multiple disease states including cancer. However, the underlying molecular mechanisms of their biogenesis and release remain unknown. We designed artificially barcoded, exosomal microRNAs (bEXOmiRs) to monitor extracellular vesicle release quantitatively using deep sequencing. We then expressed distinct pairs of CRISPR guide RNAs and bEXOmiRs, enabling identification of genes influencing bEXOmiR secretion from Cas9-edited cells. This approach uncovered genes with unrecognized roles in multivesicular endosome exocytosis, including critical roles for Wnt signaling in extracellular vesicle release regulation. Coupling bEXOmiR reporter analysis with CRISPR-Cas9 screening provides a powerful and unbiased means to study extracellular vesicle biology and for the first time, to associate a nucleic acid tag with individual membrane vesicles.

    View details for PubMedID 30556811

  • Genome-wide CRISPR Analysis Identifies Substrate-Specific Conjugation Modules in ER-Associated Degradation. Molecular cell Leto, D. E., Morgens, D. W., Zhang, L., Walczak, C. P., Elias, J. E., Bassik, M. C., Kopito, R. R. 2018

    Abstract

    The ubiquitin proteasome system (UPS) maintains the integrity of the proteome by selectively degrading misfolded or mis-assembled proteins, but the rules that govern how conformationally defective proteins in the secretory pathway are selected from the structurally and topologically diverse constellation of correctly folded membrane and secretory proteins for efficient degradation by cytosolic proteasomes is not well understood. Here, we combine parallel pooled genome-wide CRISPR-Cas9 forward genetic screening with a highly quantitative and sensitive protein turnover assay to discover a previously undescribed collaboration between membrane-embedded cytoplasmic ubiquitin E3 ligases to conjugate heterotypic branched or mixed ubiquitin (Ub) chains on substrates of endoplasmic-reticulum-associated degradation (ERAD). These findings demonstrate that parallel CRISPR analysis can be used to deconvolve highly complex cell biological processes and identify new biochemical pathways in protein quality control.

    View details for PubMedID 30581143

  • Discovery of common and rare genetic risk variants for colorectal cancer. Nature genetics Huyghe, J. R., Bien, S. A., Harrison, T. A., Kang, H. M., Chen, S., Schmit, S. L., Conti, D. V., Qu, C., Jeon, J., Edlund, C. K., Greenside, P., Wainberg, M., Schumacher, F. R., Smith, J. D., Levine, D. M., Nelson, S. C., Sinnott-Armstrong, N. A., Albanes, D., Alonso, M. H., Anderson, K., Arnau-Collell, C., Arndt, V., Bamia, C., Banbury, B. L., Baron, J. A., Berndt, S. I., Bezieau, S., Bishop, D. T., Boehm, J., Boeing, H., Brenner, H., Brezina, S., Buch, S., Buchanan, D. D., Burnett-Hartman, A., Butterbach, K., Caan, B. J., Campbell, P. T., Carlson, C. S., Castellvi-Bel, S., Chan, A. T., Chang-Claude, J., Chanock, S. J., Chirlaque, M., Cho, S. H., Connolly, C. M., Cross, A. J., Cuk, K., Curtis, K. R., de la Chapelle, A., Doheny, K. F., Duggan, D., Easton, D. F., Elias, S. G., Elliott, F., English, D. R., Feskens, E. J., Figueiredo, J. C., Fischer, R., FitzGerald, L. M., Forman, D., Gala, M., Gallinger, S., Gauderman, W. J., Giles, G. G., Gillanders, E., Gong, J., Goodman, P. J., Grady, W. M., Grove, J. S., Gsur, A., Gunter, M. J., Haile, R. W., Hampe, J., Hampel, H., Harlid, S., Hayes, R. B., Hofer, P., Hoffmeister, M., Hopper, J. L., Hsu, W., Huang, W., Hudson, T. J., Hunter, D. J., Ibanez-Sanz, G., Idos, G. E., Ingersoll, R., Jackson, R. D., Jacobs, E. J., Jenkins, M. A., Joshi, A. D., Joshu, C. E., Keku, T. O., Key, T. J., Kim, H. R., Kobayashi, E., Kolonel, L. N., Kooperberg, C., Kuhn, T., Kury, S., Kweon, S., Larsson, S. C., Laurie, C. A., Le Marchand, L., Leal, S. M., Lee, S. C., Lejbkowicz, F., Lemire, M., Li, C. I., Li, L., Lieb, W., Lin, Y., Lindblom, A., Lindor, N. M., Ling, H., Louie, T. L., Mannisto, S., Markowitz, S. D., Martin, V., Masala, G., McNeil, C. E., Melas, M., Milne, R. L., Moreno, L., Murphy, N., Myte, R., Naccarati, A., Newcomb, P. A., Offit, K., Ogino, S., Onland-Moret, N. C., Pardini, B., Parfrey, P. S., Pearlman, R., Perduca, V., Pharoah, P. D., Pinchev, M., Platz, E. A., Prentice, R. L., Pugh, E., Raskin, L., Rennert, G., Rennert, H. S., Riboli, E., Rodriguez-Barranco, M., Romm, J., Sakoda, L. C., Schafmayer, C., Schoen, R. E., Seminara, D., Shah, M., Shelford, T., Shin, M., Shulman, K., Sieri, S., Slattery, M. L., Southey, M. C., Stadler, Z. K., Stegmaier, C., Su, Y., Tangen, C. M., Thibodeau, S. N., Thomas, D. C., Thomas, S. S., Toland, A. E., Trichopoulou, A., Ulrich, C. M., Van Den Berg, D. J., van Duijnhoven, F. J., Van Guelpen, B., van Kranen, H., Vijai, J., Visvanathan, K., Vodicka, P., Vodickova, L., Vymetalkova, V., Weigl, K., Weinstein, S. J., White, E., Win, A. K., Wolf, C. R., Wolk, A., Woods, M. O., Wu, A. H., Zaidi, S. H., Zanke, B. W., Zhang, Q., Zheng, W., Scacheri, P. C., Potter, J. D., Bassik, M. C., Kundaje, A., Casey, G., Moreno, V., Abecasis, G. R., Nickerson, D. A., Gruber, S. B., Hsu, L., Peters, U. 2018

    Abstract

    To further dissect the genetic architecture of colorectal cancer (CRC), we performed whole-genome sequencing of 1,439 cases and 720 controls, imputed discovered sequence variants and Haplotype Reference Consortium panel variants into genome-wide association study data, and tested for association in 34,869 cases and 29,051 controls. Findings were followed up in an additional 23,262 cases and 38,296 controls. We discovered a strongly protective 0.3% frequency variant signal at CHD1. In a combined meta-analysis of 125,478 individuals, we identified 40 new independent signals at P<5*10-8, bringing the number of known independent signals for CRC to ~100. New signals implicate lower-frequency variants, Kruppel-like factors, Hedgehog signaling, Hippo-YAP signaling, long noncoding RNAs and somatic drivers, and support a role for immune function. Heritability analyses suggest that CRC risk is highly polygenic, and larger, more comprehensive studies enabling rare variant analysis will improve understanding of biology underlying this risk and influence personalized screening strategies and drug development.

    View details for PubMedID 30510241

  • Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens. Nature genetics Haney, M. S., Bohlen, C. J., Morgens, D. W., Ousey, J. A., Barkal, A. A., Tsui, C. K., Ego, B. K., Levin, R., Kamber, R. A., Collins, H., Tucker, A., Li, A., Vorselen, D., Labitigan, L., Crane, E., Boyle, E., Jiang, L., Chan, J., Rincon, E., Greenleaf, W. J., Li, B., Snyder, M. P., Weissman, I. L., Theriot, J. A., Collins, S. R., Barres, B. A., Bassik, M. C. 2018

    Abstract

    Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-beta aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.

    View details for PubMedID 30397336

  • CBP modulates sensitivity to dasatinib in pre-BCR+ acute lymphoblastic leukemia. Cancer research Duque-Afonso, J., Lin, C., Han, K., Morgens, D. W., Jeng, E. E., Weng, Z., Jeong, J., Wong, S. H., Zhu, L., Wei, M. C., Chae, H., Schrappe, M., Cario, G., Duyster, J., Sakamoto, K. M., Bassik, M. C., Cleary, M. L. 2018

    Abstract

    Dasatinib is a multi-tyrosine kinase inhibitor approved for treatment of Ph+ acute lymphoblastic leukemia (ALL), but its efficacy is limited by resistance. Recent preclinical studies suggest that dasatinib may be a candidate therapy in additional ALL subtypes including pre-BCR+ ALL. Here we utilized shRNA library screening and global transcriptomic analysis to identify several novel genes and pathways that may enhance dasatinib efficacy or mitigate potential resistance in human pre-BCR+ ALL. Depletion of the transcriptional co-activator CBP increased dasatinib sensitivity by activating transcription of the pre-BCR signaling pathway previously associated with dasatinib sensitivity. Acquired resistance was due in part to upregulation of alternative pathways including WNT through a mechanism suggesting transcriptional plasticity. Small molecules that disrupt CBP interactions with the CREB KID domain or beta-catenin showed promising preclinical efficacy in combination with dasatinib. These findings highlight novel modulators of sensitivity to targeted therapies in human pre-BCR+ ALL, which can be reversed by small molecules inhibitors. They also identify promising therapeutic approaches to ameliorate dasatinib sensitivity and prevent resistance in ALL.

    View details for PubMedID 30262461

  • KIF15 nanomechanics and kinesin inhibitors, with implications for cancer chemotherapeutics PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Milic, B., Chakraborty, A., Han, K., Bassik, M. C., Block, S. M. 2018; 115 (20): E4613–E4622

    Abstract

    Eg5, a mitotic kinesin, has been a target for anticancer drug development. Clinical trials of small-molecule inhibitors of Eg5 have been stymied by the development of resistance, attributable to mitotic rescue by a different endogenous kinesin, KIF15. Compared with Eg5, relatively little is known about the properties of the KIF15 motor. Here, we employed single-molecule optical-trapping techniques to define the KIF15 mechanochemical cycle. We also studied the inhibitory effects of KIF15-IN-1, an uncharacterized, commercially available, small-molecule inhibitor, on KIF15 motility. To explore the complementary behaviors of KIF15 and Eg5, we also scored the effects of small-molecule inhibitors on admixtures of both motors, using both a microtubule (MT)-gliding assay and an assay for cancer cell viability. We found that (i) KIF15 motility differs significantly from Eg5; (ii) KIF15-IN-1 is a potent inhibitor of KIF15 motility; (iii) MT gliding powered by KIF15 and Eg5 only ceases when both motors are inhibited; and (iv) pairing KIF15-IN-1 with Eg5 inhibitors synergistically reduces cancer cell growth. Taken together, our results lend support to the notion that a combination drug therapy employing both inhibitors may be a viable strategy for overcoming chemotherapeutic resistance.

    View details for PubMedID 29703754

  • A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies Nat. Genet. Breslow, D. K., Hoogendoorn, S., Kopp, A. R., Morgens, D. W., Vu, B. K., Han, K., Li, A., Hess, G. T., Bassik, M. C., Chen, J. K., V, N. M. 2018; Epub ahead of print: 460–71

    Abstract

    Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

    View details for DOI 10.1038/s41588-018-0054-7

    View details for PubMedCentralID PMC5862771

  • A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies. Nature genetics Breslow, D. K., Hoogendoorn, S., Kopp, A. R., Morgens, D. W., Vu, B. K., Kennedy, M. C., Han, K., Li, A., Hess, G. T., Bassik, M. C., Chen, J. K., Nachury, M. V. 2018; 50 (3): 460–71

    Abstract

    Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

    View details for PubMedID 29459677

  • CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity. Nature genetics Kramer, N. J., Haney, M. S., Morgens, D. W., Jovičić, A., Couthouis, J., Li, A., Ousey, J., Ma, R., Bieri, G., Tsui, C. K., Shi, Y., Hertz, N. T., Tessier-Lavigne, M., Ichida, J. K., Bassik, M. C., Gitler, A. D. 2018

    Abstract

    Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases.

    View details for PubMedID 29507424

  • CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity NATURE Burr, M. L., Sparbier, C. E., Chan, Y., Williamson, J. C., Woods, K., Beavis, P. A., Lam, E. N., Henderson, M. A., Bell, C. C., Stolzenburg, S., Gilan, O., Bloor, S., Noori, T., Morgens, D. W., Bassik, M. C., Neeson, P. J., Behren, A., Darcy, P. K., Dawson, S., Voskoboinik, I., Trapani, J. A., Cebon, J., Lehner, P. J., Dawson, M. A. 2017; 549 (7670): 101–5

    Abstract

    Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR-Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.

    View details for PubMedID 28813417

    View details for PubMedCentralID PMC5706633

  • Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens NATURE COMMUNICATIONS Morgens, D. W., Wainberg, M., Boyle, E. A., Ursu, O., Araya, C. L., Tsui, C. K., Haney, M. S., Hess, G. T., Han, K., Jeng, E. E., Li, A., Snyder, M. P., Greenleaf, W. J., Kundaje, A., Bassik, M. C. 2017; 8

    Abstract

    CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.

    View details for DOI 10.1038/ncomms15178

    View details for PubMedID 28474669

  • Population- and individual- specific regulatory variation in Sardinia NATURE GENETICS Pala, M., Zappala, Z., Marongiu, M., Li, X., Davis, J. R., Cusano, R., Crobu, F., Kukurba, K. R., Gloudemans, M. J., Reinier, F., Berutti, R., Piras, M. G., Mulas, A., Zoledziewska, M., Marongiu, M., Sorokin, E. P., Hess, G. T., Smith, K. S., Busonero, F., Maschio, A., Steri, M., Sidore, C., Sanna, S., Fiorillo, E., Bassik, M. C., Sawcer, S. J., Battle, A., Novembre, J., Jones, C., Angius, A., Abecasis, G. R., Schlessinger, D., Cucca, F., Montgomery, S. B. 2017; 49 (5): 700-?

    Abstract

    Genetic studies of complex traits have mainly identified associations with noncoding variants. To further determine the contribution of regulatory variation, we combined whole-genome and transcriptome data for 624 individuals from Sardinia to identify common and rare variants that influence gene expression and splicing. We identified 21,183 expression quantitative trait loci (eQTLs) and 6,768 splicing quantitative trait loci (sQTLs), including 619 new QTLs. We identified high-frequency QTLs and found evidence of selection near genes involved in malarial resistance and increased multiple sclerosis risk, reflecting the epidemiological history of Sardinia. Using family relationships, we identified 809 segregating expression outliers (median z score of 2.97), averaging 13.3 genes per individual. Outlier genes were enriched for proximal rare variants, providing a new approach to study large-effect regulatory variants and their relevance to traits. Our results provide insight into the effects of regulatory variants and their relationship to population history and individual genetic risk.

    View details for DOI 10.1038/ng.3840

    View details for Web of Science ID 000400051400010

    View details for PubMedID 28394350

  • Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions NATURE BIOTECHNOLOGY Han, K., Jeng, E. E., Hess, G. T., Morgens, D. W., Li, A., Bassik, M. C. 2017; 35 (5): 463-?

    Abstract

    Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers, but direct screening of all possible drug combinations is infeasible. Here we introduce a CRISPR-based double knockout (CDKO) system that improves the efficiency of combinatorial genetic screening using an effective strategy for cloning and sequencing paired single guide RNA (sgRNA) libraries and a robust statistical scoring method for calculating genetic interactions (GIs) from CRISPR-deleted gene pairs. We applied CDKO to generate a large-scale human GI map, comprising 490,000 double-sgRNAs directed against 21,321 pairs of drug targets in K562 leukemia cells and identified synthetic lethal drug target pairs for which corresponding drugs exhibit synergistic killing. These included the BCL2L1 and MCL1 combination, which was also effective in imatinib-resistant cells. We further validated this system by identifying known and previously unidentified GIs between modifiers of ricin toxicity. This work provides an effective strategy to screen synergistic drug combinations in high-throughput and a CRISPR-based tool to dissect functional GI networks.

    View details for DOI 10.1038/nbt.3834

    View details for Web of Science ID 000400809800021

    View details for PubMedID 28319085

  • Human pyrimidine nucleotide biosynthesis as a target for antiviral chemotherapy. Current opinion in biotechnology Okesli, A., Khosla, C., Bassik, M. C. 2017; 48: 127-134

    Abstract

    The development of broad-spectrum, host-acting antiviral therapies remains an important but elusive goal in anti-infective drug discovery. To replicate efficiently, viruses not only depend on their hosts for an adequate supply of pyrimidine nucleotides, but also up-regulate pyrimidine nucleotide biosynthesis in infected cells. In this review, we outline our understanding of mammalian de novo and salvage metabolic pathways for pyrimidine nucleotide biosynthesis. The available spectrum of experimental and FDA-approved drugs that modulate individual steps in these metabolic pathways is also summarized. The logic of a host-acting combination antiviral therapy comprised of inhibitors of dihydroorotate dehydrogenase and uridine/cytidine kinase is discussed.

    View details for DOI 10.1016/j.copbio.2017.03.010

    View details for PubMedID 28458037

  • Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes. Molecular cell Hess, G. T., Tycko, J., Yao, D., Bassik, M. C. 2017; 68 (1): 26–43

    Abstract

    The past several years have seen an explosion in development of applications for the CRISPR-Cas9 system, from efficient genome editing, to high-throughput screening, to recruitment of a range of DNA and chromatin-modifying enzymes. While homology-directed repair (HDR) coupled with Cas9 nuclease cleavage has been used with great success to repair and re-write genomes, recently developed base-editing systems present a useful orthogonal strategy to engineer nucleotide substitutions. Base editing relies on recruitment of cytidine deaminases to introduce changes (rather than double-stranded breaks and donor templates) and offers potential improvements in efficiency while limiting damage and simplifying the delivery of editing machinery. At the same time, these systems enable novel mutagenesis strategies to introduce sequence diversity for engineering and discovery. Here, we review the different base-editing platforms, including their deaminase recruitment strategies and editing outcomes, and compare them to other CRISPR genome-editing technologies. Additionally, we discuss how these systems have been applied in therapeutic, engineering, and research settings. Lastly, we explore future directions of this emerging technology.

    View details for PubMedID 28985508

  • Static and Dynamic DNA Loops form AP-1-Bound Activation Hubs during Macrophage Development. Molecular cell Phanstiel, D. H., Van Bortle, K., Spacek, D., Hess, G. T., Shamim, M. S., Machol, I., Love, M. I., Aiden, E. L., Bassik, M. C., Snyder, M. P. 2017; 67 (6): 1037–48.e6

    Abstract

    The three-dimensional arrangement of the human genome comprises a complex network of structural and regulatory chromatin loops important for coordinating changes in transcription during human development. To better understand the mechanisms underlying context-specific 3D chromatin structure and transcription during cellular differentiation, we generated comprehensive in situ Hi-C maps of DNA loops in human monocytes and differentiated macrophages. We demonstrate that dynamic looping events are regulatory rather than structural in nature and uncover widespread coordination of dynamic enhancer activity at preformed and acquired DNA loops. Enhancer-bound loop formation and enhancer activation of preformed loops together form multi-loop activation hubs at key macrophage genes. Activation hubs connect 3.4 enhancers per promoter and exhibit a strong enrichment for activator protein 1 (AP-1)-binding events, suggesting that multi-loop activation hubs involving cell-type-specific transcription factors represent an important class of regulatory chromatin structures for the spatiotemporal control of transcription.

    View details for PubMedID 28890333

  • Finding host targets for HIV therapy. Nature genetics Tsui, C. K., Gupta, A., Bassik, M. C. 2017; 49 (2): 175–76

    Abstract

    A CRISPR screen conducted in a CD4+ T cell leukemia line has identified host factors required for HIV infection but dispensable for cellular survival. The results highlight sulfation on the HIV co-receptor CCR5 and cellular aggregation as potential targets for therapeutic intervention.

    View details for PubMedID 28138150

  • The impact of rare variation on gene expression across tissues. Nature Li, X., Kim, Y., Tsang, E. K., Davis, J. R., Damani, F. N., Chiang, C., Hess, G. T., Zappala, Z., Strober, B. J., Scott, A. J., Li, A., Ganna, A., Bassik, M. C., Merker, J. D., Hall, I. M., Battle, A., Montgomery, S. B. 2017; 550 (7675): 239–43

    Abstract

    Rare genetic variants are abundant in humans and are expected to contribute to individual disease risk. While genetic association studies have successfully identified common genetic variants associated with susceptibility, these studies are not practical for identifying rare variants. Efforts to distinguish pathogenic variants from benign rare variants have leveraged the genetic code to identify deleterious protein-coding alleles, but no analogous code exists for non-coding variants. Therefore, ascertaining which rare variants have phenotypic effects remains a major challenge. Rare non-coding variants have been associated with extreme gene expression in studies using single tissues, but their effects across tissues are unknown. Here we identify gene expression outliers, or individuals showing extreme expression levels for a particular gene, across 44 human tissues by using combined analyses of whole genomes and multi-tissue RNA-sequencing data from the Genotype-Tissue Expression (GTEx) project v6p release. We find that 58% of underexpression and 28% of overexpression outliers have nearby conserved rare variants compared to 8% of non-outliers. Additionally, we developed RIVER (RNA-informed variant effect on regulation), a Bayesian statistical model that incorporates expression data to predict a regulatory effect for rare variants with higher accuracy than models using genomic annotations alone. Overall, we demonstrate that rare variants contribute to large gene expression changes across tissues and provide an integrative method for interpretation of rare variants in individual genomes.

    View details for PubMedID 29022581

  • Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators. Nature Liu, N., Lee, C. H., Swigut, T., Grow, E., Gu, B., Bassik, M., Wysocka, J. 2017

    Abstract

    Transposable elements (TEs) are now recognized not only as parasitic DNA, whose spread in the genome must be controlled by the host, but also as major players in genome evolution and regulation1-6. Long INterspersed Element-1 (LINE-1 or L1), the only currently autonomous mobile transposon in humans, occupies 17% of the genome and continues to generate inter- and intra-individual genetic variation, in some cases resulting in disease1-7. Nonetheless, how L1 activity is controlled and what function L1s play in host gene regulation remain incompletely understood. Here, we use CRISPR/Cas9 screening strategies in two distinct human cell lines to provide the first genome-wide survey of genes involved in L1 retrotransposition control. We identified functionally diverse genes that either promote or restrict L1 retrotransposition. These genes, often associated with human diseases, control the L1 lifecycle at transcriptional or post-transcriptional levels and in a manner that can depend on the endogenous L1 sequence, underscoring the complexity of L1 regulation. We further investigated L1 restriction by MORC2 and human silencing hub (HUSH) complex subunits MPP8 and TASOR8. HUSH/MORC2 selectively bind evolutionarily young, full-length L1s located within transcriptionally permissive euchromatic environment, and promote H3K9me3 deposition for transcriptional silencing. Interestingly, these silencing events often occur within introns of transcriptionally active genes and lead to down-regulation of host gene expression in a HUSH/MORC2-dependent manner. Together, we provide a rich resource for studies of L1 retrotransposition, elucidate a novel L1 restriction pathway, and illustrate how epigenetic silencing of TEs rewires host gene expression programs.

    View details for PubMedID 29211708

  • The mTOR Complex Controls HIV Latency CELL HOST & MICROBE Besnard, E., Hakre, S., Kampmann, M., Lim, H. W., Hosmane, N. N., Martin, A., Bassik, M. C., Verschueren, E., Battivelli, E., Chan, J., Svensson, J. P., Gramatica, A., Conrad, R. J., Ott, M., Greene, W. C., Krogan, N. J., Siliciano, R. F., Weissman, J. S., Verdin, E. 2016; 20 (6): 785-797

    Abstract

    A population of CD4 T lymphocytes harboring latent HIV genomes can persist in patients on antiretroviral therapy, posing a barrier to HIV eradication. To examine cellular complexes controlling HIV latency, we conducted a genome-wide screen with a pooled ultracomplex shRNA library and in vitro system modeling HIV latency and identified the mTOR complex as a modulator of HIV latency. Knockdown of mTOR complex subunits or pharmacological inhibition of mTOR activity suppresses reversal of latency in various HIV-1 latency models and HIV-infected patient cells. mTOR inhibitors suppress HIV transcription both through the viral transactivator Tat and via Tat-independent mechanisms. This inhibition occurs at least in part via blocking the phosphorylation of CDK9, a p-TEFb complex member that serves as a cofactor for Tat-mediated transcription. The control of HIV latency by mTOR signaling identifies a pathway that may have significant therapeutic opportunities.

    View details for DOI 10.1016/j.chom.2016.11.001

    View details for Web of Science ID 000392843500014

    View details for PubMedID 27978436

    View details for PubMedCentralID PMC5354304

  • Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nature methods Hess, G. T., Frésard, L., Han, K., Lee, C. H., Li, A., Cimprich, K. A., Montgomery, S. B., Bassik, M. C. 2016

    Abstract

    Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.

    View details for DOI 10.1038/nmeth.4038

    View details for PubMedID 27798611

  • E2A-PBX1 remodels oncogenic signaling networks in B-cell precursor acute lymphoid leukemia. Cancer research Duque-Afonso, J., Lin, C., Han, K., Wei, M. C., Feng, J., Kurzer, J., Schneidawind, C., Wong, S. H., Bassik, M. C., Cleary, M. L. 2016

    Abstract

    There is limited understanding of how signaling pathways are altered by oncogenic fusion transcription factors that drive leukemogenesis. To address this, we interrogated activated signaling pathways in a comparative analysis of mouse and human leukemias expressing the fusion protein E2A-PBX1, which is present in 5%-7% of pediatric and 50% of pre-B-cell receptor (preBCR(+)) acute lymphocytic leukemia (ALL). In this study, we describe remodeling of signaling networks by E2A-PBX1 in pre-B-ALL, which results in hyperactivation of the key oncogenic effector enzyme PLCγ2. Depletion of PLCγ2 reduced proliferation of mouse and human ALLs, including E2A-PBX1 leukemias, and increased disease-free survival after secondary transplantation. Mechanistically, E2A-PBX1 bound promoter regulatory regions and activated the transcription of its key target genes ZAP70, SYK, and LCK, which encode kinases upstream of PLCγ2. Depletion of the respective upstream kinases decreased cell proliferation and phosphorylated levels of PLCγ2 (pPLCγ2). Pairwise silencing of ZAP70, SYK, or LCK showed additive effects on cell growth inhibition, providing a rationale for combination therapy with inhibitors of these kinases. Accordingly, inhibitors such as the SRC family kinase (SFK) inhibitor dasatinib reduced pPLCγ2 and inhibited proliferation of human and mouse preBCR(+)/E2A-PBX1(+) leukemias in vitro and in vivo Furthermore, combining small-molecule inhibition of SYK, LCK, and SFK showed synergistic interactions and preclinical efficacy in the same setting. Our results show how the oncogenic fusion protein E2A-PBX1 perturbs signaling pathways upstream of PLCγ2 and renders leukemias amenable to targeted therapeutic inhibition. Cancer Res; 76(23); 6937-49. ©2016 AACR.

    View details for PubMedID 27758892

  • Bithionol blocks pathogenicity of bacterial toxins, ricin, and Zika virus SCIENTIFIC REPORTS Leonardi, W., Zilbermintz, L., Cheng, L. W., Zozaya, J., Tran, S. H., Elliott, J. H., Polukhina, K., Manasherob, R., Li, A., Chi, X., Gharaibeh, D., Kenny, T., Zamani, R., Soloveva, V., Haddow, A. D., Nasar, F., Bavari, S., Bassik, M. C., Cohen, S. N., Levitin, A., Martchenko, M. 2016; 6

    Abstract

    Diverse pathogenic agents often utilize overlapping host networks, and hub proteins within these networks represent attractive targets for broad-spectrum drugs. Using bacterial toxins, we describe a new approach for discovering broad-spectrum therapies capable of inhibiting host proteins that mediate multiple pathogenic pathways. This approach can be widely used, as it combines genetic-based target identification with cell survival-based and protein function-based multiplex drug screens, and concurrently discovers therapeutic compounds and their protein targets. Using B-lymphoblastoid cells derived from the HapMap Project cohort of persons of African, European, and Asian ancestry we identified host caspases as hub proteins that mediate the lethality of multiple pathogenic agents. We discovered that an approved drug, Bithionol, inhibits host caspases and also reduces the detrimental effects of anthrax lethal toxin, diphtheria toxin, cholera toxin, Pseudomonas aeruginosa exotoxin A, Botulinum neurotoxin, ricin, and Zika virus. Our study reveals the practicality of identifying host proteins that mediate multiple disease pathways and discovering broad-spectrum therapies that target these hub proteins.

    View details for DOI 10.1038/srep34475

    View details for Web of Science ID 000384291100001

    View details for PubMedID 27686742

    View details for PubMedCentralID PMC5043268

  • Translation readthrough mitigation NATURE Arribere, J. A., Cenik, E. S., Jain, N., Hess, G. T., Lee, C. H., Bassik, M. C., Fire, A. Z. 2016; 534 (7609): 719-?

    Abstract

    A fraction of ribosomes engaged in translation will fail to terminate when reaching a stop codon, yielding nascent proteins inappropriately extended on their C termini. Although such extended proteins can interfere with normal cellular processes, known mechanisms of translational surveillance are insufficient to protect cells from potential dominant consequences. Here, through a combination of transgenics and CRISPR–Cas9 gene editing in Caenorhabditis elegans, we demonstrate a consistent ability of cells to block accumulation of C-terminal-extended proteins that result from failure to terminate at stop codons. Sequences encoded by the 3′ untranslated region (UTR) were sufficient to lower protein levels. Measurements of mRNA levels and translation suggested a co- or post-translational mechanism of action for these sequences in C. elegans. Similar mechanisms evidently operate in human cells, in which we observed a comparable tendency for translated human 3′ UTR sequences to reduce mature protein expression in tissue culture assays, including 3′ UTR sequences from the hypomorphic ‘Constant Spring’ haemoglobin stop codon variant. We suggest that 3′ UTRs may encode peptide sequences that destabilize the attached protein, providing mitigation of unwelcome and varied translation errors.

    View details for DOI 10.1038/nature18308

    View details for Web of Science ID 000378676000044

    View details for PubMedID 27281202

    View details for PubMedCentralID PMC5054982

  • Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes NATURE BIOTECHNOLOGY Morgens, D. W., Deans, R. M., Li, A., Bassik, M. C. 2016; 34 (6): 634-636

    Abstract

    We compared the ability of short hairpin RNA (shRNA) and CRISPR/Cas9 screens to identify essential genes in the human chronic myelogenous leukemia cell line K562. We found that the precision of the two libraries in detecting essential genes was similar and that combining data from both screens improved performance. Notably, results from the two screens showed little correlation, which can be partially explained by the identification of distinct essential biological processes with each technology.

    View details for DOI 10.1038/nbt.3567

    View details for Web of Science ID 000377846400030

    View details for PubMedID 27159373

    View details for PubMedCentralID PMC4900911

  • Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification NATURE CHEMICAL BIOLOGY Deans, R. M., Morgens, D. W., Okesli, A., Pillay, S., Horlbeck, M. A., Kampmann, M., Gilbert, L. A., Li, A., Mateo, R., Smith, M., Glenn, J. S., Carette, J. E., Khosla, C., Bassik, M. C. 2016; 12 (5): 361-?

    Abstract

    Broad-spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we used parallel genome-wide high-coverage short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad-spectrum antiviral with unexplained cytotoxicity. We found that GSK983 blocked cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduced GSK983 cytotoxicity but not antiviral activity, providing an attractive new approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Our results highlight the distinct advantages and limitations of each screening method for identifying drug targets, and demonstrate the utility of parallel knockdown and knockout screens for comprehensive probing of drug activity.

    View details for DOI 10.1038/NCHEMBIO.2050

    View details for PubMedID 27018887

  • Weak base pairing in both seed and 3' regions reduces RNAi off-targets and enhances si/shRNA designs. Nucleic acids research Gu, S., Zhang, Y., Jin, L., Huang, Y., Zhang, F., Bassik, M. C., Kampmann, M., Kay, M. A. 2014; 42 (19): 12169-12176

    Abstract

    The use of RNA interference is becoming routine in scientific discovery and treatment of human disease. However, its applications are hampered by unwanted effects, particularly off-targeting through miRNA-like pathways. Recent studies suggest that the efficacy of such off-targeting might be dependent on binding stability. Here, by testing shRNAs and siRNAs of various GC content in different guide strand segments with reporter assays, we establish that weak base pairing in both seed and 3' regions is required to achieve minimal off-targeting while maintaining the intended on-target activity. The reduced off-targeting was confirmed by RNA-Seq analyses from mouse liver RNAs expressing various anti-HCV shRNAs. Finally, our protocol was validated on a large scale by analyzing results of a genome-wide shRNA screen. Compared with previously established work, the new algorithm was more effective in reducing off-targeting without jeopardizing on-target potency. These studies provide new rules that should significantly improve on siRNA/shRNA design.

    View details for DOI 10.1093/nar/gku854

    View details for PubMedID 25270879

    View details for PubMedCentralID PMC4231738

  • Functional genomics platform for pooled screening and generation of mammalian genetic interaction maps NATURE PROTOCOLS Kampmann, M., Bassik, M. C., Weissman, J. S. 2014; 9 (8): 1825-1847

    Abstract

    Systematic genetic interaction maps in microorganisms are powerful tools for identifying functional relationships between genes and for defining the function of uncharacterized genes. We have recently implemented this strategy in mammalian cells as a two-stage approach. First, genes of interest are robustly identified in a pooled genome-wide screen using complex shRNA libraries. Second, phenotypes for all pairwise combinations of 'hit' genes are measured in a double-shRNA screen and used to construct a genetic interaction map. Our protocol allows for rapid pooled screening under various conditions without a requirement for robotics, in contrast to arrayed approaches. Each round of screening can be implemented in ∼2 weeks, with additional time for analysis and generation of reagents. We discuss considerations for screen design, and we present complete experimental procedures, as well as a full computational analysis suite for the identification of hits in pooled screens and generation of genetic interaction maps. Although the protocol outlined here was developed for our original shRNA-based approach, it can be applied more generally, including to CRISPR-based approaches.

    View details for DOI 10.1038/nprot.2014.103

    View details for Web of Science ID 000340039700004

    View details for PubMedID 24992097

  • Next-Generation NAMPT Inhibitors Identified by Sequential High-Throughput Phenotypic Chemical and Functional Genomic Screens. Chemistry & biology Matheny, C. J., Wei, M. C., Bassik, M. C., Donnelly, A. J., Kampmann, M., Iwasaki, M., Piloto, O., Solow-Cordero, D. E., Bouley, D. M., Rau, R., Brown, P., McManus, M. T., Weissman, J. S., Cleary, M. L. 2013; 20 (11): 1352-1363

    Abstract

    Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of the mechanistically relevant targets remains a major experimental challenge. We report the application of sequential unbiased high-throughput chemical and ultracomplex small hairpin RNA (shRNA) screens to identify a distinctive class of inhibitors that target nicotinamide phosphoribosyl transferase (NAMPT), a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide, a crucial cofactor in many biochemical processes. The lead compound STF-118804 is a highly specific NAMPT inhibitor, improves survival in an orthotopic xenotransplant model of high-risk acute lymphoblastic leukemia, and targets leukemia stem cells. Tandem high-throughput screening using chemical and ultracomplex shRNA libraries, therefore, provides a rapid chemical genetics approach for seamless progression from small-molecule lead identification to target discovery and validation.

    View details for DOI 10.1016/j.chembiol.2013.09.014

    View details for PubMedID 24183972

  • A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility. Cell Bassik, M. C., Kampmann, M., Lebbink, R. J., Wang, S., Hein, M. Y., Poser, I., Weibezahn, J., Horlbeck, M. A., Chen, S., Mann, M., Hyman, A. A., Leproust, E. M., McManus, M. T., Weissman, J. S. 2013; 152 (4): 909–22

    Abstract

    Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.

    View details for DOI 10.1016/j.cell.2013.01.030

    View details for PubMedID 23394947

  • Rapid creation and quantitative monitoring of high coverage shRNA libraries. Nature methods Bassik, M. C., Lebbink, R. J., Churchman, L. S., Ingolia, N. T., Patena, W., LeProust, E. M., Schuldiner, M., Weissman, J. S., McManus, M. T. 2009; 6 (6): 443–45

    Abstract

    Short hairpin RNA libraries are limited by low efficacy of many shRNAs and by off-target effects, which give rise to false negatives and false positives, respectively. Here we present a strategy for rapidly creating expanded shRNA pools (approximately 30 shRNAs per gene) that are analyzed by deep sequencing (EXPAND). This approach enables identification of multiple effective target-specific shRNAs from a complex pool, allowing a rigorous statistical evaluation of true hits.

    View details for DOI 10.1038/nmeth.1330

    View details for PubMedID 19448642