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News Article | December 19, 2016

CAMBRIDGE, Mass. (Dec. 19, 2016) -- Investigators from Whitehead Institute, the Ragon Institute of MGH, MIT and Harvard and the Broad Institute of MIT and Harvard have used CRISPR-Cas9 gene-editing technology to identify three promising new targets for treatment of HIV infection. In their report receiving advance online publication in Nature Genetics, the research team describes how screening with CRISPR for human genes that are essential for HIV infection but not for cellular survival identified five genes -- three of which had not been identified in earlier studies using RNA interference. Their method can also be used to identify therapeutic targets for other viral pathogens. "We were surprised to find that there are so few host factors required for HIV infection given some of the previous literature," observes David M. Sabatini, Whitehead Institute Faculty Member and co-corresponding author of the Nature Genetics paper. "The beauty of the CRISPR-based genetic screens is the clear and robust results they yield," notes Sabatini, who is also member of the Broad Institute and Professor of Biology at Massachusetts Institute of Technology. "Current anti-HIV medications overwhelmingly target viral proteins," says Ryan J. Park of the Ragon Institute and the Broad Institute, co-lead author of the report. "Because HIV mutates so rapidly, drug-resistant strains frequently emerge, particularly when patients miss doses of their medication. Developing new drugs to target human genes required for HIV infection is a promising approach to HIV therapy, with potentially fewer opportunities for the development of resistance." Bruce Walker, director of the Ragon Institute and co-corresponding author of the Nature Genetics paper, explains, "Viruses are very small and have very few genes - HIV has only 9, while humans have more than 19,000 - so viruses commandeer human genes to make essential building blocks for their replication. Our goal was to identify human genes, also called host genes, that are essential for HIV to replicate but could be eliminated without harming a human patient." Tim Wang, a doctoral student conducting research at Whitehead Institute and the Broad Institute, and co-lead author of the report, explains, "CRISPR makes it possible to completely knock out genes at the DNA level; and our genome-wide, CRISPR-Cas9-based approach targets more than 18,500 genes, the vast majority of human protein-coding genes. Our study demonstrates how CRISPR-based screens can be applied to identify host factors critical to the survival of other viral pathogens but dispensable for host cell viability. Broad application of this method should pinpoint a novel class of potential therapeutic targets that have previously been underexplored for the treatment of infectious disease." Co-corresponding author Nir Hacohen, an institute member at the Broad Institute and director of Cancer Immunology at Massachusetts General Hospital (MGH), adds, "An important aspect of our study was to focus on human T cells, the primary targets of HIV, and to identify host genes with the most dramatic role in viral infection of T cells." Previous research has identified several host dependency factors, including two proteins required for HIV to enter CD4 T cells, the primary target of the virus: the CD4 molecule itself, to which the virus binds, and CCR5, which facilitates the binding of common HIV strains. Individuals with a particular CCR5 mutation are immune to those viral strains - indeed the only individual considered cured of HIV infection received a bone marrow transplant from a donor with that CCR5 mutation - but while therapeutic CCR5 inhibitors have been developed and are in clinical use, they can cause serious side effects. Three 2008 studies that used RNA interference (RNAi) to identify potential host dependency factors identified more than 800 possible targets; but the little overlap among the results of the studies suggested a high rate of false positive results. In addition, none of those studies was performed using the immune cells targeted by HIV, which also reduces the likelihood that the identified genes actually participate in HIV's infection of CD4 T cells. Whitehead Institute's Tim Wang explains that, "RNAi suppresses but does not completely block gene expression - which could allow a targeted gene to produce enough protein to permit HIV infection - and it also can suppress expression of additional genes besides the intended target, leading to a false positive result." Using CRISPR to screen a cell line derived from HIV-susceptible CD4 T cells identified five genes that, when inactivated, protected cells from HIV infection without affecting cellular survival. In addition to CD4 and CCR5, the screen identified genes for two enzymes -- TPST2 and SLC35B2 -- that modify the CCR5 molecule in a way that is required for the binding of HIV. An additional gene identified through the screen was ALCAM, which is involved in cell-to-cell adhesion. When CD4 T cells are exposed to low amounts of virus, as might be seen in natural transmission, loss of ALCAM was associated with striking protection from HIV infection. Park explains, "ALCAM is necessary for cell-to-cell adhesion in our cell line, allowing more efficient viral transfer from one cell to the next. In fact, we found that artificially inducing the aggregation of cells lacking ALCAM restored the cell-to-cell transmission of HIV. Further studies are needed to investigate whether targeting these genes would be toxic to humans. However, even if systemic inhibition has toxic effects, gene therapy approaches that selectively target these genes only in CD4 T cells or their precursors may avoid these toxicities, although it's important to note that gene therapy remains a challenging and potentially costly therapeutic approach." Eric S. Lander, of the Broad Institute, is a co-corresponding author of the Nature Genetics paper, along with Sabatini, Hacohen, and Walker - who is also the Phillip and Susan Ragon Professor of Medicine at Harvard Medical School, a clinician in the MGH Division of Infectious Diseases and an associate member of the Broad Institute. Additional co-authors are Dylan Koundakjian, Pedro Lamothe-Molina, Blandine Monel, Wilfredo Garcia-Beltran and Alicja Piechocka-Trocha, Ragon Institute; Judd F. Hultquist, Kathrin Schumann, Alexander Marson and Nevan J. Krogan University of California, San Francisco; Haiyan Yu, Broad Institute; and Kevin M. Krupczak, a member of the Sabatini lab at Whitehead Institute when the study was conducted. The study was supported by funds from the Ragon Institute and the Howard Hughes Medical Institute. The study, formally titled A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors, will appear via Advance Online Publication on the Nature Genetics website on 19 December 2016. 1Ragon Institute of Massachusetts General Hospital (MGH), Massachusetts Institute of Technology (MIT), and Harvard University, Cambridge, Massachusetts, USA 7Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biosciences, QB3, University of California at San Francisco (UCSF), San Francisco, California, USA 11Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, California, USA 13Department of Medicine, University of California at San Francisco, San Francisco, California, USA 19Institute of Medical Engineering and Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA The following funding acknowledgements from the authors appear at the end of the paper: This work was supported by the Howard Hughes Medical Institute (D.M.S. and B.D.W.), the National Institutes of Health (grants CA103866 (D.M.S.), F31 CA189437 (T.W.), P50 GM082250 (A.M. and N.J.K.), U19 AI106754 (J.F.H. and N.J.K.), and P01 AI090935 (N.J.K.)), the National Human Genome Research Institute (grant 2U54HG003067-10; E.S.L.), the National Science Foundation (T.W.), the MIT Whitaker Health Sciences Fund (T.W.), the UCSF Sandler Fellowship (A.M.), a gift from J. Aronov (A.M.), the UCSF MPHD T32 Training Grant (J.F.H.), and the Deutsche Forschungsgemeinschaft (grant SCHU3020/2-1; K.S.). Support was also provided by NIH-funded Centers for AIDS Research (grant P30 AI027763, UCSF Center for AIDS Research (N.J.K.) and grant P30 AI060354, Harvard University Center for AIDS Research (B.D.W.)), which are supported by the following NIH co-funding and participating Institutes and Centers: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, FIC, and OAR. D.M.S. and B.D.W. are investigators of the Howard Hughes Medical Institute. R.J.P. is a Howard Hughes Medical Institute Research Fellow.

Beltrao P.,European Bioinformatics Institute | Bork P.,Structural and Computational Biology Unit | Bork P.,Max Delbrück Center for Molecular Medicine | Krogan N.J.,University of California at San Francisco | And 3 more authors.
Molecular Systems Biology | Year: 2013

Protein post-translational modifications (PTMs) allow the cell to regulate protein activity and play a crucial role in the response to changes in external conditions or internal states. Advances in mass spectrometry now enable proteome wide characterization of PTMs and have revealed a broad functional role for a range of different types of modifications. Here we review advances in the study of the evolution and function of PTMs that were spurred by these technological improvements. We provide an overview of studies focusing on the origin and evolution of regulatory enzymes as well as the evolutionary dynamics of modification sites. Finally, we discuss different mechanisms of altering protein activity via post-translational regulation and progress made in the large-scale functional characterization of PTM function. © 2013 The Authors.

Springer M.,Harvard University | Weissman J.S.,Howard Hughes Medical Institute | Weissman J.S.,California Institute for Quantitative Biosciences | Weissman J.S.,University of California at San Francisco | Kirschner M.W.,Harvard University
Molecular Systems Biology | Year: 2010

Gene copy number variation has been discovered in humans, between related species, and in different cancer tissues, but it is unclear how much of this genomic-level variation leads to changes in the level of protein abundance. To address this, we eliminated one of the two genomic copies of 730 different genes in Saccharomyces cerevisiae and asked how often a 50% reduction in gene dosage leads to a 50% reduction in protein level. For at least 80% of genes tested, and under several environmental conditions, it does: protein levels in the heterozygous strain are close to 50% of wild type. For 5% of the genes tested, the protein levels in the heterozygote are maintained at nearly wild-type levels. These experiments show that protein levels are not, in general, directly monitored and adjusted to a desired level. Combined with fitness data, this implies that proteins are expressed at levels higher than necessary for survival. © 2010 EMBO and Macmillan Publishers Limited. All rights reserved.

Salt M.B.,University of California at San Francisco | Bandyopadhyay S.,University of California at San Francisco | Bandyopadhyay S.,California Institute for Quantitative Biosciences | McCormick F.,University of California at San Francisco
Cancer Discovery | Year: 2014

Tumors showing evidence of epithelial-to-mesenchymal transition (EMT) have been associated with metastasis, drug resistance, and poor prognosis. Heterogeneity along the EMT spectrum is observed between and within tumors. To develop effective therapeutics, a mechanistic understanding of how EMT affects the molecular requirements for proliferation is needed. We found that although cells use phosphoinositide 3-kinase (PI3K) for proliferation in both the epithelial and mesenchymal states, EMT rewires the mechanism of PI3K pathway activation. In epithelial cells, autocrine ERBB3 activation maintains PI3K signaling, whereas after EMT, downregulation of ERBB3 disrupts autocrine signaling to PI3K. Loss of ERBB3 leads to reduced serum-independent proliferation after EMT that can be rescued through reactivation of PI3K by enhanced signaling from p110α, ERBB3 reexpression, or growth factor stimulation. In vivo, we demonstrate that PIK3CA expression is upregulated in mesenchymal tumors with low levels of ERBB3. This study defines how ERBB3 downregulation after EMT affects PI3K-dependent proliferation. © 2014 American Association for Cancer Research.

Ideker T.,University of California at San Diego | Krogan N.J.,University of California at San Francisco | Krogan N.J.,California Institute for Quantitative Biosciences | Krogan N.J.,Gladstone
Molecular Systems Biology | Year: 2012

Protein and genetic interaction maps can reveal the overall physical and functional landscape of a biological system. To date, these interaction maps have typically been generated under a single condition, even though biological systems undergo differential change that is dependent on environment, tissue type, disease state, development or speciation. Several recent interaction mapping studies have demonstrated the power of differential analysis for elucidating fundamental biological responses, revealing that the architecture of an interactome can be massively re-wired during a cellular or adaptive response. Here, we review the technological developments and experimental designs that have enabled differential network mapping at very large scales and highlight biological insight that has been derived from this type of analysis. We argue that differential network mapping, which allows for the interrogation of previously unexplored interaction spaces, will become a standard mode of network analysis in the future, just as differential gene expression and protein phosphorylation studies are already pervasive in genomic and proteomic analysis. © 2012 EMBO and Macmillan Publishers Limited All rights reserved.

Heredia J.E.,University of California at San Francisco | Mukundan L.,University of California at San Francisco | Chen F.M.,University of California at San Francisco | Mueller A.A.,Stanford University | And 7 more authors.
Cell | Year: 2013

In vertebrates, activation of innate immunity is an early response to injury, implicating it in the regenerative process. However, the mechanisms by which innate signals might regulate stem cell functionality are unknown. Here, we demonstrate that type 2 innate immunity is required for regeneration of skeletal muscle after injury. Muscle damage results in rapid recruitment of eosinophils, which secrete IL-4 to activate the regenerative actions of muscle resident fibro/adipocyte progenitors (FAPs). In FAPs, IL-4/IL-13 signaling serves as a key switch to control their fate and functions. Activation of IL-4/IL-13 signaling promotes proliferation of FAPs to support myogenesis while inhibiting their differentiation into adipocytes. Surprisingly, type 2 cytokine signaling is also required in FAPs, but not in myeloid cells, for rapid clearance of necrotic debris, a process that is necessary for timely and complete regeneration of tissues. © 2013 Elsevier Inc.

Smith C.A.,University of California at San Francisco | Kortemme T.,University of California at San Francisco | Kortemme T.,California Institute for Quantitative Biosciences
Journal of Molecular Biology | Year: 2010

Protein-protein recognition, frequently mediated by members of large families of interaction domains, is one of the cornerstones of biological function. Here, we present a computational, structure-based method to predict the sequence space of peptides recognized by PDZ domains, one of the largest families of recognition proteins. As a test set, we use a considerable amount of recent phage display data that describe the peptide recognition preferences for 169 naturally occurring and engineered PDZ domains. For both wild-type PDZ domains and single point mutants, we find that 70-80% of the most frequently observed amino acids by phage display are predicted within the top five ranked amino acids. Phage display frequently identified recognition preferences for amino acids different from those present in the original crystal structure. Notably, in about half of these cases, our algorithm correctly captures these preferences, indicating that it can predict mutations that increase binding affinity relative to the starting structure. We also find that we can computationally recapitulate specificity changes upon mutation, a key test for successful forward design of protein-protein interface specificity. Across all evaluated data sets, we find that incorporation backbone sampling improves accuracy substantially, irrespective of using a crystal or NMR structure as the starting conformation. Finally, we report successful prediction of several amino acid specificity changes from blind tests in the DREAM4 peptide recognition domain specificity prediction challenge. Because the foundational methods developed here are structure based, these results suggest that the approach can be more generally applied to specificity prediction and redesign of other protein-protein interfaces that have structural information but lack phage display data. © 2010 Elsevier Ltd.

News Article | October 26, 2016

Researchers at UC San Francisco and the academically affiliated Gladstone Institutes have used a newly developed gene-editing system to find gene mutations that make human immune cells resistant to HIV infection. The team built a high-throughput cell-editing platform using a variant of CRISPR/Cas9 technology that allowed them to test how well scores of different genetic tweaks defended immune cells against HIV. The new system allows researchers to quickly modify the genetic code of freshly donated human immune cells and will hopefully accelerate the quest to finally cure HIV+ patients, the researchers said. "This is an ability HIV researchers have wanted for a long time," said postdoctoral researcher Judd F. Hultquist, PhD, one of the new paper's co-lead authors. "I hope this will take what seemed like an insurmountable task a year ago and make it something everyone can do." The research, which was published online October 25, 2016 in Cell Reports, was conducted by the laboratories of co-senior authors Nevan J. Krogan, PhD, a professor of cellular and molecular pharmacology at UCSF, director of the Quantitative Biosciences Institute (QBI) in UCSF's School of Pharmacy, and a senior investigator at the Gladstone Institutes, and Alexander Marson, MD, PhD, an assistant professor of microbiology and immunology in UCSF's School of Medicine. The research was spearheaded by Hultquist, who is in Krogan's lab, and Kathrin Schumann, PhD, a postdoctoral researcher in Marson's lab. Despite great progress made since the 1980s in the ability to treat and control HIV with antiretroviral drugs, there is still no cure for the virus, and millions of people are newly infected every year. Once the virus infiltrates a patient's immune system, it can hide indefinitely within cells' own DNA, impossible to detect or destroy with current technology. As a result, patients must continue on antiretroviral drugs for the rest of their lives. However, not everyone is susceptible to the virus. Scientists have taken inspiration from a group of individuals whose immune cells appear to be naturally resistant to HIV infection, and hope to one day edit HIV patients' immune systems to mimic the biology of these HIV-resistant individuals. "There have been lots of efforts to sequence the genomes of resistant people to discover the mutations that make them immune to the virus," Hultquist said. "But there are many different genes that could be involved: some control the virus's ability to enter immune cells, others control how the virus tricks cells into expressing its genes. Until now, there was no way to test which of these mutations actually confer resistance in primary human T-cells." Despite being the immune system's lead fighters, T cells are delicate - only able to survive outside the body for a couple weeks. They are also resistant to the viruses researchers use in other cell types to deliver DNA instructions about how to build the machinery needed for CRISPR/Cas9 gene editing. Last year, Marson and Schumann successfully used CRISPR to perform precise DNA sequence replacements in primary human T cells for the first time by prefabricating the CRISPR machinery in test tubes, then adding it to the freshly donated immune cells. "It's incredibly fast," Schumann said. "The desired editing occurs rapidly, and then the cell degrades the CRISPR machinery so it can't go on making changes. That's really important: otherwise it's like doing surgery and leaving in the scalpel." In the new paper, Schumann and Hultquist improved the technique by devising an automated system for high-throughput, parallel editing of T cells. The new approach enables the researchers to mutate different candidate genes in hundreds of thousands of T cells from healthy volunteers, expose these mutant cells to the HIV virus, then screen through the cells to find which mutations were able to prevent infection. A key feature of this system is its speed, as donated T cells can only survive outside of the body for two to three weeks. "If we want to start editing T cells and putting them back into people as a therapy," Krogan said, "I think this will be the gold standard for how to do that quickly, safely, and efficiently." The researchers used the new technique to mutate the genes CXCR4 and CCR5, which encode receptor molecules that different strains of the HIV virus use to sneak in and infect immune cells and which have been targeted in previous cell therapy trials. Inactivating either of these genes successfully blocked HIV infection of the human T cells by the relevant HIV strain. Additional experiments showed the feasibility of creating a two-layer security system for T cells by simultaneously blocking a gene the HIV virus needs to gain entry into cells and a gene the virus needs to survive and reproduce within the cell, resulting in doubly secure resistance. To demonstrate the efficiency and power of the new high-throughput technology, the researchers also developed 146 different CRISPR-based edits, each designed to deactivate one of 45 genes linked to HIV's ability to integrate into host cells. They identified several genes whose absence conferred HIV resistance, some of which had been predicted by previous studies and others that had never been directly tied to HIV infection before. 'Tip of the iceberg' for infectious disease research The researchers plan to use the new platform to identify additional weaknesses in the HIV virus's life cycle that could be exploited either by cell therapy or targeted drugs. They also want to be able to insert more subtle mutations, such as those reported in HIV-resistant individuals, which could alter cell function just enough to confer resistance but without fully deactivating the gene and impeding cell function. However, their greater hope is that the system will have much broader applications than just HIV and eventually be used in labs around the world to study the virus of their choice. "This toolkit has been a huge missing piece in infectious disease research," Marson said. "Now we have the ability to make modifications in human immune cells and right away see the effects. The potential is immense -- this is just the tip of the iceberg." Additional authors on the new paper include Jonathan M. Woo, MS, of UCSF; Michael J. McGregor, MS, of UCSF and the Gladstone Institutes; Lara Manganaro, PhD, and Viviana Simon, MD, PhD, of Mt. Sinai Icahn School of Medicine; and Jennifer Doudna, PhD, of UC Berkeley and the Howard Hughes Medical Institute. Krogan is director of the UCSF site of the California Institute for Quantitative Biosciences (QB3). Marson is a member of the Divisions of Infectious Diseases and Rheumatology in UCSF's Department of Medicine, the UCSF Diabetes Center, and the Helen Diller Family Comprehensive Cancer Center. Marson and Doudna are members of the University of California Innovative Genomics Initiative. This research was supported by UCSF; the National Institutes of Health, National Institute of Allergy and Infectious Disease, and National Institute of General Medical Sciences (R01 AI064001, P50 GM082250, U19 AI106754, P01 AI090935, CFAR, and P30 AI027763); the Deutsche Forschungsgemeinschaft (SCHU3020/2-1); the UCSF Sandler Fellowship; and a gift from Jake Aronov. Marson has filed a patent on the use of Cas9 RNPs to edit the genome of human primary T cells, and serves as an advisor to Juno Therapeutics independently from this work. About UCSF: UC San Francisco (UCSF) is a leading university dedicated to promoting health worldwide through advanced biomedical research, graduate-level education in the life sciences and health professions, and excellence in patient care. It includes top-ranked graduate schools of dentistry, medicine, nursing and pharmacy; a graduate division with nationally renowned programs in basic, biomedical, translational and population sciences; and a preeminent biomedical research enterprise. It also includes UCSF Health, which comprises top-ranked hospitals, UCSF Medical Center and UCSF Benioff Children's Hospitals in San Francisco and Oakland - and other partner and affiliated hospitals and healthcare providers throughout the Bay Area. Please visit http://www. . About the Gladstone Institutes: To ensure our work does the greatest good, the Gladstone Institutes focuses on conditions with profound medical, economic, and social impact--unsolved diseases of the brain, the heart, and the immune system. Affiliated with the University of California, San Francisco, Gladstone is an independent, nonprofit life science research organization that uses visionary science and technology to overcome disease.

Guo H.,Whitehead Institute For Biomedical Research | Guo H.,Howard Hughes Medical Institute | Ingolia N.T.,Howard Hughes Medical Institute | Ingolia N.T.,California Institute for Quantitative Biosciences | And 4 more authors.
Nature | Year: 2010

MicroRNAs (miRNAs) are endogenous g∼1/422-nucleotide RNAs that mediate important gene-regulatory events by pairing to the mRNAs of protein-coding genes to direct their repression. Repression of these regulatory targets leads to decreased translational efficiency and/or decreased mRNA levels, but the relative contributions of these two outcomes have been largely unknown, particularly for endogenous targets expressed at low-to-moderate levels. Here, we use ribosome profiling to measure the overall effects on protein production and compare these to simultaneously measured effects on mRNA levels. For both ectopic and endogenous miRNA regulatory interactions, lowered mRNA levels account for most (≥84%) of the decreased protein production. These results show that changes in mRNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization of target mRNAs is the predominant reason for reduced protein output. © 2010 Macmillan Publishers Limited. All rights reserved.

Rog O.,University of California at Berkeley | Rog O.,Howard Hughes Medical Institute | Dernburg A.F.,University of California at Berkeley | Dernburg A.F.,Howard Hughes Medical Institute | And 2 more authors.
Current Opinion in Cell Biology | Year: 2013

Meiosis is the specialized cell division cycle that produces haploid gametes to enable sexual reproduction. Reduction of chromosome number by half requires elaborate chromosome dynamics that occur in meiotic prophase to establish physical linkages between each pair of homologous chromosomes. Caenorhabditis elegans has emerged as an excellent model organism for molecular studies of meiosis, enabling investigators to combine the power of molecular genetics, cytology, and live analysis. Here we focus on recent studies that have shed light on how chromosomes find and identify their homologous partners, and the structural changes that accompany and mediate these interactions. © 2013 Elsevier Ltd.

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