Journal ArticleDOI
Haploid Genetic Screens in Human Cells Identify Host Factors Used by Pathogens
Jan E. Carette,Carla P. Guimaraes,Malini Varadarajan,Annie S. Park,Irene Wuethrich,Alzbeta Godarova,Maciej Kotecki,Brent H. Cochran,Eric Spooner,Hidde L. Ploegh,Thijn R. Brummelkamp +10 more
TLDR
Using insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8, host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects ofdiphtheria toxin and exotoxin A are identified.Abstract:
Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast.read more
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Journal ArticleDOI
Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells
Ophir Shalem,Ophir Shalem,Neville E. Sanjana,Neville E. Sanjana,Ella Hartenian,Xi-Shun Shi,David A. Scott,David A. Scott,Tarjei S. Mikkelsen,Dirk Heckl,Benjamin L. Ebert,David E. Root,John G. Doench,Feng Zhang,Feng Zhang +14 more
TL;DR: This work shows that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells, and observes a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation.
Journal ArticleDOI
Genetic Screens in Human Cells Using the CRISPR-Cas9 System
TL;DR: In this paper, a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library was described.
Genetic Screens in Human Cells Using the CRISPR-Cas9 System
TL;DR: A pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library is described and it is shown that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs.
Journal ArticleDOI
Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation
Luke A. Gilbert,Max A. Horlbeck,Britt Adamson,Jacqueline E. Villalta,Yuwen Chen,Evan H. Whitehead,Carla P. Guimaraes,Barbara Panning,Hidde L. Ploegh,Michael C. Bassik,Lei S. Qi,Martin Kampmann,Jonathan S. Weissman +12 more
TL;DR: This work identifies rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators to endogenous genes via endonuclease-deficient Cas9, which enable modulation of gene expression over a ∼1,000-fold range.
Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation
Luke A. Gilbert,Max A. Horlbeck,Britt Adamson,Jacqueline E. Villalta,Yuwen Chen,Evan H. Whitehead,Carla P. Guimaraes,Barbara Panning,Michael C. Bassik,Lei S. Qi,Martin Kampmann,Jonathan S. Weissman,Hidde L. Ploegh +12 more
TL;DR: In this article, the authors identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRisPRa) to endogenous genes via endonuclease-deficient Cas9.
References
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Journal ArticleDOI
Apoptosis by death factor.
TL;DR: This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, and Culture of Japan and by a Research Grant from the Princess Takamatsu Cancer Research Fund, and performed in part through Special Coordination Funds of the Science and Technology Agency of the Japanese Government.
Journal ArticleDOI
Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen
Abraham L. Brass,Derek M. Dykxhoorn,Yair Benita,Nan Yan,Alan Engelman,Ramnik J. Xavier,Judy Lieberman,Stephen J. Elledge +7 more
TL;DR: This article performed a large-scale small interfering RNA screen to identify host factors required by HIV-1 and identified more than 250 HIV-dependency factors (HDFs), which participate in a broad array of cellular functions and implicate new pathways in the viral life cycle.
Identification of host proteins required for HIV infection through a functional genomic screen
TL;DR: A large-scale small interfering RNA screen was performed to identify host factors required by HIV-1 and more than 250 HIV-dependency factors (HDFs) were identified, suggesting that viruses evolve in host cells that optimally perform the functions required for their life cycle.
Journal ArticleDOI
The Chemical Genomic Portrait of Yeast: Uncovering a Phenotype for All Genes
Maureen E. Hillenmeyer,Eula Fung,Jan Wildenhain,Sarah E. Pierce,Shawn Hoon,William Lee,Michael Proctor,Robert P. St.Onge,Mike Tyers,Daphne Koller,Russ B. Altman,Ronald W. Davis,Corey Nislow,Guri Giaever +13 more
TL;DR: It is found that 97% of gene deletions exhibited a measurable growth phenotype, suggesting that nearly all genes are essential for optimal growth in at least one condition.
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Jan E. Carette,Jan E. Carette,Matthijs Raaben,Anthony C. Wong,Andrew S. Herbert,Gregor Obernosterer,Gregor Obernosterer,Nirupama Mulherkar,Ana I. Kuehne,Philip J. Kranzusch,April M. Griffin,Gordon Ruthel,Paola Dal Cin,John M. Dye,Sean P. J. Whelan,Kartik Chandran,Thijn R. Brummelkamp,Thijn R. Brummelkamp +17 more