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Immune CRISPR Screening

Robert C Orchard, Meagan E Sullender, Bria F Dunlap, Dale R Balce, John G Doench, Herbert W Virgin
Noroviruses (NoVs) are a leading cause of gastroenteritis world-wide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation (CRISPRa) genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in human cells. Our screens identified with high confidence 49 genes that can inhibit MNoV infection when overexpressed. A significant number of these genes are in interferon and immune regulation signaling networks, but surprising, the majority of the genes identified are not associated with innate or adaptive immunity nor with any antiviral activity...
October 10, 2018: Journal of Virology
Patrick H Lizotte, Ruey-Long Hong, Troy A Luster, Megan E Cavanaugh, Luke J Taus, Stephen Wang, Abha Dhaneshwar, Naomi Mayman, Aaron Yang, Meghana Kulkarni, Lauren Badalucco, Erica Fitzpatrick, Hsiang-Fong Kao, Mari Kuraguchi, Mark Bittinger, Paul T Kirschmeier, Nathanael S Gray, David A Barbie, Pasi A Jänne
We developed a screening assay in which luciferized ID8 expressing OVA was cocultured with transgenic CD8+ T cells specifically recognizing the model antigen in an H-2b-restricted manner. The assay was screened with a small-molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells. Erlotinib, an EGFR inhibitor, was the top compound that enhanced T-cell killing of tumor cells. Subsequent experiments with erlotinib and additional EGFR inhibitors validated the screen results...
September 21, 2018: Cancer Immunology Research
Kyle E Watters, Christof Fellmann, Hua B Bai, Shawn M Ren, Jennifer A Doudna
Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility for synthetic genome engineering, agricultural genomics, and biomedical applications. Although bacteria harboring CRISPR-Cas9 or CRISPR-Cas3 adaptive immune systems sometimes acquire mobile genetic elements encoding anti-CRISPR proteins that inhibit Cas9, Cas3, or the DNA-binding Cascade complex, no such inhibitors have been found for CRISPR-Cas12a. Here we use a comprehensive bioinformatic and experimental screening approach to identify three different inhibitors that block or diminish CRISPR-Cas12a-mediated genome editing in human cells...
October 12, 2018: Science
Ping Zhou, Yang She, Na Dong, Peng Li, Huabin He, Alessio Borio, Qingcui Wu, Shan Lu, Xiaojun Ding, Yong Cao, Yue Xu, Wenqing Gao, Mengqiu Dong, Jingjin Ding, Da-Cheng Wang, Alla Zamyatina, Feng Shao
Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-κB signalling1 . Recent studies indicate that the bacterial metabolite D-glycero-β-D-manno-heptose 1,7-bisphosphate (HBP) can activate NF-κB signalling in host cytosol2-4 , but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-β-D-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-κB activation and cytokine expression...
September 2018: Nature
Xihong Zhao, Zhixue Yu, Zhenbo Xu
Staphylococcus aureus is a foodborne pathogen that causes food contamination and food poisoning, which poses great harm to health, agriculture and other hosts. Clustered regularly interspaced short palindromic repeats (CRISPR) are a recently discovered bacterial immune system that resists foreign genes such as phage DNA. This system inhibits the transfer of specific movable genetic elements that match the CRISPR spacer sequences, thereby preventing the spread of drug-resistant genes between pathogens. In this study, 57 CRISPR loci were screened from 38 strains of S...
2018: Frontiers in Microbiology
Dana Elster, Marie Tollot, Karin Schlegelmilch, Alessandro Ori, Andreas Rosenwald, Erik Sahai, Björn von Eyss
Yes-associated protein (YAP), the downstream transducer of the Hippo pathway, is a key regulator of organ size, differentiation and tumorigenesis. To uncover Hippo-independent YAP regulators, we performed a genome-wide CRISPR screen that identifies the transcriptional repressor protein Trichorhinophalangeal Syndrome 1 (TRPS1) as a potent repressor of YAP-dependent transactivation. We show that TRPS1 globally regulates YAP-dependent transcription by binding to a large set of joint genomic sites, mainly enhancers...
August 6, 2018: Nature Communications
Muhammad R Javed, Maria Sadaf, Temoor Ahmed, Amna Jamil, Marium Nawaz, Hira Abbas, Anam Ijaz
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR or more precisely CRISPR-Cas) system has proven to be a highly efficient and simple tool for achieving site-specific genome modifications in comparison to Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). The discovery of bacterial defense system that uses RNA-guided DNA cleaving enzymes for producing double-strand breaks along CRISPR has provided an exciting alternative to ZFNs and TALENs for gene editing & regulation, as the CRISPR-associated (Cas) proteins remain the same for different gene targets and only the short sequence of the guide RNA needs to be changed to redirect the site-specific cleavage...
August 4, 2018: Current Microbiology
Vijaya L Simhadri, Joseph McGill, Shane McMahon, Junxia Wang, Haiyan Jiang, Zuben E Sauna
The repurposing of the CRISPR/Cas microbial adaptive immune system for gene editing has resulted in an exponential rise in new technologies and promising approaches for treating numerous human diseases. While some of the approaches being currently developed involve ex vivo editing by CRISPR/Cas9, many more potential applications will require in vivo editing. The in vivo use of this technology comes with challenges, one of which is the immune response to Cas9, a protein of microbial origin. Thus, the prevalence of pre-existing antibodies to Cas9 could also be a relevant parameter...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Emily A Stout, Rosemary Sanozky-Dawes, Yong Jun Goh, Alexandra B Crawley, Todd R Klaenhammer, Rodolphe Barrangou
Lactobacillus gasseri is a human commensal which carries CRISPR-Cas, an adaptive immune system that protects the cell from invasive mobile genetic elements (MGEs). However, MGEs occasionally escape CRISPR targeting due to DNA mutations that occur in sequences involved in CRISPR interference. To better understand CRISPR escape processes, a plasmid interference assay was used to screen for mutants that escape CRISPR-Cas targeting. Plasmids containing a target sequence and a protospacer adjacent motif (PAM) were transformed for targeting by the native CRISPR-Cas system...
September 2018: Microbiology
Yoshifumi Nishikawa, Naomi Shimoda, Ragab M Fereig, Tomoya Moritaka, Kousuke Umeda, Maki Nishimura, Fumiaki Ihara, Kaoru Kobayashi, Yuu Himori, Yutaka Suzuki, Hidefumi Furuoka
Neospora caninum is a protozoan parasite closely related to Toxoplasma gondii Neosporosis caused by N. caninum is considered one of the main causes of abortion in cattle and nervous-system dysfunction in dogs, and identification of the virulence factors of this parasite is important for the development of control measures. Here, we used a luciferase reporter assay to screen the dense granule proteins genes of N. caninum , and we found that NcGRA6, NcGRA7, and NcGRA14 are involved in the activation of the NF-κB, calcium/calcineurin, and cAMP/PKA signals...
September 15, 2018: Applied and Environmental Microbiology
Ahmed Mahas, Magdy Mahfouz
In prokaryotes, CRISPR/Cas adaptive immunity systems target and destroy nucleic acids derived from invading bacteriophages and other foreign genetic elements. In eukaryotes, the native function of these systems has been exploited to combat viruses in mammals and plants. Rewired CRISPR/Cas9 and CRISPR/Cas13 systems have been used to confer resistance against DNA and RNA viruses, respectively. Here, we discuss recent approaches employing CRISPR/Cas systems to combat viruses in eukaryotes, highlight key challenges, and provide future perspectives...
July 10, 2018: Current Opinion in Virology
Conor J Kearney, Stephin J Vervoort, Simon J Hogg, Kelly M Ramsbottom, Andrew J Freeman, Najoua Lalaoui, Lizzy Pijpers, Jessica Michie, Kristin K Brown, Deborah A Knight, Vivien Sutton, Paul A Beavis, Ilia Voskoboinik, Phil K Darcy, John Silke, Joseph A Trapani, Ricky W Johnstone, Jane Oliaro
Immunotherapy has revolutionized outcomes for cancer patients, but the mechanisms of resistance remain poorly defined. We used a series of whole-genome clustered regularly interspaced short palindromic repeat (CRISPR)-based screens performed in vitro and in vivo to identify mechanisms of tumor immune evasion from cytotoxic lymphocytes [CD8+ T cells and natural killer (NK) cells]. Deletion of key genes within the tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways provided protection of tumor cells from CD8+ T cell-mediated killing and blunted antitumor immune responses in vivo...
May 18, 2018: Science Immunology
Harriet Prior, Lauren MacConnachie, Jose L Martinez, Georgina C B Nicholl, Asim A Beg
The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals...
April 30, 2018: Journal of Visualized Experiments: JoVE
Qingxiang Liu, Yaoxing Wu, Yunfei Qin, Jiajia Hu, Weihong Xie, F Xiao-Feng Qin, Jun Cui
The innate immune response conferred by type I interferons is essential for host defense against viral infection but needs to be tightly controlled to avoid immunopathology. We performed a systematic functional screening by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) knockout and overexpression to investigate the roles of the deubiquitinating enzyme (DUB) family in regulating antiviral immunity. We demonstrated that the expression of a large fraction of DUBs underwent complex temporal alteration, suggesting a dynamic program of feedback regulation...
May 2018: Science Advances
Yang Liu, Zhi-Ting Cao, Cong-Fei Xu, Zi-Dong Lu, Ying-Li Luo, Jun Wang
Inflammation is closely related to the development of many diseases and is commonly characterized by abnormal infiltration of immune cells, especially neutrophils. The current therapeutics of inflammatory diseases give little attention to direct modulation of these diseases with respect to immune cells. Nanoparticles are applied for efficient drug delivery into the disease-related immune cells, but their performance is significantly affected by their surface properties. In this study, to optimize the properties of nanoparticles for modulating neutrophils-related inflammation, we prepared a library of poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-b-PLGA)-based cationic lipid-assisted nanoparticles (CLANs) with different surface PEG density and surface charge...
July 2018: Biomaterials
Brendan V Jamison, Margaret W Thairu, Allison K Hansen
While the wealth of genomic data presently available is increasing rapidly, the advancement of functional genomics technologies for the large majority of these organisms has lagged behind. The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 system is an emerging gene-editing technology derived from a bacterial adaptive immune system that has proven highly effective in multiple model systems. Here, the CRISPR/Cas9 system was delivered into the ovarioles of the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphididae), with a new delivery method utilizing in vivo electroporation...
March 1, 2018: Journal of Insect Science
Siyuan Ding, Jonathan Diep, Ningguo Feng, Lili Ren, Bin Li, Yaw Shin Ooi, Xin Wang, Kevin F Brulois, Linda L Yasukawa, Xingnan Li, Calvin J Kuo, David A Solomon, Jan E Carette, Harry B Greenberg
Cohesin is a multi-subunit nuclear protein complex that coordinates sister chromatid separation during cell division. Highly frequent somatic mutations in genes encoding core cohesin subunits have been reported in multiple cancer types. Here, using a genome-wide CRISPR-Cas9 screening approach to identify host dependency factors and novel innate immune regulators of rotavirus (RV) infection, we demonstrate that the loss of STAG2, an important component of the cohesin complex, confers resistance to RV replication in cell culture and human intestinal enteroids...
April 16, 2018: Nature Communications
Neftali Vazquez, Lilia Sanchez, Rebecca Marks, Eduardo Martinez, Victor Fanniel, Alma Lopez, Andrea Salinas, Itzel Flores, Jesse Hirschmann, Robert Gilkerson, Erin Schuenzel, Robert Dearth, Reginald Halaby, Wendy Innis-Whitehouse, Megan Keniry
BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone...
March 14, 2018: BMC Molecular Biology
Katharina Hochheiser, Andrew J Kueh, Thomas Gebhardt, Marco J Herold
The CRISPR/Cas9-system was originally identified as part of the adaptive immune system in bacteria and has since been adapted for the genetic manipulation of eukaryotic cells. The technique is of particular value for biomedical sciences, as it enables the genetic manipulation of cell lines and primary cells as well as whole organisms with unprecedented ease and efficiency. Furthermore, the CRISPR/Cas9-technology has the potential for future therapeutic applications in the clinic. Here, we discuss the use of CRISPR/Cas9 for the genetic modification of haematopoietic cells and the generation of mouse models for immunological research...
April 2018: European Journal of Immunology
Deng Pan, Aya Kobayashi, Peng Jiang, Lucas Ferrari de Andrade, Rong En Tay, Adrienne M Luoma, Daphne Tsoucas, Xintao Qiu, Klothilda Lim, Prakash Rao, Henry W Long, Guo-Cheng Yuan, John Doench, Myles Brown, X Shirley Liu, Kai W Wucherpfennig
Many human cancers are resistant to immunotherapy, for reasons that are poorly understood. We used a genome-scale CRISPR-Cas9 screen to identify mechanisms of tumor cell resistance to killing by cytotoxic T cells, the central effectors of antitumor immunity. Inactivation of >100 genes-including Pbrm1 , Arid2 , and Brd7 , which encode components of the PBAF form of the SWI/SNF chromatin remodeling complex-sensitized mouse B16F10 melanoma cells to killing by T cells. Loss of PBAF function increased tumor cell sensitivity to interferon-γ, resulting in enhanced secretion of chemokines that recruit effector T cells...
February 16, 2018: Science
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