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Crispr primary t cell

Nicholas J Kramer, Michael S Haney, David W Morgens, Ana Jovičić, Julien Couthouis, Amy Li, James Ousey, Rosanna Ma, Gregor Bieri, C Kimberly Tsui, Yingxiao Shi, Nicholas T Hertz, Marc Tessier-Lavigne, Justin K Ichida, Michael C Bassik, Aaron D Gitler
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...
March 5, 2018: Nature Genetics
Matthew L Cooper, Jaebok Choi, Karl Staser, Julie K Ritchey, Jessica M Devenport, Kayla Eckardt, Michael P Rettig, Bing Wang, Linda G Eissenberg, Armin Ghobadi, Leah N Gehrs, Julie L Prior, Samuel Achilefu, Christopher A Miller, Catrina C Fronick, Julie O'Neal, Feng Gao, David M Weinstock, Alejandro Gutierrez, Robert S Fulton, John F DiPersio
T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here, we describe a fratricide-resistant "off-the-shelf" CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression...
February 20, 2018: Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
Toshitsugu Fujita, Fusako Kitaura, Asami Oji, Naoki Tanigawa, Miyuki Yuno, Masahito Ikawa, Ichiro Taniuchi, Hodaka Fujii
We developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology to isolate specific genomic regions while retaining their molecular interactions. In enChIP, the locus of interest is tagged with an engineered DNA-binding molecule, such as a modified form of the clustered regularly interspaced short palindromic repeats (CRISPR) system containing a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). The locus is then affinity-purified to enable identification of associated molecules...
February 26, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Sojung Kim, Taeyoung Koo, Hyeon-Gun Jee, Hee-Yeon Cho, Gyeorae Lee, Dong-Gyun Lim, Hyoung Shik Shin, Jin-Soo Kim
Here, we report that CRISPR guide RNAs (gRNAs) with a 5'-triphosphate group (5'-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5'-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5'-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4+ T cells...
February 22, 2018: Genome Research
David K Breslow, Sascha Hoogendoorn, Adam R Kopp, David W Morgens, Brandon K Vu, Margaret C Kennedy, Kyuho Han, Amy Li, Gaelen T Hess, Michael C Bassik, James K Chen, Maxence V Nachury
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...
February 19, 2018: Nature Genetics
Mara Kornete, Romina Marone, Lukas T Jeker
Adoptive cell transfer is an important approach for basic research and emerges as an effective treatment for various diseases, including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. In this article, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms in up to 25% of the transfected primary T cells...
February 14, 2018: Journal of Immunology: Official Journal of the American Association of Immunologists
Akiko Seki, Sascha Rutz
CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell-based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation...
February 7, 2018: Journal of Experimental Medicine
Sunil S Raikar, Lauren C Fleischer, Robert Moot, Andrew Fedanov, Na Yoon Paik, Kristopher A Knight, Christopher B Doering, H Trent Spencer
Relapsed T-cell malignancies have poor outcomes when treated with chemotherapy, but survival after allogeneic bone marrow transplantation (BMT) approaches 50%. A limitation to BMT is the difficulty of achieving remission prior to transplant. Chimeric antigen receptor (CAR) T-cell therapy has shown successes in B-cell malignancies. This approach is difficult to adapt for the treatment of T-cell disease due to lack of a T-lymphoblast specific antigen and the fratricide of CAR T cells that occurs with T-cell antigen targeting...
2018: Oncoimmunology
Sanjeev Noel, Sul A Lee, Mohanraj Sadasivam, Abdel R A Hamad, Hamid Rabb
Oxidant stress modifies T lymphocyte activation and function. Previous work demonstrated that murine T cell-specific kelch like-ECH-associated protein 1 (Keap1) deletion enhances antioxidant capacity and protects from experimental acute kidney injury. In this study, we used CRISPR technology to develop clinically translatable human T cell-specific KEAP1 deletion. Delivery of KEAP1 exon 2 specific Cas9:guide RNA in Jurkat T cells led to significant (∼70%) editing and upregulation of NRF2-regulated antioxidant genes NADPH dehydrogenase quinone 1 (NQO1) (up to 11-fold), heme oxygenase 1 (HO1) (up to 11-fold), and GCLM (up to 2-fold)...
January 19, 2018: Journal of Immunology: Official Journal of the American Association of Immunologists
Xiaoqiong Duan, Shilin Li, Jacinta A Holmes, Zeng Tu, Yujia Li, Dachuan Cai, Xiao Liu, Wenting Li, Chunhui Yang, Baihai Jiao, Esperance A Schaefer, Dahlene N Fusco, Shadi Salloum, Limin Chen, Wenyu Lin, Raymond T Chung
BACKGROUND: HCV infection has been shown to regulate miR-130a in patient biopsies and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and HBV replication.METHODS: We used bioinformatics software including miRanda, TargetScan, PITA and RNAhybrid to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed, or knocked down by siRNA or by CRISPR/Cas9 gRNA, respectively. Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, JFH1 HCV-infected Huh7...
January 10, 2018: Journal of Virology
Hong Sun Kim, Yu-Chih Chen, Felipe Nör, Kristy A Warner, April Andrews, Vivian P Wagner, Zhaocheng Zhang, Zhixiong Zhang, Manoela D Martins, Alexander T Pearson, Euisik Yoon, Jacques E Nör
Recent evidence suggests that the metastatic spread of head and neck squamous cell carcinomas (HNSCC) requires the function of cancer stem cells endowed with multipotency, self-renewal, and high tumorigenic potential. We demonstrated that cancer stem cells reside in perivascular niches and are characterized by high aldehyde dehydrogenase (ALDH) activity and high CD44 expression (ALDHhighCD44high) in HNSCC. Here, we hypothesize that endothelial cell-secreted interleukin-6 (IL-6) contributes to tumor progression by enhancing the migratory phenotype and survival of cancer stem cells...
November 21, 2017: Oncotarget
Kazuya Takahashi, Takeshi Inukai, Toshihiko Imamura, Mio Yano, Chihiro Tomoyasu, David M Lucas, Atsushi Nemoto, Hiroki Sato, Meixian Huang, Masako Abe, Keiko Kagami, Tamao Shinohara, Atsushi Watanabe, Shinpei Somazu, Hiroko Oshiro, Koshi Akahane, Kumiko Goi, Jiro Kikuchi, Yusuke Furukawa, Hiroaki Goto, Masayoshi Minegishi, Shotaro Iwamoto, Kanji Sugita
Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response...
2017: PloS One
Francesco Spallotta, Chiara Cencioni, Sandra Atlante, Davide Garella, Mattia Cocco, Mattia Mori, Raffaella Mastrocola, Carsten Künne, Stefan Günther, Simona Nanni, Valerio Azzimato, Sven Zukunft, Angela Kornberger, Duran Sueruen, Frank Schnutgen, Harald von Melchner, Antonella Di Stilo, Manuela Aragno, Maarten Braspenning, Wim Van Criekinge, Miles J De Blasio, Rebecca H Ritchie, Germana Zaccagnini, Fabio Martelli, Antonella Farsetti, Ingrid Fleming, Thomas Braun, Andres Beiras-Fernandez, Bruno Botta, Massimo Collino, Massimo Bertinaria, Andreas M Zeiher, Carlo Gaetano
Rationale: Human cardiac mesenchymal cells (CMSCs) are a therapeutically-relevant primary cell population. Diabetes compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. Objective: To investigate the role of α-ketoglutarate (αKG) in the epi-metabolic control of DNA demethylation in CMSCs. Methods and Results: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing and gene specific GC methylation detection revealed an accumulation of 5mC, 5hmC and 5fC in the genomic DNA of human CMSCs isolated from diabetic (D) donors (D-CMSCs)...
November 20, 2017: Circulation Research
Qiankun Wang, Shuliang Chen, Qiaoqiao Xiao, Zhepeng Liu, Shuai Liu, Panpan Hou, Li Zhou, Wei Hou, Wenzhe Ho, Chunmei Li, Li Wu, Deyin Guo
BACKGROUND: The CRISPR/Cas9 system has been widely used for genome editing in mammalian cells. CXCR4 is a co-receptor for human immunodeficiency virus type 1 (HIV-1) entry, and loss of CXCR4 function can protect cells from CXCR4 (X4)-tropic HIV-1 infection, making CXCR4 an important target for HIV-1 gene therapy. However, the large size of the CRISPR/SpCas9 system presents an obstacle to its efficient delivery into primary CD4+ T cells. Recently, a small Staphylococcus aureus Cas9 (SaCas9) has been developed as a genome editing tool can address this question...
November 15, 2017: Retrovirology
Mateusz Legut, Garry Dolton, Afsar Ali Mian, Oliver Ottmann, Andrew Sewell
Adoptive transfer of T-cells genetically modified to express a cancer-specific T-cell receptor (TCR) has shown significant therapeutic potential for both hematological and solid tumors. However, a major issue of transducing T-cells with a transgenic TCR is the pre-existing expression of TCRs in the recipient cells. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation. Mixed dimers, formed by mispairing between the endogenous and transgenic TCRs, may harbor autoreactive specificities...
November 9, 2017: Blood
Xiao Wang, Avanthi Raghavan, Derek T Peters, Evanthia E Pashos, Daniel J Rader, Kiran Musunuru
OBJECTIVE: The noncoding single-nucleotide polymorphism rs12740374 has been hypothesized to be the causal variant responsible for liver-specific modulation of SORT1(sortilin 1) expression (ie, expression quantitative trait locus) and, by extension, the association of the SORT1 locus on human chromosome 1p13 with low-density lipoprotein cholesterol levels and coronary heart disease. The goals of this study were to compare 3 different hepatocyte models in demonstrating that the rs12740374 minor allele sequence is responsible for transcriptional activation of SORT1 expression...
November 2, 2017: Arteriosclerosis, Thrombosis, and Vascular Biology
Usheer Kanjee, Christof Grüring, Mudit Chaand, Kai-Min Lin, Elizabeth Egan, Jale Manzo, Patrick L Jones, Tiffany Yu, Robert Barker, Michael P Weekes, Manoj T Duraisingh
During malaria blood-stage infections, Plasmodium parasites interact with the RBC surface to enable invasion followed by intracellular proliferation. Critical factors involved in invasion have been identified using biochemical and genetic approaches including specific knockdowns of genes of interest from primary CD34(+) hematopoietic stem cells (cRBCs). Here we report the development of a robust in vitro culture system to produce RBCs that allow the generation of gene knockouts via CRISPR/Cas9 using the immortal JK-1 erythroleukemia line...
October 31, 2017: Proceedings of the National Academy of Sciences of the United States of America
Tatsuo Miyamoto, Silvia Natsuko Akutsu, Akihiro Fukumitsu, Hiroyuki Morino, Yoshinori Masatsuna, Kosuke Hosoba, Hideshi Kawakami, Takashi Yamamoto, Kenji Shimizu, Hirofumi Ohashi, Shinya Matsuura
Primary microcephaly (MCPH) is an autosomal recessive disorder characterized by congenital reduction of head circumference. Here, we identified compound heterozygous mutations c.731 C > T (p.Ser 244 Leu) and c.2413 G > T (p.Glu 805 X) in the WDR62/MCPH2 gene, which encodes the mitotic centrosomal protein WDR62, in two siblings in a Japanese family with microcephaly using whole-exome sequencing. However, the molecular and cellular pathology of microcephaly caused by WDR62/MCPH2 mutation remains unclear...
November 15, 2017: Human Molecular Genetics
Rasmus O Bak, Daniel P Dever, Andreas Reinisch, David Cruz Hernandez, Ravindra Majeti, Matthew H Porteus
Precise and efficient manipulation of genes is crucial for understanding the molecular mechanisms that govern human hematopoiesis and for developing novel therapies for diseases of the blood and immune system. Current methods do not enable precise engineering of complex genotypes that can be easily tracked in a mixed population of cells. We describe a method to multiplex homologous recombination (HR) in human hematopoietic stem and progenitor cells and primary human T cells by combining rAAV6 donor delivery and the CRISPR/Cas9 system delivered as ribonucleoproteins (RNPs)...
September 28, 2017: ELife
Maxwell R Mumbach, Ansuman T Satpathy, Evan A Boyle, Chao Dai, Benjamin G Gowen, Seung Woo Cho, Michelle L Nguyen, Adam J Rubin, Jeffrey M Granja, Katelynn R Kazane, Yuning Wei, Trieu Nguyen, Peyton G Greenside, M Ryan Corces, Josh Tycko, Dimitre R Simeonov, Nabeela Suliman, Rui Li, Jin Xu, Ryan A Flynn, Anshul Kundaje, Paul A Khavari, Alexander Marson, Jacob E Corn, Thomas Quertermous, William J Greenleaf, Howard Y Chang
The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets...
November 2017: Nature Genetics
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