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https://www.readbyqxmd.com/read/29668111/efficient-in-planta-gene-targeting-in-tomato-using-geminiviral-replicons-and-the-crispr-cas9-system
#1
Tal Dahan-Meir, Shdema Filler-Hayut, Cathy Melamed-Bessudo, Samuel Bocobza, Henryk Czosnek, Asaph Aharoni, Avraham A Levy
Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype...
April 18, 2018: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/29668051/evidence-for-a-specific-and-critical-role-of-mitogen-activated-protein-kinase-20-in-uni-to-binucleate-transition-of-microgametogenesis-in-tomato
#2
Lifei Chen, Dandan Yang, Youwei Zhang, Limin Wu, Yaoyao Zhang, Lei Ye, Changtian Pan, Yanjun He, Li Huang, Yong-Ling Ruan, Gang Lu
Mitogen-activated protein kinases (MAPKs) regulate diverse aspects of plant growth. However, their potential role in reproductive development remains elusive. Here, we discovered an unique role of SlMPK20, a plant-specific group D MAPK, in pollen development in tomato. RNAi-mediated suppression of SlMPK20 or its knockout using CRISPR/Cas9 significantly reduced or completely abolished pollen viability, respectively, with no effects on maternal fertility. Cell biology and gene expression analyses established that SlMPK20 exerts its role specifically at the uni-to-binucleate transition during microgametogenesis...
April 18, 2018: New Phytologist
https://www.readbyqxmd.com/read/29667553/car-t-cell-therapy-a-new-era-in-cancer-immunotherapy
#3
Miliotou N Androulla, Papadopoulou C Lefkothea
BACKGROUND: Cancer is one of the leading causes of death worldwide. Over the years, a number of conventional cytotoxic approaches for neoplastic diseases has been developed. However, due to their limited effectiveness in accordance with the heterogeneity of cancer cells, there is a constant search for therapeutic approaches with improved outcome, such as immunotherapy that utilizes and enhances the normal capacity of the patient's immune system. METHODS: Chimeric Antigen Receptor (CAR) T-cell therapy involves genetic modification of patient's autologous T-cells to express a CAR specific for a tumor antigen, following by ex vivo cell expansion and re-infusion back to the patient...
April 17, 2018: Current Pharmaceutical Biotechnology
https://www.readbyqxmd.com/read/29666786/modeling-neuropsychiatric-and-neurodegenerative-diseases-with-induced-pluripotent-stem-cells
#4
REVIEW
Elizabeth A LaMarca, Samuel K Powell, Schahram Akbarian, Kristen J Brennand
Human-induced pluripotent stem cells (hiPSCs) have revolutionized our ability to model neuropsychiatric and neurodegenerative diseases, and recent progress in the field is paving the way for improved therapeutics. In this review, we discuss major advances in generating hiPSC-derived neural cells and cutting-edge techniques that are transforming hiPSC technology, such as three-dimensional "mini-brains" and clustered, regularly interspersed short palindromic repeats (CRISPR)-Cas systems. We examine specific examples of how hiPSC-derived neural cells are being used to uncover the pathophysiology of schizophrenia and Parkinson's disease, and consider the future of this groundbreaking research...
2018: Frontiers in Pediatrics
https://www.readbyqxmd.com/read/29666291/determining-the-specificity-of-cascade-binding-interference-and-primed-adaptation-in-vivo-in-the-escherichia-coli-type-i-e-crispr-cas-system
#5
Lauren A Cooper, Anne M Stringer, Joseph T Wade
In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo , for the type I-E system of Escherichia coli Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target...
April 17, 2018: MBio
https://www.readbyqxmd.com/read/29666231/next-generation-crispr-cas9-transcriptional-activation-in-drosophila-using-flysam
#6
Yu Jia, Rong-Gang Xu, Xingjie Ren, Ben Ewen-Campen, Rajendhran Rajakumar, Jonathan Zirin, Donghui Yang-Zhou, Ruibao Zhu, Fang Wang, Decai Mao, Ping Peng, Huan-Huan Qiao, Xia Wang, Lu-Ping Liu, Bowen Xu, Jun-Yuan Ji, Qingfei Liu, Jin Sun, Norbert Perrimon, Jian-Quan Ni
CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability...
April 16, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29666172/genetic-editing-of-colonic-organoids-provides-a-molecularly-distinct-and-orthotopic-preclinical-model-of-serrated-carcinogenesis
#7
Tamsin R M Lannagan, Young K Lee, Tongtong Wang, Jatin Roper, Mark L Bettington, Lochlan Fennell, Laura Vrbanac, Lisa Jonavicius, Roshini Somashekar, Krystyna Gieniec, Miao Yang, Jia Q Ng, Nobumi Suzuki, Mari Ichinose, Josephine A Wright, Hiroki Kobayashi, Tracey L Putoczki, Yoku Hayakawa, Simon J Leedham, Helen E Abud, Ömer H Yilmaz, Julie Marker, Sonja Klebe, Pratyaksha Wirapati, Siddhartha Mukherjee, Sabine Tejpar, Barbara A Leggett, Vicki L J Whitehall, Daniel L Worthley, Susan L Woods
OBJECTIVE: Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-activated kinase pathway gene, BRAF , and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein...
April 17, 2018: Gut
https://www.readbyqxmd.com/read/29666143/truncated-c-terminus-of-fibrillin-1-induces-marfanoid-progeroid-lipodystrophy-mpl-syndrome-in-rabbit
#8
Mao Chen, Bing Yao, Qiangbing Yang, Jichao Deng, Yuning Song, Tingting Sui, Lina Zhou, HaoBing Yao, Yuanyuan Xu, Hongsheng Ouyang, Daxin Pang, Zhanjun Li, Liangxue Lai
Various clinical differences have been observed between patients with the FBN1 gene mutation and those with the classical Marfan phenotype. Although FBN1 knockout (KO) or dominant-negative mutant mice are widely used as an animal model for Marfan syndrome (MFS), these mice cannot recapitulate the genotype/phenotype relationship of Marfanoid-progeroid-lipodystrophy (MPL) syndrome, which is caused by a mutation in the C-terminus of fibrillin-1, the penultimate exon of the FBN1 gene. Here, we describe the generation of a rabbit MPL model with C-terminal truncation of fibrillin-1 using a CRISPR/Cas9 system...
April 9, 2018: Disease Models & Mechanisms
https://www.readbyqxmd.com/read/29665411/iterative-genome-editing-of-escherichia-coli-for-3-hydroxypropionic-acid-production
#9
Rongming Liu, Liya Liang, Alaksh Choudhury, Marcelo C Bassalo, Andrew D Garst, Katia Tarasava, Ryan T Gill
Synthetic biology requires strategies for the targeted, efficient, and combinatorial engineering of biological sub-systems at the molecular level. Here, we report the use of the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for the rapid construction of combinatorially modified genomes. We coupled this genome engineering strategy with high-throughput phenotypic screening and selections to recursively engineer multiple traits in Escherichia coli for improved production of the platform chemical 3-hydroxypropionic acid (3HP)...
April 14, 2018: Metabolic Engineering
https://www.readbyqxmd.com/read/29665362/chd9-mediates-highly-loosened-chromatin-structure-in-growing-mouse-oocytes
#10
Masatoshi Ooga, Satoshi Fuyana, Yuki Hashioka, Wataru Fujii, Kunihiko Naito, Masataka G Suzuki, Fugaku Aoki
During oogenesis, oocytes prepare for embryonic development following fertilization. The mechanisms underlying this process are still unknown. Recently, it has been suggested that a loosened chromatin structure is involved in pluripotency and totipotency in embryonic stem (ES) cells and early preimplantation embryos, respectively. Here, we explored chromatin looseness in oocytes by fluorescence recovery after photobleaching (FRAP) using enhanced green fluorescent protein-tagged histone H2B. The results indicated that the chromatin in growing oocytes was already highly loosened to a level comparable to that in early preimplantation embryos...
April 14, 2018: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29664675/knockout-of-glucose-transporter-glut6-has-minimal-effects-on-whole-body-metabolic-physiology-in-mice
#11
Frances L Byrne, Ellen M Olzomer, Robert Brink, Kyle L Hoehn
Glucose transporter 6 (GLUT6) is a member of the facilitative glucose transporter family. GLUT6 is up-regulated in several cancers, but is not widely expressed in normal tissues. Previous studies have shown that GLUT6 knockdown kills endometrial cancer cells that express elevated levels of the protein. However, whether GLUT6 represents a viable anti-cancer drug target is unclear because the role of GLUT6 in normal metabolic physiology is unknown. Herein we generated GLUT6 knockout mice to determine how loss of GLUT6 affected whole body glucose homeostasis and metabolic physiology...
April 17, 2018: American Journal of Physiology. Endocrinology and Metabolism
https://www.readbyqxmd.com/read/29663920/advances-in-the-engineering-of-the-gene-editing-enzymes-and-the-genomes-understanding-and-handling-the-off-target-effects-of-crispr-cas9
#12
Yufang Yin, Qian Wang, Li Xiao, Fengjiao Wang, Zhuo Song, Cuilan Zhou, Xuan Liu, Chungen Xing, Nongyue He, Kai Li, Yan Feng, Jia Zhang
In the past decades, significant progresses have been achieved in genetic engineering of nucleases. Among the genetically engineered nucleases, zinc finger nucleases, transcription activator-like (TAL) effector nucleases, and CRIPSPR/Cas9 system form a new field of gene editing. The gene editing efficiency or targeting effect and the off-target effect are the two major determinant factors in evaluating the usefulness of a new enzyme. Engineering strategies in improving these gene editing enzymes, particularly in minimizing their off-target effects, are the focus of this paper...
March 1, 2018: Journal of Biomedical Nanotechnology
https://www.readbyqxmd.com/read/29663663/modular-ligation-extension-of-guide-rna-operons-lego-for-multiplexed-dcas9-regulation-of-metabolic-pathways-in-saccharomyces-cerevisiae
#13
Matthew Deaner, Allison Holzman, Hal S Alper
Metabolic engineering typically utilizes a suboptimal step-wise gene target optimization approach to parse a highly connected and regulated cellular metabolism. While the endonuclease-null CRISPR/Cas system has enabled gene expression perturbations without genetic modification, it has been mostly limited to small sets of gene targets in eukaryotes due to inefficient methods to assemble and express large sgRNA operons. In this work, we develop a TEF1p-tRNA expression system and demonstrate that the use of tRNAs as splicing elements flanking sgRNAs provides higher efficiency than both Pol III and ribozyme-based expression across a variety of single sgRNA and multiplexed contexts...
April 16, 2018: Biotechnology Journal
https://www.readbyqxmd.com/read/29662840/activation-of-the-nlrp3-inflammasome-pathway-by-uropathogenic-escherichia-coli-is-virulence-factor-dependent-and-influences-colonization-of-bladder-epithelial-cells
#14
Isak Demirel, Alexander Persson, Annelie Brauner, Eva Särndahl, Robert Kruse, Katarina Persson
The NLRP3 inflammasome and IL-1β release have recently been suggested to be important for the progression of urinary tract infection (UTI). However, much is still unknown regarding the interaction of UPEC and the NLRP3 inflammasome. The purpose of this study was to elucidate what virulence factors uropathogenic Escherichia coli (UPEC) use to modulate NLRP3 inflammasome activation and subsequent IL-1β release and the role of NLRP3 for UPEC colonization of bladder epithelial cells. The bladder epithelial cell line 5637, CRISPR/Cas9 generated NLRP3, caspase-1 and mesotrypsin deficient cell lines and transformed primary bladder epithelial cells (HBLAK) were stimulated with UPEC isolates and the non-pathogenic MG1655 strain...
2018: Frontiers in Cellular and Infection Microbiology
https://www.readbyqxmd.com/read/29662651/p27-kip1-regulates-alpha-synuclein-expression
#15
Edurne Gallastegui, Carla Domuro, Joan Serratosa, Alejandra Larrieux, Laura Sin, Jonatan Martinez, Arnaud Besson, José Manuel Morante-Redolat, Serena Orlando, Rosa Aligue, Isabel Fariñas, María Jesús Pujol, Oriol Bachs
Alpha-synuclein (α-SYN) is the main component of anomalous protein aggregates (Lewy bodies) that play a crucial role in several neurodegenerative diseases (synucleinopathies) like Parkinson's disease and multiple system atrophy. However, the mechanisms involved in its transcriptional regulation are poorly understood. We investigated here the role of the cyclin-dependent kinase (Cdk) inhibitor and transcriptional regulator p27Kip1 (p27) in the regulation of α-SYN expression. We observed that selective deletion of p27 by CRISPR/Cas9 technology in neural cells resulted in increased levels of α-SYN...
March 27, 2018: Oncotarget
https://www.readbyqxmd.com/read/29662178/defining-essential-genes-for-human-pluripotent-stem-cells-by-crispr-cas9-screening-in-haploid-cells
#16
Atilgan Yilmaz, Mordecai Peretz, Aviram Aharony, Ido Sagi, Nissim Benvenisty
The maintenance of pluripotency requires coordinated expression of a set of essential genes. Using our recently established haploid human pluripotent stem cells (hPSCs), we generated a genome-wide loss-of-function library targeting 18,166 protein-coding genes to define the essential genes in hPSCs. With this we could allude to an intrinsic bias of essentiality across cellular compartments, uncover two opposing roles for tumour suppressor genes and link autosomal-recessive disorders with growth-retardation phenotypes to early embryogenesis...
April 16, 2018: Nature Cell Biology
https://www.readbyqxmd.com/read/29662124/stag2-deficiency-induces-interferon-responses-via-cgas-sting-pathway-and-restricts-virus-infection
#17
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
https://www.readbyqxmd.com/read/29661863/-vibrio-cholerae-outer-membrane-vesicles-inhibit-bacteriophage-infection
#18
Tamara Reyes-Robles, Rebecca S Dillard, Lynne S Cairns, Cecilia A Silva-Valenzuela, Max Housman, Afsar Ali, Elizabeth R Wright, Andrew Camilli
Novel preventatives could help in efforts to limit Vibrio cholerae infection and the spread of cholera. Bacteriophage (or phage) treatment has been proposed to be an alternative intervention, given the rapid replication of virulent phages, prey specificity, and relative ease of finding new virulent phages. Phage tropism is dictated in part by the presence of phage receptors on the bacterial surface. While many phages that can kill V. cholerae have been isolated, whether this pathogen is able to defend itself by neutralizing phage binding is unknown...
April 16, 2018: Journal of Bacteriology
https://www.readbyqxmd.com/read/29661844/plexin-a4-plexin-d1-complexes-convey-semaphorin-3c-signals-to-induce-cytoskeletal-collapse-in-the-absence-of-neuropilins
#19
Tatyana Smolkin, Inbal Nir-Zvi, Nerri Duvshani, Yelena Mumblat, Ofra Kessler, Gera Neufeld
Class-3 semaphorin guidance factors bind to receptor complexes containing neuropilin and plexin receptors. A semaphorin may bind to several receptor complexes containing somewhat different constituents, resulting in diverse effects on cell migration. U87MG glioblastoma cells express both neuropilins and the four class-A plexins. They respond by cytoskeletal collapse and cell contraction to sema3A or sema3B but fail to contract in response to Sema3C, Sema3D, Sema3G or sema3E even when class-A plexins are over-expressed in the cells...
April 16, 2018: Journal of Cell Science
https://www.readbyqxmd.com/read/29661831/differential-effects-of-estrogen-receptor-beta-isoforms-on-glioblastoma-progression
#20
Jinyou Liu, Gangadhara R Sareddy, Mei Zhou, Suryavathi Viswanadhapalli, Xiaonan Li, Zhao Lai, Rajeshwar R Tekmal, Andrew J Brenner, Ratna K Vadlamudi
The estrogen receptor beta (ERβ) functions as a tumor suppressor in glioblastoma cells (GBM). However, the in vivo significance of endogenous ERβ and the roles of its isoforms in GBM are incompletely understood. Using ERβ isoform-specific PCR screening, we found that GBM cells predominantly express ERβ1 and ERβ5, along with low levels of ERβ2 and ERβ4. We observed greater ERβ5 expression in higher grades of glioma than in lower grades. In CRISPR-based ERβ knockout (KO) cells and ERβ KO cells uniquely expressing ERβ1, ERβ1 significantly reduced proliferation...
April 16, 2018: Cancer Research
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