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Inducible crispr

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https://www.readbyqxmd.com/read/28644125/exploiting-crispr-cas-to-manipulate-enterococcus-faecalis-populations
#1
Karthik Hullahalli, Marinelle Rodrigues, Kelli L Palmer
CRISPR-Cas provides a barrier to horizontal gene transfer in prokaryotes. It was previously observed that functional CRISPR-Cas systems are absent from multidrug-resistant (MDR) Enterococcus faecalis, which only possess an orphan CRISPR locus, termed CRISPR2, lacking cas genes. It was of interest to investigate how the interplay between CRISPR-Cas genome defense and antibiotic selection for mobile genetic elements shapes E. faecalis populations. Here, we demonstrate that CRISPR2 can be reactivated for genome defense in MDR strains...
June 23, 2017: ELife
https://www.readbyqxmd.com/read/28643258/crispr-cas9-mediated-targeted-knockin-of-exogenous-reporter-genes-in-zebrafish
#2
Atsuo Kawahara
Genome editing technologies such as ZFN, TALEN, and CRISPR/Cas9 efficiently induce DNA double-stranded breaks (DSBs) at a targeted genomic locus, often resulting in a frameshift-mediated target gene disruption. It remains difficult to perform targeted integration of exogenous genes by genome editing technologies. DSBs can be restored through DNA repair mechanisms, such as non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination (HR). It is well known that HR facilitates homology-dependent integration of donor DNA template into a targeted locus...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28642749/rna-seq-reveals-enhanced-sugar-metabolism-in-streptococcus-mutans-co-cultured-with-candida-albicans-within-mixed-species-biofilms
#3
Jinzhi He, Dongyeop Kim, Xuedong Zhou, Sang-Joon Ahn, Robert A Burne, Vincent P Richards, Hyun Koo
Early childhood caries (ECC), which can lead to rampant tooth-decay that is painful and costly to treat, is one of the most prevalent infectious diseases affecting children worldwide. Previous studies support that interactions between Streptococcus mutans and Candida albicans are associated with the pathogenesis of ECC. The presence of Candida enhances S. mutans growth, fitness and accumulation within biofilms in vitro, although the molecular basis for these behaviors is undefined. Using an established co-cultivation biofilm model and RNA-Seq, we investigated how C...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28640964/targeted-mutagenesis-in-tetraploid-switchgrass-panicum-virgatum-l-using-crispr-cas9
#4
Yang Liu, Paul Merrick, Zhengzhi Zhang, Chonghui Ji, Bing Yang, Shui-Zhang Fei
The CRISPR/Cas9 system has become a powerful tool for targeted mutagenesis. Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that has been designated as a model biomass crop by the U.S. Department of Energy. The self infertility and high ploidy level make it difficult to study gene function or improve germplasm. To overcome these constraints, we explored the feasibility of using CRISPR/Cas9 for targeted mutagenesis in a tetraploid cultivar 'Alamo' switchgrass. We first developed a transient assay by which a non-functional green fluorescent protein gene containing a 1 bp frameshift insertion in its 5' coding region was successfully mutated by a Cas9/sgRNA complex resulting in its restored function...
June 22, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28640207/reverse-gyrase-functions-in-genome-integrity-maintenance-by-protecting-dna-breaks-in-vivo
#5
Wenyuan Han, Xu Feng, Qunxin She
Reverse gyrase introduces positive supercoils to circular DNA and is implicated in genome stability maintenance in thermophiles. The extremely thermophilic crenarchaeon Sulfolobus encodes two reverse gyrase proteins, TopR1 (topoisomerase reverse gyrase 1) and TopR2, whose functions in thermophilic life remain to be demonstrated. Here, we investigated the roles of TopR1 in genome stability maintenance in S. islandicus in response to the treatment of methyl methanesulfonate (MMS), a DNA alkylation agent. Lethal MMS treatment induced two successive events: massive chromosomal DNA backbone breakage and subsequent DNA degradation...
June 22, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28638383/non-canonical-glucocorticoid-receptor-transactivation-of-gilz-by-alcohol-suppresses-cell-inflammatory-response
#6
Hang Pong Ng, Scott Jennings, Jack Wang, Patricia E Molina, Steve Nelson, Guoshun Wang
Acute alcohol exposure suppresses cell inflammatory response. The underlying mechanism has not been fully defined. Here we report that alcohol was able to activate glucocorticoid receptor (GR) signaling in the absence of glucocorticoids (GCs) and upregulated glucocorticoid-induced leucine zipper (gilz), a prominent GC-responsive gene. Such a non-canonical activation of GR was not blocked by mifepristone, a potent GC competitor. The proximal promoter of gilz, encompassing five GC-responsive elements (GREs), was incorporated and tested in a luciferase reporter system...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/28636533/microrna-149-suppresses-hepatic-inflammatory-response-through-antagonizing-stat3-signaling-pathway
#7
Qiqi Zhang, Jia Su, Ziwei Wang, Hui Qi, Zeyong Ge, Zhijun Li, Wei-Dong Chen, Yan-Dong Wang
Chronic inflammation is increasingly recognized as an important component of tumorigenesis and metabolic diseases. The roles of microRNA149* (miRNA149*) in inflammation remain poorly understood. Here, we demonstrate that miR-149* is a suppressor of STAT3-mediated inflammation. MiR-149*-/- mice were generated with CRISPR/CAS9 technique. In a lipopolysaccharide (LPS)-induced inflammation model, miR-149*-/- mice show more severe liver injury and inflammation, compared with wild-type (WT) mice. MiR-149*-/- mice also displayed elevated messenger RNA (mRNA) levels of interleukin (IL)-6, inducible nitric oxide synthase (iNOS), complement C3 (C3) and IL-4 in response to LPS...
June 16, 2017: Oncotarget
https://www.readbyqxmd.com/read/28630470/overcoming-evolved-resistance-to-population-suppressing-homing-based-gene-drives
#8
John M Marshall, Anna Buchman, Héctor M Sánchez C, Omar S Akbari
The recent development of a CRISPR-Cas9-based homing system for the suppression of Anopheles gambiae is encouraging; however, with current designs, the slow emergence of homing-resistant alleles is expected to result in suppressed populations rapidly rebounding, as homing-resistant alleles have a significant fitness advantage over functional, population-suppressing homing alleles. To explore this concern, we develop a mathematical model to estimate tolerable rates of homing-resistant allele generation to suppress a wild population of a given size...
June 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28629934/recent-advances-in-leishmania-reverse-genetics-manipulating-a-manipulative-parasite
#9
REVIEW
Samuel M Duncan, Nathaniel G Jones, Jeremy C Mottram
In this review we describe the expanding repertoire of molecular tools with which to study gene function in Leishmania. Specifically we review the tools available for studying functions of essential genes, such as plasmid shuffle and DiCre, as well as the rapidly expanding portfolio of available CRISPR/Cas9 approaches for large scale gene knockout and endogenous tagging. We include detail on approaches that allow the direct manipulation of RNA using RNAi and protein levels via Tet or DiCre induced overexpression and destabilization domain mediated degradation...
June 16, 2017: Molecular and Biochemical Parasitology
https://www.readbyqxmd.com/read/28628795/knockout-of-the-nogo-b-gene-attenuates-tumor-growth-and-metastasis-in-hepatocellular-carcinoma
#10
Bo Zhu, Shaobo Chen, Xiaoding Hu, Xiaofeng Jin, Yichen Le, Lihuan Cao, Zhonghua Yuan, Zhen Lin, Songmin Jiang, Lichun Sun, Long Yu
Human hepatocellular carcinoma (HCC) is a malignant cancer. It is a challenge to develop anti-HCC drugs due to HCC's extreme aggressiveness and with the sensitivity of the liver to show severe adverse effects. More importantly, the precise mechanisms causing HCC pathogenicity are not known. Our previous study disclosed Nogo-B as a reticulon 4 (Rtn4) family member. In the present study, we first identified that Nogo-B played a critical role in HCC progression. We found, via in vitro and in vivo assays, that Nogo-B was expressed aberrantly in primary HCC tumor tissues and immortal HCC cells but was relatively scarce in the normal liver tissues or cells...
June 16, 2017: Neoplasia: An International Journal for Oncology Research
https://www.readbyqxmd.com/read/28628606/splicing-stimulates-sirna-formation-at-drosophila-dna-double-strand-breaks
#11
Karin Merk, Marco Breinig, Romy Böttcher, Stefan Krebs, Helmut Blum, Michael Boutros, Klaus Förstemann
DNA double-strand breaks trigger the production of locus-derived siRNAs in fruit flies, human cells and plants. At least in flies, their biogenesis depends on active transcription running towards the break. Since siRNAs derive from a double-stranded RNA precursor, a major question is how broken DNA ends can generate matching sense and antisense transcripts. We performed a genome-wide RNAi-screen in cultured Drosophila cells, which revealed that in addition to DNA repair factors, many spliceosome components are required for efficient siRNA generation...
June 19, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28625156/heteroduplex-cleavage-assay-for-screening-of-probable-zygosities-resulting-from-crispr-mutations-in-diploid-single-cell-lines
#12
Kyle D Luttgeharm, Kit-Sum Wong, Steve Siembieda
The most common gene editing methods, such as CRISPR, involve random repair of an induced double-stranded DNA break through the non-homologous end joining (NHEJ) repair pathway, resulting in small insertions/deletions. In diploid cells, these mutations can take on one of three zygosities: monoallelic, diallelic heterozygous, or diallelic homozygous. While many advances have been made in CRISPR delivery systems and gene editing efficiency, little work has been done to streamline detection of gene editing events...
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28623169/mutation-of-the-inhibitory-ethanol-site-in-gabaa-%C3%AF-1-receptors-promotes-tolerance-to-ethanol-induced-motor-incoordination
#13
Yuri A Blednov, Cecilia M Borghese, Carlos I Ruiz, Madeline A Cullins, Adriana Da Costa, Elizabeth A Osterndorff-Kahanek, Gregg E Homanics, R Adron Harris
Genes encoding the ρ1/2 subunits of GABAA receptors have been associated with alcohol (ethanol) dependence in humans, and ρ1 was also shown to regulate some of the behavioral effects of ethanol in animal models. Ethanol inhibits GABA-mediated responses in wild-type (WT) ρ1, but not ρ1(T6'Y) mutant receptors expressed in Xenopus laevis oocytes, indicating the presence of an inhibitory site for ethanol in the second transmembrane helix. In this study, we found that ρ1(T6'Y) receptors expressed in oocytes display overall normal responses to GABA, the endogenous GABA modulator (zinc), and partial agonists (β-alanine and taurine)...
June 13, 2017: Neuropharmacology
https://www.readbyqxmd.com/read/28620317/comparative-and-experimental-studies-on-the-genes-altered-by-chronic-hypoxia-in-human-brain-microendothelial-cells
#14
Eugenia Mata-Greenwood, Dipali Goyal, Ravi Goyal
Background : Hypoxia inducible factor 1 alpha (HIF1A) is a master regulator of acute hypoxia; however, with chronic hypoxia, HIF1A levels return to the normoxic levels. Importantly, the genes that are involved in the cell survival and viability under chronic hypoxia are not known. Therefore, we tested the hypothesis that chronic hypoxia leads to the upregulation of a core group of genes with associated changes in the promoter DNA methylation that mediates the cell survival under hypoxia. Results : We examined the effect of chronic hypoxia (3 days; 0...
2017: Frontiers in Physiology
https://www.readbyqxmd.com/read/28619709/loss-of-fam46c-promotes-cell-survival-in-myeloma
#15
Yuan Xiao Zhu, Chang-Xin Shi, Laura A Bruins, Patrick Jedlowski, Xuewei Wang, K Martin Kortüm, Moulun Luo, Jonathan Ahmann, Esteban Braggio, A Keith Stewart
FAM46C is one of the most recurrently mutated genes in multiple myeloma (MM), however its role in disease pathogenesis has not been determined. Here we demonstrate that wild type (WT) FAM46C overexpression induces substantial cytotoxicity in MM cells. In contrast, FAM46C mutations found in MM patients abrogate this cytotoxicity, indicating a survival advantage conferred by the FAM46C mutant phenotype. WT FAM46C overexpression downregulated IRF4, CEBPB, and MYC and upregulated immunoglobulin (Ig) light chain and HSPA5/BIP Furthermore, pathway analysis suggests that enforced FAM46C expression activated the unfolded protein response (UPR) pathway and induced mitochondrial dysfunction...
June 15, 2017: Cancer Research
https://www.readbyqxmd.com/read/28619647/using-crispr-cas9-to-generate-gene-corrected-autologous-ipscs-for-the-treatment-of-inherited-retinal-degeneration
#16
Erin R Burnight, Manav Gupta, Luke A Wiley, Kristin R Anfinson, Audrey Tran, Robinson Triboulet, Jeremy M Hoffmann, Darcey L Klaahsen, Jeaneen L Andorf, Chunhua Jiao, Elliott H Sohn, Malavika K Adur, Jason W Ross, Robert F Mullins, George Q Daley, Thorsten M Schlaeger, Edwin M Stone, Budd A Tucker
Patient-derived induced pluripotent stem cells (iPSCs) hold great promise for autologous cell replacement. However, for many inherited diseases, treatment will likely require genetic repair pre-transplantation. Genome editing technologies are useful for this application. The purpose of this study was to develop CRISPR-Cas9-mediated genome editing strategies to target and correct the three most common types of disease-causing variants in patient-derived iPSCs: (1) exonic, (2) deep intronic, and (3) dominant gain of function...
June 12, 2017: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/28616754/sexually-dimorphic-distribution-of-prokr2-neurons-revealed-by-the-prokr2-cre-mouse-model
#17
Zaid Mohsen, Hosung Sim, David Garcia-Galiano, Xingfa Han, Nicole Bellefontaine, Thomas L Saunders, Carol F Elias
Prokineticin receptor 2 (PROKR2) is predominantly expressed in the mammalian central nervous system. Loss-of-function mutations of PROKR2 in humans are associated with Kallmann syndrome due to the disruption of gonadotropin releasing hormone neuronal migration and deficient olfactory bulb morphogenesis. PROKR2 has been also implicated in the neuroendocrine control of GnRH neurons post-migration and other physiological systems. However, the brain circuitry and mechanisms associated with these actions have been difficult to investigate mainly due to the widespread distribution of Prokr2-expressing cells, and the lack of animal models and molecular tools...
June 14, 2017: Brain Structure & Function
https://www.readbyqxmd.com/read/28615323/generic-membrane-spanning-features-endow-ire1%C3%AE-with-responsiveness-to-membrane-aberrancy
#18
Nozomu Kono, Niko Amin-Wetzel, David Ron
Altered cellular lipid composition activates the endoplasmic reticulum unfolded protein response (UPR) and UPR signaling effects important changes in lipid metabolism. Secondary effects on protein folding homeostasis likely contribute to UPR activation, but deletion of the unfolded protein stress-sensing luminal domain of the UPR transducers PERK and IRE1α does not abolish their responsiveness to lipid perturbation. This finding suggests that PERK and IRE1α also directly recognize the membrane aberrancy wrought by lipid perturbation...
June 14, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28615073/crispr-cas9-mediated-genome-editing-induces-exon-skipping-by-alternative-splicing-or-exon-deletion
#19
Haiwei Mou, Jordan L Smith, Lingtao Peng, Hao Yin, Jill Moore, Xiao-Ou Zhang, Chun-Qing Song, Ankur Sheel, Qiongqiong Wu, Deniz M Ozata, Yingxiang Li, Daniel G Anderson, Charles P Emerson, Erik J Sontheimer, Melissa J Moore, Zhiping Weng, Wen Xue
CRISPR is widely used to disrupt gene function by inducing small insertions and deletions. Here, we show that some single-guide RNAs (sgRNAs) can induce exon skipping or large genomic deletions that delete exons. For example, CRISPR-mediated editing of β-catenin exon 3, which encodes an autoinhibitory domain, induces partial skipping of the in-frame exon and nuclear accumulation of β-catenin. A single sgRNA can induce small insertions or deletions that partially alter splicing or unexpected larger deletions that remove exons...
June 14, 2017: Genome Biology
https://www.readbyqxmd.com/read/28613254/crispr-cas9-mediated-correction-of-the-fancd1-gene-in-primary-patient-cells
#20
Karolina Skvarova Kramarzova, Mark J Osborn, Beau R Webber, Anthony P DeFeo, Amber N McElroy, Chong Jai Kim, Jakub Tolar
Fanconi anemia (FA) is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such that gene expression remains under the endogenous control mechanisms. This has been accomplished to date only in transformed cells or their reprogrammed induced pluripotent stem cell counterparts; however, it has not yet been reported in primary patient cells...
June 14, 2017: International Journal of Molecular Sciences
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