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Inducible crispr

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https://www.readbyqxmd.com/read/29150930/photoautotrophic-production-of-macular-pigment-in-a-chlamydomonas-reinhardtii-strain-generated-by-using-dna-free-crispr-cas9-rnp-mediated-mutagenesis
#1
Kwangryul Baek, Jihyeon Yu, Jooyeon Jeong, Sang Jun Sim, Sangsu Bae, EonSeon Jin
Lutein and zeaxanthin are dietary carotenoids reported to be protective against age-related macular degeneration. Recently, the green alga Chlamydomonas reinhardtii has received attention as a photosynthetic cell factory, but the potential of this alga for carotenoid production has not yet been evaluated. In this study, we selected the C. reinhardtii CC-4349 strain as the best candidate among seven laboratory strains tested for carotenoid production. A knock-out mutant of the zeaxanthin epoxidase gene induced by preassembled DNA-free CRISPR-Cas9 ribonucleoproteins in the CC-4349 strain had a significantly higher zeaxanthin content (56-fold) and productivity (47-fold) than the wild type without the reduction in lutein level...
November 18, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/29150004/dynamics-of-indel-profiles-induced-by-various-crispr-cas9-delivery-methods
#2
Michael Kosicki, Sandeep S Rajan, Flaminia C Lorenzetti, Hans H Wandall, Yoshiki Narimatsu, Emmanouil Metzakopian, Eric P Bennett
The introduction of CRISPR/Cas9 gene editing in mammalian cells is a scientific breakthrough, which has greatly affected basic research and gene therapy. The simplicity and general access to CRISPR/Cas9 reagents has in an unprecedented manner "democratized" gene targeting in biomedical research, enabling genetic engineering of any gene in any cell, tissue, organ, and organism. The ability for fast, precise, and efficient profiling of the double-stranded break induced insertions and deletions (indels), mediated by any of the available programmable nucleases, is paramount to any given gene targeting approach...
2017: Progress in Molecular Biology and Translational Science
https://www.readbyqxmd.com/read/29145633/the-initiation-propagation-and-dynamics-of-crispr-spycas9-r-loop-complex
#3
Yan Zeng, Yang Cui, Yong Zhang, Yanruo Zhang, Meng Liang, Hui Chen, Jie Lan, Guangtao Song, Jizhong Lou
CRISPR-Cas9 system has been widely used for efficient genome editing. Although the structures of Cas9 protein in complex with single-guided RNA (sgRNA) and target DNA have been resolved, the molecular details about the formation of Cas9 endonuclease R-loop structure remain elusive. Here we examine the DNA cleavage activities of Streptococcus pyogenes Cas9 (SpyCas9) and its mutants using various target sequences and study the conformational dynamics of R-loop structure during target binding using single-molecule fluorescence energy transfer (smFRET) technique...
November 14, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29141633/genome-modification-of-cxcr4-by-staphylococcus-aureus-cas9-renders-cells-resistance-to-hiv-1-infection
#4
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
https://www.readbyqxmd.com/read/29141013/indcaps-a-tool-for-designing-screening-primers-for-crispr-cas9-mutagenesis-events
#5
Charles Hodgens, Zachary L Nimchuk, Joseph J Kieber
Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles...
2017: PloS One
https://www.readbyqxmd.com/read/29140568/crispr-cas9-based-safe-harbor-gene-editing-in-rhesus-ipscs
#6
Ravi Chandra Yada, John W Ostrominski, Ilker Tunc, So Gun Hong, Jizhong Zou, Cynthia E Dunbar
NHP iPSCs provide a unique opportunity to test safety and efficacy of iPSC-derived therapies in clinically relevant NHP models. To monitor these cells in vivo, there is a need for safe and efficient labeling methods. Gene insertion into genomic safe harbors (GSHs) supports reliable transgene expression while minimizing the risk the modification poses to the host genome or target cell. Specifically, this protocol demonstrates targeting of the adeno-associated virus site 1 (AAVS1), one of the most widely used GSH loci in the human genome, with CRISPR/Cas9, allowing targeted marker or therapeutic gene insertion in rhesus macaque induced pluripotent stem cells (RhiPSCs)...
November 15, 2017: Current Protocols in Stem Cell Biology
https://www.readbyqxmd.com/read/29136157/depletion-of-recombination-specific-co-factors-by-the-c-terminal-mutant-of-the-activation-induced-cytidine-deaminase-causes-the-dominant-negative-effect-on-class-switch-recombination
#7
Azza Al Ismail, Afzal Husain, Maki Kobayashi, Tasuku Honjo, Nasim A Begum
Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes. Studies on in vitro mutagenized AID as well as its mutations in human patients with Hyper-IgM (HIGM)-syndrome type II revealed that C-terminal AID mutations were defective in CSR whereas their DNA cleavage and SHM activities remained intact. The C-terminal mutants of AID were speculated to exert the dominant negative effect on wild type WT AID whereas its mechanism remains unknown...
November 10, 2017: International Immunology
https://www.readbyqxmd.com/read/29133788/regulation-of-angiotensin-ii-actions-by-enhancers-and-super-enhancers-in-vascular-smooth-muscle-cells
#8
Sadhan Das, Parijat Senapati, Zhuo Chen, Marpadga A Reddy, Rituparna Ganguly, Linda Lanting, Varun Mandi, Anita Bansal, Amy Leung, Selena Zhang, Ye Jia, Xiwei Wu, Dustin E Schones, Rama Natarajan
Angiotensin II (AngII) promotes hypertension and atherosclerosis by activating growth-promoting and pro-inflammatory gene expression in vascular smooth muscle cells (VSMCs). Enhancers and super-enhancers (SEs) play critical roles in driving disease-associated gene expression. However, enhancers/SEs mediating VSMC dysfunction remain uncharacterized. Here, we show that AngII alters vascular enhancer and SE repertoires in cultured VSMCs in vitro, ex vivo, and in AngII-infused mice aortas in vivo. AngII-induced enhancers/SEs are enriched in binding sites for signal-dependent transcription factors and dependent on key signaling kinases...
November 13, 2017: Nature Communications
https://www.readbyqxmd.com/read/29133306/quaking-inhibits-doxorubicin-mediated-cardiotoxicity-through-regulation-of-cardiac-circular-rna-expression
#9
Shashi K Gupta, Ankita Garg, Christian Bär, Shambhabi Chatterjee, Ariana Foinquinos, Hendrik Milting, Katrin Streckfuss-Bömeke, Jan Fiedler, Thomas Thum
Rationale: RNA-binding proteins (RBPs) have been described to be expressed and regulated in various organs including the heart. Little is known about the role of RBPs in heart failure induced by the chemotherapy drug doxorubicin and their interaction with circular RNAs. Objective: We aimed to identify key RBPs involved in doxorubicin-mediated heart failure and to elucidate their function. Methods and Results: Global transcriptome profiling from murine myocardium exposed to doxorubicin identified five differentially expressed RBPs...
November 13, 2017: Circulation Research
https://www.readbyqxmd.com/read/29130512/hepatocyte-generated-27-hydroxycholesterol-promotes-the-growth-of-melanoma-by-activation-of-estrogen-receptor-alpha
#10
Wei Tian, Wenxiao Pang, Yao Ge, Xiaomeng He, Duowei Wang, Xianjing Li, Hui Hou, Dewang Zhou, Shuang Feng, Zhen Chen, Yong Yang
Cholesterol plays an important role in maintaining normal physiological function of human body. However, excessive intake will induce a series of diseases including cancer. For melanoma, the relationship between hypercholesterolemia and its incidence remains unknown. The cholesterol metabolite 27-hydroxy cholesterol (27-HC) catalyzed by CYP27A1 has been reported to activate estrogen receptor (ER). As studies have indicated that melanoma expresses ER, we designed experiments to explore whether 27-HC could link hypercholesterolemia and melanoma...
November 11, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/29130076/crispr-correction-of-a-homozygous-low-density-lipoprotein-receptor-mutation-in-familial-hypercholesterolemia-induced-pluripotent-stem-cells
#11
Linda Omer, Elizabeth A Hudson, Shirong Zheng, James B Hoying, Yuan Shan, Nolan L Boyd
Familial hypercholesterolemia (FH) is a hereditary disease primarily due to mutations in the low-density lipoprotein receptor (LDLR) that lead to elevated cholesterol and premature development of cardiovascular disease. Homozygous FH patients (HoFH) with two dysfunctional LDLR alleles are not as successfully treated with standard hypercholesterol therapies, and more aggressive therapeutic approaches to control cholesterol levels must be considered. Liver transplant can resolve HoFH, and hepatocyte transplantation has shown promising results in animals and humans...
November 2017: Hepatol Commun
https://www.readbyqxmd.com/read/29128401/h2s-protects-lipopolysaccharide-induced-inflammation-by-blocking-nf%C3%AE%C2%BAb-transactivation-in-endothelial-cells
#12
Caitlyn Bourque, Yanjie Zhang, Ming Fu, Mélanie Racine, Adam Greasley, Yanxi Pei, Lingyun Wu, Rui Wang, Guangdong Yang
Hydrogen sulfide (H2S) is a novel gasotransmitter and acts as a multifunctional regulator in various cellular functions. Past studies have demonstrated a significant role of H2S and its generating enzyme cystathionine gamma-lyase (CSE) in the cardiovascular system. Lipopolysaccharide (LPS), a major pathogenic factor, is known to initiate the inflammatory immune response. The cross talk between LPS-induced inflammation and the CSE/H2S system in vascular cells has not yet been elucidated in detail. Here we showed that LPS decreased CSE mRNA and protein expression in human endothelial cells and blocked H2S production in mouse aorta tissues...
November 8, 2017: Toxicology and Applied Pharmacology
https://www.readbyqxmd.com/read/29126790/translational-research-exploring-and-creating-genetic-diversity
#13
REVIEW
Pierre Jacob, Adi Avni, Abdelhafid Bendahmane
The crop selection process has created a genetic bottleneck ultimately restricting breeding output. Wild relatives of major crops as well as the so-called 'neglected plant' species represent a reservoir of genetic diversity that remains underutilized. These species could be used as a tool to discover new alleles of agronomic interest or could be the target of breeding programs. Targeted induced local lesions in the genome (TILLING) can be used to translate in neglected crops what has been discovered in major crops and reciprocally...
November 7, 2017: Trends in Plant Science
https://www.readbyqxmd.com/read/29124278/targeted-gene-editing-in-human-pluripotent-stem-cells-using-site-specific-nucleases
#14
Sylvia Merkert, Ulrich Martin
Introduction of induced pluripotent stem cell (iPSC) technology and site-directed nucleases brought a major breakthrough in the development of regenerative therapies and biomedical research. With the advancement of ZFNs, TALENs, and the CRISPR/Cas9 technology, straightforward and precise manipulation of the genome of human pluripotent stem cells (PSC) became possible, allowing relatively easy and fast generation of gene knockouts, integration of transgenes, or even introduction of single nucleotide changes for correction or introduction of disease-specific mutations...
November 10, 2017: Advances in Biochemical Engineering/biotechnology
https://www.readbyqxmd.com/read/29122551/crispr-mediated-deletion-of-the-pecam-1-cytoplasmic-domain-increases-receptor-lateral-mobility-and-strengthens-endothelial-cell-junctional-integrity
#15
Danying Liao, Heng Mei, Yu Hu, Debra K Newman, Peter J Newman
AIMS: PECAM-1 is an abundant endothelial cell surface receptor that becomes highly enriched at endothelial cell-cell junctions, where it functions to mediate leukocyte transendothelial migration, sense changes in shear and flow, and maintain the vascular permeability barrier. Homophilic interactions mediated by the PECAM-1 extracellular domain are known to be required for PECAM-1 to perform these functions; however, much less is understood about the role of its cytoplasmic domain in these processes...
November 6, 2017: Life Sciences
https://www.readbyqxmd.com/read/29116071/-crispr-cas9-editing-based-modeling-of-hypoxia-in-renal-cancer-cells
#16
N A Zhigalova, S V Zhenilo, A V Artemov, E B Prokhortchouk
Uncontrolled growth in the cell mass of malignant tumors induces intensive angiogenesis. However, the demands of the cancer cells for nutrients and oxygen remain only partially met. Hypoxia is a process that accompanies malignant transformation and evokes changes in the DNA methylation profile in solid tumors. To a certain extent, these changes, including the hypermethylation of tumor suppressor gene promoters, are related to the decrease in the activity of Tet proteins under the conditions of oxygen and free radical deficit...
September 2017: Molekuliarnaia Biologiia
https://www.readbyqxmd.com/read/29113998/rhoc-regulates-actin-remodeling-to-form-phagosomes-during-fc%C3%AE-r-mediated-phagocytosis
#17
Youhei Egami, Katsuhisa Kawai, Nobukazu Araki
Phagosome formation is a complicated process that requires spatiotemporally regulated actin reorganization. We found that RhoC GTPase is a critical regulator of FcγR-mediated phagocytosis in macrophages. Our live-cell imaging revealed that RhoC, but not RhoA, is recruited to phagocytic cups engulfing IgG-opsonized erythrocytes (IgG-Es). RhoC silencing by RNAi, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated RhoC knockout and the expression of dominant-negative or dominant-active RhoC mutants suppressed the phagocytosis of IgG-Es...
November 7, 2017: Journal of Cell Science
https://www.readbyqxmd.com/read/29112727/application-of-crispr-interference-for-metabolic-engineering-of-the-heterocyst-forming-multicellular-cyanobacterium-anabaena-sp-pcc-7120
#18
Akiyoshi Higo, Atsuko Isu, Yuki Fukaya, Shigeki Ehira, Toru Hisabori
Anabaena sp. PCC 7120 (A. 7120) is a heterocyst-forming multicellular cyanobacterium that performs nitrogen fixation. This cyanobacterium has been extensively studied as a model for multicellularity in prokaryotic cells. We have been interested in photosynthetic production of nitrogenous compounds using A. 7120. However, the lack of efficient gene repression tools has limited its usefulness. We originally developed an artificial endogenous gene repression method in this cyanobacterium using small antisense RNA (Higo A...
November 3, 2017: Plant & Cell Physiology
https://www.readbyqxmd.com/read/29107083/p63-and-p73-repress-cxcr5-chemokine-receptor-gene-expression-in-p53-deficient-mcf-7-breast-cancer-cells-during-genotoxic-stress
#19
Nikita A Mitkin, Alisa M Muratova, George V Sharonov, Kirill V Korneev, Ekaterina N Sviriaeva, Dmitriy Mazurov, Anton M Schwartz, Dmitry V Kuprash
Many types of chemotherapeutic agents induce of DNA-damage that is accompanied by activation of p53 tumor suppressor, a key regulator of tumor development and progression. In our previous study we demonstrated that p53 could repress CXCR5 chemokine receptor gene in MCF-7 breast cancer cells via attenuation of NFkB activity. In this work we aimed to determine individual roles of p53 family members in the regulation of CXCR5 gene expression under genotoxic stress. DNA-alkylating agent methyl methanesulfonate caused a reduction in CXCR5 expression not only in parental MCF-7 cells but also in MCF-7-p53off cells with CRISPR/Cas9-mediated inactivation of the p53 gene...
November 6, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29103811/crispr-cas9-based-plant-genome-editing-significance-opportunities-and-recent-advances
#20
Neelam Soda, Lokesh Verma, Jitender Giri
Precise genome editing is a quantum leap in the field of plant sciences. Clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas9 protein have emerged as a powerful tool for precise genome editing. CRISPR-Cas9 system introduces small heritable mutations (indels) in the genome of an organism. This system also enables precise gene characterization in plants with complex genomes. Besides, it offers new opportunities of trait stacking, where addition of desirable traits or removal of undesirable traits can be achieved simultaneously in a single event...
October 27, 2017: Plant Physiology and Biochemistry: PPB
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