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https://www.readbyqxmd.com/read/28418635/enhancing-protein-production-yield-from-cho-cells-by-crispr-interference-crispri
#1
Chih-Che Shen, Li-Yu Sung, Shih-Yeh Lin, Mei-Wei Lin, Yu-Chen Hu
CHO cell is an important host for biopharmaceutical production. Generation of stable CHO cell typically requires co-integration of dhfr and foreign gene into chromosomes and subsequent methotrexate (MTX) selection for co-amplification of dhfr and foreign gene. CRISPR interference (CRISPRi) is an emerging system that effectively suppresses gene transcription through the coordination of dCas9 protein and guide RNA (gRNA). However, CRISPRi has yet to be exploited in CHO cells. Here we constructed vectors expressing the functional CRISPRi system and proved effective CRISPRi-mediated suppression of dhfr transcription in CHO cells...
April 18, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28410976/genome-editing-via-delivery-of-cas9-ribonucleoprotein
#2
Mark DeWitt, Jacob E Corn, Dana Carroll
The CRISPR-Cas genome editing system is very powerful. The format of the CRISPR reagents and the means of delivery are often important factors in targeting efficiency. Delivery of recombinant Cas9 protein and guide RNA (gRNA) as a preformed ribonucleoprotein (RNP) complex has recently emerged as a powerful and general approach to genome editing. Here we outline methods to produce and deliver Cas9 RNPs. A donor DNA carrying desired sequence changes can also be included to program precise sequence introduction or replacement...
April 11, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28390761/the-aav-mediated-and-rna-guided-crispr-cas9-system-for-gene-therapy-of-dmd-and-bmd
#3
REVIEW
Jing-Zhang Wang, Peng Wu, Zhi-Min Shi, Yan-Li Xu, Zhi-Jun Liu
Mutations in the dystrophin gene (Dmd) result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), which afflict many newborn boys. In 2016, Brain and Development published several interesting articles on DMD treatment with antisense oligonucleotide, kinase inhibitor, and prednisolone. Even more strikingly, three articles in the issue 6271 of Science in 2016 provide new insights into gene therapy of DMD and BMD via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)...
April 5, 2017: Brain & Development
https://www.readbyqxmd.com/read/28366931/hydrocortisone-induced-parkin-prevents-dopaminergic-cell-death-via-creb-pathway-in-parkinson-s-disease-model
#4
Sangwoo Ham, Yun-Il Lee, Minkyung Jo, Hyojung Kim, Hojin Kang, Areum Jo, Gum Hwa Lee, Yun Jeong Mo, Sang Chul Park, Yun Song Lee, Joo-Ho Shin, Yunjong Lee
Dysfunctional parkin due to mutations or post-translational modifications contributes to dopaminergic neurodegeneration in Parkinson's disease (PD). Overexpression of parkin provides protection against cellular stresses and prevents dopamine cell loss in several PD animal models. Here we performed an unbiased high-throughput luciferase screening to identify chemicals that can increase parkin expression. Among promising parkin inducers, hydrocortisone possessed the most favorable profiles including parkin induction ability, cell protection ability, and physicochemical property of absorption, distribution, metabolism, and excretion (ADME) without inducing endoplasmic reticulum stress...
April 3, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28356547/mechanisms-of-ips-cell-generation-and-beyond
#5
Keisuke Kaji
The generation of induced pluripotent stem cells (iPSCs) achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc, transformed our classical views of the cellular epigenetic landscape and delivered a new concept for cell and tissue engineering. In addition to iPSCs, several other cell types have also been generated by master transcription factor (TF)-mediated transdifferentiation. However, the critical molecular mechanisms amongst diverse cellular identity changes are not well understood. Through the investigation of reprogramming mechanisms, we recently revealed that over-expression of constitutive active Smad3 boosted not only iPSC generation, but also 3 other master TF-mediated conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons...
2017: Keio Journal of Medicine
https://www.readbyqxmd.com/read/28325301/increased-expression-of-laminin-subunit-alpha-1-chain-by-dcas9-vp160
#6
Arnaud Perrin, Joël Rousseau, Jacques P Tremblay
Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adult human muscle, but laminin subunit alpha 1 (LAMA1) gene is expressed only during embryogenesis...
March 17, 2017: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/28303677/examination-of-crispr-cas9-design-tools-and-the-effect-of-target-site-accessibility-on-cas9-activity
#7
Ciaran M Lee, Timothy H Davis, Gang Bao
The recent adaptation of the CRISPR/Cas9 system for targeted genome engineering has led to its widespread applications in many fields worldwide. In order to better understand the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design these studies have spawned a plethora of gRNA design tools with algorithms based solely on direct or indirect sequence features...
March 16, 2017: Experimental Physiology
https://www.readbyqxmd.com/read/28298224/transcriptional-reprogramming-in-yeast-using-dcas9-and-combinatorial-grna-strategies
#8
Emil D Jensen, Raphael Ferreira, Tadas Jakočiūnas, Dushica Arsovska, Jie Zhang, Ling Ding, Justin D Smith, Florian David, Jens Nielsen, Michael K Jensen, Jay D Keasling
BACKGROUND: Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site...
March 15, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28279800/crispr-cas9-system-driven-site-specific-selection-pressure-on-herpes-simplex-virus-genomes
#9
Zhihua Li, Yanwei Bi, Hongjian Xiao, Le Sun, Yuan Ren, Yadong Li, Chen Chen, Wei Cun
The CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system has been widely used for viral genome editing, transcription regulation and chromosomal localization in eukaryotic cells. In this study, a guide RNA (gRNA) that specifically recognizes HSV-1 viral genomes was used in the CRISPR-Cas9 system to inhibit viral replication. This inhibition could be achieved with both wild type Cas9 protein and Cas9 nickase (D10A). By targeting viral genomes containing sequences recognized by the gRNA, the CRISPR-Cas9 system distinguished between different viral genome sequences and provided single nucleotide-specific selection pressure to significantly change the proportions of viruses in a mixed viral pool...
March 6, 2017: Virus Research
https://www.readbyqxmd.com/read/28263296/guidescan-software-for-improved-single-and-paired-crispr-guide-rna-design
#10
Alexendar R Perez, Yuri Pritykin, Joana A Vidigal, Sagar Chhangawala, Lee Zamparo, Christina S Leslie, Andrea Ventura
We present GuideScan software for the design of CRISPR guide RNA libraries that can be used to edit coding and noncoding genomic regions. GuideScan produces high-density sets of guide RNAs (gRNAs) for single- and paired-gRNA genome-wide screens. We also show that the trie data structure of GuideScan enables the design of gRNAs that are more specific than those designed by existing tools.
April 2017: Nature Biotechnology
https://www.readbyqxmd.com/read/28258147/genome-editing-in-clostridium-saccharoperbutylacetonicum-n1-4-using-crispr-cas9-system
#11
Shaohua Wang, Sheng Dong, Pixiang Wang, Yong Tao, Yi Wang
Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the CRISPR-Cas9 system. First, the functionality of the CRISPR-Cas9 system previously customized for C. beijerinckii was evaluated in C. saccharoperbutylacetonicum by targeting on pta and buk, two essential genes for acetate and butyrate production, respectively...
March 3, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28250269/inhibition-of-crispr-cas9-mediated-genome-engineering-by-a-type-i-interferon-induced-reduction-in-guide-rna-expression
#12
Mitsuhiro Machitani, Fuminori Sakurai, Keisaku Wakabayashi, Kosuke Nakatani, Kazuo Takayama, Masashi Tachibana, Hiroyuki Mizuguchi
Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome engineering technology is a powerful tool for generation of cells and animals with engineered mutations in their genomes. In order to introduce the CRISPR/Cas9 system into target cells, nonviral and viral vectors are often used; however, such vectors trigger innate immune responses associated with production of type I interferons (IFNs). We have recently demonstrated that type I IFNs inhibit short-hairpin RNA-mediated gene silencing, which led us to hypothesize that type I IFNs may also inhibit CRISPR/Cas9-mediated genome mutagenesis...
2017: Biological & Pharmaceutical Bulletin
https://www.readbyqxmd.com/read/28228480/a-stable-but-reversible-integrated-surrogate-reporter-for-assaying-crispr-cas9-stimulated-homology-directed-repair
#13
Yahong Wen, Grace Liao, Thomas Pritchard, Ting-Ting Zhao, Jon P Connelly, Shondra M Pruett-Miller, Valerie Blanc, Nicholas O Davidson, Blair B Madison
The discovery and application of CRISPR/Cas9 technology for genome editing has greatly accelerated targeted mutagenesis in a variety of organisms. CRISPR/Cas9-mediated site-specific cleavage is typically exploited for the generation of insertions or deletions (indels) after aberrant dsDNA repair via the endogenous non-homology end-joining (NHEJ) pathway or, alternatively, for enhancing homology-directed repair to facilitate the generation of a specific mutation (or "knock-in"). However, there is a need for efficient cellular assays that can measure Cas9/guide RNA activity...
April 14, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28179526/a-sequence-independent-unstructured-internal-ribosome-entry-site-is-responsible-for-internal-expression-of-the-coat-protein-of-turnip-crinkle-virus
#14
Jared May, Philip Johnson, Huma Saleem, Anne E Simon
To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5' cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher-order RNA structure, an unstructured sequence of about 84 nucleotides (nt) immediately upstream of the Turnip crinkle virus (TCV) coat protein (CP) open reading frame (ORF) has been found to promote internal expression of the CP from the genomic RNA (gRNA) both in vitro and in vivo An absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing...
April 15, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28177825/enhancement-of-single-guide-rna-transcription-for-efficient-crispr-cas-based-genomic-engineering
#15
Kumiko Ui-Tei, Shohei Maruyama, Yuko Nakano
Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein...
January 26, 2017: Genome Génome / Conseil National de Recherches Canada
https://www.readbyqxmd.com/read/28176813/a-combinational-crispr-cas9-gene-editing-approach-can-halt-hiv-replication-and-prevent-viral-escape
#16
Robert Jan Lebbink, Dorien C M de Jong, Femke Wolters, Elisabeth M Kruse, Petra M van Ham, Emmanuel J H J Wiertz, Monique Nijhuis
HIV presents one of the highest evolutionary rates ever detected and combination antiretroviral therapy is needed to overcome the plasticity of the virus population and control viral replication. Conventional treatments lack the ability to clear the latent reservoir, which remains the major obstacle towards a cure. Novel strategies, such as CRISPR/Cas9 gRNA-based genome-editing, can permanently disrupt the HIV genome. However, HIV genome-editing may accelerate viral escape, questioning the feasibility of the approach...
February 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28124028/optimized-crispr-cas9-genome-editing-for-leishmania-and-its-use-to-target-a-multigene-family-induce-chromosomal-translocation-and-study-dna-break-repair-mechanisms
#17
Wen-Wei Zhang, Patrick Lypaczewski, Greg Matlashewski
CRISPR-Cas9-mediated genome editing has recently been adapted for Leishmania spp. parasites, the causative agents of human leishmaniasis. We have optimized this genome-editing tool by selecting for cells with CRISPR-Cas9 activity through cotargeting the miltefosine transporter gene; mutation of this gene leads to miltefosine resistance. This cotargeting strategy integrated into a triple guide RNA (gRNA) expression vector was used to delete all 11 copies of the A2 multigene family; this was not previously possible with the traditional gene-targeting method...
January 2017: MSphere
https://www.readbyqxmd.com/read/28123598/lentiviral-crispr-cas9-vector-mediated-mir-21-gene-editing-inhibits-the-epithelial-to-mesenchymal-transition-in-ovarian-cancer-cells
#18
Wenying Huo, Guannan Zhao, Jinggang Yin, Xuan Ouyang, Yinan Wang, Chuanhe Yang, Baojing Wang, Peixin Dong, Zhixiang Wang, Hidemichi Watari, Edward Chaum, Lawrence M Pfeffer, Junming Yue
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing...
2017: Journal of Cancer
https://www.readbyqxmd.com/read/28119062/baculovirus-based-genome-editing-in-primary-cells
#19
Maysam Mansouri, Zahra Ehsaei, Verdon Taylor, Philipp Berger
Genome editing in eukaryotes became easier in the last years with the development of nucleases that induce double strand breaks in DNA at user-defined sites. CRISPR/Cas9-based genome editing is currently one of the most powerful strategies. In the easiest case, a nuclease (e.g. Cas9) and a target defining guide RNA (gRNA) are transferred into a target cell. Non-homologous end joining (NHEJ) repair of the DNA break following Cas9 cleavage can lead to inactivation of the target gene. Specific repair or insertion of DNA with Homology Directed Repair (HDR) needs the simultaneous delivery of a repair template...
January 22, 2017: Plasmid
https://www.readbyqxmd.com/read/28116670/an-inducible-crispr-on-system-for-controllable-gene-activation-in-human-pluripotent-stem-cells
#20
Jianying Guo, Dacheng Ma, Rujin Huang, Jia Ming, Min Ye, Kehkooi Kee, Zhen Xie, Jie Na
Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line...
January 23, 2017: Protein & Cell
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