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https://www.readbyqxmd.com/read/29231142/crispr-cas-in-medicinal-chemistry-applications-and-regulatory-concerns
#1
Aliuska Duardo-Sanchez
A rapid search in scientific publication's databases shows how the use of CRISPR-Cas genome editions' technique has considerably expanded, and its growing importance, in modern molecular biology. Just in pub-med platform, the search of the term gives more than 3000 results. Specifically, in Drug Discovery, Medicinal Chemistry and Chemical Biology in general CRISPR method may have multiple applications. Some of these applications are: resistance-selection studies of antimalarial lead organic compounds; investigation of druggability; development of animal models for chemical compounds testing, etc...
December 11, 2017: Current Topics in Medicinal Chemistry
https://www.readbyqxmd.com/read/29230929/application-of-protoplast-technology-to-crispr-cas9-mutagenesis-from-single-cell-mutation-detection-to-mutant-plant-regeneration
#2
Choun-Sea Lin, Chen-Tran Hsu, Ling-Hung Yang, Lan-Ying Lee, Jin-Yuan Fu, Qiao-Wei Cheng, Fu-Hui Wu, Han Chen-Wei Hsiao, Yesheng Zhang, Ru Zhang, Wan-Jung Chang, Chen-Ting Yu, Wen Wang, Li-Jen Liao, Stanton B Gelvin, Ming-Che Shih
Plant protoplasts are useful for assessing the efficiency of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated Protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency...
December 12, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/29230095/crispr-offinder-a-crispr-guide-rna-design-and-off-target-searching-tool-for-user-defined-protospacer-adjacent-motif
#3
Changzhi Zhao, Xiaoguo Zheng, Wubin Qu, Guanglei Li, Xinyun Li, Yi-Liang Miao, Xiaosong Han, Xiangdong Liu, Zhenhua Li, Yunlong Ma, Qianzhi Shao, Haiwei Li, Fei Sun, Shengsong Xie, Shuhong Zhao
Designing efficient and specific CRISPR single-guide RNAs (sgRNAs) is vital for the successful application of CRISPR technology. Currently, a growing number of new RNA-guided endonucleases with a different protospacer adjacent motif (PAM) have been discovered, suggesting the necessity to develop a versatile tool for designing sgRNA to meet the requirement of different RNA-guided DNA endonucleases. Here, we report the development of a flexible sgRNA design program named "CRISPR-offinder". Support for user-defined PAM and sgRNA length was provided to increase the targeting range and specificity...
2017: International Journal of Biological Sciences
https://www.readbyqxmd.com/read/29229993/therapeutic-relevance-of-the-pp2a-b55-inhibitory-kinase-mastl-greatwall-in-breast-cancer
#4
Mónica Álvarez-Fernández, María Sanz-Flores, Belén Sanz-Castillo, María Salazar-Roa, David Partida, Elisabet Zapatero-Solana, H Raza Ali, Eusebio Manchado, Scott Lowe, Todd VanArsdale, David Shields, Carlos Caldas, Miguel Quintela-Fandino, Marcos Malumbres
PP2A is a major tumor suppressor whose inactivation is frequently found in a wide spectrum of human tumors. In particular, deletion or epigenetic silencing of genes encoding the B55 family of PP2A regulatory subunits is a common feature of breast cancer cells. A key player in the regulation of PP2A/B55 phosphatase complexes is the cell cycle kinase MASTL (also known as Greatwall). During cell division, inhibition of PP2A-B55 by MASTL is required to maintain the mitotic state, whereas inactivation of MASTL and PP2A reactivation is required for mitotic exit...
December 11, 2017: Cell Death and Differentiation
https://www.readbyqxmd.com/read/29229813/human-genetic-variation-alters-crispr-cas9-on-and-off-targeting-specificity-at-therapeutically-implicated-loci
#5
Samuel Lessard, Laurent Francioli, Jessica Alfoldi, Jean-Claude Tardif, Patrick T Ellinor, Daniel G MacArthur, Guillaume Lettre, Stuart H Orkin, Matthew C Canver
The CRISPR-Cas9 nuclease system holds enormous potential for therapeutic genome editing of a wide spectrum of diseases. Large efforts have been made to further understanding of on- and off-target activity to assist the design of CRISPR-based therapies with optimized efficacy and safety. However, current efforts have largely focused on the reference genome or the genome of cell lines to evaluate guide RNA (gRNA) efficiency, safety, and toxicity. Here, we examine the effect of human genetic variation on both on- and off-target specificity...
December 11, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29229728/cas9-enhances-bacterial-virulence-by-repressing-the-regr-transcriptional-regulator-in-streptococcus-agalactiae
#6
Ke Ma, Qing Cao, Su Luo, Zhaofei Wang, Guangjin Liu, Chengping Lu, Yongjie Liu
Clustered regularly interspaced palindromic repeats (CRISPR) and its associated cas genes have been demonstrated to regulate self-genes and virulence in many pathogens. In this study, we found that inactivation of cas9 caused reduced adhesion and intracellular survival of the piscine Streptococcus agalactiae strain GD201008-001 and significantly decreased the virulence of this strain in zebrafish and mice. Further investigation indicated that the regR transcriptional regulator was upregulated in the Δcas9 mutant...
December 11, 2017: Infection and Immunity
https://www.readbyqxmd.com/read/29229685/the-essential-role-of-phospho-t38-cpi-17-in-the-maintenance-of-physiological-blood-pressure-using-genetically-modified-mice
#7
Qunhui Yang, Wataru Fujii, Noriyuki Kaji, Shigeru Kakuta, Kodai Kada, Masayoshi Kuwahara, Hirokazu Tsubone, Hiroshi Ozaki, Masatoshi Hori
PKC-potentiated phosphorylation-dependent inhibitory protein of protein phosphatase 1 (CPI-17), an endogenous myosin phosphatase inhibitory protein, is considered a key molecule for Ca2+ sensitization of the contractile apparatus. Here, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 to generate CPI-17-deficient [knockout (KO)] and threonine 38 (T38)-phospho-resistant mice [Threonine mutant into Alanine (TA)], and then effects of CPI-17 on vascular contractility in vitro and mean blood pressure (MBP) in vivo were investigated...
December 11, 2017: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://www.readbyqxmd.com/read/29228730/wisp2-disruption-represses-cxcr4-expression-and-inhibits-bmscs-homing-to-injured-liver
#8
Dan Qin, Yi Yan, Bian Hu, Wanpo Zhang, Hanmin Li, Xiaodong Li, Shenghui Liu, Depeng Dai, Xiongji Hu, Xingxu Huang, Lisheng Zhang
Liver regeneration/repair is a compensatory regrowth following acute liver failure, and bone marrow-derived mesenchyme stem cell (BMSC) transplantation is an effective therapy that promotes liver regeneration/repair. Wnt1 inducible signaling pathway protein 2 (Wisp2) is highly expressed in BMSCs, however, its function remains unclear. In this work, we used clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein -9 nuclease (CRISPR/Cas9) genome editing technology to knockdown Wisp2 in BMSCs, and these modified cells were then transplanted into rats which were induced by the 2-AAF/PH...
November 17, 2017: Oncotarget
https://www.readbyqxmd.com/read/29228332/prespacer-processing-and-specific-integration-in-a-type-i-a-crispr-system
#9
Clare Rollie, Shirley Graham, Christophe Rouillon, Malcolm F White
The CRISPR-Cas system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. Adaptation is dependent on the Cas1 and Cas2 proteins along with varying accessory proteins. Here we analyse the process in Sulfolobus solfataricus, showing that while Cas1 and Cas2 catalyze spacer integration in vitro, host factors are required for specificity. Specific integration also requires at least 400 bp of the leader sequence, and is dependent on the presence of hydrolysable ATP, suggestive of an active process that may involve DNA remodelling...
December 8, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29228209/cd38-knockout-suppresses-tumorigenesis-in-mice-and-clonogenic-growth-of-human-lung-cancer-cells
#10
Xiangning Bu, Jiro Kato, Julie A Hong, Maria J Merino, David S Schrump, Frances E Lund, Joel Moss
The ectodomain of the plasma membrane ecto-enzyme CD38 functions as both an NAD glycohydrolase and an ADP-ribosyl cyclase by catalyzing, respectively, the conversion of NAD to nicotinamide and ADP-ribose or cyclic ADP-ribose. CD38 is attracting particular attention in cancer therapy. An anti-CD38 monoclonal antibody (daratumumab) was approved for treatment of patients with multiple myeloma. However, the role of CD38 in non-hematological malignancies has not been explored. Previously, we reported that ADP-ribose-acceptor hydrolase (ARH)-1 deficiency in mice was associated with tumor development...
December 8, 2017: Carcinogenesis
https://www.readbyqxmd.com/read/29228005/rna-seq-reveals-transcriptome-changes-in-goats-following-myostatin-gene-knockout
#11
Lamei Wang, Bei Cai, Shiwei Zhou, Haijing Zhu, Lei Qu, Xiaolong Wang, Yulin Chen
Myostatin (MSTN) is a powerful negative regulator of skeletal muscle mass in mammalian species that is primarily expressed in skeletal muscles, and mutations of its encoding gene can result in the double-muscling trait. In this study, the CRISPR/Cas9 technique was used to edit MSTN in Shaanbei Cashmere goats and generate knockout animals. RNA sequencing was used to determine and compare the transcriptome profiles of the muscles from three wild-type (WT) goats, three fibroblast growth factor 5 (FGF5) knockout goats (FGF5+/- group) and three goats with disrupted expression of both the FGF5 and MSTN genes (FM+/- group)...
2017: PloS One
https://www.readbyqxmd.com/read/29226180/cytosolic-and-nuclear-delivery-of-crispr-cas9-ribonucleoprotein-for-gene-editing-using-arginine-functionalized-gold-nanoparticles
#12
Rubul Mout, Vincent M Rotello
In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in an E-tag and usually n = 15 or 20), C-terminus nuclear localizing signal (NLS), and a C-terminus 6xHis-tag. [Cas9En hereafter] To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant Cas9En, and sgRNA)...
October 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29226078/identification-and-in%C3%A2-silico-structural-analysis-of-gallus-gallus-protein-arginine-methyltransferase-4-prmt4
#13
Hannah Berberich, Felix Terwesten, Sinja Rakow, Peeyush Sahu, Caroline Bouchard, Marion Meixner, Sjaak Philipsen, Peter Kolb, Uta-Maria Bauer
Protein arginine methyltransferase 4 (PRMT4) is an essential epigenetic regulator of fundamental and conserved processes during vertebrate development, such as pluripotency and differentiation. Surprisingly, PRMT4 homologs have been identified in nearly all vertebrate classes except the avian genome. This raises the possibility that in birds PRMT4 functions are taken over by other PRMT family members. Here, we reveal the existence of a bona fide PRMT4 homolog in the chicken, Gallus gallus. Using a biochemical approach, we initially purified a putative chicken PRMT4 protein and thus provided the first evidence for the presence of an endogenous PRMT4-specific enzymatic activity toward histone H3 arginine 17 (H3R17) in avian cells...
December 2017: FEBS Open Bio
https://www.readbyqxmd.com/read/29225130/on-the-g-protein-coupling-selectivity-of-the-native-a2b-adenosine-receptor
#14
Zhan-Guo Gao, Asuka Inoue, Kenneth A Jacobson
A2B adenosine receptor (A2BAR) activation induces Gs-dependent cyclic AMP accumulation. However, A2BAR G protein-coupling to other signaling events, e.g. ERK1/2 and calcium, is not well documented. We explored Gi, Gq/11 and Gs coupling in 1321N1 astrocytoma, HEK293, and T24 bladder cancer cells endogenously expressing human A2BAR, using NECA or nonnucleoside BAY60-6583 as agonist, selective Gi, Gs and Gq/11 blockers, and CRISPR/Cas9-based Gq- and Gs-null HEK293 cells. In HEK293 cells, A2BAR-mediated ERK1/2 activity occurred via both Gi and Gs, but not Gq/11...
December 7, 2017: Biochemical Pharmacology
https://www.readbyqxmd.com/read/29224783/in%C3%A2-vivo-target-gene-activation-via-crispr-cas9-mediated-trans-epigenetic-modulation
#15
Hsin-Kai Liao, Fumiyuki Hatanaka, Toshikazu Araoka, Pradeep Reddy, Min-Zu Wu, Yinghui Sui, Takayoshi Yamauchi, Masahiro Sakurai, David D O'Keefe, Estrella Núñez-Delicado, Pedro Guillen, Josep M Campistol, Cheng-Jang Wu, Li-Fan Lu, Concepcion Rodriguez Esteban, Juan Carlos Izpisua Belmonte
Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling...
November 30, 2017: Cell
https://www.readbyqxmd.com/read/29224076/next-generation-sequencing-of-genome-wide-crispr-screens
#16
Edwin H Yau, Tariq M Rana
Genome-wide functional genomic screens utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system have proven to be a powerful tool for systematic genomic perturbation in mammalian cells and provide an alternative to previous screens utilizing RNA interference technology. The wide availability of these libraries through public plasmid repositories as well as the decreasing cost and speed in quantifying these screens using high-throughput next-generation sequencing (NGS) allows for the adoption of the technology in a variety of laboratories interested in diverse biologic questions...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29223949/-will-the-crispr-cas9-system-change-the-genome-of-humanity
#17
EDITORIAL
Damasia Becú-Villalobos
No abstract text is available yet for this article.
2017: Medicina
https://www.readbyqxmd.com/read/29223140/collagen-induced-arthritis-analysis-in-rhbdf2-knockout-mouse
#18
Min-Young Lee, Ju-Seong Kang, Ryeo-Eun Go, Yong-Sub Byun, Young Jin Wi, Kyung-A Hwang, Jae-Hoon Choi, Hyoung-Chin Kim, Kyung-Chul Choi, Ki-Hoan Nam
Rhomboid family member 2 gene (Rhbdf2) is an inactive homologue lacking essential catalytic residues of rhomboid intramembrane serine proteases. The protein is necessary for maturation of tumor necrosis factor-alpha (TNF-α) converting enzyme, which is the molecule responsible for the release of TNF-α. In this study, Rhbdf2 knockout (KO) mice were produced by CRISPR/CAS9. To see the effects of the failure of TNF-α release induced by Rhbdf2 gene KO, collagen-induced arthritis (CIA), which is the representative TNF-α related disease, was induced in the Rhbdf2 mutant mouse using chicken collagen type II...
December 8, 2017: Biomolecules & Therapeutics
https://www.readbyqxmd.com/read/29222528/structure-and-dynamics-of-cas9-hnh-domain-catalytic-state
#19
Zhicheng Zuo, Jin Liu
The bacterial CRISPR-Cas9 immune system has been harnessed as a powerful and versatile genome-editing tool and holds immense promise for future therapeutic applications. Despite recent advances in understanding Cas9 structures and its functional mechanism, little is known about the catalytic state of the Cas9 HNH nuclease domain, and identifying how the divalent metal ions affect the HNH domain conformational transition remains elusive. A deeper understanding of Cas9 activation and its cleavage mechanism can enable further optimization of Cas9-based genome-editing specificity and efficiency...
December 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29222508/crispr-cpf1-mediates-efficient-homology-directed-repair-and-temperature-controlled-genome-editing
#20
Miguel A Moreno-Mateos, Juan P Fernandez, Romain Rouet, Charles E Vejnar, Maura A Lane, Emily Mis, Mustafa K Khokha, Jennifer A Doudna, Antonio J Giraldez
Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms...
December 8, 2017: Nature Communications
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