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CRISPR/Cas9

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https://www.readbyqxmd.com/read/29238045/ripk3-promotes-adenovirus-type-5-activity
#1
Melanie Weigert, Alex Binks, Suzanne Dowson, Elaine Y L Leung, Dmitris Athineos, Xinzi Yu, Margaret Mullin, Josephine B Walton, Clare Orange, Darren Ennis, Karen Blyth, Stephen W G Tait, Iain A McNeish
Oncolytic adenoviral mutants infect human malignant cells and replicate selectively within them. This induces direct cytotoxicity that can also trigger profound innate and adaptive immune responses. However, the mechanism by which adenoviruses produce cell death remains uncertain. We previously suggested that type 5 adenoviruses, including the E1A CR2 deletion mutant dl922-947, might induce a novel form of programmed death resembling necroptosis. Here we have investigated the roles of core necrosis proteins RIPK1, RIPK3 and MLKL in the cytotoxicity of dl922-947 and other adenovirus serotypes...
December 13, 2017: Cell Death & Disease
https://www.readbyqxmd.com/read/29237052/multimode-drug-inducible-crispr-cas9-devices-for-transcriptional-activation-and-genome-editing
#2
Jia Lu, Chen Zhao, Yingze Zhao, Jingfang Zhang, Yue Zhang, Li Chen, Qiyuan Han, Yue Ying, Shuai Peng, Runna Ai, Yu Wang
Precise investigation and manipulation of dynamic biological processes often requires molecular modulation in a controlled inducible manner. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) has emerged as a versatile tool for targeted gene editing and transcriptional programming. Here, we designed and vigorously optimized a series of Hybrid drug Inducible CRISPR/Cas9 Technologies (HIT) for transcriptional activation by grafting a mutated human estrogen receptor (ERT2) to multiple CRISPR/Cas9 systems, which renders them 4-hydroxytamoxifen (4-OHT) inducible for the access of genome...
December 9, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29237010/crispr-cas9-mediated-modulation-of-splicing-efficiency-reveals-short-splicing-isoform-of-xist-rna-is-sufficient-to-induce-x-chromosome-inactivation
#3
Minghui Yue, Yuya Ogawa
Alternative splicing of mRNA precursors results in multiple protein variants from a single gene and is critical for diverse cellular processes and development. Xist encodes a long noncoding RNA which is a central player to induce X-chromosome inactivation in female mammals and has two major splicing variants: long and short isoforms of Xist RNA. Although a differentiation-specific and a female-specific expression of Xist isoforms have been reported, the functional role of each Xist RNA isoform is largely unexplored...
December 9, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29236701/anti-leukemic-activity-of-bortezomib-and-carfilzomib-on-b-cell-precursor-all-cell-lines
#4
Kazuya Takahashi, Takeshi Inukai, Toshihiko Imamura, Mio Yano, Chihiro Tomoyasu, David M Lucas, Atsushi Nemoto, Hiroki Sato, Meixian Huang, Masako Abe, Keiko Kagami, Tamao Shinohara, Atsushi Watanabe, Shinpei Somazu, Hiroko Oshiro, Koshi Akahane, Kumiko Goi, Jiro Kikuchi, Yusuke Furukawa, Hiroaki Goto, Masayoshi Minegishi, Shotaro Iwamoto, Kanji Sugita
Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response...
2017: PloS One
https://www.readbyqxmd.com/read/29235471/ctcf-driven-terra-transcription-facilitates-completion-of-telomere-dna-replication
#5
Kate Beishline, Olga Vladimirova, Stephen Tutton, Zhuo Wang, Zhong Deng, Paul M Lieberman
Telomere repeat DNA forms a nucleo-protein structure that can obstruct chromosomal DNA replication, especially under conditions of replication stress. Transcription of telomere repeats can initiate at subtelomeric CTCF-binding sites to generate telomere repeat-encoding RNA (TERRA), but the role of transcription, CTCF, and TERRA in telomere replication is not known. Here, we have used CRISPR/Cas9 gene editing to mutate CTCF-binding sites at the putative start site of TERRA transcripts for a class of subtelomeres...
December 13, 2017: Nature Communications
https://www.readbyqxmd.com/read/29235467/in-situ-functional-dissection-of-rna-cis-regulatory-elements-by-multiplex-crispr-cas9-genome-engineering
#6
Qianxin Wu, Quentin R V Ferry, Toni A Baeumler, Yale S Michaels, Dimitrios M Vitsios, Omer Habib, Roland Arnold, Xiaowei Jiang, Stefano Maio, Bruno R Steinkraus, Marta Tapia, Paolo Piazza, Ni Xu, Georg A Holländer, Thomas A Milne, Jin-Soo Kim, Anton J Enright, Andrew R Bassett, Tudor A Fulga
RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs...
December 13, 2017: Nature Communications
https://www.readbyqxmd.com/read/29234114/probing-the-requirement-for-cd38-in-retinoic-acid-induced-hl-60-cell-differentiation-with-a-small-molecule-dimerizer-and-genetic-knockout
#7
Robert J MacDonald, Jonathan H Shrimp, Hong Jiang, Lu Zhang, Hening Lin, Andrew Yen
CD38 is an ectoenzyme and receptor with key physiological roles. It metabolizes NAD+ to adenosine diphosphate ribose (ADPR) and cyclic ADPR, regulating several processes including calcium signalling. CD38 is both a positive and negative prognostic indicator in leukaemia. In all-trans retinoic acid (RA)-induced differentiation of acute promyelocytic leukaemia and HL-60 cells, CD38 is one of the earliest and most prominently upregulated proteins known. CD38 overexpression enhances differentiation, while morpholino- and siRNA-induced knockdown diminishes it...
December 12, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29234093/crispr-cas9-microinjection-in-oocytes-disables-pancreas-development-in-sheep
#8
Marcela Vilarino, Sheikh Tamir Rashid, Fabian Patrik Suchy, Bret Roberts McNabb, Talitha van der Meulen, Eli J Fine, Syed Ahsan, Nurlybek Mursaliyev, Vittorio Sebastiano, Santiago Sain Diab, Mark O Huising, Hiromitsu Nakauchi, Pablo J Ross
One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos...
December 12, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29233979/extracellular-vesicles-from-human-induced-pluripotent-stem-cell-derived-mesenchymal-stromal-cells-hipsc-mscs-protect-against-renal-ischemia-reperfusion-injury-via-delivering-specificity-protein-sp1-and-transcriptional-activating-of-sphingosine-kinase-1-and
#9
Xiaodong Yuan, Dawei Li, Xiaosong Chen, Conghui Han, Longmei Xu, Tao Huang, Zhen Dong, Ming Zhang
Renal ischemia-reperfusion is a main cause of acute kidney injury (AKI), which is associated with high mortality. Here we show that extracellular vesicles (EVs) secreted from hiPSC-MSCs play a critical role in protection against renal I/R injury. hiPSC-MSCs-EVs can fuse with renal cells and deliver SP1 into target cells, subsequently active SK1 expression and increase S1P formation. Chromatin immunoprecipitation (ChIP) analyses and luciferase assay were used to confirm SP1 binds directly to the SK1 promoter region and promote promoter activity...
December 11, 2017: Cell Death & Disease
https://www.readbyqxmd.com/read/29233915/highly-efficient-site-specific-mutagenesis-in-malaria-mosquitoes-using-crispr
#10
Ming Li, Omar S Akbari, Bradley J White
Anopheles mosquitoes transmit at least 200 million annual malaria infections worldwide. Despite considerable genomic resources, mechanistic understanding of biological processes in Anopheles has been hampered by a lack of tools for reverse genetics. Here, we report successful application of the CRISPR/Cas9 system for highly efficient, site-specific mutagenesis in the diverse malaria vectors Anopheles albimanus, Anopheles coluzzii, and Anopheles funestus When guide RNAs and Cas9 protein are injected at high concentration, germline mutations are common and usually bi-allelic allowing for the rapid creation of stable, mutant lines for reverse genetic analysis...
December 12, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/29233900/systematic-crispr-cas9-mediated-modifications-of-plasmodium-yoelii-apiap2-genes-reveal-functional-insights-into-parasite-development
#11
Cui Zhang, Zhenkui Li, Huiting Cui, Yuanyuan Jiang, Zhenke Yang, Xu Wang, Han Gao, Cong Liu, Shujia Zhang, Xin-Zhuan Su, Jing Yuan
Malaria parasites have a complex life cycle with multiple developmental stages in mosquito and vertebrate hosts, and different developmental stages express unique sets of genes. Unexpectedly, many transcription factors (TFs) commonly found in eukaryotic organisms are absent in malaria parasites; instead, a family of genes encoding proteins similar to the plant Apetala2 (ApiAP2) transcription factors is expanded in the parasites. Several malaria ApiAP2 genes have been shown to play a critical role in parasite development; however, the functions of the majority of the ApiAP2 genes remain to be elucidated...
December 12, 2017: MBio
https://www.readbyqxmd.com/read/29232857/mir-34a-regulates-sperm-motility-in-zebrafish
#12
Wenjie Guo, Binyue Xie, Shuting Xiong, Xufang Liang, Jian-Fang Gui, Jie Mei
Increasing attention has been focused on the role of microRNAs in post-transcription regulation during spermatogenesis. Recently, the miR-34 family has been shown to be involved in the spermatogenesis, but the clear function of the miR-34 family in spermatogenesis is still obscure. Here we analyzed the function of miR-34a, a member of the miR-34 family, during spermatogenesis using miR-34a knockout zebrafish generated by the clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) system...
December 10, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/29230929/application-of-protoplast-technology-to-crispr-cas9-mutagenesis-from-single-cell-mutation-detection-to-mutant-plant-regeneration
#13
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/29229993/therapeutic-relevance-of-the-pp2a-b55-inhibitory-kinase-mastl-greatwall-in-breast-cancer
#14
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
#15
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/29228730/wisp2-disruption-represses-cxcr4-expression-and-inhibits-bmscs-homing-to-injured-liver
#16
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/29228209/cd38-knockout-suppresses-tumorigenesis-in-mice-and-clonogenic-growth-of-human-lung-cancer-cells
#17
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
#18
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
#19
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
#20
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
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