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CRISPR CAS9 human

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https://www.readbyqxmd.com/read/28726781/autophagy-inhibition-reduces-chemoresistance-and-tumorigenic-potential-of-human-ovarian-cancer-stem-cells
#1
Anna Pagotto, Giorgia Pilotto, Elena Laura Mazzoldi, Maria Ornella Nicoletto, Simona Frezzini, Anna Pastò, Alberto Amadori
Epithelial ovarian cancer (EOC) is one of the most malignant gynecological tumors with a high mortality rate owing to tumor relapse after anticancer therapies. It is widely accepted that a rare tumor cell population, known as cancer stem cells (CSC), is responsible for tumor progression and relapse; intriguingly, these cells are able to survive nutrient starvation (such as in vitro culture in the absence of glucose) and chemotherapy treatment. Recent data also indicated that chemotherapy resistance is associated with autophagy activation...
July 20, 2017: Cell Death & Disease
https://www.readbyqxmd.com/read/28723575/crispr-mediated-integration-of-large-gene-cassettes-using-aav-donor-vectors
#2
Rasmus O Bak, Matthew H Porteus
The CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno-associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR). However, the maximum AAV packaging capacity of ∼4.5 kb limits the donor size. Here, we overcome this constraint by showing that two co-transduced AAV vectors can serve as donors during consecutive HR events for the integration of large transgenes. Importantly, the method involves a single-step procedure applicable to primary cells with relevance to therapeutic genome editing...
July 18, 2017: Cell Reports
https://www.readbyqxmd.com/read/28721587/genetically-engineered-cell-lines-for-%C3%AE-1-antitrypsin-expression
#3
Qianqian Ji, Caiping Guo, Chen Xie, Yingdan Wu, Pei Zhang, Hui Li, Yongjun Lu
OBJECTIVES: To establish genetically modified cell lines that can produce functional α1-antitrypsin (AAT), by CRISPR/Cas9-assisted homologous recombination. RESULTS: α1-Antitrypsin deficiency (AATD) is a monogenic heritable disease that often results in lungs and liver damage. Current augmentation therapy is expensive and in short of supply. To develop a safer and more effective therapeutic strategy for AATD, we integrated the AAT gene (SERPINA1, NG_008290.1) into the AAVS1 locus of human cell line HEK293T and assessed the safety and efficacy of CRISPR/Cas9 on producing potential therapeutic cell lines...
July 18, 2017: Biotechnology Letters
https://www.readbyqxmd.com/read/28720717/sting-is-an-essential-mediator-of-the-ku70-mediated-production-of-ifn-%C3%AE-1-in-response-to-exogenous-dna
#4
Hongyan Sui, Ming Zhou, Hiromi Imamichi, Xiaoli Jiao, Brad T Sherman, H Clifford Lane, Tomozumi Imamichi
We previously identified Ku70, a subunit of a DNA repair protein complex, as a cytosolic DNA sensor that induces the production of interferon-λ1 (IFN-λ1) by human primary cells and cell lines. IFN-λ1 is a type III IFN and has similar antiviral activity to that of the type I IFNs (IFN-α and IFN-β). We observed that human embryonic kidney (HEK) 293T cells, which are deficient in the innate immune adaptor protein STING (stimulator of IFN genes), did not produce IFN-λ1 in response to DNA unless they were reconstituted with STING...
July 18, 2017: Science Signaling
https://www.readbyqxmd.com/read/28716076/klf8-regulates-left-right-asymmetric-patterning-through-modulation-of-kupffer-s-vesicle-morphogenesis-and-spaw-expression
#5
Che-Yi Lin, Ming-Yuan Tsai, Yu-Hsiu Liu, Yu-Fen Lu, Yi-Chung Chen, Yun-Ren Lai, Hsin-Chi Liao, Huang-Wei Lien, Chung-Hsiang Yang, Chang-Jen Huang, Sheng-Ping L Hwang
BACKGROUND: Although vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer's vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM...
July 17, 2017: Journal of Biomedical Science
https://www.readbyqxmd.com/read/28714864/an-erythroid-specific-atp2b4-enhancer-mediates-red-blood-cell-hydration-and-malaria-susceptibility
#6
Samuel Lessard, Emily Stern Gatof, Mélissa Beaudoin, Patrick G Schupp, Falak Sher, Adnan Ali, Sukhpal Prehar, Ryo Kurita, Yukio Nakamura, Esther Baena, Jonathan Ledoux, Delvac Oceandy, Daniel E Bauer, Guillaume Lettre
The lack of mechanistic explanations for many genotype-phenotype associations identified by GWAS precludes thorough assessment of their impact on human health. Here, we conducted an expression quantitative trait locus (eQTL) mapping analysis in erythroblasts and found erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of red blood cells (rbc). The same SNPs were previously associated with mean corpuscular hemoglobin concentration (MCHC) and susceptibility to severe malaria infection. We showed that Atp2b4-/- mice demonstrate increased MCHC, confirming ATP2B4 as the causal gene at this GWAS locus...
July 17, 2017: Journal of Clinical Investigation
https://www.readbyqxmd.com/read/28712454/crispr-cas9-mediated-scanning-for-regulatory-elements-required-for-hprt1-expression-via-thousands-of-large-programmed-genomic-deletions
#7
Molly Gasperini, Gregory M Findlay, Aaron McKenna, Jennifer H Milbank, Choli Lee, Melissa D Zhang, Darren A Cusanovich, Jay Shendure
The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion ("ScanDel"). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder...
July 1, 2017: American Journal of Human Genetics
https://www.readbyqxmd.com/read/28710427/the-essential-role-of-tap73-in-bortezomib-induced-apoptosis-in-p53-deficient-colorectal-cancer-cells
#8
Yasamin Dabiri, Sara Kalman, Clara-Marie Gürth, Jee Young Kim, Viola Mayer, Xinlai Cheng
Mutations in the tumor suppressor p53 are among the most highly occurring events in colorectal cancer (CRC). Such mutations have been shown to influence the sensitivity of cancer cells to chemotherapeutic agents. However their impact on the efficacy of the proteasomal inhibitor bortezomib remains controversial. We thus re-evaluated the toxicity of bortezomib in the CRC cell lines HCT116 wt (wild-type) and its p53-/- clone. Transient resistance to bortezomib treatment was observed in p53-null cells that was later accompanied by an increase in levels and nuclear translocation of TAp73, an isoform of the p53-homologue p73, as well as induction of apoptosis...
July 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28710008/identifying-synthetic-lethal-targets-using-crispr-cas9-system
#9
Jaspreet Kaur Dhanjal, Navaneethan Radhakrishnan, Durai Sundar
Synthetic lethality occurs when co-occurrence of two genetic events is unfavorable for the survival of the cell or organism. The conventional approach of high throughput screening of synthetic lethal targets using chemical compounds has been replaced by RNAi technology. CRISPR/Cas9, an RNA guided endonuclease system is the most recent technology for this work. Here, we have discussed the major considerations involved in designing a CRISPR/Cas9 based screening experiment for identification of synthetic lethal targets...
July 11, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28706995/disabling-cas9-by-an-anti-crispr-dna-mimic
#10
Jiyung Shin, Fuguo Jiang, Jun-Jie Liu, Nicolas L Bray, Benjamin J Rauch, Seung Hyun Baik, Eva Nogales, Joseph Bondy-Denomy, Jacob E Corn, Jennifer A Doudna
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established...
July 2017: Science Advances
https://www.readbyqxmd.com/read/28702647/an-enhanced-htert-promoter-driven-crispr-cas9-system-selectively-inhibits-the-progression-of-bladder-cancer-cells
#11
Xinbo Huang, Chengle Zhuang, Changshui Zhuang, Tiefu Xiong, Yawen Li, Yaoting Gui
The current therapies for treating tumors are lacking in efficacy and specificity. Synthetic biology principles may bring some new possible methods for curing cancer. Here we present a synthetic logic circuit based on the CRISPR/Cas9 system. The CRISPR/Cas9 technology has been applied in many biological fields, including cancer research. In this study, the expression of Cas9 nuclease was controlled indirectly by an enhanced hTERT promoter using the GAL4/upstream activating sequence (UAS) binding system. Cas9 was driven by 5XUAS, single guide RNA (sgRNA) was used to target mutant or wild-type HRAS, and the fusion gene GAL4-P65 was driven by the enhanced hTERT promoter...
July 12, 2017: Molecular BioSystems
https://www.readbyqxmd.com/read/28694465/generation-of-complement-protein-c3-deficient-pigs-by-crispr-cas9-mediated-gene-targeting
#12
Wei Zhang, Guan Wang, Ying Wang, Yong Jin, Lihua Zhao, Qiang Xiong, Lining Zhang, Lisha Mou, Rongfeng Li, Haiyuan Yang, Yifan Dai
Complement protein C3 is the pivotal component of the complement system. Previous studies have demonstrated that C3 has implications in various human diseases and exerts profound functions under certain conditions. However, the delineation of pathological and physiological roles of C3 has been hampered by the insufficiency of suitable animal models. In the present study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the C3 gene in porcine fetal fibroblasts...
July 10, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28694259/high-efficiency-non-mosaic-crispr-mediated-knock-in-and-mutations-in-f0-xenopus
#13
Yetki Aslan, Emmanuel Tadjuidje, Aaron M Zorn, Sang-Wook Cha
The revolution in CRISPR-mediated genome editing has enabled the mutation and insertion of virtually any DNA sequence, particularly in cell culture where selection can be used to recover relatively rare homologous recombination events. The efficient use of this technology in animal models still presents a number of challenges including the time to establish mutant lines, mosaic gene editing in founder animals, and low homologous recombination rates. Here we report a method for CRISPR-mediated genome editing in Xenopus oocytes with homology-dependent repair (HDR) that provides efficient non-mosaic targeted insertion of small DNA fragments of 40-50 nucleotides, in 4...
July 10, 2017: Development
https://www.readbyqxmd.com/read/28692648/zebrafish-slc30a10-deficiency-revealed-a-novel-compensatory-mechanism-of-atp2c1-in-maintaining-manganese-homeostasis
#14
Zhidan Xia, Jiayu Wei, Yingniang Li, Jia Wang, Wenwen Li, Kai Wang, Xiaoli Hong, Lu Zhao, Caiyong Chen, Junxia Min, Fudi Wang
Recent studies found that mutations in the human SLC30A10 gene, which encodes a manganese (Mn) efflux transporter, are associated with hypermanganesemia with dystonia, polycythemia, and cirrhosis (HMDPC). However, the relationship between Mn metabolism and HMDPC is poorly understood, and no specific treatments are available for this disorder. Here, we generated two zebrafish slc30a10 mutant lines using the CRISPR/Cas9 system. Compared to wild-type animals, mutant adult animals developed significantly higher systemic Mn levels, and Mn accumulated in the brain and liver of mutant embryos in response to exogenous Mn...
July 10, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28690838/recent-advances-in-high-throughput-approaches-to-dissect-enhancer-function
#15
REVIEW
David Santiago-Algarra, Lan T M Dao, Lydie Pradel, Alexandre España, Salvatore Spicuglia
The regulation of gene transcription in higher eukaryotes is accomplished through the involvement of transcription start site (TSS)-proximal (promoters) and -distal (enhancers) regulatory elements. It is now well acknowledged that enhancer elements play an essential role during development and cell differentiation, while genetic alterations in these elements are a major cause of human disease. Many strategies have been developed to identify and characterize enhancers. Here, we discuss recent advances in high-throughput approaches to assess enhancer activity, from the well-established massively parallel reporter assays to the recent clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based technologies...
2017: F1000Research
https://www.readbyqxmd.com/read/28688920/single-cell-rna-sequencing-reveals-an-altered-gene-expression-pattern-as-a-result-of-crispr-cas9-mediated-deletion-of-gene-33-mig6-and-chronic-exposure-to-hexavalent-chromium-in-human-lung-epithelial-cells
#16
Soyoung Park, Xiaowen Zhang, Cen Li, Changhong Yin, Jiangwei Li, John T Fallon, Weihua Huang, Dazhong Xu
Gene 33 (Mig6, ERRFI1) is an adaptor protein with multiple cellular functions. We recently reported that depletion of this protein promotes lung epithelial cell transformation induced by hexavalent chromium [Cr(VI)]. However, the early molecular events that mediate this process are not clear. In the present study, we used single-cell RNA sequencing to compare gene expression profiles between BEAS-2B lung epithelial cells chronically exposed to a sublethal dose of Cr(VI) with or without CRISPR/cas9-mediated deletion of Gene 33...
July 5, 2017: Toxicology and Applied Pharmacology
https://www.readbyqxmd.com/read/28687620/kindlin-2-regulates-the-growth-of-breast-cancer-tumors-by-activating-csf-1-mediated-macrophage-infiltration
#17
Khalid Sossey-Alaoui, Elzbieta Pluskota, Katarzyna Bialkowska, Dorota Szpak, Yvonne Parker, Chevaun Morrison, Daniel J Lindner, William P Schiemann, Edward F Plow
Interplay between tumor cells and host cells in the tumor microenvironment dictates the development of all cancers. In breast cancer, malignant cells educate host macrophages to adopt a pro-tumorigenic phenotype. In this study, we show how the integrin regulatory protein kindlin-2 (FERMT2) promotes metastatic progression of breast cancer through the recruitment and subversion of host macrophages. Kindlin-2 expression was elevated in BC biopsy tissues where its levels correlated with reduced patient survival...
July 7, 2017: Cancer Research
https://www.readbyqxmd.com/read/28687616/post-transcriptional-regulation-of-parg-mrna-by-hur-facilitates-dna-repair-and-resistance-to-parp-inhibitors
#18
Saswati N Chand, Mahsa Zarei, Matthew J Schiewer, Akshay R Kamath, Carmella Romeo, Shruti Lal, Joseph A Cozzitorto, Avinoam Nevler, Laura Scolaro, Eric Londin, Wei Jiang, Nicole Meisner-Kober, Michael J Pishvaian, Karen E Knudsen, Charles J Yeo, John M Pascal, Jordan M Winter, Jonathan R Brody
The majority of pancreatic ductal adenocarcinomas (PDA) rely on the mRNA stability factor HuR (ELAV-L1) to drive cancer growth and progression. Here we show that CRISPR-Cas9-mediated silencing of the HuR locus increases the relative sensitivity of PDA cells to PARP inhibitors (PARPi). PDA cells treated with PARPi stimulated translocation of HuR from the nucleus to the cytoplasm, specifically promoting stabilization of a new target, polyADP-ribose glycohydrolase (PARG) mRNA, by binding a unique sequence embedded in its 3' untranslated region (UTR)...
July 7, 2017: Cancer Research
https://www.readbyqxmd.com/read/28685575/biodegradable-amino-ester-nanomaterials-for-cas9-mrna-delivery-in-vitro-and-in-vivo
#19
Xinfu Zhang, Bin Li, Xiao Luo, Weiyu Zhao, Justin Jiang, Chengxiang Zhang, Min Gao, Xiaofang Chen, Yizhou Dong
Efficient and safe delivery of the CRISPR/Cas system is one of the key challenges for genome-editing applications in humans. Herein, we designed and synthesized a series of biodegradable lipidlike compounds containing ester groups for the delivery of mRNA-encoding Cas9. Two lead materials, termed N-methyl-1,3-propanediamine (MPA)-A and MPA-Ab, showed a tunable rate of biodegradation. MPA-A with linear ester chains was degraded dramatically faster than MPA-Ab with branched ester chains in the presence of esterase or in wild-type mice...
July 19, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28684413/a-crispr-cas9-approach-reveals-that-the-polymerase-activity-of-dna-polymerase-%C3%AE-is-dispensable-for-hiv-1-infection-in-dividing-and-nondividing-cells
#20
Russell W Goetze, Dong-Hyun Kim, Raymond F Schinazi, Baek Kim
Retrovirus integration into the host genome relies on several host enzymes, potentially including DNA polymerase β (Pol β). However, whether human Pol β; is essential for lentivirus replication in human cells is unclear. Here, we abolished Pol β expression by targeting its DNA polymerase domain with CRISPR/Cas9 in human monocytic THP-1 cells to investigate Pol β's role in HIV-1 transduction in both dividing and nondividing macrophage stages of THP-1 cells. Pol β-knockout was confirmed by enhanced sensitivity to methyl methanesulfonate - induced DNA damage...
July 6, 2017: Journal of Biological Chemistry
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