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Inducible crispr

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https://www.readbyqxmd.com/read/29340096/crispr-cas9-mediated-reversibly-immortalized-mouse-bone-marrow-stromal-stem-cells-bmscs-retain-multipotent-features-of-mesenchymal-stem-cells-mscs
#1
Xue Hu, Li Li, Xinyi Yu, Ruyi Zhang, Shujuan Yan, Zongyue Zeng, Yi Shu, Chen Zhao, Xingye Wu, Jiayan Lei, Yasha Li, Wenwen Zhang, Chao Yang, Ke Wu, Ying Wu, Liping An, Shifeng Huang, Xiaojuan Ji, Cheng Gong, Chengfu Yuan, Linghuan Zhang, Wei Liu, Bo Huang, Yixiao Feng, Bo Zhang, Rex C Haydon, Hue H Luu, Russell R Reid, Michael J Lee, Jennifer Moriatis Wolf, Zebo Yu, Tong-Chuan He
Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic progenitor cells that can undergo self-renewal and differentiate into multi-lineages. Bone marrow stromal stem cells (BMSCs) represent one of the most commonly-used MSCs. In order to overcome the technical challenge of maintaining primary BMSCs in long-term culture, here we seek to establish reversibly immortalized mouse BMSCs (imBMSCs). By exploiting CRISPR/Cas9-based homology-directed-repair (HDR) mechanism, we target SV40T to mouse Rosa26 locus and efficiently immortalize mouse BMSCs (i...
December 19, 2017: Oncotarget
https://www.readbyqxmd.com/read/29333573/simultaneous-site-directed-mutagenesis-of-duplicated-loci-in-soybean-using-a-single-guide-rna
#2
Yuhei Kanazashi, Aya Hirose, Ippei Takahashi, Masafumi Mikami, Masaki Endo, Sakiko Hirose, Seiichi Toki, Akito Kaga, Ken Naito, Masao Ishimoto, Jun Abe, Tetsuya Yamada
Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T2 seeds. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD)...
January 15, 2018: Plant Cell Reports
https://www.readbyqxmd.com/read/29331077/optimized-paired-sgrna-cas9-cloning-and-expression-cassette-triggers-high-efficiency-multiplex-genome-editing-in-kiwifruit
#3
Zupeng Wang, Shuaibin Wang, Dawei Li, Qiong Zhang, Li Li, Caihong Zhong, Yifei Liu, Hongwen Huang
Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing...
January 13, 2018: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/29330306/an-rb-family-independent-e2f3-transcription-factor-variant-impairs-stat5-signaling-and-mammary-gland-remodeling-during-pregnancy-in-mice
#4
Yang Liao, Wei Du
E2F transcription factors are regulated by binding to the retinoblastoma (Rb) tumor suppressor family of proteins. Previously, we reported an E2FLQ mutation that disrupts the binding with Rb proteins without affecting E2F's transcriptional activity. We also showed that mouse embryonic fibroblasts (MEFs) with an E2F3LQ mutation exhibit increased E2F activity and more rapid cell proliferation. In this report, we analyzed E2F3LQ mice to further characterize the in vivo consequences of Rb family-independent E2F3 activity...
January 12, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29330288/epigenetically-regulated-chromosome-14q32-mirna-cluster-induces-metastasis-and-predicts-poor-prognosis-in-lung-adenocarcinoma-patients
#5
Margarita Gonzalez-Vallinas, Manuel Rodriguez-Paredes, Marco Albrecht, Carsten Sticht, Damian Stichel, Julian Gutekunst, Adriana Pitea, Steffen Sass, Francisco J Sánchez-Rivera, Justo L Bermejo, Jennifer Schmitt, Carolina De La Torre, Arne Warth, Fabian Theis, Nikola Mueller, Norbert Gretz, Thomas Muley, Michael Meister, Darjus F Tschaharganeh, Peter Schirmacher, Franziska Matthäus, Kai Breuhahn
Most lung cancer deaths are related to metastases, which indicates the necessity of detecting and inhibiting tumor cell dissemination. Here, we aimed to identify microRNAs (miRNAs) involved in metastasis of lung adenocarcinoma as prognostic biomarkers and therapeutic targets. To that end, lymph node metastasis-associated miRNAs were identified in The Cancer Genome Atlas (TCGA) lung adenocarcinoma patient cohort (sequencing data; n=449) and subsequently validated by RT-qPCR in an independent clinical cohort (n=108)...
January 12, 2018: Molecular Cancer Research: MCR
https://www.readbyqxmd.com/read/29329593/targeting-ectodysplasin-promotor-by-crispr-dcas9-effector-effectively-induces-the-reprogramming-of-human-bone-marrow-derived-mesenchymal-stem-cells-into-sweat-gland-like-cells
#6
Sujing Sun, Jun Xiao, Jiahui Huo, Zhijun Geng, Kui Ma, Xiaoyan Sun, Xiaobing Fu
BACKGROUND: Patients with a deep burn injury are characterized by losing the function of perspiration and being unable to regenerate the sweat glands. Because of their easy accession, multipotency, and lower immunogenicity, bone marrow-derived mesenchymal stem cells (BM-MSCs) represent as an ideal biological source for cell therapy. The aim of this study was to identify whether targeting the promotor of ectodysplasin (EDA) by CRISPR/dCas9-effector (dCas9-E) could induce the BM-MSCs to differentiate into sweat gland-like cells (SGCs)...
January 12, 2018: Stem Cell Research & Therapy
https://www.readbyqxmd.com/read/29326299/targeting-mutant-kras-with-crispr-cas9-controls-tumor-growth
#7
Hyongbum Henry Kim, Wonjoo Kim, Sangeun Lee, Han Sang Kim, Minjung Song, Yong Hoon Cha, Young-Hoon Kim, Jeonghong Shin, Eun-Seo Lee, Yeonsoo Joo, Jae J Song, Eun Ju Choi, Jae W Choi, Jinu Lee, Moonkyung Kang, Jong In Yook, Min Goo Lee, Yeon-Soo Kim, Soonmyung Paik
KRAS is the most frequently mutated oncogene in human tumors and its activating mutations represents important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here we harnessed CRISPR to specifically target mutant KRAS alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA...
January 11, 2018: Genome Research
https://www.readbyqxmd.com/read/29325827/efficient-oligo-nucleotide-mediated-crispr-cas9-gene-editing-in-aspergilli
#8
Christina S Nødvig, Jakob B Hoof, Martin E Kogle, Zofia D Jarczynska, Jan Lehmbeck, Dorte K Klitgaard, Uffe H Mortensen
CRISPR-Cas9 technologies are revolutionizing fungal gene editing. Here we show that survival of specific Cas9/sgRNA mediated DNA double strand breaks (DSBs) depends on the non-homologous end-joining, NHEJ, DNA repair pathway and we use this observation to develop a tool to assess protospacer efficiency in Aspergillus nidulans. Moreover, we show that in NHEJ deficient strains, highly efficient marker-free gene targeting can be performed. Indeed, we show that even single-stranded oligo nucleotides efficiently works as repair templates of specific Cas9/sgRNA induced DNA DSBs in A...
January 8, 2018: Fungal Genetics and Biology: FG & B
https://www.readbyqxmd.com/read/29325430/rapid-and-sensitive-assessment-of-globin-chains-for-gene-and-cell-therapy-of-haemoglobinopathies
#9
Constantinos Christos Loucari, Petros Patsali, Thamar B van Dijk, Coralea Stephanou, Panayiota Papasavva, Maria Zanti, Ryo Kurita, Yukio Nakamura, Soteroulla Christou, Maria Sitarou, Sjaak Philipsen, Carsten Werner Lederer, Marina Kleanthous
The β-haemoglobinopathies sickle cell anaemia and β-thalassaemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains, because it indicates the level of anaemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer either from long run times, high sample requirements or inability to separate murine from human β-globin chains...
January 12, 2018: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/29324767/glass-promotes-the-differentiation-of-neuronal-and-non-neuronal-cell-types-in-the-drosophila-eye
#10
Carolyn A Morrison, Hao Chen, Tiffany Cook, Stuart Brown, Jessica E Treisman
Transcriptional regulators can specify different cell types from a pool of equivalent progenitors by activating distinct developmental programs. The Glass transcription factor is expressed in all progenitors in the developing Drosophila eye, and is maintained in both neuronal and non-neuronal cell types. Glass is required for neuronal progenitors to differentiate as photoreceptors, but its role in non-neuronal cone and pigment cells is unknown. To determine whether Glass activity is limited to neuronal lineages, we compared the effects of misexpressing it in neuroblasts of the larval brain and in epithelial cells of the wing disc...
January 11, 2018: PLoS Genetics
https://www.readbyqxmd.com/read/29323274/apobec3-induces-mutations-during-repair-of-crispr-cas9-generated-dna-breaks
#11
Liqun Lei, Hongquan Chen, Wei Xue, Bei Yang, Bian Hu, Jia Wei, Lijie Wang, Yiqiang Cui, Wei Li, Jianying Wang, Lei Yan, Wanjing Shang, Jimin Gao, Jiahao Sha, Min Zhuang, Xingxu Huang, Bin Shen, Li Yang, Jia Chen
The APOBEC-AID family of cytidine deaminase prefers single-stranded nucleic acids for cytidine-to-uridine deamination. Single-stranded nucleic acids are commonly involved in the DNA repair system for breaks generated by CRISPR-Cas9. Here, we show in human cells that APOBEC3 can trigger cytidine deamination of single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through homology-directed repair (HDR) of Cas9-generated double-strand breaks. In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks in human cells can be further processed to yield mutations mainly involving insertions or deletions (indels)...
January 2018: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/29321585/site-specific-randomization-of-the-endogenous-genome-by-a-regulatable-crispr-cas9-piggybac-system-in-human-cells
#12
Kentaro Ishida, Huaigeng Xu, Noriko Sasakawa, Mandy Siu Yu Lung, Julia Alexandra Kudryashev, Peter Gee, Akitsu Hotta
Randomized mutagenesis at an endogenous chromosomal locus is a promising approach for protein engineering, functional assessment of regulatory elements, and modeling genetic variations. In mammalian cells, however, it is challenging to perform site-specific single-nucleotide substitution with single-stranded oligodeoxynucleotide (ssODN) donor templates due to insufficient homologous recombination and the infeasibility of positive selection. Here, we developed a DNA transposon based CRISPR-Cas9 regulated transcription and nuclear shuttling (CRONUS) system that enables the stable transduction of CRISPR-Cas9/sgRNA in broad cell types, but avoids undesired genome cleavage in the absence two chemical inducing molecules...
January 10, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29320733/dna-unwinding-is-the-primary-determinant-of-crispr-cas9-activity
#13
Shanzhong Gong, Helen Hong Yu, Kenneth A Johnson, David W Taylor
Bacterial adaptive immunity utilizes RNA-guided surveillance complexes comprising Cas proteins together with CRISPR RNAs (crRNAs) to target foreign nucleic acids for destruction. Cas9, a type II CRISPR-Cas effector complex, can be programed with a single-guide RNA that base pairs with the target strand of dsDNA, displacing the non-target strand to create an R-loop, where the HNH and the RuvC nuclease domains cleave opposing strands. While many structural and biochemical studies have shed light on the mechanism of Cas9 cleavage, a clear unifying model has yet to emerge...
January 9, 2018: Cell Reports
https://www.readbyqxmd.com/read/29317080/effective-regeneration-of-dystrophic-muscle-using-autologous-ipsc-derived-progenitors-with-crispr-cas9-mediated-precise-correction
#14
Mackenzie Hagan, Muhammad Ashraf, Il-Man Kim, Neal L Weintraub, Yaoliang Tang
Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disease caused by a lack of dystrophin, which eventually leads to apoptosis of muscle cells and impaired muscle contractility. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) gene editing of induced pluripotent stem cells (IPSC) offers the potential to correct the DMD gene defect and create healthy IPSC for autologous cell transplantation without causing immune activation. However, IPSC carry a risk of tumor formation, which can potentially be mitigated by differentiation of IPSC into myogenic progenitor cells (MPC)...
January 2018: Medical Hypotheses
https://www.readbyqxmd.com/read/29316444/force-generation-via-%C3%AE-cardiac-myosin-titin-and-%C3%AE-actinin-drives-cardiac-sarcomere-assembly-from-cell-matrix-adhesions
#15
Anant Chopra, Matthew L Kutys, Kehan Zhang, William J Polacheck, Calvin C Sheng, Rebeccah J Luu, Jeroen Eyckmans, J Travis Hinson, Jonathan G Seidman, Christine E Seidman, Christopher S Chen
Truncating mutations in the sarcomere protein titin cause dilated cardiomyopathy due to sarcomere insufficiency. However, it remains mechanistically unclear how these mutations decrease sarcomere content in cardiomyocytes. Utilizing human induced pluripotent stem cell-derived cardiomyocytes, CRISPR/Cas9, and live microscopy, we characterize the fundamental mechanisms of human cardiac sarcomere formation. We observe that sarcomerogenesis initiates at protocostameres, sites of cell-extracellular matrix adhesion, where nucleation and centripetal assembly of α-actinin-2-containing fibers provide a template for the fusion of Z-disk precursors, Z bodies, and subsequent striation...
January 8, 2018: Developmental Cell
https://www.readbyqxmd.com/read/29316264/heme-oxygenase-1-affects-generation-and-spontaneous-cardiac-differentiation-of-induced-pluripotent-stem-cells
#16
Jacek Stepniewski, Tomasz Pacholczak, Aniela Skrzypczyk, Maciej Ciesla, Agata Szade, Krzysztof Szade, Romain Bidanel, Agnieszka Langrzyk, Radoslaw Grochowski, Felix Vandermeeren, Neli Kachamakova-Trojanowska, Mateusz Jez, Grazyna Drabik, Mahito Nakanishi, Alicja Jozkowicz, Jozef Dulak
Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1-/- fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1+/+ cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3σ was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency...
January 9, 2018: IUBMB Life
https://www.readbyqxmd.com/read/29312555/the-relationship-between-dna-methylation-and-reprimo-gene-expression-in-gastric-cancer-cells
#17
Junzhong Lai, Hanze Wang, Qianping Luo, Shanlu Huang, Shujin Lin, Yansong Zheng, Qi Chen
Reprimo (RPRM) is a tumor suppressor involved in the development of a number of malignant tumors including gastric cancer which is highly related to its gene hypermethylation. However, the regulation of RPRM gene expression by DNA methylation in gastric cancer is not well understood. We examined the RPRM gene methylation in gastric cancer tissues or plasma samples by bisulfite sequencing, and investigated the relationship between DNA methylation and the RPRM gene expression by quantitative reverse transcription-PCR and Western blotting...
December 12, 2017: Oncotarget
https://www.readbyqxmd.com/read/29311693/combined-shrna-over-crispr-cas9-as-a-methodology-to-detect-off-target-effects-and-a-potential-compensatory-mechanism
#18
Liat Peretz, Elazar Besser, Renana Hajbi, Natania Casden, Dan Ziv, Nechama Kronenberg, Liat Ben Gigi, Sahar Sweetat, Saleh Khawaled, Rami Aqeilan, Oded Behar
Inhibition of genes is a powerful approach to study their function. While RNA interference is a widely used method to achieve this goal, mounting evidence indicates that such an approach is prone to off-target effects. An alternative approach to gene function inhibition is genetic mutation, such as the CRISPR/cas9 method. A recent report, however, demonstrated that genetic mutation and inhibition of gene expression do not always give corresponding results. This can be explained by off-target effects, but it was recently shown, at least in one case, that these differences are the result of a compensatory mechanism induced only by genetic mutation...
January 8, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29311600/depdc5-deficiency-contributes-to-resistance-to-leucine-starvation-via-p62-accumulation-in-hepatocellular-carcinoma
#19
Yuki Mizuno, Shu Shimada, Yoshimitsu Akiyama, Shuichi Watanabe, Tomomi Aida, Kosuke Ogawa, Hiroaki Ono, Yusuke Mitsunori, Daisuke Ban, Atsushi Kudo, Shigeki Arii, Shoji Yamaoka, Minoru Tanabe, Shinji Tanaka
Decrease in blood concentration of branched-chain amino acids, especially leucine, is known to promote liver carcinogenesis in patients with chronic liver disease, but the mechanism is unclear. We herein established hepatocellular carcinoma (HCC) cells knocked out for DEPDC5 by using the CRISPR/Cas9 system, and elucidated that cell viability of the DEPDC5 knockout (DEPDC5-KO) cells was higher than that of the DEPDC5 wild-type (DEPDC5-WT) under leucine starvation. Considering that autophagy deficiency might be involved in acquired resistance to leucine deprivation, we observed reduction of LC3-II followed by accumulation of p62 in the DEPDC5-KO, which induced reactive oxygen species (ROS) tolerance...
January 8, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29311364/isg15-induced-il-10-is-a-novel-anti-inflammatory-myeloid-axis-disrupted-during-active-tuberculosis
#20
Paula Fernandes Dos Santos, Johan Van Weyenbergh, Murilo Delgobo, Daniel de Oliveira Patricio, Brian J Ferguson, Rodrigo Guabiraba, Tim Dierckx, Soraya Maria Menezes, André Báfica, Daniel Santos Mansur
IFN-stimulated gene 15 (ISG15) deficiency in humans leads to severe IFNopathies and mycobacterial disease, the latter being previously attributed to its extracellular cytokine-like activity. In this study, we demonstrate a novel role for secreted ISG15 as an IL-10 inducer, unique to primary human monocytes. A balanced ISG15-induced monocyte/IL-10 versus lymphoid/IFN-γ expression, correlating with p38 MAPK and PI3K signaling, was found using targeted in vitro and ex vivo systems analysis of human transcriptomic datasets...
January 8, 2018: Journal of Immunology: Official Journal of the American Association of Immunologists
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