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Crispr screen

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https://www.readbyqxmd.com/read/28628606/splicing-stimulates-sirna-formation-at-drosophila-dna-double-strand-breaks
#1
Karin Merk, Marco Breinig, Romy Böttcher, Stefan Krebs, Helmut Blum, Michael Boutros, Klaus Förstemann
DNA double-strand breaks trigger the production of locus-derived siRNAs in fruit flies, human cells and plants. At least in flies, their biogenesis depends on active transcription running towards the break. Since siRNAs derive from a double-stranded RNA precursor, a major question is how broken DNA ends can generate matching sense and antisense transcripts. We performed a genome-wide RNAi-screen in cultured Drosophila cells, which revealed that in addition to DNA repair factors, many spliceosome components are required for efficient siRNA generation...
June 19, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28625976/a-genome-wide-crispr-screen-identifies-genes-critical-for-resistance-to-flt3-inhibitor-ac220
#2
Panpan Hou, Chao Wu, Yuchen Wang, Rui Qi, Dheeraj Bhavanasi, Zhixiang Zuo, Cedric Dos Santos, Shuliang Chen, Yu Chen, Hong Zheng, Hong Wang, Alexander E Perl, Deyin Guo, Jian Huang
Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. The mechanisms underlying drug resistance in AML are poorly understood. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are the most common molecular abnormality in AML. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3-ITD-positive and -negative AML patients and as maintenance therapy...
June 16, 2017: Cancer Research
https://www.readbyqxmd.com/read/28625679/phenotype-databases-for-genetic-screens-in-human-cells
#3
Benedikt Rauscher, Erica Valentini, Ulrike Hardeland, Michael Boutros
Genetic screens are powerful tools to identify components that make up biological systems. Perturbations introduced by methods such as RNA interference (RNAi) or CRISPR/Cas9-mediated genome editing lead to biological phenotypes that can be examined to understand the molecular function of genes in the cell. Over the years, many of such experiments have been conducted providing a wealth of knowledge about genotype-to-phenotype relationships. These data are a rich source of information and it is in a common interest to make them available in a simplified and integrated format...
June 15, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28625156/heteroduplex-cleavage-assay-for-screening-of-probable-zygosities-resulting-from-crispr-mutations-in-diploid-single-cell-lines
#4
Kyle D Luttgeharm, Kit-Sum Wong, Steve Siembieda
The most common gene editing methods, such as CRISPR, involve random repair of an induced double-stranded DNA break through the non-homologous end joining (NHEJ) repair pathway, resulting in small insertions/deletions. In diploid cells, these mutations can take on one of three zygosities: monoallelic, diallelic heterozygous, or diallelic homozygous. While many advances have been made in CRISPR delivery systems and gene editing efficiency, little work has been done to streamline detection of gene editing events...
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28611215/genome-wide-crispr-screen-identifies-hnrnpl-as-a-prostate-cancer-dependency-regulating-rna-splicing
#5
Teng Fei, Yiwen Chen, Tengfei Xiao, Wei Li, Laura Cato, Peng Zhang, Maura B Cotter, Michaela Bowden, Rosina T Lis, Shuang G Zhao, Qiu Wu, Felix Y Feng, Massimo Loda, Housheng Hansen He, X Shirley Liu, Myles Brown
Alternative RNA splicing plays an important role in cancer. To determine which factors involved in RNA processing are essential in prostate cancer, we performed a genome-wide CRISPR/Cas9 knockout screen to identify the genes that are required for prostate cancer growth. Functional annotation defined a set of essential spliceosome and RNA binding protein (RBP) genes, including most notably heterogeneous nuclear ribonucleoprotein L (HNRNPL). We defined the HNRNPL-bound RNA landscape by RNA immunoprecipitation coupled with next-generation sequencing and linked these RBP-RNA interactions to changes in RNA processing...
June 13, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28607761/multiplex-gene-regulation-by-crispr-ddcpf1
#6
Xiaochun Zhang, Jingman Wang, Qiuxiang Cheng, Xuan Zheng, Guoping Zhao, Jin Wang
The clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 system has been widely applied in both transcriptional regulation and epigenetic studies. However, for multiple targets, independent expression of multiple single guide RNAs (sgRNAs) is needed, which is less convenient. To address the problem, we employed a DNase-dead Cpf1 mutant (ddCpf1) for multiplex gene regulation. We demonstrated that ddCpf1 alone could be employed for gene repression in Escherichia coli, and the repression was more effective with CRISPR RNAs (crRNAs) specifically targeting to the template strand of its target genes, which was different from that of dCas9...
2017: Cell Discovery
https://www.readbyqxmd.com/read/28600547/modeling-congenital-hyperinsulinism-with-abcc8-deficient-human-embryonic-stem-cells-generated-by-crispr-cas9
#7
Dongsheng Guo, Haikun Liu, Aynisahan Ruzi, Ge Gao, Abbas Nasir, Yanli Liu, Fan Yang, Feima Wu, Guosheng Xu, Yin-Xiong Li
Congenital hyperinsulinism (CHI) is a rare genetic disorder characterized by excess insulin secretion, which results in hypoglycemia. Mutation of sulfonylurea receptor 1 (SUR1), encoded by the ABCC8 gene, is the main cause of CHI. Here, we captured the phenotype of excess insulin secretion through pancreatic differentiation of ABCC8-deficient stem cells generated by the CRISPR/Cas9 system. ABCC8-deficient insulin-producing cells secreted higher insulin than their wild-type counterparts, and the excess insulin secretion was rescued by nifedipine, octreotide and nicorandil...
June 9, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28599149/an-optimized-lentiviral-vector-system-for-conditional-rnai-and-efficient-cloning-of-microrna-embedded-short-hairpin-rna-libraries
#8
Felix F Adams, Dirk Heckl, Thomas Hoffmann, Steven R Talbot, Arnold Kloos, Felicitas Thol, Michael Heuser, Johannes Zuber, Axel Schambach, Adrian Schwarzer
RNA interference (RNAi) and CRISPR-Cas9-based screening systems have emerged as powerful and complementary tools to unravel genetic dependencies through systematic gain- and loss-of-function studies. In recent years, a series of technical advances helped to enhance the performance of virally delivered RNAi. For instance, the incorporation of short hairpin RNAs (shRNAs) into endogenous microRNA contexts (shRNAmiRs) allows the use of Tet-regulated promoters for synchronous onset of gene knockdown and precise interrogation of gene dosage effects...
September 2017: Biomaterials
https://www.readbyqxmd.com/read/28590031/progress-and-application-of-crispr-cas-technology-in-biological-and-biomedical-investigation
#9
Jiachen Lin, Yangzhong Zhou, Jiaqi Liu, Jia Chen, Weisheng Chen, Sen Zhao, Zhihong Wu, Nan Wu
Based on the tremendous progress of understanding of the CRISPR-Cas systems, the application CRISPR/Cas technology has been extended into increasing scenarios for biological and biomedical investigation. The potency of gene editing has been greatly improved by the rapid development of engineered Cas9 variants and modified CRISPR platforms. As advanced sequencing technology identified vast causative genetic basis for human diseases, CRISPR toolkits are now able to mediate precise genetic disruption or correction in vitro and in vivo...
June 7, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/28588028/the-lysine-methyltransferase-smyd2-methylates-the-kinase-domain-of-type-ii-receptor-bmpr2-and-stimulates-bone-morphogenetic-protein-signaling
#10
Shuman Gao, Zhiqiang Wang, Wencai Wang, Xueli Hu, Peilin Chen, Jiwen Li, Xin Hua Feng, Jiemin Wong, James X Du
Lysine methylation of chromosomal and nuclear proteins is a well-known mechanism of epigenetic regulation, but relatively little is known about the role of this protein modification in signal transduction. Using an RNAi-based functional screening of the SMYD family of lysine methyltransferases (KMTs), we identified SMYD2 as a KMT essential for robust bone morphogenic protein (BMP)- but not TGFβ-induced target gene expression in HaCaT keratinocyte cells. A role for SMYD2 in BMP-induced gene expression was confirmed by shRNA knockdown and CRISPR/Cas9-mediated knockout of SMYD2...
June 6, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28587596/a-systematic-evaluation-of-nucleotide-properties-for-crispr-sgrna-design
#11
Pei Fen Kuan, Scott Powers, Shuyao He, Kaiqiao Li, Xiaoyu Zhao, Bo Huang
BACKGROUND: CRISPR is a versatile gene editing tool which has revolutionized genetic research in the past few years. Optimizing sgRNA design to improve the efficiency of target/DNA cleavage is critical to ensure the success of CRISPR screens. RESULTS: By borrowing knowledge from oligonucleotide design and nucleosome occupancy models, we systematically evaluated candidate features computed from a number of nucleic acid, thermodynamic and secondary structure models on real CRISPR datasets...
June 6, 2017: BMC Bioinformatics
https://www.readbyqxmd.com/read/28581500/hyperactivation-of-hush-complex-function-by-charcot-marie-tooth-disease-mutation-in-morc2
#12
Iva A Tchasovnikarova, Richard T Timms, Christopher H Douse, Rhys C Roberts, Gordon Dougan, Robert E Kingston, Yorgo Modis, Paul J Lehner
Dominant mutations in the MORC2 gene have recently been shown to cause axonal Charcot-Marie-Tooth (CMT) disease, but the cellular function of MORC2 is poorly understood. Here, through a genome-wide CRISPR-Cas9-mediated forward genetic screen, we identified MORC2 as an essential gene required for epigenetic silencing by the HUSH complex. HUSH recruits MORC2 to target sites in heterochromatin. We exploited a new method, differential viral accessibility (DIVA), to show that loss of MORC2 results in chromatin decompaction at these target loci, which is concomitant with a loss of H3K9me3 deposition and transcriptional derepression...
June 5, 2017: Nature Genetics
https://www.readbyqxmd.com/read/28581493/crispr-stop-gene-silencing-through-base-editing-induced-nonsense-mutations
#13
Cem Kuscu, Mahmut Parlak, Turan Tufan, Jiekun Yang, Karol Szlachta, Xiaolong Wei, Rashad Mammadov, Mazhar Adli
CRISPR-Cas9-induced DNA damage may have deleterious effects at high-copy-number genomic regions. Here, we use CRISPR base editors to knock out genes by changing single nucleotides to create stop codons. We show that the CRISPR-STOP method is an efficient and less deleterious alternative to wild-type Cas9 for gene-knockout studies. Early stop codons can be introduced in ∼17,000 human genes. CRISPR-STOP-mediated targeted screening demonstrates comparable efficiency to WT Cas9, which indicates the suitability of our approach for genome-wide functional screenings...
June 5, 2017: Nature Methods
https://www.readbyqxmd.com/read/28573017/a-crispr-cas9-high-throughput-genome-editing-toolkit-for-kinetoplastids
#14
Tom Beneke, Ross Madden, Laura Makin, Jessica Valli, Jack Sunter, Eva Gluenz
Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit...
May 2017: Royal Society Open Science
https://www.readbyqxmd.com/read/28569207/seed-effect-modeling-improves-the-consistency-of-genome-wide-loss-of-function-screens-and-identifies-synthetic-lethal-vulnerabilities-in-cancer-cells
#15
Alok Jaiswal, Gopal Peddinti, Yevhen Akimov, Krister Wennerberg, Sergey Kuznetsov, Jing Tang, Tero Aittokallio
BACKGROUND: Genome-wide loss-of-function profiling is widely used for systematic identification of genetic dependencies in cancer cells; however, the poor reproducibility of RNA interference (RNAi) screens has been a major concern due to frequent off-target effects. Currently, a detailed understanding of the key factors contributing to the sub-optimal consistency is still a lacking, especially on how to improve the reliability of future RNAi screens by controlling for factors that determine their off-target propensity...
June 1, 2017: Genome Medicine
https://www.readbyqxmd.com/read/28558381/aldolase-b-overexpression-is-associated-with-poor-prognosis-and-promotes-tumor-progression-by-epithelial-mesenchymal-transition-in-colorectal-adenocarcinoma
#16
Qingguo Li, Yaqi Li, Junyan Xu, Sheng Wang, Ye Xu, Xinxiang Li, Sanjun Cai
BACKGROUND: Glycolysis is considered to be the root of cancer development and progression, which involved a multi-step enzymatic reaction. Our study aimed at figuring out which glycolysis enzyme participates in the development of colorectal cancer and its possible mechanisms. METHODS: We firstly screened out Aldolase B (ALDOB) by performing qRT-PCR arrays of glycolysis-related genes in five paired liver metastasis and primary colorectal tissues, and further detected ALDOB protein with immunohistochemistry in tissue microarray (TMA) consisting of 229 samples from stage I-III colorectal cancer patients...
2017: Cellular Physiology and Biochemistry
https://www.readbyqxmd.com/read/28558198/engineered-crispr-systems-for-next-generation-gene-therapies
#17
Michael Pineda, Farzaneh Moghadam, Mo R Ebrahimkhani, Samira Kiani
An ideal in vivo gene therapy platform provides safe, reprogrammable, and precise strategies which modulate cell and tissue gene regulatory networks with a high temporal and spatial resolution. Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adoptive immune system, and its CRISPR-associated protein 9 (Cas9), have gained attention for the ability to target and modify DNA sequences on demand with unprecedented flexibility and precision. The precision and programmability of Cas9 is derived from its complexation with a guide-RNA (gRNA) that is complementary to a desired genomic sequence...
June 7, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28557353/generation-of-carbamoyl-phosphate-synthetase-1-reporter-cell-lines-for-the-assessment-of-ammonia-metabolism
#18
Yi Wang, Le Chang, Jiahui Zhai, Qiao Wu, Donggen Wang, Yunfang Wang
Both primary hepatocytes and stem cells-derived hepatocyte-like cells (HLCs) are major sources for bioartificial liver (BAL). Maintenance of hepatocellular functions and induction of functional maturity of HLCs are critical for BAL's support effect. It remains difficult to assess and improve detoxification functions inherent to hepatocytes, including ammonia clearance. Here, we aim to assess ammonia metabolism and identify ammonia detoxification enhancer by developing an imaging strategy. In hepatoma cell line HepG2, and immortalized hepatic cell line LO2, carbamoyl phosphate synthetase 1 (CPS1) gene, the first enzyme of ammonia-eliminating urea cycle, was labelled with fluorescence protein via CRISPR/Cas9 system...
May 30, 2017: Journal of Cellular and Molecular Medicine
https://www.readbyqxmd.com/read/28543738/crispr-editing-in-biological-and-biomedical-investigation
#19
Xing-Da Ju, Jing Xu, Zhong Sheng Sun
The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR--associated protein) system, a prokaryotic RNA-based adaptive immune system against viral infection, is emerging as a powerful genome editing tool in broad research areas. To further improve and expand its functionality, various CRISPR delivery strategies have been tested and optimized, and key CRISPR system components such as Cas protein have been engineered with different purposes. Benefiting from more in-depth understanding and further development of CRISPR, versatile CRISPR-based platforms for genome editing have been rapidly developed to advance investigations in biology and biomedicine...
May 20, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/28541456/crisprcloud-a-secure-cloud-based-pipeline-for-crispr-pooled-screen-deconvolution
#20
Hyun-Hwan Jeong, Seon Young Kim, Maxime Wc Rousseaux, Huda Y Zoghbi, Zhandong Liu
Summary: We present a user-friendly, cloud-based, data analysis pipeline for the deconvolution of pooled screening data. This tool, CRISPRcloud, serves a dual purpose of extracting, clustering and analyzing raw next generation sequencing files derived from pooled screening experiments while at the same time presenting them in a user-friendly way on a secure web-based platform. Moreover, CRISPRcloud serves as a useful web-based analysis pipeline for reanalysis of pooled CRISPR screening datasets...
May 24, 2017: Bioinformatics
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