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https://www.readbyqxmd.com/read/28228480/a-stable-but-reversible-integrated-surrogate-reporter-for-assaying-crispr-cas9-stimulated-homology-directed-repair
#1
Yahong Wen, Grace Liao, Thomas Pritchard, Ting-Ting Zhao, Jon P Connelly, Shondra M Pruett-Miller, Valerie Blanc, Nicholas O Davidson, Blair B Madison
The discovery and application of CRISPR/Cas9 technology for genome editing has greatly accelerated targeted mutagenesis in a variety of organisms. CRISPR/Cas9-mediated site-specific cleavage is typically exploited for the generation of insertions or deletions (indels) following aberrant dsDNA repair via the endogenous non-homology end-joining (NHEJ) pathway, or alternatively, for enhancing homology directed repair (HDR) to facilitate the generation of a specific mutation (or knock-in). However, there is a need for efficient cellular assays that can measure Cas9/guide RNA (gRNA) activity...
February 22, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28218837/rapid-and-efficient-genome-editing-in-staphylococcus-aureus-by-using-an-engineered-crispr-cas9-system
#2
Weizhong Chen, Yifei Zhang, Won-Sik Yeo, Taeok Bae, Quanjiang Ji
Staphylococcus aureus, a major human pathogen, has been the cause of serious infectious diseases with a high mortality rate. Although genetics is a key means to study S. aureus physiology, such as drug resistance and pathogenesis, genetic manipulation in S. aureus is always time consuming and labor intensive. Here, we report a CRISPR/Cas9 system (pCasSA) for rapid and efficient genome editing, including gene deletion, insertion and single-base substitution mutation in S. aureus. The designed pCasSA system is amenable to assembly of spacers and repair arms by Golden Gate assembly and Gibson assembly, respectively, enabling rapid construction of the plasmids for editing...
February 20, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28215525/explicit-modeling-of-sirna-dependent-on-and-off-target-repression-improves-the-interpretation-of-screening-results
#3
Andrea Riba, Mario Emmenlauer, Amy Chen, Frederic Sigoillot, Feng Cong, Christoph Dehio, Jeremy Jenkins, Mihaela Zavolan
RNAi is broadly used to map gene regulatory networks, but the identification of genes that are responsible for the observed phenotypes is challenging, as small interfering RNAs (siRNAs) simultaneously downregulate the intended on targets and many partially complementary off targets. Additionally, the scarcity of publicly available control datasets hinders the development and comparative evaluation of computational methods for analyzing the data. Here, we introduce PheLiM (https://github.com/andreariba/PheLiM), a method that uses predictions of siRNA on- and off-target downregulation to infer gene-specific contributions to phenotypes...
February 22, 2017: Cell Systems
https://www.readbyqxmd.com/read/28211892/high-throughput-in-situ-cell-electroporation-microsystem-for-parallel-delivery-of-single-guide-rnas-into-mammalian-cells
#4
Shengtai Bian, Yicen Zhou, Yawei Hu, Jing Cheng, Xiaofang Chen, Youchun Xu, Peng Liu
Arrayed genetic screens mediated by the CRISPR/Cas9 technology with single guide RNA (sgRNA) libraries demand a high-throughput platform capable of transfecting diverse cell types at a high efficiency in a genome-wide scale for detection and analysis of sophisticated cellular phenotypes. Here we developed a high-throughput in situ cell electroporation (HiCEP) microsystem which leveraged the superhydrophobic feature of the microwell array to achieve individually controlled conditions in each microwell and coupled an interdigital electrode array chip with the microwells in a modular-based scheme for highly efficient delivery of exogenous molecules into cells...
February 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28207002/detecting-natural-adaptation-of-the-streptococcus-thermophilus-crispr-cas-systems-in-research-and-classroom-settings
#5
Alexander P Hynes, Marie-Laurence Lemay, Luc Trudel, Hélène Deveau, Michel Frenette, Denise M Tremblay, Sylvain Moineau
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been adapted into a powerful genome-editing tool. The basis for the flexibility of the tool lies in the adaptive nature of CRISPR-Cas as a bacterial immune system. Here, we describe a protocol to experimentally demonstrate the adaptive nature of this bacterial immune system by challenging the model organism for the study of CRISPR adaptation, Streptococcus thermophilus, with phages in order to detect natural CRISPR immunization...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28207001/genome-editing-using-facs-enrichment-of-nuclease-expressing-cells-and-indel-detection-by-amplicon-analysis
#6
Lindsey A Lonowski, Yoshiki Narimatsu, Anjum Riaz, Catherine E Delay, Zhang Yang, Francesco Niola, Katarzyna Duda, Elke A Ober, Henrik Clausen, Hans H Wandall, Steen H Hansen, Eric P Bennett, Morten Frödin
This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28205168/crispr-cas9-mediated-generation-of-niemann-pick-c1-knockout-cell-line
#7
Ximing Du, Ivan Lukmantara, Hongyuan Yang
Generating a cholesterol storage phenotype of Niemann-Pick Type C (NPC) disease is important for investigating the mechanisms of intracellular cholesterol trafficking, as well as screening drugs for potential treatment of NPC disease. The use of the CRISPR/Cas9 technology to knockout specific genes within the genome of mammals has become routine in the past few years. Here, we describe a protocol for producing a cellular NPC cholesterol storage phenotype in HeLa cells using the CRISPR-Cas9 system to disrupt the NPC1 gene...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28202911/what-rheumatologists-need-to-know-about-crispr-cas9
#8
REVIEW
Gary J Gibson, Maozhou Yang
CRISPR/Cas9 genome editing technology has taken the research world by storm since its use in eukaryotes was first proposed in 2012. Publications describing advances in technology and new applications have continued at an unrelenting pace since that time. In this Review, we discuss the application of CRISPR/Cas9 for creating gene mutations - the application that initiated the current avalanche of interest - and new developments that have largely answered initial concerns about its specificity and ability to introduce new gene sequences...
February 9, 2017: Nature Reviews. Rheumatology
https://www.readbyqxmd.com/read/28192788/metabolic-gatekeeper-function-of-b-lymphoid-transcription-factors
#9
Lai N Chan, Zhengshan Chen, Daniel Braas, Jae-Woong Lee, Gang Xiao, Huimin Geng, Kadriye Nehir Cosgun, Christian Hurtz, Seyedmehdi Shojaee, Valeria Cazzaniga, Hilde Schjerven, Thomas Ernst, Andreas Hochhaus, Steven M Kornblau, Marina Konopleva, Miles A Pufall, Giovanni Cazzaniga, Grace J Liu, Thomas A Milne, H Phillip Koeffler, Theodora S Ross, Isidro Sánchez-García, Arndt Borkhardt, Keith R Yamamoto, Ross A Dickins, Thomas G Graeber, Markus Müschen
B-lymphoid transcription factors, such as PAX5 and IKZF1, are critical for early B-cell development, yet lesions of the genes encoding these transcription factors occur in over 80% of cases of pre-B-cell acute lymphoblastic leukaemia (ALL). The importance of these lesions in ALL has, until now, remained unclear. Here, by combining studies using chromatin immunoprecipitation with sequencing and RNA sequencing, we identify a novel B-lymphoid program for transcriptional repression of glucose and energy supply...
February 13, 2017: Nature
https://www.readbyqxmd.com/read/28181494/microenvironment-derived-factors-driving-metastatic-plasticity-in-melanoma
#10
Isabella S Kim, Silja Heilmann, Emily R Kansler, Yan Zhang, Milena Zimmer, Kajan Ratnakumar, Robert L Bowman, Theresa Simon-Vermot, Myles Fennell, Ralph Garippa, Liang Lu, William Lee, Travis Hollmann, Joao B Xavier, Richard M White
Cellular plasticity is a state in which cancer cells exist along a reversible phenotypic spectrum, and underlies key traits such as drug resistance and metastasis. Melanoma plasticity is linked to phenotype switching, where the microenvironment induces switches between invasive/MITF(LO) versus proliferative/MITF(HI) states. Since MITF also induces pigmentation, we hypothesize that macrometastatic success should be favoured by microenvironments that induce a MITF(HI)/differentiated/proliferative state. Zebrafish imaging demonstrates that after extravasation, melanoma cells become pigmented and enact a gene expression program of melanocyte differentiation...
February 9, 2017: Nature Communications
https://www.readbyqxmd.com/read/28178529/atxn1l-cic-and-ets-transcription-factors-modulate-sensitivity-to-mapk-pathway-inhibition
#11
Belinda Wang, Elsa Beyer Krall, Andrew James Aguirre, Miju Kim, Hans Ragnar Widlund, Mihir Bhavik Doshi, Ewa Sicinska, Rita Sulahian, Amy Goodale, Glenn Spencer Cowley, Federica Piccioni, John Gerard Doench, David Edward Root, William Chun Hahn
Intrinsic resistance and RTK-RAS-MAPK pathway reactivation has limited the effectiveness of MEK and RAF inhibitors (MAPKi) in RAS- and RAF-mutant cancers. To identify genes that modulate sensitivity to MAPKi, we performed genome-scale CRISPR-Cas9 loss-of-function screens in two KRAS mutant pancreatic cancer cell lines treated with the MEK1/2 inhibitor trametinib. Loss of CIC, a transcriptional repressor of ETV1, ETV4, and ETV5, promoted survival in the setting of MAPKi in cancer cells derived from several lineages...
February 7, 2017: Cell Reports
https://www.readbyqxmd.com/read/28176758/lncrna-ak023948-is-a-positive-regulator-of-akt
#12
Pratirodh Koirala, Jianguo Huang, Tsui-Ting Ho, Fangting Wu, Xianfeng Ding, Yin-Yuan Mo
Despite the overwhelming number of human long non-coding RNAs (lncRNAs) reported so far, little is known about their physiological functions for the majority of them. The present study uses a CRISPR/Cas9-based synergistic activation mediator (SAM) system to identify potential lncRNAs capable of regulating AKT activity. Among lncRNAs identified from this screen, we demonstrate that AK023948 is a positive regulator for AKT. Knockout of AK023948 suppresses, whereas rescue with AK023948 restores the AKT activity...
February 8, 2017: Nature Communications
https://www.readbyqxmd.com/read/28175410/140%C3%A2-genome-wide-crispr-cas9-knockout-screens-in-human-glioblastoma-identify-genetic-vulnerabilities
#13
Imran Noorani
No abstract text is available yet for this article.
August 1, 2016: Neurosurgery
https://www.readbyqxmd.com/read/28174584/an-efficient-visual-screen-for-crispr-cas9-activity-in-arabidopsis-thaliana
#14
Florian Hahn, Otho Mantegazza, André Greiner, Peter Hegemann, Marion Eisenhut, Andreas P M Weber
The CRISPR/Cas9 system enables precision editing of the genome of the model plant Arabidopsis thaliana and likely of any other organism. Tools and methods for further developing and optimizing this widespread and versatile system in Arabidopsis would hence be welcomed. Here, we designed a generic vector system that can be used to clone any sgRNA sequence in a plant T-DNA vector containing an ubiquitously expressed Cas9 gene. With this vector, we explored two alternative marker systems for tracking Cas9-mediated gene-editing in vivo: BIALAPHOS RESISTANCE (BAR) and GLABROUS1 (GL1)...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28168222/tgprelid-a-mitochondrial-protein-linked-to-multidrug-resistance-in-the-parasite-toxoplasma-gondii
#15
Victoria Jeffers, Edwin T Kamau, Ananth R Srinivasan, Jonathan Harper, Preethi Sankaran, Sarah E Post, Joseph M Varberg, William J Sullivan, Jon P Boyle
New drugs to control infection with the protozoan parasite Toxoplasma gondii are needed as current treatments exert toxic side effects on patients. Approaches to develop novel compounds for drug development include screening of compound libraries and targeted inhibition of essential cellular pathways. We identified two distinct compounds that display inhibitory activity against the parasite's replicative stage: F3215-0002, which we previously identified during a compound library screen, and I-BET151, an inhibitor of bromodomains, the "reader" module of acetylated lysines...
January 2017: MSphere
https://www.readbyqxmd.com/read/28167502/a-genetic-interaction-analysis-identifies-cancer-drivers-that-modify-egfr-dependency
#16
Sida Liao, Teresa Davoli, Yumei Leng, Mamie Z Li, Qikai Xu, Stephen J Elledge
A large number of cancer drivers have been identified through tumor sequencing efforts, but how they interact and the degree to which they can substitute for each other have not been systematically explored. To comprehensively investigate how cancer drivers genetically interact, we searched for modifiers of epidermal growth factor receptor (EGFR) dependency by performing CRISPR, shRNA, and expression screens in a non-small cell lung cancer (NSCLC) model. We elucidated a broad spectrum of tumor suppressor genes (TSGs) and oncogenes (OGs) that can genetically modify proliferation and survival of cancer cells when EGFR signaling is altered...
January 15, 2017: Genes & Development
https://www.readbyqxmd.com/read/28166383/efficient-genome-editing-in-the-oomycete-phytophthora-sojae-using-crispr-cas9
#17
Yufeng Fang, Linkai Cui, Biao Gu, Felipe Arredondo, Brett M Tyler
Phytophthora is a filamentous fungus-like microorganism, but belongs to the oomycetes, in the kingdom Stramenopila. Phytophthora species are notorious as plant destroyers, causing multibillion-dollar damage to agriculture and natural ecosystems worldwide annually. For a long time, genome editing has been unattainable in oomycetes, because of their extremely low rate of homologous recombination. The recent implementation of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system in the soybean pathogen Phytophthora sojae, an experimental model for oomycetes, has opened up a powerful new research capability for the oomycete community...
February 6, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28165460/programmable-transcriptional-repression-in-mycobacteria-using-an-orthogonal-crispr-interference-platform
#18
Jeremy M Rock, Forrest F Hopkins, Alejandro Chavez, Marieme Diallo, Michael R Chase, Elias R Gerrick, Justin R Pritchard, George M Church, Eric J Rubin, Christopher M Sassetti, Dirk Schnappinger, Sarah M Fortune
The development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic manipulations in Mycobacterium tuberculosis. CRISPR interference (CRISPRi) promises to be a robust, easily engineered and scalable platform for regulated gene silencing. However, in M. tuberculosis, the existing Streptococcus pyogenes Cas9-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. To address these limitations, we screened eleven diverse Cas9 orthologues and identified four that are broadly functional for targeted gene knockdown in mycobacteria...
February 6, 2017: Nature Microbiology
https://www.readbyqxmd.com/read/28162770/gene-essentiality-profiling-reveals-gene-networks-and-synthetic-lethal-interactions-with-oncogenic-ras
#19
Tim Wang, Haiyan Yu, Nicholas W Hughes, Bingxu Liu, Arek Kendirli, Klara Klein, Walter W Chen, Eric S Lander, David M Sabatini
The genetic dependencies of human cancers widely vary. Here, we catalog this heterogeneity and use it to identify functional gene interactions and genotype-dependent liabilities in cancer. By using genome-wide CRISPR-based screens, we generate a gene essentiality dataset across 14 human acute myeloid leukemia (AML) cell lines. Sets of genes with correlated patterns of essentiality across the lines reveal new gene relationships, the essential substrates of enzymes, and the molecular functions of uncharacterized proteins...
February 1, 2017: Cell
https://www.readbyqxmd.com/read/28155635/genome-wide-functional-analysis-using-the-barcode-sequence-alignment-and-statistical-analysis-barcas-tool
#20
Jihyeob Mun, Dong-Uk Kim, Kwang-Lae Hoe, Seon-Young Kim
BACKGROUND: Pooled library screen analysis using shRNAs or CRISPR-Cas9 hold great promise to genome-wide functional studies. While pooled library screens are effective tools, erroneous barcodes can potentially be generated during the production of many barcodes. However, no current tools can distinguish erroneous barcodes from PCR or sequencing errors in a data preprocessing step. RESULTS: We developed the Barcas program, a specialized program for the mapping and analysis of multiplexed barcode sequencing (barcode-seq) data...
December 23, 2016: BMC Bioinformatics
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