Browse
Submit Data
Databases
API
Help

Dataset Information

23 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

DrugTargetSeqR: a genomics- and CRISPR-Cas9-based method to analyze drug targets.

ABSTRACT: To identify physiological targets of drugs and bioactive small molecules, we developed an approach, named DrugTargetSeqR, which combines high-throughput sequencing, computational mutation discovery and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based genome editing. We applied this approach to ispinesib and YM155, drugs that have undergone clinical trials as anticancer agents, and uncovered mechanisms of action and identified genetic and epigenetic mechanisms likely to cause drug resistance in human cancer cells.

SUBMITTER: Kasap C

PROVIDER: S-EPMC4123312 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

DrugTargetSeqR: a genomics- and CRISPR-Cas9-based method to analyze drug targets.

Kasap Corynn C Elemento Olivier O Kapoor Tarun M TM

Nature chemical biology 20140615 8

To identify physiological targets of drugs and bioactive small molecules, we developed an approach, named DrugTargetSeqR, which combines high-throughput sequencing, computational mutation discovery and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based genome editing. We applied this approach to ispinesib and YM155, drugs that have undergone clinical trials as anticancer agents, and uncovered mechanisms of action and identified genetic and epigenetic mechanisms likely ...[more]

PMID: 24929528

Similar Datasets

A pre-screening FISH-based method to detect CRISPR/Cas9 off-targets in mouse embryonic stem cells.

Project description:The clustered regularly interspaced short palindromic repeat (CRISPR)/associated 9 (Cas9) technology has been recently added to the tools allowing efficient and easy DNA targeting, representing a very promising approach to gene engineering. Using the CRISPR/Cas9 system we have driven the integration of exogenous DNA sequences to the X-linked Hprt gene of mouse embryonic stem cells. We show here that a simple fluorescence in situ hybridization (FISH)-based strategy allows the detection and the frequency evaluation of non-specific integrations of a given plasmid. FISH analysis revealed that these integrations do not match the software predicted off-target loci. We conclude that the frequency of these CRISPR-mediated off-target DNA cuts is negligible, since, due to the occurrence of spontaneous double-strand breaks, we observed more aspecific plasmid integrations than those corresponding to predicted off-target sites.

| S-EPMC4513284 | biostudies-other

Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains.

Project description:CRISPR-Cas9 genome editing technology holds great promise for discovering therapeutic targets in cancer and other diseases. Current screening strategies target CRISPR-Cas9-induced mutations to the 5' exons of candidate genes, but this approach often produces in-frame variants that retain functionality, which can obscure even strong genetic dependencies. Here we overcome this limitation by targeting CRISPR-Cas9 mutagenesis to exons encoding functional protein domains. This generates a higher proportion of null mutations and substantially increases the potency of negative selection. We also show that the magnitude of negative selection can be used to infer the functional importance of individual protein domains of interest. A screen of 192 chromatin regulatory domains in murine acute myeloid leukemia cells identifies six known drug targets and 19 additional dependencies. A broader application of this approach may allow comprehensive identification of protein domains that sustain cancer cells and are suitable for drug targeting.

| S-EPMC4529991 | biostudies-literature

Short-Homology-Mediated CRISPR/Cas9-Based Method for Genome Editing in Fission Yeast.

Project description:The CRISPR/Cas9 system enables the editing of genomes of numerous organisms through the induction of the double-strand breaks (DSB) at specific chromosomal targets. We improved the CRISPR/Cas9 system to ease the direct introduction of a point mutation or a tagging sequence into the chromosome by combining it with the noncanonical homology-directed DNA repair (HDR) based genome editing in fission yeast. We constructed convenient cloning vectors, which possessed a guide RNA (gRNA) expression module, or the humanized Streptococcus pyogenes Cas9 gene that is expressed under the control of an inducible promoter to avoid the needless expression, or both a gRNA and Cas9 gene. Using this system, we attempted the short-homology-mediated genome editing and found that the HDR pathway provides high-frequency genome editing at target loci without the need of a long donor DNA. Using short oligonucleotides, we successfully introduced point mutations into two target genes at high frequency. We also precisely integrated the sequences for epitope and GFP tagging using donor DNA possessing short homology into the target loci, which enabled us to obtain cells expressing N-terminally tagged fusion proteins. This system could expedite genome editing in fission yeast, and could be applicable to other organisms.

| S-EPMC6469419 | biostudies-other

CRISPR/CAS9-based DNA damage response screens reveal gene-drug interactions.

Project description:DNA damage response (DDR) is critically important for cell survival, genome maintenance, and its defect has been exploited therapeutically in cancer treatment. Many DDR-targeting agents have been generated and have entered the clinic and/or clinical trials. In order to provide a global and unbiased view of DDR network, we designed a focused CRISPR library targeting 365 DDR genes and performed CRISPR screens on the responses to several DDR inhibitors and DNA-damaging agents in 293A cells. With these screens, we determined responsive pathways enriched under treatment with different types of small-molecule agents. Additionally, we showed that POLE3/4-deficient cells displayed enhanced sensitivity to an ATR inhibitor, a PARP inhibitor, and camptothecin. Moreover, by performing DDR screens in isogenic TP53 wild-type and TP53 knock-out cell lines, our results suggest that the performance of our CRISPR DDR dropout screens is independent of TP53 status. Collectively, our findings indicate that CRISPR DDR screens can be used to identify potential targets of small-molecule drugs and reveal that TP53 status does not affect the outcome of these screens.

| S-EPMC7034363 | biostudies-literature

A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish.

Project description:The zebrafish is a popular model organism for studying development and disease, and genetically modified zebrafish provide an essential tool for functional genomic studies. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9, and they have included descriptions of a variety of tools and methods for guide RNA synthesis and mutant identification. However, most of the published techniques are not readily scalable to increase throughput. We recently described a CRISPR/Cas9-based high-throughput mutagenesis and phenotyping pipeline in zebrafish. Here, we present a complete workflow for this pipeline, including target selection; cloning-free single-guide RNA (sgRNA) synthesis; microinjection; validation of the target-specific activity of the sgRNAs; founder screening to identify germline-transmitting mutations by fluorescence PCR; determination of the exact lesion by Sanger or next-generation sequencing (including software for analysis); and genotyping in the F1 or subsequent generations. Using these methods, sgRNAs can be evaluated in 3 d, zebrafish germline-transmitting mutations can be identified within 3 months and stable lines can be established within 6 months. Realistically, two researchers can target tens to hundreds of genes per year using this protocol.

| S-EPMC5630457 | biostudies-literature

Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

Project description:Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

| S-EPMC4385744 | biostudies-literature

A CRISPR/Cas9-riboswitch-Based Method for Downregulation of Gene Expression in Trypanosoma cruzi.

Project description:Few genetic tools were available to work with Trypanosoma cruzi until the recent introduction of the CRISPR/Cas9 technique for gene knockout, gene knock-in, gene complementation, and endogenous gene tagging. Riboswitches are naturally occurring self-cleaving RNAs (ribozymes) that can be ligand-activated. Results from our laboratory recently demonstrated the usefulness of the glmS ribozyme from Bacillus subtilis, which has been shown to control reporter gene expression in response to exogenous glucosamine, for gene silencing in Trypanosoma brucei. In this work we used the CRISPR/Cas9 system for endogenously tagging T. cruzi glycoprotein 72 (TcGP72) and vacuolar proton pyrophosphatase (TcVP1) with the active (glmS) or inactive (M9) ribozyme. Gene tagging was confirmed by PCR and protein downregulation was verified by western blot analyses. Further phenotypic characterization was performed by immunofluorescence analysis and quantification of growth in vitro. Our results indicate that the method was successful in silencing the expression of both genes without the need of glucosamine in the medium, suggesting that T. cruzi produces enough levels of endogenous glucosamine 6-phosphate to stimulate the glmS ribozyme activity under normal growth conditions. This method could be useful to obtain knockdowns of essential genes in T. cruzi and to validate potential drug targets in this parasite.

| S-EPMC7056841 | biostudies-literature

HIV-CRISPR: A CRISPR/Cas9 Screening Method to Identify Genes Affecting HIV Replication.

Project description:Screening with CRISPR/Cas9 technology has already led to significant discoveries in the fields of cancer biology, cell biology and virology. Because of the relatively low false discovery rates and the ability to perform high-throughput, pooled approaches, it has rapidly become the assay of choice for screening studies, including whole-genome screens. Here, we describe a CRISPR screening protocol that allows for efficient screening of the entire life cycle of HIV-1 through packaging of the HIV-CRISPR lentiviral genomes by infecting HIV-1 virus in trans.

| S-EPMC7842688 | biostudies-literature

CRISPR Cas9 screens for drug resistance in AML cell lines

Project description:Acute myeloid leukaemia (AML) continues to have a very high mortality and its treatment hasnot changed for more than 20 years.Here, we propose to apply genomic-wide screens to directly identify therapeutic vulnerabilitiesof AML using AML cells lines with pre-defined somatic mutations. Our studies will be basedon genome-wide CRISPR/CAs9 gRNA screens and will be used to identify drivers of drugresistance to standard chemotherapies.This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

2015-02-03 | E-ERAD-335 | biostudies-arrayexpress

H2O2-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies.

Project description:Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H2O2 to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H2O2 content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H2O2 and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H2O2 by 3,3'-diaminobenzidine (DAB) staining suggested that H2O2 could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H2O2-based method. Moreover, HyB induced overproduction of H2O2 in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice.

| S-EPMC6720670 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data