Browse
Submit Data
Databases
API
Help

Dataset Information

89 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

JACKS: joint analysis of CRISPR/Cas9 knockout screens.

ABSTRACT: Genome-wide CRISPR/Cas9 knockout screens are revolutionizing mammalian functional genomics. However, their range of applications remains limited by signal variability from different guide RNAs that target the same gene, which confounds gene effect estimation and dictates large experiment sizes. To address this problem, we report JACKS, a Bayesian method that jointly analyzes screens performed with the same guide RNA library. Modeling the variable guide efficacies greatly improves hit identification over processing a single screen at a time and outperforms existing methods. This more efficient analysis gives additional hits and allows designing libraries with a 2.5-fold reduction in required cell numbers without sacrificing performance compared to current analysis standards.

SUBMITTER: Allen F

PROVIDER: S-EPMC6396427 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

JACKS: joint analysis of CRISPR/Cas9 knockout screens.

Allen Felicity F Behan Fiona F Khodak Anton A Iorio Francesco F Yusa Kosuke K Garnett Mathew M Parts Leopold L

Genome research 20190123 3

Genome-wide CRISPR/Cas9 knockout screens are revolutionizing mammalian functional genomics. However, their range of applications remains limited by signal variability from different guide RNAs that target the same gene, which confounds gene effect estimation and dictates large experiment sizes. To address this problem, we report JACKS, a Bayesian method that jointly analyzes screens performed with the same guide RNA library. Modeling the variable guide efficacies greatly improves hit identificat ...[more]

PMID: 30674557

Similar Datasets

Protocol of CRISPR-Cas9 knockout screens for identifying ferroptosis regulators.

Project description:Ferroptosis, an iron-dependent programmed cell death triggered by excessive lipid peroxidation, has shown promising therapeutic potentials in human diseases. Here, we describe a protocol of a CRISPR-Cas9 loss-of-function screen to identify regulators in response to different inducers of ferroptosis. We emphasize the steps of library amplification, drug treatment, high-throughput sequencing preparation, and bioinformatics analysis using model-based analysis of genome-wide CRISPR-Cas9 knockout (MAGeCK). We also present a method to discover the regulators of ferroptosis and verify the potential targets efficiently. For complete details on use and execution of this protocol, please refer to Yang et al. (2023).1.

| S-EPMC10730373 | biostudies-literature

MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens.

Project description:We propose the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method for prioritizing single-guide RNAs, genes and pathways in genome-scale CRISPR/Cas9 knockout screens. MAGeCK demonstrates better performance compared with existing methods, identifies both positively and negatively selected genes simultaneously, and reports robust results across different experimental conditions. Using public datasets, MAGeCK identified novel essential genes and pathways, including EGFR in vemurafenib-treated A375 cells harboring a BRAF mutation. MAGeCK also detected cell type-specific essential genes, including BCR and ABL1, in KBM7 cells bearing a BCR-ABL fusion, and IGF1R in HL-60 cells, which depends on the insulin signaling pathway for proliferation.

| S-EPMC4290824 | biostudies-other

Improved design and analysis of CRISPR knockout screens.

Project description:MotivationGenome-wide clustered, regularly interspaced, short palindromic repeat (CRISPR)-Cas9 screen has been widely used to interrogate gene functions. However, the rules to design better libraries beg further refinement.ResultsWe found single guide RNA (sgRNA) outliers are characterized by higher G-nucleotide counts, especially in regions distal from the PAM motif and are associated with stronger off-target activities. Furthermore, using non-targeting sgRNAs as negative controls lead to strong bias, which can be mitigated by using sgRNAs targeting multiple 'safe harbor' regions. Custom-designed screens confirmed our findings and further revealed that 19 nt sgRNAs consistently gave the best signal-to-noise ratio. Collectively, our analysis motivated the design of a new genome-wide CRISPR/Cas9 screen library and uncovered some intriguing properties of the CRISPR-Cas9 system.Availability and implementationThe MAGeCK workflow is available open source at https://bitbucket.org/liulab/mageck_nest under the MIT license.Supplementary informationSupplementary data are available at Bioinformatics online.

| S-EPMC6247926 | biostudies-literature

Kinome-wide CRISPR-Cas9 knockout screens revealed PLK1 as a therapeutic target for osteosarcoma.

Project description:Osteosarcoma is the most common malignant bone tumor, tending to be aggressive and recurrent. The therapeutic development for treating osteosarcoma has been largely hampered by the lack of effective and specific targets. Using kinome-wide CRISPR-Cas9 knockout screens, we systematically revealed a cohort of kinases essential for the survival and growth of human osteosarcoma cells, in which Polo-like kinase 1 (PLK1) appeared as a specific prominent hit. PLK1 knockout substantially inhibited proliferation of osteosarcoma cells in vitro and the tumor growth of osteosarcoma xenograft in vivo. Volasertib, a potent experimental PLK1 inhibitor, can effectively inhibit the growth of the osteosarcoma cell lines in vitro. It can also disrupt the development of tumors in the patient-derived xenograft (PDX) models in vivo. Furthermore, we confirmed that the mode of action (MoA) of volasertib is primarily mediated by the cell-cycle arrest and apoptosis triggered by DNA damage. As PLK1 inhibitors are entering phase III clinical trials, our findings provide important insights into the efficacy and MoA of the relevant therapeutic approach for combating osteosarcoma.

| S-EPMC10328921 | biostudies-literature

De novo identification of essential protein domains from CRISPR-Cas9 tiling-sgRNA knockout screens.

Project description:High-throughput CRISPR-Cas9 knockout screens using a tiling-sgRNA design permit in situ evaluation of protein domain function. Here, to facilitate de novo identification of essential protein domains from such screens, we propose ProTiler, a computational method for the robust mapping of CRISPR knockout hyper-sensitive (CKHS) regions, which refer to the protein regions associated with a strong sgRNA dropout effect in the screens. Applied to a published CRISPR tiling screen dataset, ProTiler identifies 175 CKHS regions in 83 proteins. Of these CKHS regions, more than 80% overlap with annotated Pfam domains, including all of the 15 known drug targets in the dataset. ProTiler also reveals unannotated essential domains, including the N-terminus of the SWI/SNF subunit SMARCB1, which is validated experimentally. Surprisingly, the CKHS regions are negatively correlated with phosphorylation and acetylation sites, suggesting that protein domains and post-translational modification sites have distinct sensitivities to CRISPR-Cas9 mediated amino acids loss.

| S-EPMC6778102 | biostudies-literature

Analysis of CRISPR-Cas9 screens identifies genetic dependencies in melanoma.

Project description:Targeting the MAPK signaling pathway has transformed the treatment of metastatic melanoma. CRISPR-Cas9 genetic screens provide a genome-wide approach to uncover novel genetic dependencies that might serve as therapeutic targets. Here, we analyzed recently reported CRISPR-Cas9 screens comparing data from 28 melanoma cell lines and 313 cell lines of other tumor types in order to identify fitness genes related to melanoma. We found an average of 1,494 fitness genes in each melanoma cell line. We identified 33 genes, inactivation of which specifically reduced the fitness of melanoma. This set of tumor type-specific genes includes established melanoma fitness genes as well as many genes that have not previously been associated with melanoma growth. Several genes encode proteins that can be targeted using available inhibitors. We verified that genetic inactivation of DUSP4 and PPP2R2A reduces the proliferation of melanoma cells. DUSP4 encodes an inhibitor of ERK, suggesting that further activation of MAPK signaling activity through its loss is selectively deleterious to melanoma cells. Collectively, these data present a resource of genetic dependencies in melanoma that may be explored as potential therapeutic targets.

| S-EPMC7818247 | biostudies-literature

Genome-wide CRISPR-Cas9 knockout screens identify DNMT1 as a druggable dependency in sonic hedgehog medulloblastoma.

Project description:Sonic hedgehog subgroup of medulloblastoma (SHH-MB) is characterized by aberrant activation of the SHH signaling pathway. An inhibition of the positive SHH regulator Smoothened (SMO) has demonstrated promising clinical efficacy. Yet, primary and acquired resistance to SMO inhibitors limit their efficacy. An understanding of underlying molecular mechanisms of resistance to therapy is warranted to bridge this unmet need. Here, we make use of genome-wide CRISPR-Cas9 knockout screens in murine SMB21 and human DAOY cells, in order to unravel genetic dependencies and drug-related genetic interactors that could serve as alternative therapeutic targets for SHH-MB. Our screens reinforce SMB21 cells as a faithful model system for SHH-MB, as opposed to DAOY cells, and identify members of the epigenetic machinery including DNA methyltransferase 1 (DNMT1) as druggable targets in SHH-dependent tumors. We show that Dnmt1 plays a crucial role in normal murine cerebellar development and is required for SHH-MB growth in vivo. Additionally, DNMT1 pharmacological inhibition alone and in combination with SMO inhibition effectively inhibits tumor growth in murine and human SHH-MB cell models and prolongs survival of SHH-MB mouse models by inhibiting SHH signaling output downstream of SMO. In conclusion, our data highlight the potential of inhibiting epigenetic regulators as a novel therapeutic avenue in SMO-inhibitor sensitive as well as resistant SHH-MBs.

| S-EPMC11304869 | biostudies-literature

Cas-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cas9.

Project description:MotivationCRISPR-derived RNA guided endonucleases (RGENs) have been widely used for both gene knockout and knock-in at the level of single or multiple genes. RGENs are now available for forward genetic screens at genome scale, but single guide RNA (sgRNA) selection at this scale is difficult.ResultsWe develop an online tool, Cas-Database, a genome-wide gRNA library design tool for Cas9 nucleases from Streptococcus pyogenes (SpCas9). With an easy-to-use web interface, Cas-Database allows users to select optimal target sequences simply by changing the filtering conditions. Furthermore, it provides a powerful way to select multiple optimal target sequences from thousands of genes at once for the creation of a genome-wide library. Cas-Database also provides a web application programming interface (web API) for advanced bioinformatics users.Availability and implementationFree access at http://www.rgenome.net/cas-database/Contactsangsubae@hanyang.ac.kr or jskim01@snu.ac.krSupplementary informationSupplementary data are available at Bioinformatics online.

| S-EPMC4920116 | biostudies-literature

Genome-scale CRISPR-Cas9 knockout screens for enterovirus D68 and rhinovirus C15

Project description:H1-HeLa cells were stably transduced with lentiCas9-Blast (Addgene, Plasmid #52962) and subsequently selected using blasticidin to generate constitutively expressing Cas9 H1-HeLa cells. A single Cas9-expressing H1-HeLa clone was then transduced with lentivirus without a selection marker to stably express CDHR3 C529Y (H1-HeLa+CDHR3). A single CDHR3-expressing H1-HeLa clone was then chosen based on RT-qPCR of CHDR3 expression and RV-C15 RNA levels for mutagenesis. 300 million of the H1-HeLa cells constitutively expressing CDHR3 and Cas9 were transduced with the lentiGuide-Puro from the GeCKO v2 library at a MOI of 0.3. Cells were selected using puromycin and heterogeneous H1-HeLa knockout cell populations were subsequently pooled together. The CRISPR genetic screens were started 10 days post transduction. >1000-fold coverage of mutagenized cells (libraries A and B) was infected with either RV-C15 (MOI=1 PFU/cell) or EV-D68 Missouri (MOI=1 PFU/cell). RV-C15 infection was repeated for an additional round at 6 days post-infection. As soon as appearance of visibly viable colonies was observed, populations of virus-resistant cells were pooled and harvested. Uninfected starting populations of mutagenized cells were used as the unselected reference. Total genomic DNA from both virus-resistant and uninfected cells was respectively extracted using QIAamp DNA Mini Kit (Qiagen). The inserted guide RNA sequences were retrieved from the genomic DNA by PCR amplification. The PCR products were then purified and subjected to NextSeq platform (Illumina) next-generation sequencing.

2019-07-10 | E-MTAB-8125 | biostudies-arrayexpress

Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.

Project description:Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.

| S-EPMC5800952 | biostudies-literature

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