Homo sapiens Mus musculus
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ABSTRACT:
PROVIDER: PRJNA1310217 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Machine learning-based prediction of the activity and specificity of Cas9 variants in gene editing
2024-08-22 | GSE231840 | GEO
Project description:Off-target (OT) analysis of different guide RNAs targeting PKLR gene. GUIDE-Seq analyses were done in HEK293 cells stably expressing Cas9 transfected with gRNA complexes, generated using Alt-R® CRISPR-Cas9 crRNA XT and Alt-R®CRISPR-Cas9 tracrRNA, and transfected with a dsODN tag
2021-04-13 | GSE171935 | GEO
Project description:Multiplex genotyping method to validate the multiallelic genome editing outcomes using machine learning-assisted long-read sequencing
| PRJDB11499 | ENA
Project description:Genome-wide knockout or knockdown screens have become powerful tools for the investigation of genotype-to-phenotype relationships. In bacteria, these screens commonly rely on transcriptional repression by dCas9, gene knockouts through Cas9 editing or random transposon mutagenesis, but depending on the technique, suffer from incomplete gene silencing, low editing efficiencies or they require massive library sizes. Here, we take a distinct approach with base editing to introduce premature stop codons or mutate start codons in Escherichia coli using a ScCas9 nickase derived base editor (ScBE3) that exhibits flexible PAM recognition. We then derive guide design rules by applying machine learning to a gene essentiality screen conducted in E. coli. For further improvement, we combined base-editing with Cas9-induced cleavage of the unedited cell fraction. The efficiency of this dual system was validated through a screen of conditionally essential E. coli genes. This improved setup that decouples the gene editing from the screening leads to more efficient guide depletion and confirmed previously published conditionally essential genes. Overall, base editing represents a useful tool for genome-wide knockout screens in bacteria and will eventually enable genome-wide knockout screens in a broader range of bacterial species to study their diverse genetics.
2024-02-05 | GSE225335 | GEO
Project description:Systematic mapping of genetic interactions and interrogation of the functions of sizeable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR-based screening system for combinatorial genetic manipulation that employs co-expression of Cas9 and Cas12a nucleases and machine learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named CHyMErA (Cas Hybrid for Multiplexed Editing and Screening Applications), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive genetic interactions and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemo-genetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for genetic interaction mapping and the functional analysis of sizeable genomic regions, such as alternative exons.
2020-01-28 | GSE144281 | GEO
Project description:Systematic mapping of genetic interactions and interrogation of the functions of sizeable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR-based screening system for combinatorial genetic manipulation that employs co-expression of Cas9 and Cas12a nucleases and machine learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named CHyMErA (Cas Hybrid for Multiplexed Editing and Screening Applications), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive genetic interactions and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemo-genetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for genetic interaction mapping and the functional analysis of sizeable genomic regions, such as alternative exons.
2020-02-29 | GSE144078 | GEO
Project description:RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from ~10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide 1.sgRNA1-6 binding sites were identified with ChipSeq by using HA antibody (there are 2 replicates for sgRNA1-3, one sample for sgRNA4-6,one control without sgRNA) 2.PCR products which amplifies " off-target genomic sites" were deep sequenced in the presence of WT Cas9+sgRNA or WT Cas9 alone( unique adaptor was used for each sgRNA and mixed for multiplex run)
2014-05-18 | E-GEOD-55887 | biostudies-arrayexpress
Project description:The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5’NGG3’ protospacer adjacent motifs (PAM). Examination of dmCas9 binding sites using two Trp53 targeting sgRNAs in Arf -/- MEF cell line (mouse).
2014-08-29 | E-GEOD-57650 | biostudies-arrayexpress
Project description:Machine learning-based prediction of the activity and specificity of Cas9 variants in gene editing
| PRJNA968010 | ENA