Targeted sequencing by a CRISPR-assistant target enrichment-sequencing (CATE-seq)
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA491192 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7832320_1.fastq.gz | Fastqsanger.gz | |||
| SRR7832320_2.fastq.gz | Fastqsanger.gz | |||
| SRR7832321_1.fastq.gz | Fastqsanger.gz | |||
| SRR7832321_2.fastq.gz | Fastqsanger.gz | |||
| SRR7832315_1.fastq.gz | Fastqsanger.gz |
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Project description:Targeted sequencing of genome is essential to the basic biological research and biomedicine. Therefore, various targeted enrichment methods have been developed for this end, in which various hybridization-based methods have been most widely used. However, the current hybridization-based methods (both on solid or in solution) are still restricted by the intrinsic shortcomings of nucleic acid hybridization. In this study, by combining an engineered dCas9-sgRNA with widely used magnetic isolation, we developed a new strategy to enrich target genomic DNAs in high efficiency. In this strategy, target DNA was firstly specifically recognized and bound by a complex of dCas9 and capture sgRNA (csgRNA). The DNA-dCas9-csgRNA complex was then captured on magnetic beads through the annealing of elongated 3′ end of csgRNA with single-stranded capture oligonucleotides coupled on streptavidin-coated magnetic beads. We thus named the strategy as CRISPR-assistant target enrichment (CATE). The enriched DNAs can be analyzed by the next generation sequencing (NGS). We thus named the technique as CATE-seq. Used the technique, we successfully enriched 35 target exons of 6 genes in 6 cell lines by using 54 csgRNA. We found that the target genomic DNA fragments such as exons could be efficiently enriched and analyzed by CATE-seq. The technique has several significant advantages over the current hybridization-based methods, including high simplicity, specificity, sensitivity, and throughput. This study therefore provides a new powerful tool for targeted sequencing.
2018-09-15 | GSE119994 | GEO
Project description:Igk locus capture-based target enrichment sequencing
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Project description:Identification of important, functional small RNA (sRNA) species is presently hampered by the lack of reliable and sensitive methods to isolate and characterize them. We have developed a method termed Target-Enrichment of small RNAs (TEsR), which enables targeted sequencing of rare sRNAs and diverse precursor and mature forms of sRNAs not detectable by current standard sRNA sequencing methods. It is based on the amplification of full length sRNA molecules, production of biotinylated RNA probes, hybridization to one or multiple targeted RNA, removal of non-targeted sRNAs, and sequencing. By this approach, target sRNAs can be enriched by a factor of 500-30,000 while maintaining strand specificity. TEsR enriches for sRNAs irrespective of length or different molecular features, such as the presence or absence of a 5’-cap or of secondary structures or abundance levels, and allows the detection of the complete sequence (including sequence variants, 5’ and 3’ ends) of precursors, intermediate and mature forms, and all this quantitatively.
2017-08-22 | GSE102845 | GEO
Project description:Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest. Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest.
2010-09-12 | E-GEOD-22024 | biostudies-arrayexpress
Project description:We performed a large-scale genome-wide characterisation of indels generated following editing with CRISPR/Cas9. We used pools of sgRNAs and performed targeted capture and sequencing of the edited regions in HepG2 cells.
2018-12-13 | E-MTAB-7095 | biostudies-arrayexpress
Project description:Target enrichment sequencing of head and neck cell lines
| PRJNA869767 | ENA
Project description:We report the use of short biotinylated oligonucleotides for the targeted pulldown of potential CRISPR/Cas9 off-target loci in condition and control to measure the frequency of off target editing
2017-09-14 | GSE95370 | GEO
Project description:HERVK HML-2 Target Enrichment pilot experiment
| PRJEB21477 | ENA
Project description:We sought to characterise how chromatin states affected CRISPR/Cas9 activity and the induced indel profiles. We used two different doses of Trichostatin and two different doses of a EZH2 inhibitor to modulate the chromatin state and then performed targeted DNA-seq of selected sgRNA target regions to identify the indels.
2018-12-13 | E-MTAB-7091 | biostudies-arrayexpress
Project description:Single-cell CRISPR screens allow for the exploration of mammalian gene function and genetic regulatory networks, but their utility has been limited in part by their reliance on indirect sgRNA indexing. Here, we present direct capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct capture Perturb-seq enables the detection of multiple distinct sgRNAs expressed from a single vector within individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries. We demonstrate that this approach allows high-throughput investigations of genetic interactions, and we leverage this ability to dissect epistatic interactions between cholesterol biogenesis and DNA repair. We also show that targeting individual genes with multiple sgRNAs per cell improves the efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Lastly, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.
2020-03-03 | GSE146194 | GEO