Cas9:sgRNA in vitro selection libraries
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ABSTRACT:
PROVIDER: PRJNA212837 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.
2017-12-25 | E-MTAB-6310 | biostudies-arrayexpress
Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.
2017-12-15 | E-MTAB-6357 | biostudies-arrayexpress
Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.
2017-12-15 | E-MTAB-6358 | biostudies-arrayexpress
Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.
2017-12-15 | E-MTAB-6356 | biostudies-arrayexpress
Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.
2017-12-15 | E-MTAB-6355 | biostudies-arrayexpress
Project description:By a robust unbiased ChIP-seq approach, we demonstrated that CRISPR/Cas9 had crRNA-specific off-target binding activities in human genome. However, most of those binding off-targets could not be efficiently cleaved both in vivo and in vitro which suggested the cleavage off-target activity of CRISPR/Cas9 in human genome is very limited. We provided a valuable tool to further investigate the molecular mechanism of CRISPR/Cas9 and to optimize its in vivo targeting sgRNA binding sites were identified with ChipSeq by using GFP antibody (there are 2 replicates for egfa-t1 sgRNA,emx1 sgRNA and control without sgRNA in Hek293T cells, one egfa-t1 sgRNA,emx1 sgRNA and control without sgRNA in HeLaS3 cells)
2014-06-18 | E-GEOD-58511 | biostudies-arrayexpress
Project description:The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.
2016-05-01 | E-MTAB-4143 | biostudies-arrayexpress
Project description:Genome-wide CRISPR-Cas9 knockout screen using TKOv1 sgRNA library was performed in isogenic RBM10-proficient and RBM10-deficient HCC827 cells.
2023-03-04 | E-MTAB-12724 | biostudies-arrayexpress
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:CRISPR interference (CRISPRi) genetic screens use programmable repression of gene expression to systematically explore questions in cell biology and genetics. However, wider adoption of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and lack of consensus on the choice of CRISPRi effector proteins. Here, we address these challenges to present next-generation CRISPRi sgRNA libraries and effectors. First, we combine empiric sgRNA selection with a dual sgRNA library design to generate an ultra-compact, highly active CRISPRi sgRNA library. Next, we rigorously compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an optimal balance between strong on-target knockdown and minimal nonspecific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines which stably express Zim3-dCas9 and demonstrate robust on-target knockdown across these cell lines. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.
2022-06-04 | GSE205310 | GEO