CRISPR-Cas9 knockdown of RBM48 in K562 cells
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ABSTRACT:
PROVIDER: PRJNA658070 | ENA |
REPOSITORIES: ENA
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Project description:We recreated the t(7;12) translocation in K562 cells by CRISPR/Cas9 to understand its effects on haematopoietic cells, which is of relevance to understand how this cytogenetic abnormalities causes and promotes acute leukaemia in infants. Wild-type K562 were edited by electroporation of ribonucleoprotein complexes consisting of Cas9 enzyme and two guide RNAs targeting patient-specific breakpoint loci. K562 electroporated with Cas9 enzyme only were used as control. Edited K562 harbouring the t(7;12) were single-cell cloned to obtain homogeneous populations (hereby referred to as K562-t(7;12)). We performed RNA sequencing analysis of K562-t(7;12) compared to K562 control to uncover transcriptional changes associated with the translocation.
2022-09-06 | E-MTAB-11851 | biostudies-arrayexpress
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Project description:Identifying putative transcription factor target genes by combining CRISPR/Cas9-based transcriptional activation with RNAseq in Drosophila S2R+ cells. This study focuses on the transcription factors Twist and Snail, singly and together. RNA from Drosophila cells following CRISPR/Cas9-based activation of Twist, Snail, or Twist and Snail together, compared with non-targeting sgRNA. Two biological replicates for each experiment
2015-08-10 | E-GEOD-71430 | biostudies-arrayexpress
Project description:U12-type or minor (U12) introns are spliced by a distinct minor spliceosome and are found in the vast majority of multicellular eukaryotes, including plants and animals. Although U12 introns constitute less than 0.5% of all introns in many species, minor intron containing genes (MIGs) are important for organismal growth, development and pathology. We recently reported that maize RNA Binding Motif Protein 48 (RBM48) is required for U12-type intron splicing. Maize rbm48 mutants have genome-wide defects of U12 intron splicing, leading to abnormal endosperm cell differentiation and proliferation. To investigate whether RBM48 mediated U12 splicing is conserved between maize and humans, we generated a CRISPR/Cas9-mediated RBM48 functional knockout of the human RBM48 ortholog (RBM48 FunKO) in human K-562 cells.
2020-08-20 | GSE156471 | GEO
Project description:Based on the hypothesis that, enhancing the local concentration of donor oligos could increase the correction rates, we generated and tested novel CRISPR-Cas9 systems, in which the DNA repair template is covalently conjugated to Cas9 (RNPD system). To validate our results from the HEK293T reporter cells, we here tested our approach at different endogenous genomic loci and in different cell types. We first targeted the human beta globin (HBB) locus in the K562 cell line, and analyzed correction- and editing frequencies using next generation sequencing (NGS). Next we targeted the Rosa26 and proprotein convertase subtilisin/kexin type 9 (Pcsk9) locus in mouse embryonic stem cells (mESCs). Here, RNPD system was always compared to Cas9 SNAP-tag fusion proteins with uncoupled donor oligos. To also directly compare the engineered RNPD system to the classical CRISPR-Cas9 system, we performed experiments where we used wild-type Cas9 with the uncoupled donor oligos as a control. We therefore targeted the fluorescent reporter locus as well as the endogenous loci HBB, empty spiracles homeobox 1 (EMX1), and C-X-C chemokine receptor type 4 (CXCR4) in HEK293T cells. Finally, we performed the analysis of three computationally predicted off-target sites of the reporter locus.
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Project description:Stau2 knockdown in human bcCML cells (K562)"
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Project description:HUB162 (chr11:42,878,450-42,883,450, hg38) was identified as an essential locus in the quiescent region in the K562 cells, and HUB161 (chr11:42,873,450-42,878,450, hg38) as a nonessential one. We used Arima Hi-C to study chromosomal structure after CRISPR-Cas9 deletion of either HUB162 (Ess) or HUB161 (Noness)
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Project description:Summary: Ribonuclease Inhibitor (RI also known as Rnh1) is a 50 kDa, ubiquitously expressed leucine-rich repeat (LRR) protein. It is localized in cytosol and binds to pancreatic-type ribonucleases and inhibit their function. However, the entire biological role for Rnh1 is unknown. We generated RNH1 knock out K562 cells by CRISPR/Cas9 method. Here we studied differential gene expression from wild type and RNH1 knock out K562 cells by RNA-Seq analysis. Overall design: Total RNA was isolated from wild type and RNH1 deficient K562 cells.
2017-03-05 | E-MTAB-5162 | 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:Genome-wide CRISPR-Cas9 knockout screen using TKOv1 sgRNA library performed in isogenic RBM10-proficient and RBM10-deficient HCC827 cells.
2024-03-01 | E-MTAB-13626 | biostudies-arrayexpress