A genome-wide CRISPR/Cas9 screen to identify genes required for transgene silencing in human cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE95458 | GEO | 2017/05/29
SECONDARY ACCESSION(S): PRJNA377246
REPOSITORIES: GEO
Similar Datasets
Project description: Not available
| S-EPMC12660324 | biostudies-literature
Project description:The molecular basis of transgene susceptibility to silencing is poorly characterized in plants, thus we evaluated several transgene design parameters as means to reduce heritable sRNA-mediated transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposable element (TE)-like silencing of canola-biased PUFA transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three Bacillus thuringiensis (Bt) crystal proteins tested the impact of CDSs recoding using different codon bias tables. Transgenes with the higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.
2017-10-30 | GSE103037 | GEO
Project description:The molecular basis of transgene susceptibility to silencing is poorly characterized in plants, thus we evaluated several transgene design parameters as means to reduce heritable sRNA-mediated transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposable element (TE)-like silencing of canola-biased PUFA transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three Bacillus thuringiensis (Bt) crystal proteins tested the impact of CDSs recoding using different codon bias tables. Transgenes with the higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.
2017-10-30 | GSE76406 | GEO
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:Crop development relies on the function of transgenes to instill favorable traits and enhance crop yield, but the effectiveness of these traits are constantly threatened by transgene silencing. The reason why certain transgenes are targeted by silencing pathways, while others remain highly expressed and durable, has remained a major question for decades, in part due to the lack of technologies to study the initiation of transgene silencing. We aimed to develop two technologies to identify the trigger that leads to transgene silencing: one to visualize the precise developmental time point of transgene silencing in a model plant and crop, and the second to identify all transcripts produced from a transgene in high depth without RNA fragmentation. By combining these two new methods, and Random Forest machine learning, we identified a cleaved aberrant RNA that accumulates to high levels in a small interfering RNA deficient mutant and in wild-type closely correlates with the onset of RNA interference, ultimately initiating transgene silencing. We show that the production of the aberrant RNA is likely due to ribosome stalling and No-Go RNA decay due to a rare enrichment of positively charged amino acids directly adjacent to the site of RNA cleavage.
2026-05-27 | GSE296082 | GEO
Project description:Crop development relies on the function of transgenes to instill favorable traits and enhance crop yield, but the effectiveness of these traits are constantly threatened by transgene silencing. The reason why certain transgenes are targeted by silencing pathways, while others remain highly expressed and durable, has remained a major question for decades, in part due to the lack of technologies to study the initiation of transgene silencing. We aimed to develop two technologies to identify the trigger that leads to transgene silencing: one to visualize the precise developmental time point of transgene silencing in a model plant and crop, and the second to identify all transcripts produced from a transgene in high depth without RNA fragmentation. By combining these two new methods, and Random Forest machine learning, we identified a cleaved aberrant RNA that accumulates to high levels in a small interfering RNA deficient mutant and in wild-type closely correlates with the onset of RNA interference, ultimately initiating transgene silencing. We show that the production of the aberrant RNA is likely due to ribosome stalling and No-Go RNA decay due to a rare enrichment of positively charged amino acids directly adjacent to the site of RNA cleavage.
2026-05-27 | GSE280623 | GEO
Project description:Crop development relies on the function of transgenes to instill favorable traits and enhance crop yield, but the effectiveness of these traits are constantly threatened by transgene silencing. The reason why certain transgenes are targeted by silencing pathways, while others remain highly expressed and durable, has remained a major question for decades, in part due to the lack of technologies to study the initiation of transgene silencing. We aimed to develop two technologies to identify the trigger that leads to transgene silencing: one to visualize the precise developmental time point of transgene silencing in a model plant and crop, and the second to identify all transcripts produced from a transgene in high depth without RNA fragmentation. By combining these two new methods, and Random Forest machine learning, we identified a cleaved aberrant RNA that accumulates to high levels in a small interfering RNA deficient mutant and in wild-type closely correlates with the onset of RNA interference, ultimately initiating transgene silencing. We show that the production of the aberrant RNA is likely due to ribosome stalling and No-Go RNA decay due to a rare enrichment of positively charged amino acids directly adjacent to the site of RNA cleavage.
2026-05-27 | GSE280624 | GEO
Project description:Crop development relies on the function of transgenes to instill favorable traits and enhance crop yield, but the effectiveness of these traits are constantly threatened by transgene silencing. The reason why certain transgenes are targeted by silencing pathways, while others remain highly expressed and durable, has remained a major question for decades, in part due to the lack of technologies to study the initiation of transgene silencing. We aimed to develop two technologies to identify the trigger that leads to transgene silencing: one to visualize the precise developmental time point of transgene silencing in a model plant and crop, and the second to identify all transcripts produced from a transgene in high depth without RNA fragmentation. By combining these two new methods, and Random Forest machine learning, we identified a cleaved aberrant RNA that accumulates to high levels in a small interfering RNA deficient mutant and in wild-type closely correlates with the onset of RNA interference, ultimately initiating transgene silencing. We show that the production of the aberrant RNA is likely due to ribosome stalling and No-Go RNA decay due to a rare enrichment of positively charged amino acids directly adjacent to the site of RNA cleavage.
2026-05-27 | GSE280625 | GEO
Project description:Epigenetic differences between transgenes and endogenous genes promote transgene silencing in plants
| PRJNA639789 | ENA
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