Gene expression changes of CRISPR/Cas13d-mediated HoxB13 knockdown in 293FT cells and LNCaP95 mouse liver metastasis
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE264062 | GEO | 2025/05/14
REPOSITORIES: GEO
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Project description:To examine the potential off-target effects by CRISPR/Cas13d-mediated CTSL knockdown in 293FT cells
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Project description:Gene expression changes of CRISPR/Cas13d-mediated HoxB13 knockdown in 293FT cells and LNCaP95 mouse liver metastasis
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Project description:Gene expression profiling by CRISPR/Cas13d-mediated CTSL knockdown in 293FT cells
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Project description:Circular RNAs (circRNAs) are widely expressed, but their functions remain largely unknown. To study circRNAs in a high-throughput manner, short hairpin RNA (shRNA) screens1 have recently been used to deplete circRNAs by targeting their unique back-splicing junction (BSJ) sites. Here, we report frequent discrepancies between shRNA-mediated circRNA knockdown efficiency and the corresponding biological effect, raising pressing concerns about the robustness of shRNA screening for circRNA functional characterization. To address this issue, we leveraged the CRISPR/Cas13d system2 for functional study of circRNAs by optimizing the strategy for designing single guide RNAs to deplete circRNAs. We then performed shRNA and CRISPR/Cas13d parallel screenings and demonstrated that shRNA-mediated circRNA screening yielded a high rate of false positive phenotypes. Furthermore, using a CRISPR/Cas13d screening library targeting over 2,500 human hepatocellular carcinomas (HCC) related circRNAs, we identified a group of circRNAs, whose inhibition increased the therapeutic efficacy of sorafenib, an approved HCC drug. Collectively, these data demonstrate that CRISPR/Cas13d system is an effective approach to study the function of circRNAs in a high-throughput manner.
2020-12-06 | GSE162720 | GEO
Project description:Early embryonic development is driven by maternal gene products deposited into the oocyte. Due to the lack of reliable knockdown strategies, maternal mRNA functions have remained elusive. CRISPR-Cas13 systems have recently been employed to induce RNA degradation in yeast, plants and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal model system. Here, we show that CRISPR-Cas13d is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that both zygotically-expressed and maternally-provided transcripts are efficiently targeted, resulting in an 85% average decrease in transcript level and the recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, sea anemone and mouse embryos. All together our results demonstrate that CRISPR-Cas13d is an efficient knockdown platform to understand gene function in animal embryos.
2022-01-21 | GSE135884 | GEO
Project description:We performed an experimental Cas13d-SARScov2 genome-wide screen to identify gRNAs that would allow Cas13d to degrade the viral RNA. We built mCherry reporter plasmids that express mCherry with a 3kb 3'UTR deriving from the SARScov2 genome. In total we designed 11 reporters covering the entire plus strand of the viral genome and 11 other reporters covering the entire minus strand. Each of the 22 mCherry reporter plasmids carries a U6 expression cassette containing a Cas13d gRNA that targets the 3'UTR of the mCherry reporter. Each reporter is represented by a pool of reporters each containing a different gRNA that targets mCherry 3'UTR for a total average of ~300 gRNA per 3'UTR. The entire pool of 22 reporters, each with a pool of ~300 different gRNAs constitutes a comprehensive set ~6,500 reporters (~ 6,500 different gRNAs) that allowed us to interrogate the entire SARScov2 plus and minus strand viral RNA for regions of vulnerability and targetability. In order to specifically interrogate Cas13d activity an remove the biases that would be introduced in the reporter expression by the presence of a large 3kb 3'UTR we used a case (presence of Cas13d) control (absence of Cas13d) design. Briefly, the ~6,500 reporters were lentiviral transduced in RKO cells, the cells were split in 2 populations, 1 population was transduced with Cas13d and the other serving as control did not. The population expressing Cas13d was FACS sorted in low mCherry (efficient gRNAs) and high mCherry (un-efficient gRNAs) in 2 biological replicates and the genomic DNA of these populations was extracted, gRNAs were PCR amplified and sequenced. For the population that did not express Cas13d, a low mCherry, one high mCherry and unsorted population were sequenced as control libraries.
2023-10-29 | E-MTAB-11111 | biostudies-arrayexpress
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2025-01-10 | GSE275631 | GEO
Project description:Cas13 is a unique family of CRISPR endonucleases exhibiting programmable binding and cleavage of RNAs and is a strong candidate for eukaryotic RNA knockdown in the laboratory and the clinic. However, sequence-specific binding of Cas13 to the target RNA unleashes non-specific bystander RNA cleavage, or collateral activity, which may confound knockdown experiments and raises concerns for therapeutic applications. Although conserved across orthologs and robust in cell-free and bacterial environments, the extent of collateral activity in mammalian cells remains disputed. Here, we investigate Cas13d collateral activity in the context of an RNA-targeting therapy for myotonic dystrophy type 1, a disease caused by a transcribed long CTG repeat expansion. We find that when targeting CUGn RNA in HeLa and other cell lines, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis pathways. We also observe collateral effects when targeting other repetitive and unique transgenic sequences, and we provide evidence for collateral activity when targeting highly expressed endogenous transcripts. To minimize collateral activity for repeat-targeting Cas13d therapeutics, we introduce gRNA excision for negative-autoregulatory optimization (GENO), a simple strategy that leverages crRNA processing to control Cas13d expression and is easily integrated into an AAV gene therapy. We argue that thorough assessment of collateral activity is necessary when applying Cas13d in mammalian cells and that implementation of GENO illustrates the advantages of compact and universally robust regulatory systems for Cas-based gene therapies.
2021-12-23 | GSE191329 | GEO
Project description:To identify potential cofactors of HOXB13 in suppressing lipogenic programs in prostate cancer cells, we performed tandem affinity purification followed by mass spectrometry analysis of WT and G84E HOXB13 expressed in LNCaP cells. Out of the HOXB13-enriched proteins are previously reported interactors such as AR and its cofactors FOXA1, GATA2, and NKX3. However, these interactions were not disrupted by G84E as compared to WT HOXB13. Interestingly, we found strong interactions of HOXB13 with HDAC1/3 and their corepressors NCoR1/2 and TBL1X. Notably, these interactions were drastically reduced by G84E mutation.
2023-03-11 | PXD030810 | Pride
Project description:Treatment induced-resistance of CRPC is an imminent undesirable outcome in patients. Tissue and lineage-specific super-enhancers (SEs) determine cell fate and plasticity during development and disease respectively. However, the identity and function of CRPC-specific SEs (CSEs) regulated genes is unknown. Herein we report the lysine 13 acetylation of the prostate-enriched transcription factor HOXB13 (acK13-HOXB13) mediated by the histone acetyl transferase (HAT) CBP/p300 as a critical mechanism of CSE establishment. Mechanistically, acK13-HOXB13 establishes the CRPC enhanceosome comprising chromatin remodeling bromo-domain proteins SMARCA2/BAZ2B and the HAT p300/CBP which enable histone and non-histone protein acetylation at CSEs. Such CSEs sprout at tyrosine kinase genes encoding ACK1/TNK2, VEGFA, and ANGPT2/ANGPTL3 to increase pathogenic output in primary human tumors. These tyrosine kinase mediated signaling cascades establish robust networks to conduce growth, survival and androgen-bypass. Consistently, the loss of function acK13-HOXB13 mutants show significant reduction of proliferation, spheroid formation, and xenograft tumor growth that correlates with the high sensitivity to the AR-antagonist Enzalutamide. Targeting HOXB13 acetylation mediated CRPC-SE establishment at critical tyrosine kinase genes could therefore have significant clinical implications in preventing PC recurrence.
2022-09-22 | GSE169132 | GEO