Transcriptional response to collateral RNA cleavage by LbuCas13a
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE220759 | GEO | 2023/01/19
REPOSITORIES: GEO
Similar Datasets
Project description:Transcriptional response to collateral RNA cleavage by LbuCas13a
| PRJNA911286 | ENA
Project description:The intrinsic collateral cleavage activity of Cas13d,which refers to the gRNA-independent degradation of bystander RNA, significantly limits its therapeutic potential. To extensively evaluate the collateral effects of hpCas13d genome-wide, we conducted RNA-seq analysis of the transcriptome in HEK293T cells after the transfections with dCas13d, wtCas13d, hpCas13d, and hfCas13d . Compared to dCas13d, wtCas13d with a PPIA gRNA (reported to induce the significant collateral cleavage) resulted in significant downregulations of thousands genes. In sharp contrast, hpCas13d and hfCas13d showed much less off-target genes, respectively. These findings demonstrate that hpCas13d exhibits reduced collateral activity, rendering it suitable for in vivo applications.
2024-02-28 | GSE260500 | GEO
Project description:The CRISPR/Cas13 system has garnered attention as a potential tool for RNA editing. However, the degree of collateral activity among various Cas13 orthologs and their cytotoxic effects in mammalian cells remain contentious, potentially impacting their applications. In this study, we observed differential collateral activities for LwaCas13a and RfxCas13d in 293T and U87 cells by applying both sensitive dual-fluorescence (mRuby/GFP) reporter and quantifiable dual-luciferase (Fluc/Rluc) reporter, with LwaCas13a displaying notable activity contrary to previous reports. However, significant collateral RNA cleavage exerted only a modest impact on cell viability. Furthermore, the collateral activity of LwaCas13a mildly impeded but did not arrest, porcine embryo development. Our findings reveal that distinct collateral RNA cleavage by Cas13 slightly suppresses mammalian cell proliferation and embryo development. This could account for the lack of reported collateral effects in numerous prior studies and offers new insights into the implications of the collateral activity of Cas13 for clinical application.
2024-01-25 | GSE254168 | 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:Identification of collateral RNA cleavage mediated by Type VI CRISPR-Cas system from Leptotrichia shahiii
| PRJNA758987 | ENA
Project description:We investigated the in vivo and in vitro specificity of collateral RNA degradation mediated by LshCas13a protein encoded by Type VI CRISPR-Cas system derived from L. shahii. In our experimental system, we used Escherichia coli strain harboring Type VI spacer matching inducible transcript ("targeting" cells) or strain that does not encode spacers matching any E. coli transcripts ("nontargeting" cells). After the induction of the target transcription, cells were collected, total RNA samples were extracted from the cells and subjected to high throughput RNA sequencing with a specific protocol allowing to capture RNA degradation products. To investigate the influence of RNase toxins encoded in E. coli genome on the observed RNA degradation pattern, we repeated the described experiment on E. coli strain lacking ten known ribonuclease-encoding toxin-antitoxin loci. To investigate in vitro specificity of LshCas13a protein, we analyzed products of the degradation on total E. coli RNA by purified LshCas13a supplemented with crRNA and targeted or nontargeted transcripts. The obtained samples were processed as it was described above. All experiments were independently performed in triplicate. To detect transcript degradation sites, the obtained read pairs were filtered using trimmomatic tool and then mapped onto reference sequences using bowtie2. Next, for each nucleotide position of each strand of reference sequences the number of 5’ ends of aligned fragments were counted producing corresponding tables. The differences between the numbers of mapped 5’ ends in targeting and nontargeting samples were analyzed using edgeR package. Using this data, we identified target preferences for collateral RNA degradation by LshCas13a effectors.
2022-08-31 | GSE183061 | GEO
Project description:Genomic analysis of control and collateral arteries was used to investigate mechanisms responsible for impaired collateral growth in the Spontaneously Hypertensive rat, an animal model of essential hypertension with increased oxidative and nitrosative stress and metabolic abnormalities. A fundamental difference was observed in the overall expression pattern in SHR vs WKY collaterals. Redox related genes with altered expression included cyba (the gene encoding p22phox), superoxide dismutase 3, and thioredoxin reductases 1 and 2. Cystatin C, hevin, angiotensinogen, and the angiotensin type 1b receptor (AT1bR) had altered collateral expression that was confirmed by RT-PCR. These molecules are known to have significant roles in other types of arterial remodeling. Of specific interest was the AT1bR which exhibited up-regulation in WKY collaterals, but down-regulation in SHR. A remarkable increase in AT1R protein was observed in WKY but not SHR collaterals. Pharmacological blockade of the AT1R with losartan prevented collateral luminal expansion in WKY. In SHR, captopril restored redox status assessed by cyba expression and nitric oxide concentration, prevented collateral AT1bR down-regulation and re-established the capacity for collateral growth. These results indicate that redox-status significantly alters flow-mediated transcriptional regulation and demonstrate that increased flow-related expression/activation of the AT1R is required for normal collateral luminal expansion but is altered by chronic oxidative and/or nitrosative stress in hypertensive rats. Keywords: gene expression two strains: two vessel types
2010-12-15 | E-GEOD-19524 | biostudies-arrayexpress
Project description:Gene Expression Profiling of Severed Rat Medial Collateral Ligament at 1, 2, 4, 7,1 0, and 14 days Following Injury with Control and Cultured Ligament Fibroblasts and Rat Universal Reference RNA; The aim of this study was to assess the genes involved in the repair of the the dense connective tissue of the a rat ligament in order to provide targets for improvement in healing. Rat whole genome microarrays (Agilent) were used in this study and Cy3 and Cy5 labeled total RNA was extracted and labeled with Cy3 or Cy5 prior to fragmentation and hybridization. Experiment Overall Design: To conserve microarrays to allow maximum replicates, two samples either Cy3 or Cy5 labeled were hybridized to each microarry. Total RNA from each rat medial collateral ligament was labeled and hybrided to a microarray. The fluorescence intensity for each column minus the background was extracted from each microarray for submission to BRB Array tools for statistical analysis.
2008-04-05 | E-GEOD-10720 | biostudies-arrayexpress
Project description:using peripheral blood monocytes to identify marker genes for an extensively grown coronary collateral circulation. We used microarrays to detail the global programme of gene expression underlying collateralization and identified distinct classes of differently regulated genes during this process. Experiment Overall Design: Collateral flow index (CFI) was obtained invasively by angioplasty pressure sensor guidewire, a group of patients with coronary artery disease CAD and a group without CAD were selected for peripheral monocyte isolation, RNA extraction and hybridization on Affymetrix microarrays.
2008-10-26 | E-GEOD-7638 | biostudies-arrayexpress