Rapid cell-free characterization of multi-subunit CRISPR effectors and transposons
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA744274 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:We report the PAMs of diverse type I-E CRISPR- Cas systems and the type I-C and the type I-F1 CRISPR-Cas systems from Xanthomonas albilineans. Furthermore, we report PAMs of two type I-B CRISPR transposons (CASTs) and the Vibrio cholerae type I-F CAST. For identification of the PAMs, we used a cell-free TXTL-based PAM screen we named PAM-DETECT. By adding a 5N randomized PAM library and plasmids encoding for Cascade genes and gRNAs, recognized PAMs were bound by Cascade and protected from cleavage by a restriction enzyme that has it's recognition site within the target region. By amplifying the non-cleaved target plasmid, we used next-generation sequencing to analyze the enrichment of functional PAMs of the studied CRISPR-Cas systems. We additionally assessed the insertion sites of crRNA-dependent and crRNA-independent transposition of the Rippkaea orientalis type I-B CAST in TXTL and E. coli.
2022-02-24 | GSE179614 | GEO
Project description:SINE transposons in multiple lepidopteran insects Raw sequence reads
| PRJNA665855 | ENA
Project description:Vesper bats (family Vespertilionidae) experienced a rapid adaptive radiation beginning around 36 mya that resulted in the second most species rich mammalian family. Coincident with that radiation was an initial burst of DNA transposon activity that has continued into the present. Deep sequencing of small RNAs from the vespertilionid, Eptesicus fuscus, as well as dog and horse revealed that substantial numbers of novel bat miRNAs are derived from DNA transposons unique to vespertilionids. In fact, 35.9% of Eptesicus-specific miRNAs derive from DNA transposons compared to 2.2 and 5.9% of dog- and horse-specific miRNAs, respectively and targets of several miRNAs are identifiable. Timing of the DNA transposon expansion and the introduction of novel miRNAs coincides remarkably well with the rapid diversification of the family Vespertilionidae. We suggest that the rapid and repeated perturbation of regulatory networks by the introduction of many novel miRNA loci was a factor in the rapid radiation. A testicular tissue sample from dog, horse, and two different Eptesicus fuscus individuals. Four samples total.
2014-05-19 | E-GEOD-47593 | biostudies-arrayexpress
Project description:The piRNA pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. piRNAs are maternally deposited and required for proper transposon silencing in adult offspring. However, a long-standing question in the field is the precise function of maternally deposited piRNAs and its associated factors during embryogenesis. Here, we probe the spatio-temporal expression patterns of several piRNA pathway components during early stages of development. Amongst those, factors required for transcriptional gene silencing (TGS) showed ubiquitous abundance in somatic and pole cells throughout the first half of embryogenesis. We further analysed the transcriptomes of various embryo stages and correlated these with the presence of selected chromatin marks. We found that a number of transposon families show bursts of transcription during early embryonic stages. Transposons heavily targeted by maternally deposited piRNAs accumulated repressive chromatin marks following their spike in expression. Furthermore, depletion of maternally deposited Piwi protein in early embryos resulted in increased expression of transposons targeted by inherited piRNAs and was accompanied by a strong loss of repressive chromatin marks at coding sequences. Overall, our data suggests a pivotal role for the piRNA pathway in transposon defence during Drosophila embryogenesis in somatic cells.
2021-07-26 | PXD022409 | Pride
Project description:Vesper bats (family Vespertilionidae) experienced a rapid adaptive radiation beginning around 36 mya that resulted in the second most species rich mammalian family. Coincident with that radiation was an initial burst of DNA transposon activity that has continued into the present. Deep sequencing of small RNAs from the vespertilionid, Eptesicus fuscus, as well as dog and horse revealed that substantial numbers of novel bat miRNAs are derived from DNA transposons unique to vespertilionids. In fact, 35.9% of Eptesicus-specific miRNAs derive from DNA transposons compared to 2.2 and 5.9% of dog- and horse-specific miRNAs, respectively and targets of several miRNAs are identifiable. Timing of the DNA transposon expansion and the introduction of novel miRNAs coincides remarkably well with the rapid diversification of the family Vespertilionidae. We suggest that the rapid and repeated perturbation of regulatory networks by the introduction of many novel miRNA loci was a factor in the rapid radiation.
2014-05-19 | GSE47593 | GEO
Project description:Here we utilize a forward genetics approach in Neurospora crassa to identify novel effectors of Polycomb repression. We recovered two mutant alleles of a gene (NCU04278), which we determined encodes a PRC2 accessory subunit (PAS). PAS is not essential for all H3K27 methylation, but rather its absence leads to losses of H3K27 methylation concentrated near chromosome ends.
2020-03-30 | GSE140787 | GEO
Project description:p53 directly represses human LINE1 transposons
| PRJNA667740 | ENA
Project description:Emergence of induced pluripotent stem cells (iPSC) technology has paved novel routes for regenerative medicine. iPSCs offer the possibilities of disease modeling, drug toxicity studies as well as cell replacement therapies by autologous transplantation. Classical protocols of iPSC generation harness infection by retro- or lenti-viruses. Although such integrating viruses represent very robust tools for reprogramming, the presence of viral transgenes in iPSCs is deleterious as it holds the risk of insertional mutagenesis leading to malignant transformation. Moreover, remaining reprogramming transgenes have been shown to affect the differentiation potential of iPSCs. More recently, alternative protocols have been explored to derive transgene-free iPSC, including use of transposons, mRNA transfection, episomal plasmid transfection, and infection with non-integrating viruses such as Sendai virus. However, the utility of such protocols remains limited due to low efficiency and narrow range of cell specificity. In this study we aim at combining the robustness of lentiviral reprogramming with the high efficacy of Cre recombinase protein transduction to readily delete reprogramming transgenes from iPSCs. We demonstrate rapid generation of transgene-free human iPSCs by excising the loxP-flanked reprogramming cassette employing direct delivery of biologically active Cre protein. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs do resemble more to human ESCs and has better differentiation potential than iPSCs before Cre transduction. Our study provides a simple, rapid and robust protocol for the generation of superior transgene-free iPSCs suitable for disease modeling, tissue engineering and cell replacement therapies. mRNA extracted from human Fibroblasts (AR1034ZIMA), human Embryonic Stem Cell line I3 (hES I3), three human induced Pluripotent Stem Cell clones 1, 1.2 and 1.4 (fl-ARiPS cl1, del-ARiPS cl 1.2, del-ARiPS cl1.4) has been hybridized on Illumina Human HT-12 (version 4 revision 2) arrays for genome wide expression analysis. Samples were run at least as duplicate technical replicates. Differential gene expression analysis has been performed on the grouped expression data with the human embryonic stem cells (hES I3) group as the reference.
2014-03-11 | E-GEOD-55725 | biostudies-arrayexpress
Project description:Emergence of induced pluripotent stem cells (iPSC) technology has paved novel routes for regenerative medicine. iPSCs offer the possibilities of disease modeling, drug toxicity studies as well as cell replacement therapies by autologous transplantation. Classical protocols of iPSC generation harness infection by retro- or lenti-viruses. Although such integrating viruses represent very robust tools for reprogramming, the presence of viral transgenes in iPSCs is deleterious as it holds the risk of insertional mutagenesis leading to malignant transformation. Moreover, remaining reprogramming transgenes have been shown to affect the differentiation potential of iPSCs. More recently, alternative protocols have been explored to derive transgene-free iPSC, including use of transposons, mRNA transfection, episomal plasmid transfection, and infection with non-integrating viruses such as Sendai virus. However, the utility of such protocols remains limited due to low efficiency and narrow range of cell specificity. In this study we aim at combining the robustness of lentiviral reprogramming with the high efficacy of Cre recombinase protein transduction to readily delete reprogramming transgenes from iPSCs. We demonstrate rapid generation of transgene-free human iPSCs by excising the loxP-flanked reprogramming cassette employing direct delivery of biologically active Cre protein. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs do resemble more to human ESCs and has better differentiation potential than iPSCs before Cre transduction. Our study provides a simple, rapid and robust protocol for the generation of superior transgene-free iPSCs suitable for disease modeling, tissue engineering and cell replacement therapies.
2014-03-11 | GSE55725 | GEO
Project description:Pack-TYPE transposons are a unique class of potentially mobile non-autonomous elements that can capture, merge and relocate fragments of chromosomal DNA. It has been postulated that their activity accelerates the evolution of host genes. However, this important presumption is based only on the sequences of currently inactive Pack-TYPE transposons and the acquisition of chromosomal DNA has not been recorded in real time. Analysing the DNA copy number variation in hypomethylated Arabidopsis lines, we have now for the first time witnessed the mobilization of novel Pack-TYPE elements related to the CACTA transposon family, over several plant generations. Remarkably, these elements can insert into genes as closely spaced direct repeats and they frequently undergo incomplete excisions, resulting in the deletion of one of the end sequences. These properties suggest a mechanism of efficient acquisition of genic DNA residing between neighbouring Pack-TYPE transposons and its subsequent mobilization. Our work documents crucial steps in the formation of in vivo novel Pack-TYPE transposons, and thus the possible mechanism of gene shuffling mediated by this type of mobile element.
2018-11-22 | GSE120571 | GEO