Ontology highlight
ABSTRACT:
PROVIDER: PRJNA227019 | ENA |
REPOSITORIES: ENA
Similar Datasets
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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.
2018-06-04 | E-MTAB-6808 | biostudies-arrayexpress
Project description:Species within the human pathogenic Cryptococcus species complex are major threats to public health, causing about one million infections globally each year. Cryptococcus amylolentus is the most closely known related species of the pathogenic Cryptococcus species complex, and it is non-pathogenic. Additionally, while pathogenic Cryptococcus species have bipolar mating systems with a single large MAT locus that represents a derived state in Basidiomycetes, C. amylolentus has a tetrapolar mating system with two MAT loci (P/R and HD) located on different chromosomes. Thus, studying C. amylolentus will shed light on the origin and evolution of pathogenesis, as well as the transition from tetrapolar to bipolar mating systems in the pathogenic Cryptococcus species. In this study, we sequenced, assembled, and annotated the genomes of two C. amylolentus isolates, CBS6039 and CBS6273, which are sexual and interfertile. Genome comparison between the two C. amylolentus isolates identified the boundaries and the complete gene contents of the P/R and HD MAT loci. Also, bioinformatics and ChIP-seq analyses showed that C. amylolentus has regional centromeres that are enriched with species-specific transposable and repetitive elements, similar to the centromeric structures in the pathogenic Cryptococcus species. Additionally, we found that while neither of the P/R and HD loci in C. amylolentus is physically linked to its centromere, both MAT loci exhibit centromere linkage in meiosis, suggesting the presence of recombinational suppressors and/or epistatic gene interactions in the inter MAT-CEN regions. Furthermore, genomic comparison between C. amylolentus and pathogenic Cryptococcus species provides evidence that chromosomal rearrangements mediated by intercentromeric recombination have occurred after the two lineages split from their common ancestor. We propose a model in which the evolution of the bipolar mating system was initiated by an ectopic recombination event mediated by repetitive elements located within the centromeric regions and shared between chromosomes. This translocation brought the P/R and HD loci onto the same chromosome, and was followed by chromosomal rearrangements that resulted in the two MAT loci becoming physically linked and eventually fused to form the single contiguous MAT locus that is now extant in the pathogenic Cryptococcus species.
2018-04-08 | GSE95128 | GEO