Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1003443 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR25570240_1.fastq.gz | Fastqsanger.gz | |||
| SRR25570240_2.fastq.gz | Fastqsanger.gz | |||
| SRR25570241_1.fastq.gz | Fastqsanger.gz | |||
| SRR25570241_2.fastq.gz | Fastqsanger.gz | |||
| SRR25570242_1.fastq.gz | Fastqsanger.gz |
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Project description:We report the integration site specificity of Cas1–Cas2 into target plasmid DNA encoding a CRISPR locus. The goal of this study is to determine the effect of IHF on target site selection of Cas1–Cas2.
2016-05-27 | GSE79992 | GEO
Project description:Bacteria defend themselves from viral infection using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this immune strategy by instead leveraging DNA synthesis, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems execute an unprecedented immunity mechanism that involves de novo gene synthesis via rolling-circle reverse transcription of a non-coding RNA (ncRNA). Unbiased profiling of RT-associated RNA and DNA ligands in DRT2-expressing cells revealed that reverse transcription generates concatenated cDNA repeats through programmed template jumping on the ncRNA. The presence of phage then triggers second-strand cDNA synthesis, leading to the production of long double-stranded DNA. Remarkably, this DNA product is efficiently transcribed, generating messenger RNAs that encode a stop codon-less, never-ending ORF (neo) whose translation causes potent growth arrest. Phylogenetic analyses and screening of diverse DRT2 homologs further revealed broad conservation of rolling-circle reverse transcription and Neo protein function. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation, and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA.
2024-06-18 | MSV000095071 | MassIVE
Project description:We report the sites of protospacer integration into plasmids by Cas1-Cas2. The goal of this study is to determine the role of sequence elements on protospacer integration. Methods: Cas1-Cas2 integration products were fragmented, end-repaired, adapter ligated, PCR amplified, and analyzed by Illumina sequencing. Protospacer-containing reads were selected by Cutadapt and mapped to the plasmid with Bowtie.
2015-01-30 | E-GEOD-64552 | biostudies-arrayexpress
Project description:Bacteria defend themselves from viral predation using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this immune strategy by instead leveraging DNA synthesis, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems execute an unprecedented immunity mechanism that involves de novo gene synthesis via rolling-circle reverse transcription of a non-coding RNA (ncRNA). Unbiased profiling of RT-associated RNA and DNA ligands uncovered constitutive synthesis of concatenated cDNA repeats through programmed template jumping on the ncRNA. The presence of phage then triggers second-strand cDNA synthesis, leading to the production of long double-stranded DNA. Remarkably, this nascent ‘gene’ is efficiently transcribed, generating messenger RNAs that encode a stop codon-less, never-ending ORF (neo) whose translation causes potent growth arrest. Phylogenetic analyses and screening of diverse DRT2 homologs further revealed broad conservation of rolling-circle reverse transcription and Neo protein function. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation, and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA.
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Project description:Bacteria defend themselves from viral predation using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this immune strategy by instead leveraging DNA synthesis, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems execute an unprecedented immunity mechanism that involves de novo gene synthesis via rolling-circle reverse transcription of a non-coding RNA (ncRNA). Unbiased profiling of RT-associated RNA and DNA ligands uncovered constitutive synthesis of concatenated cDNA repeats through programmed template jumping on the ncRNA. The presence of phage then triggers second-strand cDNA synthesis, leading to the production of long double-stranded DNA. Remarkably, this nascent ‘gene’ is efficiently transcribed, generating messenger RNAs that encode a stop codon-less, never-ending ORF (neo) whose translation causes potent growth arrest. Phylogenetic analyses and screening of diverse DRT2 homologs further revealed broad conservation of rolling-circle reverse transcription and Neo protein function. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation, and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA.
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Project description:Prokaryotic Cas1-Cas2 protein complexes generate adaptive immunity to mobile genetic elements (MGEs), by capture and integration of MGE DNA in to CRISPR sites. De novo immunity relies on Cas1-Cas2 targeting MGE DNA, without the aid of pre-existing immunity complexes, through mechanisms of ‘naive adaptation’ that are not clear. Using E. coli we show that the chaperone DnaK inhibits Cas1-Cas2 from DNA binding and integration, and that DnaK expression prevents naïve adaptation from chromosomal self-targeting. We show that that inhibition of naïve adaptation is reversible by eliminating DnaK from cells, by mutation of the DnaK substrate binding domain, and by expression of an MGE (phage )protein. We show that a fluorescently labelled Cas1 fusion can be visualised in living cells. Formation of foci depends on active DNA replication, and that the number of foci per cell is much increased in cells lacking DnaK. We discuss a model in which DnaK provides a mechanism for restraining naïve adaptation from self-targeting. This restraint is released once MGE DNA is present in the cell.
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