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Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements

ABSTRACT: Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements

PROVIDER: PRJNA844083 | ENA |

REPOSITORIES: ENA

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Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements

Project description:We explored how Cas9-induced double-strand breaks (DSBs) on Ty1 produce genomic alterations in the diploid yeast Saccharomyces cerevisiae. Following Cas9 induction, we observed a significant elevation of chromosome rearrangements (large deletions and duplications), loss of heterozygosity (gene conversions, crossovers, and break-induced replication), and aneuploidy. Almost all of the chromosomal rearrangements reflect the repairing of DSBs at Ty1 elements by homologous recombination.

2022-06-01 | GSE205199 | GEO

Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements.

Project description:Although homologous recombination between transposable elements can drive genomic evolution in yeast by facilitating chromosomal rearrangements, the details of the underlying mechanisms are not fully clarified. In the genome of the yeast Saccharomyces cerevisiae, the most common class of transposon is the retrotransposon Ty1. Here, we explored how Cas9-induced double-strand breaks (DSBs) directed to Ty1 elements produce genomic alterations in this yeast species. Following Cas9 induction, we observed a significant elevation of chromosome rearrangements such as deletions, duplications and translocations. In addition, we found elevated rates of mitotic recombination, resulting in loss of heterozygosity. Using Southern analysis coupled with short- and long-read DNA sequencing, we revealed important features of recombination induced in retrotransposons. Almost all of the chromosomal rearrangements reflect the repair of DSBs at Ty1 elements by non-allelic homologous recombination; clustered Ty elements were hotspots for chromosome rearrangements. In contrast, a large proportion (about three-fourths) of the allelic mitotic recombination events have breakpoints in unique sequences. Our analysis suggests that some of the latter events reflect extensive processing of the broken ends produced in the Ty element that extend into unique sequences resulting in break-induced replication. Finally, we found that haploid and diploid strain have different preferences for the pathways used to repair double-stranded DNA breaks. Our findings demonstrate the importance of DNA lesions in retrotransposons in driving genome evolution.

| S-EPMC9879454 | biostudies-literature

Detecting non-canonically-targeted SpCas9 generated DSBs in human cells by LAM-HTGTS

Project description:We identified robust cleavage by non-canonically-targeted SpCas9 that target protospacer with a few bases from the canonical NGG PAM in biochemistry assay. To determine whether this spaced SpCas9 cleavage exists in human cells, we employed high-throughput genome-wide translocation sequencing (LAM-HTGTS) using SpCas9 with three independent canonical sgRNAs served as bait (bait-A, bait-L and bait-D) to compared the cleavage activities of canonically-targeted and non-canonically-targeted SpCas9 in eight locus (RAG1B-D, RAG1K-O) in chromosome 11. We found that almost all 1bp-target shifted SpCas9 generated substantial DSBs as the canonical controls. While detected less frequently, 2bp-targeted shifted SpCas9 generated significant amounts of DSBs in RAG1D and RAG1L locus. These findings underscore the spaced PAM-mediated DSBs occurred in living cells.

2022-02-28 | GSE192459 | GEO

Detecting non-canonically-targeted SpCas9 generated DSBs in human cells by LAM-HTGTS

Project description:Detecting non-canonically-targeted SpCas9 generated DSBs in human cells by LAM-HTGTS

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Acinetobacter sp. TY1

Project description:Acinetobacter sp. TY1 Genome sequencing

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Retroelement Ty1 Integrase interactome identified using tandem chromatin affinity purification ( TChAP) after in vivo crosslink in yeast Saccharomyces cerevisiae

Project description:To identify yeast proteins associated with Ty1 integrase (IN) that could regulate Ty1 replication, we co-purified IN partners using the tandem chromatin affinity purification procedure after in vivo cross-link (TChAP), which we developed previously (Nguyen et al. 2014). We first identified RNA Pol I and Pol III complexes and also a small subset of additional evolutionary conserved complexes, including PAF1 (Polymerase-Associated Factor 1),FACT (FAcilitates Chromatin Transcription), the proteasome and the CK2 kinase. We next confirmed that CK2 interacts with Ty1 integrase in vivo and repress Ty1 retromobility. We showed that Ty1 IN is a substrate of CK2 in vitro and identified 12 phosphorylated residues. In vivo approaches showed that only part of the protein was phosphorylated in the cells and did not demonstrate any direct evidence between Ty1 IN phosphorylation and retromobility inhibition.

2022-11-17 | PXD027420 | Pride

TY1-H2A-ChIP-seq datasets of Trypanosoma brucei

Project description:Analysis of the genome-wide distribution of the histone H2A, using TY1-H2A as a Proxy

2020-02-21 | GSE145625 | GEO

Spo11-oligo mapping of DSBs in an SK1 x S288C F1 hybrid strain

Project description:The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are non-randomly distributed across the genome. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to map the distribution of meiotic DSBs in an SK1 x S88C F1 hybrid strain of Saccharomyces cerevisiae.

2017-07-13 | GSE101339 | GEO

P. aeruginosa TY1-PA3898 ChIP-seq

Project description:P. aeruginosa TY1-PA3898 ChIP-seq

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Retrotransposon Ty1 integration targets specifically positioned asymmetric nucleosomal DNA segments in tRNA hotspots

Project description:Using a TIP-seq protocol (specifically isolating transposon insertion junctions) we determined that the Ty1 retrotransposon targets tRNA genes and, in particular, we determined that the transposon inserts into nucleosomal DNA in an asymmetric pattern.

2012-01-01 | GSE33986 | GEO

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