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  1. Article ; Online: SpEDIT

    Sito Torres-Garcia / Lorenza Di Pompeo / Luke Eivers / Baptiste Gaborieau / Sharon A. White / Alison L. Pidoux / Paulina Kanigowska / Imtiyaz Yaseen / Yizhi Cai / Robin C. Allshire

    Wellcome Open Research, Vol

    A fast and efficient CRISPR/Cas9 method for fission yeast [version 1; peer review: 2 approved]

    2020  Volume 5

    Abstract: The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a ... ...

    Abstract The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong adh1 promoter. To overcome these problems we have developed an improved system, SpEDIT, that uses a synthesised Cas9 sequence codon-optimised for S. pombe expressed from the medium strength adh15 promoter. The SpEDIT system exhibits a flexible modular design where the sgRNA is fused to the 3’ end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence. Lastly, the inclusion of sites for the BsaI type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The SpEDIT system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the ade6+ or ura4+ genes by transformation of cells from asynchronous cultures. SpEDIT also permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the SpEDIT system displayed reduced toxicity compared to currently available S. pombe editing systems. Thus, SpEDIT provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.
    Keywords Medicine ; R ; Science ; Q
    Subject code 570
    Language English
    Publishing date 2020-11-01T00:00:00Z
    Publisher Wellcome
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Large domains of heterochromatin direct the formation of short mitotic chromosome loops

    Maximilian H Fitz-James / Pin Tong / Alison L Pidoux / Hakan Ozadam / Liyan Yang / Sharon A White / Job Dekker / Robin C Allshire

    eLife, Vol

    2020  Volume 9

    Abstract: During mitosis chromosomes reorganise into highly compact, rod-shaped forms, thought to consist of consecutive chromatin loops around a central protein scaffold. Condensin complexes are involved in chromatin compaction, but the contribution of other ... ...

    Abstract During mitosis chromosomes reorganise into highly compact, rod-shaped forms, thought to consist of consecutive chromatin loops around a central protein scaffold. Condensin complexes are involved in chromatin compaction, but the contribution of other chromatin proteins, DNA sequence and histone modifications is less understood. A large region of fission yeast DNA inserted into a mouse chromosome was previously observed to adopt a mitotic organisation distinct from that of surrounding mouse DNA. Here, we show that a similar distinct structure is common to a large subset of insertion events in both mouse and human cells and is coincident with the presence of high levels of heterochromatic H3 lysine nine trimethylation (H3K9me3). Hi-C and microscopy indicate that the heterochromatinised fission yeast DNA is organised into smaller chromatin loops than flanking euchromatic mouse chromatin. We conclude that heterochromatin alters chromatin loop size, thus contributing to the distinct appearance of heterochromatin on mitotic chromosomes.
    Keywords chromosome structure ; heterochromatin ; condensin ; loop extrusion ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 571
    Language English
    Publishing date 2020-09-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: The RFTS domain of Raf2 is required for Cul4 interaction and heterochromatin integrity in fission yeast.

    Sharon A White / Alessia Buscaino / Luis Sanchez-Pulido / Chris P Ponting / Matthew W Nowicki / Robin C Allshire

    PLoS ONE, Vol 9, Iss 8, p e

    2014  Volume 104161

    Abstract: Centromeric heterochromatin assembly in fission yeast is critical for faithful chromosome segregation at mitosis. Its assembly requires a concerted pathway of events whereby the RNA interference (RNAi) pathway guides H3K9 methylation to target sequences. ...

    Abstract Centromeric heterochromatin assembly in fission yeast is critical for faithful chromosome segregation at mitosis. Its assembly requires a concerted pathway of events whereby the RNA interference (RNAi) pathway guides H3K9 methylation to target sequences. H3K9 methylation, a hallmark of heterochromatin structure, is mediated by the single histone methyltransferase Clr4 (equivalent to metazoan Suv3-9), a component of the CLRC complex. Loss of or defects in CLRC components disrupts heterochromatin formation due to loss of H3K9 methylation, thus an intact, fully functional CLRC complex is required for heterochromatin integrity. Despite its importance, little is known about the contribution of the CLRC component Raf2 to H3K9 methylation and heterochromatin assembly. We demonstrate that Raf2 is concentrated at centromeres and contrary to other analyses, we find that loss of Raf2 does not affect CENP-ACnp1 localisation or recruitment to centromeres. Our sequence alignments show that Raf2 contains a Replication Foci Targeting Sequence (RFTS) domain homologous to the RFTS domain of the human DNA methyltransferase DNMT1. We show that the Raf2 RFTS domain is required for centromeric heterochromatin formation as its mutation disrupts H3K9 methylation but not the processing of centromeric transcripts into small interfering RNAs (siRNAs) by the RNAi pathway. Analysis of biochemical interactions demonstrates that the RFTS domain mediates an interaction between Raf2 and the CLRC component Cul4. We conclude that the RFTS domain of Raf2 is a protein interaction module that plays an important role in heterochromatin formation at centromeres.
    Keywords Medicine ; R ; Science ; Q
    Subject code 570
    Language English
    Publishing date 2014-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article: A systematic genetic screen identifies new factors influencing centromeric heterochromatin integrity in fission yeast

    Bayne, Elizabeth H / Dominika A Bijos / Flavia de Lima Alves / Juri Rappsilber / Robin C Allshire / Sharon A White

    Genome biology. 2014 Oct., v. 15, no. 10

    2014  

    Abstract: BACKGROUND: Heterochromatin plays important roles in the regulation and stability of eukaryotic genomes. Both heterochromatin components and pathways that promote heterochromatin assembly, including RNA interference, RNAi, are broadly conserved between ... ...

    Abstract BACKGROUND: Heterochromatin plays important roles in the regulation and stability of eukaryotic genomes. Both heterochromatin components and pathways that promote heterochromatin assembly, including RNA interference, RNAi, are broadly conserved between the fission yeast Schizosaccharomyces pombe and humans. As a result, fission yeast has emerged as an important model system for dissecting mechanisms governing heterochromatin integrity. Thus far, over 50 proteins have been found to contribute to heterochromatin assembly at fission yeast centromeres. However, previous studies have not been exhaustive, and it is therefore likely that further factors remain to be identified. RESULTS: To gain a more complete understanding of heterochromatin assembly pathways, we have performed a systematic genetic screen for factors required for centromeric heterochromatin integrity. In addition to known RNAi and chromatin modification components, we identified several proteins with previously undescribed roles in heterochromatin regulation. These included both known and newly characterised splicing-associated proteins, which are required for proper processing of centromeric transcripts by the RNAi pathway, and COP9 signalosome components Csn1 and Csn2, whose role in heterochromatin assembly can be explained at least in part by a role in the Ddb1-dependent degradation of the heterochromatin regulator Epe1. CONCLUSIONS: This work has revealed new factors involved in RNAi-directed heterochromatin assembly in fission yeast. Our findings support and extend previous observations that implicate components of the splicing machinery as a platform for RNAi, and demonstrate a novel role for the COP9 signalosome in heterochromatin regulation.
    Keywords centromeres ; genome ; heterochromatin ; humans ; RNA interference ; Schizosaccharomyces pombe
    Language English
    Dates of publication 2014-10
    Size p. 481.
    Publishing place Springer-Verlag
    Document type Article
    ZDB-ID 2040529-7
    ISSN 1474-760X ; 1465-6914 ; 1465-6906
    ISSN (online) 1474-760X ; 1465-6914
    ISSN 1465-6906
    DOI 10.1186/s13059-014-0481-4
    Database NAL-Catalogue (AGRICOLA)

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