Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast
The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of yeast cloning strategies, a new approach combining two already described methods, Fusion cloning and CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by the fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves the cloning efficacy.
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Subjects: | U60 - Sciences de la vie et de la Terre, génie génétique, génome, levure, Saccharomyces cerevisiae, génomique, Mycoplasma, http://aims.fao.org/aos/agrovoc/c_15974, http://aims.fao.org/aos/agrovoc/c_3224, http://aims.fao.org/aos/agrovoc/c_8480, http://aims.fao.org/aos/agrovoc/c_6724, http://aims.fao.org/aos/agrovoc/c_92382, http://aims.fao.org/aos/agrovoc/c_5020, |
Online Access: | http://agritrop.cirad.fr/608090/ http://agritrop.cirad.fr/608090/7/608090.pdf |
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dig-cirad-fr-6080902024-03-07T19:00:08Z http://agritrop.cirad.fr/608090/ http://agritrop.cirad.fr/608090/ Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast. Guesdon Gabrielle, Gourgues Géraldine, Rideau Fabien, Ipoutcha Thomas, Manso-Silvan Lucia, Jules Matthieu, Sirand-Pugnet Pascal, Blanchard Alain, Lartigue Carole. 2023. ACS Synthetic Biology, 12 (11) : 3252-3266.https://doi.org/10.1021/acssynbio.3c00248 <https://doi.org/10.1021/acssynbio.3c00248> Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast Guesdon, Gabrielle Gourgues, Géraldine Rideau, Fabien Ipoutcha, Thomas Manso-Silvan, Lucia Jules, Matthieu Sirand-Pugnet, Pascal Blanchard, Alain Lartigue, Carole eng 2023 ACS Synthetic Biology U60 - Sciences de la vie et de la Terre génie génétique génome levure Saccharomyces cerevisiae génomique Mycoplasma http://aims.fao.org/aos/agrovoc/c_15974 http://aims.fao.org/aos/agrovoc/c_3224 http://aims.fao.org/aos/agrovoc/c_8480 http://aims.fao.org/aos/agrovoc/c_6724 http://aims.fao.org/aos/agrovoc/c_92382 http://aims.fao.org/aos/agrovoc/c_5020 The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of yeast cloning strategies, a new approach combining two already described methods, Fusion cloning and CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by the fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves the cloning efficacy. article info:eu-repo/semantics/article Journal Article info:eu-repo/semantics/publishedVersion http://agritrop.cirad.fr/608090/7/608090.pdf text Cirad license info:eu-repo/semantics/restrictedAccess https://agritrop.cirad.fr/mention_legale.html https://doi.org/10.1021/acssynbio.3c00248 10.1021/acssynbio.3c00248 info:eu-repo/semantics/altIdentifier/doi/10.1021/acssynbio.3c00248 info:eu-repo/semantics/altIdentifier/purl/https://doi.org/10.1021/acssynbio.3c00248 info:eu-repo/semantics/dataset/purl/https://www.ncbi.nlm.nih.gov/bioproject/PRJNA910329/ info:eu-repo/grantAgreement///ANR-18-CE44-0003//(FRA) Construction et transplantation de génomes semi-synthétiques de Bacillus subtilis, vers le développement de la prochaine génération de châssis bactériens/Bacillus2-0 |
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U60 - Sciences de la vie et de la Terre génie génétique génome levure Saccharomyces cerevisiae génomique Mycoplasma http://aims.fao.org/aos/agrovoc/c_15974 http://aims.fao.org/aos/agrovoc/c_3224 http://aims.fao.org/aos/agrovoc/c_8480 http://aims.fao.org/aos/agrovoc/c_6724 http://aims.fao.org/aos/agrovoc/c_92382 http://aims.fao.org/aos/agrovoc/c_5020 U60 - Sciences de la vie et de la Terre génie génétique génome levure Saccharomyces cerevisiae génomique Mycoplasma http://aims.fao.org/aos/agrovoc/c_15974 http://aims.fao.org/aos/agrovoc/c_3224 http://aims.fao.org/aos/agrovoc/c_8480 http://aims.fao.org/aos/agrovoc/c_6724 http://aims.fao.org/aos/agrovoc/c_92382 http://aims.fao.org/aos/agrovoc/c_5020 |
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U60 - Sciences de la vie et de la Terre génie génétique génome levure Saccharomyces cerevisiae génomique Mycoplasma http://aims.fao.org/aos/agrovoc/c_15974 http://aims.fao.org/aos/agrovoc/c_3224 http://aims.fao.org/aos/agrovoc/c_8480 http://aims.fao.org/aos/agrovoc/c_6724 http://aims.fao.org/aos/agrovoc/c_92382 http://aims.fao.org/aos/agrovoc/c_5020 U60 - Sciences de la vie et de la Terre génie génétique génome levure Saccharomyces cerevisiae génomique Mycoplasma http://aims.fao.org/aos/agrovoc/c_15974 http://aims.fao.org/aos/agrovoc/c_3224 http://aims.fao.org/aos/agrovoc/c_8480 http://aims.fao.org/aos/agrovoc/c_6724 http://aims.fao.org/aos/agrovoc/c_92382 http://aims.fao.org/aos/agrovoc/c_5020 Guesdon, Gabrielle Gourgues, Géraldine Rideau, Fabien Ipoutcha, Thomas Manso-Silvan, Lucia Jules, Matthieu Sirand-Pugnet, Pascal Blanchard, Alain Lartigue, Carole Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast |
description |
The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of yeast cloning strategies, a new approach combining two already described methods, Fusion cloning and CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by the fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves the cloning efficacy. |
format |
article |
topic_facet |
U60 - Sciences de la vie et de la Terre génie génétique génome levure Saccharomyces cerevisiae génomique Mycoplasma http://aims.fao.org/aos/agrovoc/c_15974 http://aims.fao.org/aos/agrovoc/c_3224 http://aims.fao.org/aos/agrovoc/c_8480 http://aims.fao.org/aos/agrovoc/c_6724 http://aims.fao.org/aos/agrovoc/c_92382 http://aims.fao.org/aos/agrovoc/c_5020 |
author |
Guesdon, Gabrielle Gourgues, Géraldine Rideau, Fabien Ipoutcha, Thomas Manso-Silvan, Lucia Jules, Matthieu Sirand-Pugnet, Pascal Blanchard, Alain Lartigue, Carole |
author_facet |
Guesdon, Gabrielle Gourgues, Géraldine Rideau, Fabien Ipoutcha, Thomas Manso-Silvan, Lucia Jules, Matthieu Sirand-Pugnet, Pascal Blanchard, Alain Lartigue, Carole |
author_sort |
Guesdon, Gabrielle |
title |
Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast |
title_short |
Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast |
title_full |
Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast |
title_fullStr |
Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast |
title_full_unstemmed |
Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast |
title_sort |
combining fusion of cells with crispr-cas9 editing for the cloning of large dna fragments or complete bacterial genomes in yeast |
url |
http://agritrop.cirad.fr/608090/ http://agritrop.cirad.fr/608090/7/608090.pdf |
work_keys_str_mv |
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