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CReasPy-Cloning: a method for simultaneous cloning and engineering of megabase-sized genomes in yeast using the CRISPR-Cas9 system.

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dc.contributor.author Ruiz, Estelle
dc.contributor.author Talenton, Vincent
dc.contributor.author Dubrana, Marie-Pierre
dc.contributor.author Guesdon, Gabrielle
dc.contributor.author Lluch-Senar, Maria 1982-
dc.contributor.author Salin, Franck
dc.contributor.author Sirand-Pugnet, Pascal
dc.contributor.author Arfi, Yonathan
dc.contributor.author Lartigue, Carole
dc.date.accessioned 2020-03-17T07:28:54Z
dc.date.available 2020-03-17T07:28:54Z
dc.date.issued 2019
dc.identifier.citation Ruiz E, Talenton V, Dubrana MP, Guesdon G, Lluch-Senar M, Salin F, Sirand-Pugnet P, Arfi Y, Lartigue C. CReasPy-Cloning: a method for simultaneous cloning and engineering of megabase-sized genomes in yeast using the CRISPR-Cas9 system. ACS Synth Biol. 2019; 8(11):2547-57. DOI: 10.1021/acssynbio.9b00224
dc.identifier.issn 2161-5063
dc.identifier.uri http://hdl.handle.net/10230/43900
dc.description.abstract Over the past decade, a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or "cloning") their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria, and algae. The cloning step requires the insertion of yeast genetic elements in the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast's highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (<50) number of clones, allowing for significantly shortened genome engineering cycle times.
dc.description.sponsorship This work is part of the European MiniCell project “A model-driven approach to minimal cell engineering for medical therapy” selected by ANR, in the frame of the ERASynBio second Joint Call for Transnational Research Projects (No. ANR-15-SYNB-0001-04). It has also been supported by the National Science Foundation (Grant No. IOS-1110151) and the European Union’s Horizon 2020 research and innovation program under Grant Agreement No. 634942.
dc.format.mimetype application/pdf
dc.language.iso eng
dc.publisher American Chemical Society (ACS)
dc.relation.ispartof ACS Synth Biol. 2019; 8(11):2547-57
dc.rights © 2019 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
dc.rights.uri http://pubs.acs.org/page/policy/authorchoice_termsofuse.html
dc.title CReasPy-Cloning: a method for simultaneous cloning and engineering of megabase-sized genomes in yeast using the CRISPR-Cas9 system.
dc.type info:eu-repo/semantics/article
dc.identifier.doi http://dx.doi.org/10.1021/acssynbio.9b00224
dc.subject.keyword CRISPR-Cas9
dc.subject.keyword Saccharomyces cerevisiae
dc.subject.keyword Genome cloning
dc.subject.keyword Genome editing
dc.subject.keyword Genome transplantation
dc.subject.keyword Mycoplasma
dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/634942
dc.rights.accessRights info:eu-repo/semantics/openAccess
dc.type.version info:eu-repo/semantics/publishedVersion


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