Reshuffling yeast chromosomes with CRISPR/Cas9
Genome engineering is a powerful approach to study how chromosomal architecture impacts phenotypes. However, quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. We report a novel application of the CRISPR/C...
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| creator | Fleiss, Aubin O'Donnell, Samuel Fournier, Téo Lu, Wenqing Agier, Nicolas Delmas, Stéphane Schacherer, Joseph Fischer, Gilles |
| description | Genome engineering is a powerful approach to study how chromosomal architecture impacts phenotypes. However, quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. We report a novel application of the CRISPR/Cas9 technology allowing to generate with high efficiency both uniquely targeted and multiple concomitant reciprocal translocations in the yeast genome. Targeted translocations are constructed by inducing two double-strand breaks on different chromosomes and forcing the trans-chromosomal repair through homologous recombination by chimerical donor DNAs. Multiple translocations are generated from the induction of several DSBs in LTR repeated sequences and promoting repair using endogenous uncut LTR copies as template. All engineered translocations are markerless and scarless. Targeted translocations are produced at base pair resolution and can be sequentially generated one after the other. Multiple translocations result in a large diversity of karyotypes and are associated in many instances with the formation of unanticipated segmental duplications. To test the phenotypic impact of translocations, we first recapitulated in a lab strain the SSU1/ECM34 translocation providing increased sulphite resistance to wine isolates. Surprisingly, the same translocation in a laboratory strain resulted in decreased sulphite resistance. However, adding the repeated sequences that are present in the SSU1 promoter of the resistant wine strain induced sulphite resistance in the lab strain, yet to a lower level than that of the wine isolate, implying that additional polymorphisms also contribute to the phenotype. These findings illustrate the advantage brought by our technique to untangle the phenotypic impacts of structural variations from confounding effects of base substitutions. Secondly, we showed that strains with multiple translocations, even those devoid of unanticipated segmental duplications, display large phenotypic diversity in a wide range of environmental conditions, showing that simply reconfiguring chromosome architecture is sufficient to provide fitness advantages in stressful growth conditions. |
| doi_str_mv | 10.1371/journal.pgen.1008332 |
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However, quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. We report a novel application of the CRISPR/Cas9 technology allowing to generate with high efficiency both uniquely targeted and multiple concomitant reciprocal translocations in the yeast genome. Targeted translocations are constructed by inducing two double-strand breaks on different chromosomes and forcing the trans-chromosomal repair through homologous recombination by chimerical donor DNAs. Multiple translocations are generated from the induction of several DSBs in LTR repeated sequences and promoting repair using endogenous uncut LTR copies as template. All engineered translocations are markerless and scarless. Targeted translocations are produced at base pair resolution and can be sequentially generated one after the other. Multiple translocations result in a large diversity of karyotypes and are associated in many instances with the formation of unanticipated segmental duplications. To test the phenotypic impact of translocations, we first recapitulated in a lab strain the SSU1/ECM34 translocation providing increased sulphite resistance to wine isolates. Surprisingly, the same translocation in a laboratory strain resulted in decreased sulphite resistance. However, adding the repeated sequences that are present in the SSU1 promoter of the resistant wine strain induced sulphite resistance in the lab strain, yet to a lower level than that of the wine isolate, implying that additional polymorphisms also contribute to the phenotype. These findings illustrate the advantage brought by our technique to untangle the phenotypic impacts of structural variations from confounding effects of base substitutions. Secondly, we showed that strains with multiple translocations, even those devoid of unanticipated segmental duplications, display large phenotypic diversity in a wide range of environmental conditions, showing that simply reconfiguring chromosome architecture is sufficient to provide fitness advantages in stressful growth conditions.</description><identifier>ISSN: 1553-7404</identifier><identifier>ISSN: 1553-7390</identifier><identifier>EISSN: 1553-7404</identifier><identifier>DOI: 10.1371/journal.pgen.1008332</identifier><identifier>PMID: 31465441</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Anion Transport Proteins - genetics ; Artificial chromosomes ; Biochemistry, Molecular Biology ; Biology ; Biology and Life Sciences ; Biotechnology ; Chromosome translocations ; Chromosomes ; Chromosomes, Fungal - genetics ; CRISPR ; CRISPR-Cas Systems ; CRISPR-Cas technology ; Deoxyribonucleic acid ; DNA ; DNA Shuffling - methods ; Engineering and Technology ; Environmental conditions ; Ethanol ; Fitness ; Gene Editing - methods ; Gene expression ; Genetic research ; Genome editing ; Genome, Fungal - genetics ; Genomes ; Genomics ; Growth conditions ; Homologous recombination ; Karyotypes ; Laboratories ; Life Sciences ; Mammals ; Methods ; Mutation ; Phenotypes ; Physical Sciences ; Promoter Regions, Genetic - genetics ; RNA polymerase ; Saccharomyces cerevisiae - genetics ; Saccharomyces cerevisiae Proteins - genetics ; Supervision ; Translocation, Genetic ; Wine ; Yeast</subject><ispartof>PLoS genetics, 2019-08, Vol.15 (8), p.e1008332-e1008332</ispartof><rights>COPYRIGHT 2019 Public Library of Science</rights><rights>2019 Fleiss et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><rights>2019 Fleiss et al 2019 Fleiss et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c619t-caa1a63f4567734a542e3a4f710a6c35861b86ed64cce7de4b2546e6e79000473</citedby><cites>FETCH-LOGICAL-c619t-caa1a63f4567734a542e3a4f710a6c35861b86ed64cce7de4b2546e6e79000473</cites><orcidid>0000-0003-2306-3138 ; 0000-0003-0126-4537 ; 0000-0003-2447-1993 ; 0000-0001-5732-2682 ; 0000-0001-5523-7548 ; 0000-0002-4860-6728 ; 0000-0002-6606-6884</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738639/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738639/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79569,79570</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31465441$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.sorbonne-universite.fr/hal-02303598$$DView record in HAL$$Hfree_for_read</backlink></links><search><creatorcontrib>Fleiss, Aubin</creatorcontrib><creatorcontrib>O'Donnell, Samuel</creatorcontrib><creatorcontrib>Fournier, Téo</creatorcontrib><creatorcontrib>Lu, Wenqing</creatorcontrib><creatorcontrib>Agier, Nicolas</creatorcontrib><creatorcontrib>Delmas, Stéphane</creatorcontrib><creatorcontrib>Schacherer, Joseph</creatorcontrib><creatorcontrib>Fischer, Gilles</creatorcontrib><title>Reshuffling yeast chromosomes with CRISPR/Cas9</title><title>PLoS genetics</title><addtitle>PLoS Genet</addtitle><description>Genome engineering is a powerful approach to study how chromosomal architecture impacts phenotypes. However, quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. We report a novel application of the CRISPR/Cas9 technology allowing to generate with high efficiency both uniquely targeted and multiple concomitant reciprocal translocations in the yeast genome. Targeted translocations are constructed by inducing two double-strand breaks on different chromosomes and forcing the trans-chromosomal repair through homologous recombination by chimerical donor DNAs. Multiple translocations are generated from the induction of several DSBs in LTR repeated sequences and promoting repair using endogenous uncut LTR copies as template. All engineered translocations are markerless and scarless. Targeted translocations are produced at base pair resolution and can be sequentially generated one after the other. Multiple translocations result in a large diversity of karyotypes and are associated in many instances with the formation of unanticipated segmental duplications. To test the phenotypic impact of translocations, we first recapitulated in a lab strain the SSU1/ECM34 translocation providing increased sulphite resistance to wine isolates. Surprisingly, the same translocation in a laboratory strain resulted in decreased sulphite resistance. However, adding the repeated sequences that are present in the SSU1 promoter of the resistant wine strain induced sulphite resistance in the lab strain, yet to a lower level than that of the wine isolate, implying that additional polymorphisms also contribute to the phenotype. These findings illustrate the advantage brought by our technique to untangle the phenotypic impacts of structural variations from confounding effects of base substitutions. Secondly, we showed that strains with multiple translocations, even those devoid of unanticipated segmental duplications, display large phenotypic diversity in a wide range of environmental conditions, showing that simply reconfiguring chromosome architecture is sufficient to provide fitness advantages in stressful growth conditions.</description><subject>Anion Transport Proteins - genetics</subject><subject>Artificial chromosomes</subject><subject>Biochemistry, Molecular Biology</subject><subject>Biology</subject><subject>Biology and Life Sciences</subject><subject>Biotechnology</subject><subject>Chromosome translocations</subject><subject>Chromosomes</subject><subject>Chromosomes, Fungal - genetics</subject><subject>CRISPR</subject><subject>CRISPR-Cas Systems</subject><subject>CRISPR-Cas technology</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA Shuffling - methods</subject><subject>Engineering and Technology</subject><subject>Environmental conditions</subject><subject>Ethanol</subject><subject>Fitness</subject><subject>Gene Editing - methods</subject><subject>Gene expression</subject><subject>Genetic research</subject><subject>Genome editing</subject><subject>Genome, Fungal - genetics</subject><subject>Genomes</subject><subject>Genomics</subject><subject>Growth conditions</subject><subject>Homologous recombination</subject><subject>Karyotypes</subject><subject>Laboratories</subject><subject>Life Sciences</subject><subject>Mammals</subject><subject>Methods</subject><subject>Mutation</subject><subject>Phenotypes</subject><subject>Physical Sciences</subject><subject>Promoter Regions, Genetic - genetics</subject><subject>RNA polymerase</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Saccharomyces cerevisiae Proteins - genetics</subject><subject>Supervision</subject><subject>Translocation, Genetic</subject><subject>Wine</subject><subject>Yeast</subject><issn>1553-7404</issn><issn>1553-7390</issn><issn>1553-7404</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNptkm-LEzEQxhdRvPP0G4gWBNEX7eXfJps3QinqFQpK1dchm53sbkk3Ndk9uW9v1u4d7eGrhMlvnpl5Mln2GqMFpgJf7_wQOu0Whxq6BUaooJQ8yS5xntO5YIg9PblfZC9i3CFE80KK59kFxYznjOHLbLGF2AzWurarZ3egYz8zTfB7H_0e4uxP2zez1Xb94_v2eqWjfJk9s9pFeDWdV9mvL59_rm7mm29f16vlZm44lv3caI01p5blXAjKdM4IUM2swEhzk7rguCw4VJwZA6ICVpKcceAgJEKICXqVvT3qHpyPaho1KkIkZoIXhCdifSQqr3fqENq9DnfK61b9C_hQKx361jhQhJd5gQhIWyEmsZE5oxRbKa3UqORF0vo0VRvKPVQGuj5odyZ6_tK1jar9reKCFpzKJPDxKNA8SrtZbtQYQ4Qm82VxixP7YSoW_O8BYq_2bTTgnO7AD-OMBcXp5_go--4R-n8nJqrWadi2sz71aEZRteTJTcKS3Yl6f0I1oF3fRO-GvvVdPAfZETTBxxjAPgyEkRo3774JNW6emjYvpb059fAh6X7V6F_35NEG</recordid><startdate>20190801</startdate><enddate>20190801</enddate><creator>Fleiss, Aubin</creator><creator>O'Donnell, Samuel</creator><creator>Fournier, Téo</creator><creator>Lu, Wenqing</creator><creator>Agier, Nicolas</creator><creator>Delmas, Stéphane</creator><creator>Schacherer, Joseph</creator><creator>Fischer, Gilles</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QP</scope><scope>7QR</scope><scope>7SS</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>P64</scope><scope>PHGZM</scope><scope>PHGZT</scope><scope>PIMPY</scope><scope>PJZUB</scope><scope>PKEHL</scope><scope>PPXIY</scope><scope>PQEST</scope><scope>PQGLB</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>RC3</scope><scope>7X8</scope><scope>1XC</scope><scope>VOOES</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0003-2306-3138</orcidid><orcidid>https://orcid.org/0000-0003-0126-4537</orcidid><orcidid>https://orcid.org/0000-0003-2447-1993</orcidid><orcidid>https://orcid.org/0000-0001-5732-2682</orcidid><orcidid>https://orcid.org/0000-0001-5523-7548</orcidid><orcidid>https://orcid.org/0000-0002-4860-6728</orcidid><orcidid>https://orcid.org/0000-0002-6606-6884</orcidid></search><sort><creationdate>20190801</creationdate><title>Reshuffling yeast chromosomes with CRISPR/Cas9</title><author>Fleiss, Aubin ; O'Donnell, Samuel ; Fournier, Téo ; Lu, Wenqing ; Agier, Nicolas ; Delmas, Stéphane ; Schacherer, Joseph ; Fischer, Gilles</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c619t-caa1a63f4567734a542e3a4f710a6c35861b86ed64cce7de4b2546e6e79000473</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Anion Transport Proteins - genetics</topic><topic>Artificial chromosomes</topic><topic>Biochemistry, Molecular Biology</topic><topic>Biology</topic><topic>Biology and Life Sciences</topic><topic>Biotechnology</topic><topic>Chromosome translocations</topic><topic>Chromosomes</topic><topic>Chromosomes, Fungal - genetics</topic><topic>CRISPR</topic><topic>CRISPR-Cas Systems</topic><topic>CRISPR-Cas technology</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA Shuffling - methods</topic><topic>Engineering and Technology</topic><topic>Environmental conditions</topic><topic>Ethanol</topic><topic>Fitness</topic><topic>Gene Editing - methods</topic><topic>Gene expression</topic><topic>Genetic research</topic><topic>Genome editing</topic><topic>Genome, Fungal - genetics</topic><topic>Genomes</topic><topic>Genomics</topic><topic>Growth conditions</topic><topic>Homologous recombination</topic><topic>Karyotypes</topic><topic>Laboratories</topic><topic>Life Sciences</topic><topic>Mammals</topic><topic>Methods</topic><topic>Mutation</topic><topic>Phenotypes</topic><topic>Physical Sciences</topic><topic>Promoter Regions, Genetic - genetics</topic><topic>RNA polymerase</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Saccharomyces cerevisiae Proteins - genetics</topic><topic>Supervision</topic><topic>Translocation, Genetic</topic><topic>Wine</topic><topic>Yeast</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Fleiss, Aubin</creatorcontrib><creatorcontrib>O'Donnell, Samuel</creatorcontrib><creatorcontrib>Fournier, Téo</creatorcontrib><creatorcontrib>Lu, Wenqing</creatorcontrib><creatorcontrib>Agier, Nicolas</creatorcontrib><creatorcontrib>Delmas, Stéphane</creatorcontrib><creatorcontrib>Schacherer, Joseph</creatorcontrib><creatorcontrib>Fischer, Gilles</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Oncogenes and Growth Factors Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest Central (New)</collection><collection>ProQuest One Academic (New)</collection><collection>Publicly Available Content Database</collection><collection>ProQuest Health & Medical Research Collection</collection><collection>ProQuest One Academic Middle East (New)</collection><collection>ProQuest One Health & Nursing</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Applied & Life Sciences</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - 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However, quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. We report a novel application of the CRISPR/Cas9 technology allowing to generate with high efficiency both uniquely targeted and multiple concomitant reciprocal translocations in the yeast genome. Targeted translocations are constructed by inducing two double-strand breaks on different chromosomes and forcing the trans-chromosomal repair through homologous recombination by chimerical donor DNAs. Multiple translocations are generated from the induction of several DSBs in LTR repeated sequences and promoting repair using endogenous uncut LTR copies as template. All engineered translocations are markerless and scarless. Targeted translocations are produced at base pair resolution and can be sequentially generated one after the other. Multiple translocations result in a large diversity of karyotypes and are associated in many instances with the formation of unanticipated segmental duplications. To test the phenotypic impact of translocations, we first recapitulated in a lab strain the SSU1/ECM34 translocation providing increased sulphite resistance to wine isolates. Surprisingly, the same translocation in a laboratory strain resulted in decreased sulphite resistance. However, adding the repeated sequences that are present in the SSU1 promoter of the resistant wine strain induced sulphite resistance in the lab strain, yet to a lower level than that of the wine isolate, implying that additional polymorphisms also contribute to the phenotype. These findings illustrate the advantage brought by our technique to untangle the phenotypic impacts of structural variations from confounding effects of base substitutions. Secondly, we showed that strains with multiple translocations, even those devoid of unanticipated segmental duplications, display large phenotypic diversity in a wide range of environmental conditions, showing that simply reconfiguring chromosome architecture is sufficient to provide fitness advantages in stressful growth conditions.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>31465441</pmid><doi>10.1371/journal.pgen.1008332</doi><orcidid>https://orcid.org/0000-0003-2306-3138</orcidid><orcidid>https://orcid.org/0000-0003-0126-4537</orcidid><orcidid>https://orcid.org/0000-0003-2447-1993</orcidid><orcidid>https://orcid.org/0000-0001-5732-2682</orcidid><orcidid>https://orcid.org/0000-0001-5523-7548</orcidid><orcidid>https://orcid.org/0000-0002-4860-6728</orcidid><orcidid>https://orcid.org/0000-0002-6606-6884</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Anion Transport Proteins - genetics Artificial chromosomes Biochemistry, Molecular Biology Biology Biology and Life Sciences Biotechnology Chromosome translocations Chromosomes Chromosomes, Fungal - genetics CRISPR CRISPR-Cas Systems CRISPR-Cas technology Deoxyribonucleic acid DNA DNA Shuffling - methods Engineering and Technology Environmental conditions Ethanol Fitness Gene Editing - methods Gene expression Genetic research Genome editing Genome, Fungal - genetics Genomes Genomics Growth conditions Homologous recombination Karyotypes Laboratories Life Sciences Mammals Methods Mutation Phenotypes Physical Sciences Promoter Regions, Genetic - genetics RNA polymerase Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae Proteins - genetics Supervision Translocation, Genetic Wine Yeast |
| title | Reshuffling yeast chromosomes with CRISPR/Cas9 |
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