Targeted genome editing of sweet orange using Cas9/sgRNA.
Identifieur interne : 000273 ( PubMed/Checkpoint ); précédent : 000272; suivant : 000274Targeted genome editing of sweet orange using Cas9/sgRNA.
Auteurs : Hongge Jia [États-Unis] ; Nian Wang [États-Unis]Source :
- PloS one [ 1932-6203 ] ; 2014.
English descriptors
- KwdEn :
- MESH :
- chemical , genetics : RNA, Guide.
- genetics : Citrus sinensis.
- methods : Gene Targeting, Genetic Engineering.
- Clustered Regularly Interspaced Short Palindromic Repeats.
Abstract
Genetic modification, including plant breeding, has been widely used to improve crop yield and quality, as well as to increase disease resistance. Targeted genome engineering is expected to contribute significantly to future varietal improvement, and genome editing technologies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9/single guide RNA (sgRNA) have already been successfully used to genetically modify plants. However, to date, there has been no reported use of any of the current genome editing approaches in sweet orange, an important fruit crop. In this study, we first developed a novel tool, Xcc-facilitated agroinfiltration, for enhancing transient protein expression in sweet orange leaves. We then successfully employed Xcc-facilitated agroinfiltration to deliver Cas9, along with a synthetic sgRNA targeting the CsPDS gene, into sweet orange. DNA sequencing confirmed that the CsPDS gene was mutated at the target site in treated sweet orange leaves. The mutation rate using the Cas9/sgRNA system was approximately 3.2 to 3.9%. Off-target mutagenesis was not detected for CsPDS-related DNA sequences in our study. This is the first report of targeted genome modification in citrus using the Cas9/sgRNA system-a system that holds significant promise for the study of citrus gene function and for targeted genetic modification.
DOI: 10.1371/journal.pone.0093806
PubMed: 24710347
Affiliations:
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Targeted genome editing of sweet orange using Cas9/sgRNA.</title><author><name sortKey="Jia, Hongge" sort="Jia, Hongge" uniqKey="Jia H" first="Hongge" last="Jia">Hongge Jia</name><affiliation wicri:level="2"><nlm:affiliation>Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, Florida, United States of America.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, Florida</wicri:regionArea><placeName><region type="state">Floride</region></placeName></affiliation></author><author><name sortKey="Wang, Nian" sort="Wang, Nian" uniqKey="Wang N" first="Nian" last="Wang">Nian Wang</name><affiliation wicri:level="2"><nlm:affiliation>Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, Florida, United States of America.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, Florida</wicri:regionArea><placeName><region type="state">Floride</region></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2014">2014</date><idno type="RBID">pubmed:24710347</idno><idno type="pmid">24710347</idno><idno type="doi">10.1371/journal.pone.0093806</idno><idno type="wicri:Area/PubMed/Corpus">000349</idno><idno type="wicri:Area/PubMed/Curation">000349</idno><idno type="wicri:Area/PubMed/Checkpoint">000349</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Targeted genome editing of sweet orange using Cas9/sgRNA.</title><author><name sortKey="Jia, Hongge" sort="Jia, Hongge" uniqKey="Jia H" first="Hongge" last="Jia">Hongge Jia</name><affiliation wicri:level="2"><nlm:affiliation>Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, Florida, United States of America.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, Florida</wicri:regionArea><placeName><region type="state">Floride</region></placeName></affiliation></author><author><name sortKey="Wang, Nian" sort="Wang, Nian" uniqKey="Wang N" first="Nian" last="Wang">Nian Wang</name><affiliation wicri:level="2"><nlm:affiliation>Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, Florida, United States of America.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, Florida</wicri:regionArea><placeName><region type="state">Floride</region></placeName></affiliation></author></analytic><series><title level="j">PloS one</title><idno type="eISSN">1932-6203</idno><imprint><date when="2014" type="published">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Citrus sinensis (genetics)</term><term>Clustered Regularly Interspaced Short Palindromic Repeats</term><term>Gene Targeting (methods)</term><term>Genetic Engineering (methods)</term><term>RNA, Guide (genetics)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>RNA, Guide</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Citrus sinensis</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Gene Targeting</term><term>Genetic Engineering</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Clustered Regularly Interspaced Short Palindromic Repeats</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Genetic modification, including plant breeding, has been widely used to improve crop yield and quality, as well as to increase disease resistance. Targeted genome engineering is expected to contribute significantly to future varietal improvement, and genome editing technologies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9/single guide RNA (sgRNA) have already been successfully used to genetically modify plants. However, to date, there has been no reported use of any of the current genome editing approaches in sweet orange, an important fruit crop. In this study, we first developed a novel tool, Xcc-facilitated agroinfiltration, for enhancing transient protein expression in sweet orange leaves. We then successfully employed Xcc-facilitated agroinfiltration to deliver Cas9, along with a synthetic sgRNA targeting the CsPDS gene, into sweet orange. DNA sequencing confirmed that the CsPDS gene was mutated at the target site in treated sweet orange leaves. The mutation rate using the Cas9/sgRNA system was approximately 3.2 to 3.9%. Off-target mutagenesis was not detected for CsPDS-related DNA sequences in our study. This is the first report of targeted genome modification in citrus using the Cas9/sgRNA system-a system that holds significant promise for the study of citrus gene function and for targeted genetic modification.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">24710347</PMID><DateCreated><Year>2014</Year><Month>4</Month><Day>8</Day></DateCreated><DateCompleted><Year>2015</Year><Month>01</Month><Day>23</Day></DateCompleted><DateRevised><Year>2015</Year><Month>8</Month><Day>6</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Electronic">1932-6203</ISSN><JournalIssue CitedMedium="Internet"><Volume>9</Volume><Issue>4</Issue><PubDate><Year>2014</Year></PubDate></JournalIssue><Title>PloS one</Title><ISOAbbreviation>PLoS ONE</ISOAbbreviation></Journal><ArticleTitle>Targeted genome editing of sweet orange using Cas9/sgRNA.</ArticleTitle><Pagination><MedlinePgn>e93806</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1371/journal.pone.0093806</ELocationID><Abstract><AbstractText>Genetic modification, including plant breeding, has been widely used to improve crop yield and quality, as well as to increase disease resistance. Targeted genome engineering is expected to contribute significantly to future varietal improvement, and genome editing technologies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9/single guide RNA (sgRNA) have already been successfully used to genetically modify plants. However, to date, there has been no reported use of any of the current genome editing approaches in sweet orange, an important fruit crop. In this study, we first developed a novel tool, Xcc-facilitated agroinfiltration, for enhancing transient protein expression in sweet orange leaves. We then successfully employed Xcc-facilitated agroinfiltration to deliver Cas9, along with a synthetic sgRNA targeting the CsPDS gene, into sweet orange. DNA sequencing confirmed that the CsPDS gene was mutated at the target site in treated sweet orange leaves. The mutation rate using the Cas9/sgRNA system was approximately 3.2 to 3.9%. Off-target mutagenesis was not detected for CsPDS-related DNA sequences in our study. This is the first report of targeted genome modification in citrus using the Cas9/sgRNA system-a system that holds significant promise for the study of citrus gene function and for targeted genetic modification.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Jia</LastName><ForeName>Hongge</ForeName><Initials>H</Initials><AffiliationInfo><Affiliation>Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, Florida, United States of America.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Wang</LastName><ForeName>Nian</ForeName><Initials>N</Initials><AffiliationInfo><Affiliation>Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, Florida, United States of America.</Affiliation></AffiliationInfo></Author></AuthorList><Language>ENG</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2014</Year><Month>Apr</Month><Day>07</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>PLoS One</MedlineTA><NlmUniqueID>101285081</NlmUniqueID><ISSNLinking>1932-6203</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D017394">RNA, Guide</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><CommentsCorrectionsList><CommentsCorrections RefType="Cites"><RefSource>Methods Mol Biol. 2013;1069:25-42</RefSource><PMID Version="1">23996306</PMID></CommentsCorrections><CommentsCorrections RefType="Cites"><RefSource>Nat Biotechnol. 2013 Aug;31(8):691-3</RefSource><PMID Version="1">23929340</PMID></CommentsCorrections><CommentsCorrections RefType="Cites"><RefSource>Plant Mol Biol. 2013 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UI="D005818" MajorTopicYN="N">Genetic Engineering</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="Y">methods</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D017394" MajorTopicYN="N">RNA, Guide</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading></MeshHeadingList><OtherID Source="NLM">PMC3977896</OtherID></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2013</Year><Month>12</Month><Day>11</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2014</Year><Month>3</Month><Day>6</Day></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2014</Year><Month>4</Month><Day>9</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2014</Year><Month>4</Month><Day>9</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2015</Year><Month>1</Month><Day>24</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>epublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">24710347</ArticleId><ArticleId IdType="doi">10.1371/journal.pone.0093806</ArticleId><ArticleId IdType="pii">PONE-D-13-52039</ArticleId><ArticleId IdType="pmc">PMC3977896</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>États-Unis</li></country><region><li>Floride</li></region></list><tree><country name="États-Unis"><region name="Floride"><name sortKey="Jia, Hongge" sort="Jia, Hongge" uniqKey="Jia H" first="Hongge" last="Jia">Hongge Jia</name></region><name sortKey="Wang, Nian" sort="Wang, Nian" uniqKey="Wang N" first="Nian" last="Wang">Nian Wang</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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