Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression.
Identifieur interne : 000170 ( Main/Exploration ); précédent : 000169; suivant : 000171Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression.
Auteurs : Lu Long ; Dan-Dan Guo ; Wei Gao ; Wen-Wen Yang ; Li-Pan Hou ; Xiao-Nan Ma ; Yu-Chen Miao ; Jose Ramon Botella ; Chun-Peng SongSource :
- Plant methods [ 1746-4811 ] ; 2018.
Abstract
Background
When developing CRISPR/Cas9 systems for crops, it is crucial to invest time characterizing the genome editing efficiency of the CRISPR/Cas9 cassettes, especially if the transformation system is difficult or time-consuming. Cotton is an important crop for the production of fiber, oil, and biofuel. However, the cotton stable transformation is usually performed using
Results
In this study, we provide a new system to evaluate and validate the efficiency of CRISPR/Cas9 cassettes in cotton using a transient expression system. By using this system, we could select the most effective CRISPR/Cas9 cassettes before the stable transformation. We have also optimized the existing cotton CRISPR/Cas9 system to achieve vastly improved mutagenesis efficiency by incorporating an endogenous
Conclusions
This study provides essential improvements to maximize CRISPR/Cas9-mediated mutation efficiency by reducing risk and workload for the application of CRISPR/Cas9 approaches in the targeted mutagenesis of cotton.
DOI: 10.1186/s13007-018-0353-0
PubMed: 30305839
PubMed Central: PMC6169012
Affiliations:
Links toward previous steps (curation, corpus...)
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sort="Hou, Li Pan" uniqKey="Hou L" first="Li-Pan" last="Hou">Li-Pan Hou</name><affiliation><nlm:affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</nlm:affiliation><wicri:noCountry code="subField">475004 Henan People's Republic of China</wicri:noCountry></affiliation></author><author><name sortKey="Ma, Xiao Nan" sort="Ma, Xiao Nan" uniqKey="Ma X" first="Xiao-Nan" last="Ma">Xiao-Nan Ma</name><affiliation><nlm:affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</nlm:affiliation><wicri:noCountry code="subField">475004 Henan People's Republic of China</wicri:noCountry></affiliation></author><author><name sortKey="Miao, Yu Chen" sort="Miao, Yu Chen" uniqKey="Miao Y" first="Yu-Chen" last="Miao">Yu-Chen 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code="subField">QLD 4072 Australia</wicri:noCountry></affiliation></author><author><name sortKey="Song, Chun Peng" sort="Song, Chun Peng" uniqKey="Song C" first="Chun-Peng" last="Song">Chun-Peng Song</name><affiliation><nlm:affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</nlm:affiliation><wicri:noCountry code="subField">475004 Henan People's Republic of China</wicri:noCountry></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2018">2018</date><idno type="RBID">pubmed:30305839</idno><idno type="pmid">30305839</idno><idno type="doi">10.1186/s13007-018-0353-0</idno><idno type="pmc">PMC6169012</idno><idno type="wicri:Area/Main/Corpus">000150</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Corpus" wicri:corpus="PubMed">000150</idno><idno 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Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</nlm:affiliation><wicri:noCountry code="subField">475004 Henan People's Republic of China</wicri:noCountry></affiliation></author><author><name sortKey="Gao, Wei" sort="Gao, Wei" uniqKey="Gao W" first="Wei" last="Gao">Wei Gao</name><affiliation><nlm:affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</nlm:affiliation><wicri:noCountry code="subField">475004 Henan People's Republic of China</wicri:noCountry></affiliation></author><author><name sortKey="Yang, Wen Wen" sort="Yang, Wen Wen" uniqKey="Yang W" first="Wen-Wen" last="Yang">Wen-Wen Yang</name><affiliation><nlm:affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</nlm:affiliation><wicri:noCountry code="subField">475004 Henan People's Republic of China</wicri:noCountry></affiliation></author><author><name sortKey="Hou, Li Pan" sort="Hou, Li Pan" uniqKey="Hou L" first="Li-Pan" last="Hou">Li-Pan Hou</name><affiliation><nlm:affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</nlm:affiliation><wicri:noCountry code="subField">475004 Henan People's Republic of China</wicri:noCountry></affiliation></author><author><name sortKey="Ma, Xiao Nan" sort="Ma, Xiao Nan" uniqKey="Ma X" first="Xiao-Nan" last="Ma">Xiao-Nan Ma</name><affiliation><nlm:affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</nlm:affiliation><wicri:noCountry code="subField">475004 Henan People's Republic of China</wicri:noCountry></affiliation></author><author><name sortKey="Miao, Yu Chen" sort="Miao, Yu Chen" uniqKey="Miao Y" first="Yu-Chen" last="Miao">Yu-Chen Miao</name><affiliation><nlm:affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</nlm:affiliation><wicri:noCountry code="subField">475004 Henan People's Republic of China</wicri:noCountry></affiliation></author><author><name sortKey="Botella, Jose Ramon" sort="Botella, Jose Ramon" uniqKey="Botella J" first="Jose Ramon" last="Botella">Jose Ramon Botella</name><affiliation><nlm:affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</nlm:affiliation><wicri:noCountry code="subField">475004 Henan People's Republic of China</wicri:noCountry></affiliation><affiliation><nlm:affiliation>2School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD 4072 Australia.</nlm:affiliation><wicri:noCountry code="subField">QLD 4072 Australia</wicri:noCountry></affiliation></author><author><name sortKey="Song, Chun Peng" sort="Song, Chun Peng" uniqKey="Song C" first="Chun-Peng" last="Song">Chun-Peng Song</name><affiliation><nlm:affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</nlm:affiliation><wicri:noCountry code="subField">475004 Henan People's Republic of China</wicri:noCountry></affiliation></author></analytic><series><title level="j">Plant methods</title><idno type="ISSN">1746-4811</idno><imprint><date when="2018" type="published">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><b>Background</b></p><p>When developing CRISPR/Cas9 systems for crops, it is crucial to invest time characterizing the genome editing efficiency of the CRISPR/Cas9 cassettes, especially if the transformation system is difficult or time-consuming. Cotton is an important crop for the production of fiber, oil, and biofuel. However, the cotton stable transformation is usually performed using </p></div><div type="abstract" xml:lang="en"><p><b>Results</b></p><p>In this study, we provide a new system to evaluate and validate the efficiency of CRISPR/Cas9 cassettes in cotton using a transient expression system. By using this system, we could select the most effective CRISPR/Cas9 cassettes before the stable transformation. We have also optimized the existing cotton CRISPR/Cas9 system to achieve vastly improved mutagenesis efficiency by incorporating an endogenous </p></div><div type="abstract" xml:lang="en"><p><b>Conclusions</b></p><p>This study provides essential improvements to maximize CRISPR/Cas9-mediated mutation efficiency by reducing risk and workload for the application of CRISPR/Cas9 approaches in the targeted mutagenesis of cotton.</p></div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">30305839</PMID><DateRevised><Year>2020</Year><Month>09</Month><Day>30</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Print">1746-4811</ISSN><JournalIssue CitedMedium="Print"><Volume>14</Volume><PubDate><Year>2018</Year></PubDate></JournalIssue><Title>Plant methods</Title><ISOAbbreviation>Plant Methods</ISOAbbreviation></Journal><ArticleTitle>Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression.</ArticleTitle><Pagination><MedlinePgn>85</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1186/s13007-018-0353-0</ELocationID><Abstract><AbstractText Label="Background" NlmCategory="UNASSIGNED">When developing CRISPR/Cas9 systems for crops, it is crucial to invest time characterizing the genome editing efficiency of the CRISPR/Cas9 cassettes, especially if the transformation system is difficult or time-consuming. Cotton is an important crop for the production of fiber, oil, and biofuel. However, the cotton stable transformation is usually performed using <i>Agrobacterium tumefaciens</i> taking between 8 and 12 months to generate T<sub>0</sub> plants. Furthermore, cotton is a heterotetraploid and targeted mutagenesis is considered to be difficult as many genes are duplicated in this complex genome. The application of CRISPR/Cas9 in cotton is severely hampered by the long and technically challenging genetic transformation process, making it imperative to maximize its efficiency.</AbstractText><AbstractText Label="Results" NlmCategory="UNASSIGNED">In this study, we provide a new system to evaluate and validate the efficiency of CRISPR/Cas9 cassettes in cotton using a transient expression system. By using this system, we could select the most effective CRISPR/Cas9 cassettes before the stable transformation. We have also optimized the existing cotton CRISPR/Cas9 system to achieve vastly improved mutagenesis efficiency by incorporating an endogenous <i>GhU6</i> promoter that increases sgRNA expression levels over the <i>Arabidopsis AtU6</i>-<i>29</i> promoter. The 300 bp <i>GhU6.3</i> promoter was cloned and validated using the transient expression system. When sgRNAs were expressed under the control of the <i>GhU6.3</i> promoter in CRISPR/Cas9 cassettes, expression levels were 6-7 times higher than those provided by the <i>AtU6</i>-<i>29</i> promoter and CRISPR/Cas9-mediated mutation efficiency was improved 4-6 times.</AbstractText><AbstractText Label="Conclusions" NlmCategory="UNASSIGNED">This study provides essential improvements to maximize CRISPR/Cas9-mediated mutation efficiency by reducing risk and workload for the application of CRISPR/Cas9 approaches in the targeted mutagenesis of cotton.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y" EqualContrib="Y"><LastName>Long</LastName><ForeName>Lu</ForeName><Initials>L</Initials><AffiliationInfo><Affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</Affiliation><Identifier Source="ISNI">0000 0000 9139 560X</Identifier><Identifier Source="GRID">grid.256922.8</Identifier></AffiliationInfo></Author><Author ValidYN="Y" EqualContrib="Y"><LastName>Guo</LastName><ForeName>Dan-Dan</ForeName><Initials>DD</Initials><AffiliationInfo><Affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</Affiliation><Identifier Source="ISNI">0000 0000 9139 560X</Identifier><Identifier Source="GRID">grid.256922.8</Identifier></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Gao</LastName><ForeName>Wei</ForeName><Initials>W</Initials><AffiliationInfo><Affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</Affiliation><Identifier Source="ISNI">0000 0000 9139 560X</Identifier><Identifier Source="GRID">grid.256922.8</Identifier></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Yang</LastName><ForeName>Wen-Wen</ForeName><Initials>WW</Initials><AffiliationInfo><Affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</Affiliation><Identifier Source="ISNI">0000 0000 9139 560X</Identifier><Identifier Source="GRID">grid.256922.8</Identifier></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Hou</LastName><ForeName>Li-Pan</ForeName><Initials>LP</Initials><AffiliationInfo><Affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</Affiliation><Identifier Source="ISNI">0000 0000 9139 560X</Identifier><Identifier Source="GRID">grid.256922.8</Identifier></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Ma</LastName><ForeName>Xiao-Nan</ForeName><Initials>XN</Initials><AffiliationInfo><Affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</Affiliation><Identifier Source="ISNI">0000 0000 9139 560X</Identifier><Identifier Source="GRID">grid.256922.8</Identifier></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Miao</LastName><ForeName>Yu-Chen</ForeName><Initials>YC</Initials><AffiliationInfo><Affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</Affiliation><Identifier Source="ISNI">0000 0000 9139 560X</Identifier><Identifier Source="GRID">grid.256922.8</Identifier></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Botella</LastName><ForeName>Jose Ramon</ForeName><Initials>JR</Initials><AffiliationInfo><Affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</Affiliation><Identifier Source="ISNI">0000 0000 9139 560X</Identifier><Identifier Source="GRID">grid.256922.8</Identifier></AffiliationInfo><AffiliationInfo><Affiliation>2School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD 4072 Australia.</Affiliation><Identifier Source="ISNI">0000 0000 9320 7537</Identifier><Identifier Source="GRID">grid.1003.2</Identifier></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Song</LastName><ForeName>Chun-Peng</ForeName><Initials>CP</Initials><AffiliationInfo><Affiliation>1State Key Laboratory of Cotton Biology, Henan Key Laboratory of Plant Stress Biology, School of Life Science, Henan University, Kaifeng, 475004 Henan People's Republic of China.</Affiliation><Identifier Source="ISNI">0000 0000 9139 560X</Identifier><Identifier Source="GRID">grid.256922.8</Identifier></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2018</Year><Month>10</Month><Day>03</Day></ArticleDate></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>Plant Methods</MedlineTA><NlmUniqueID>101245798</NlmUniqueID><ISSNLinking>1746-4811</ISSNLinking></MedlineJournalInfo><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">CRISPR/Cas9</Keyword><Keyword MajorTopicYN="N">Genome editing</Keyword><Keyword MajorTopicYN="N">Target mutagenesis</Keyword><Keyword MajorTopicYN="N">Transient expression</Keyword><Keyword MajorTopicYN="N">U6 promoter</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2018</Year><Month>05</Month><Day>30</Day></PubMedPubDate><PubMedPubDate 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first="Wei" last="Gao">Wei Gao</name><name sortKey="Guo, Dan Dan" sort="Guo, Dan Dan" uniqKey="Guo D" first="Dan-Dan" last="Guo">Dan-Dan Guo</name><name sortKey="Hou, Li Pan" sort="Hou, Li Pan" uniqKey="Hou L" first="Li-Pan" last="Hou">Li-Pan Hou</name><name sortKey="Long, Lu" sort="Long, Lu" uniqKey="Long L" first="Lu" last="Long">Lu Long</name><name sortKey="Ma, Xiao Nan" sort="Ma, Xiao Nan" uniqKey="Ma X" first="Xiao-Nan" last="Ma">Xiao-Nan Ma</name><name sortKey="Miao, Yu Chen" sort="Miao, Yu Chen" uniqKey="Miao Y" first="Yu-Chen" last="Miao">Yu-Chen Miao</name><name sortKey="Song, Chun Peng" sort="Song, Chun Peng" uniqKey="Song C" first="Chun-Peng" last="Song">Chun-Peng Song</name><name sortKey="Yang, Wen Wen" sort="Yang, Wen Wen" uniqKey="Yang W" first="Wen-Wen" last="Yang">Wen-Wen Yang</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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