CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations
Identifieur interne : 000461 ( Pmc/Curation ); précédent : 000460; suivant : 000462CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations
Auteurs : Changtian Pan [République populaire de Chine] ; Lei Ye [République populaire de Chine] ; Li Qin [République populaire de Chine] ; Xue Liu [République populaire de Chine] ; Yanjun He [République populaire de Chine] ; Jie Wang [République populaire de Chine] ; Lifei Chen [République populaire de Chine] ; Gang Lu [République populaire de Chine]Source :
- Scientific Reports [ 2045-2322 ] ; 2016.
Abstract
The CRISPR/Cas9 system has successfully been used in various organisms for precise targeted gene editing. Although it has been demonstrated that CRISPR/Cas9 system can induce mutation in tomato plants, the stability of heredity in later generations and mutant specificity induced by the CRISPR/Cas9 system in tomato plants have not yet been elucidated in detail. In this study, two genes,
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DOI: 10.1038/srep24765
PubMed: 27097775
PubMed Central: 4838866
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations</title><author><name sortKey="Pan, Changtian" sort="Pan, Changtian" uniqKey="Pan C" first="Changtian" last="Pan">Changtian Pan</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Ye, Lei" sort="Ye, Lei" uniqKey="Ye L" first="Lei" last="Ye">Lei Ye</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Qin, Li" sort="Qin, Li" uniqKey="Qin L" first="Li" last="Qin">Li Qin</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Liu, Xue" sort="Liu, Xue" uniqKey="Liu X" first="Xue" last="Liu">Xue Liu</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="He, Yanjun" sort="He, Yanjun" uniqKey="He Y" first="Yanjun" last="He">Yanjun He</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Wang, Jie" sort="Wang, Jie" uniqKey="Wang J" first="Jie" last="Wang">Jie Wang</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Chen, Lifei" sort="Chen, Lifei" uniqKey="Chen L" first="Lifei" last="Chen">Lifei Chen</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Lu, Gang" sort="Lu, Gang" uniqKey="Lu G" first="Gang" last="Lu">Gang Lu</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">27097775</idno><idno type="pmc">4838866</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838866</idno><idno type="RBID">PMC:4838866</idno><idno type="doi">10.1038/srep24765</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000462</idno><idno type="wicri:Area/Pmc/Curation">000461</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations</title><author><name sortKey="Pan, Changtian" sort="Pan, Changtian" uniqKey="Pan C" first="Changtian" last="Pan">Changtian Pan</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Ye, Lei" sort="Ye, Lei" uniqKey="Ye L" first="Lei" last="Ye">Lei Ye</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Qin, Li" sort="Qin, Li" uniqKey="Qin L" first="Li" last="Qin">Li Qin</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Liu, Xue" sort="Liu, Xue" uniqKey="Liu X" first="Xue" last="Liu">Xue Liu</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="He, Yanjun" sort="He, Yanjun" uniqKey="He Y" first="Yanjun" last="He">Yanjun He</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Wang, Jie" sort="Wang, Jie" uniqKey="Wang J" first="Jie" last="Wang">Jie Wang</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Chen, Lifei" sort="Chen, Lifei" uniqKey="Chen L" first="Lifei" last="Chen">Lifei Chen</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Lu, Gang" sort="Lu, Gang" uniqKey="Lu G" first="Gang" last="Lu">Gang Lu</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The CRISPR/Cas9 system has successfully been used in various organisms for precise targeted gene editing. Although it has been demonstrated that CRISPR/Cas9 system can induce mutation in tomato plants, the stability of heredity in later generations and mutant specificity induced by the CRISPR/Cas9 system in tomato plants have not yet been elucidated in detail. In this study, two genes, <italic>SlPDS</italic> and <italic>SlPIF4</italic>, were used for testing targeted mutagenesis in tomato plants through an <italic>Agrobacterium tumefaciens</italic>-mediated transformation method. A high mutation frequency was observed in all tested targets in the T0 transgenic tomato plants, with an average frequency of 83.56%. Clear albino phenotypes were observed for the <italic>psd</italic> mutants. High frequencies of homozygous and biallelic mutants were detected even in T0 plants. The majority of the detected mutations were 1- to 3-nucleotide deletions, followed by 1-bp insertions. The target mutations in the T0 lines were stably transmitted to the T1 and T2 generations, without new modifications or revision. Off-target activities associated with <italic>SlPDS</italic> and <italic>SlPIF4</italic> were also evaluated by sequencing the putative off-target sites, and no clear off-target events were detected. Our results demonstrate that the CRISPR/Cas9 system is an efficient tool for generating stable and heritable modifications in tomato plants.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Gaj, T" uniqKey="Gaj T">T. Gaj</name></author><author><name sortKey="Gersbach, C A" uniqKey="Gersbach C">C. A. Gersbach</name></author><author><name sortKey="Barbas, C R" uniqKey="Barbas C">C. R. Barbas</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Voytas, D F" uniqKey="Voytas D">D. F. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Sci Rep</journal-id><journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type="epub">2045-2322</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">27097775</article-id><article-id pub-id-type="pmc">4838866</article-id><article-id pub-id-type="pii">srep24765</article-id><article-id pub-id-type="doi">10.1038/srep24765</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Pan</surname><given-names>Changtian</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Ye</surname><given-names>Lei</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Qin</surname><given-names>Li</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Xue</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>He</surname><given-names>Yanjun</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Jie</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Lifei</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Lu</surname><given-names>Gang</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><aff id="a1"><label>1</label><institution>Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture</institution>, Hangzhou 310058,<country>China</country></aff><aff id="a2"><label>2</label><institution>Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University</institution>, Hangzhou 310058,<country>China</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>glu@zju.edu.cn</email></corresp></author-notes><pub-date pub-type="epub"><day>21</day><month>04</month><year>2016</year></pub-date><pub-date pub-type="collection"><year>2016</year></pub-date><volume>6</volume><elocation-id>24765</elocation-id><history><date date-type="received"><day>18</day><month>01</month><year>2016</year></date><date date-type="accepted"><day>04</day><month>04</month><year>2016</year></date></history><permissions><copyright-statement>Copyright © 2016, Macmillan Publishers Limited</copyright-statement><copyright-year>2016</copyright-year><copyright-holder>Macmillan Publishers Limited</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract><p>The CRISPR/Cas9 system has successfully been used in various organisms for precise targeted gene editing. Although it has been demonstrated that CRISPR/Cas9 system can induce mutation in tomato plants, the stability of heredity in later generations and mutant specificity induced by the CRISPR/Cas9 system in tomato plants have not yet been elucidated in detail. In this study, two genes, <italic>SlPDS</italic> and <italic>SlPIF4</italic>, were used for testing targeted mutagenesis in tomato plants through an <italic>Agrobacterium tumefaciens</italic>-mediated transformation method. A high mutation frequency was observed in all tested targets in the T0 transgenic tomato plants, with an average frequency of 83.56%. Clear albino phenotypes were observed for the <italic>psd</italic> mutants. High frequencies of homozygous and biallelic mutants were detected even in T0 plants. The majority of the detected mutations were 1- to 3-nucleotide deletions, followed by 1-bp insertions. The target mutations in the T0 lines were stably transmitted to the T1 and T2 generations, without new modifications or revision. Off-target activities associated with <italic>SlPDS</italic> and <italic>SlPIF4</italic> were also evaluated by sequencing the putative off-target sites, and no clear off-target events were detected. Our results demonstrate that the CRISPR/Cas9 system is an efficient tool for generating stable and heritable modifications in tomato plants.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>Albinism phenotype of <italic>SlPDS</italic> transgenic plants in T0 generation.</title><p>(<bold>A</bold>) WT (contain T-DNA). (<bold>B</bold>,<bold>C</bold>) chimeric mutant. (<bold>D</bold>) biallelic mutant. Mutant B, C and D show albino phenotype to varying degrees.</p></caption><graphic xlink:href="srep24765-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>Detecting of target mutations by T7 endonuclease I (T7E1) assay.</title><p>The target fragments were amplified by PCR from genomic DNA which was extracted from independent transgenic plants leaves. #1–13 represents 13 independent transgenic plants of sgRNA2-<italic>SlPIF4.</italic> Arrows indicate the digested fragments by T7E1. +: PCR products were added. −: no PCR products were added.</p></caption><graphic xlink:href="srep24765-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>Pattern and frequency of CRISPR/Cas9-mediated mutations.</title><p>The graph assembles the sequencing data of the four target sites in T0 and T1 transgenic plants. Left inset shows the frequency of insertion (i), deletion (d) and combined (c) mutation type. Right inset exhibits the occurrence rate of different mutation length. In x-axis: i#, number of bases insertion at target site; d#, number of bases deletion at target site; c#, combined mutations.</p></caption><graphic xlink:href="srep24765-f3"></graphic></fig><table-wrap position="float" id="t1"><label>Table 1</label><caption><title>Percentage of T0 transgenic plants examined with mutations and GC content of sgRNAs.</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50">Vector</th><th align="center" valign="top" charoff="50">Target gene</th><th align="center" valign="top" charoff="50">sgRNA</th><th align="center" valign="top" charoff="50">No. of lines</th><th align="center" valign="top" charoff="50">No. of lines with mutations</th><th align="center" valign="top" charoff="50">Mutation rate</th><th align="center" valign="top" charoff="50">sgRNA GC content</th></tr></thead><tbody valign="top"><tr><td rowspan="2" align="left" valign="middle" charoff="50">AtU6-sgRNA-AtUBQ-Cas9</td><td align="center" valign="top" charoff="50"><italic>SlPDS</italic></td><td align="center" valign="top" charoff="50">sgRNA1</td><td align="center" valign="top" charoff="50">22</td><td align="center" valign="top" charoff="50">16</td><td align="center" valign="top" charoff="50">72.70%</td><td align="center" valign="top" charoff="50">40.00%</td></tr><tr><td align="center" valign="top" charoff="50"><italic>SlPDS</italic></td><td align="center" valign="top" charoff="50">sgRNA2</td><td align="center" valign="top" charoff="50">7</td><td align="center" valign="top" charoff="50">7</td><td align="center" valign="top" charoff="50">100.00%</td><td align="center" valign="top" charoff="50">55.00%</td></tr><tr><td rowspan="2" align="left" valign="middle" charoff="50">AtU6-sgRNA-2 × CaMV 35S-Cas9</td><td align="center" valign="top" charoff="50"><italic>SlPIF4</italic></td><td align="center" valign="top" charoff="50">sgRNA1</td><td align="center" valign="top" charoff="50">25</td><td align="center" valign="top" charoff="50">21</td><td align="center" valign="top" charoff="50">84.00%</td><td align="center" valign="top" charoff="50">60.00%</td></tr><tr><td align="center" valign="top" charoff="50"><italic>SlPIF4</italic></td><td align="center" valign="top" charoff="50">sgRNA2</td><td align="center" valign="top" charoff="50">19</td><td align="center" valign="top" charoff="50">17</td><td align="center" valign="top" charoff="50">89.47%</td><td align="center" valign="top" charoff="50">50.00%</td></tr><tr><td align="left" valign="top" charoff="50">Total</td><td align="center" valign="top" charoff="50">2</td><td align="center" valign="top" charoff="50">4</td><td align="center" valign="top" charoff="50">73</td><td align="center" valign="top" charoff="50">61</td><td align="center" valign="top" charoff="50">83.56%</td><td align="center" valign="top" charoff="50">51.25%</td></tr></tbody></table></table-wrap><table-wrap position="float" id="t2"><label>Table 2</label><caption><title>Detected zygosity of T0 independent transgenic lines of sg1/2-<italic>SlPDS</italic> and sg1/2-<italic>SlPIF4</italic>.</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col></colgroup><thead valign="bottom"><tr><th rowspan="2" align="left" valign="top" charoff="50">Target gene</th><th rowspan="2" align="center" valign="top" charoff="50">sites</th><th rowspan="2" align="center" valign="top" charoff="50">No. of examed lines</th><th colspan="4" align="center" valign="top" charoff="50">Zygosity<xref ref-type="fn" rid="t2-fn1">$</xref><hr></hr></th></tr><tr><th align="center" valign="top" charoff="50">Homozygote</th><th align="center" valign="top" charoff="50">Biallele</th><th align="center" valign="top" charoff="50">Chimera</th><th align="center" valign="top" charoff="50">WT<xref ref-type="fn" rid="t2-fn2">#</xref></th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50"><italic>SlPDS</italic></td><td align="center" valign="top" charoff="50">sgRNA1</td><td align="center" valign="top" charoff="50">21</td><td align="center" valign="top" charoff="50">0</td><td align="center" valign="top" charoff="50">0</td><td align="center" valign="top" charoff="50">15(71.4%)</td><td align="center" valign="top" charoff="50">6(28.6%)</td></tr><tr><td align="left" valign="top" charoff="50"><italic>SlPDS</italic></td><td align="center" valign="top" charoff="50">sgRNA2</td><td align="center" valign="top" charoff="50">7</td><td align="center" valign="top" charoff="50">0</td><td align="center" valign="top" charoff="50">1 (14.3%)</td><td align="center" valign="top" charoff="50">6 (85.7%)</td><td align="center" valign="top" charoff="50">0</td></tr><tr><td align="left" valign="top" charoff="50"><italic>SlPIF4</italic></td><td align="center" valign="top" charoff="50">sgRNA1</td><td align="center" valign="top" charoff="50">25</td><td align="center" valign="top" charoff="50">2 (8.0%)</td><td align="center" valign="top" charoff="50">3 (12.0%)</td><td align="center" valign="top" charoff="50">16 (60.0%)</td><td align="center" valign="top" charoff="50">4 (16.0%)</td></tr><tr><td align="left" valign="top" charoff="50"><italic>SlPIF4</italic></td><td align="center" valign="top" charoff="50">sgRNA2</td><td align="center" valign="top" charoff="50">19</td><td align="center" valign="top" charoff="50">3 (15.8%)</td><td align="center" valign="top" charoff="50">5 (26.3%)</td><td align="center" valign="top" charoff="50">9 (47.4%)</td><td align="center" valign="top" charoff="50">2 (10.5%)</td></tr><tr><td align="left" valign="top" charoff="50">Total</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">72</td><td align="center" valign="top" charoff="50">5 (6.9%)</td><td align="center" valign="top" charoff="50">9 (12.5%)</td><td align="center" valign="top" charoff="50">46 (63.9%)</td><td align="center" valign="top" charoff="50">12 (16.7%)</td></tr></tbody></table><table-wrap-foot><fn id="t2-fn1"><p><sup>$</sup>The zygosoty of homozygote, biallele and chimera in T0 plant lines were putative.</p></fn><fn id="t2-fn2"><p><sup>#</sup>WT, wild-type sequence without mutations detected at target sites.</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t3"><label>Table 3</label><caption><title>Segregation patterns of CRISPR/Cas9-medicated targeted mutagenesis during the T0 to T1 generation.</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col></colgroup><thead valign="bottom"><tr><th rowspan="2" align="left" valign="top" charoff="50">Target gene</th><th rowspan="2" align="center" valign="top" charoff="50">sgRNA</th><th rowspan="2" align="center" valign="top" charoff="50">Line<xref ref-type="fn" rid="t3-fn1">#</xref></th><th colspan="2" align="center" valign="top" charoff="50">T0<hr></hr></th><th colspan="2" align="center" valign="top" charoff="50">T1<hr></hr></th></tr><tr><th align="center" valign="top" charoff="50">Zygosity<xref ref-type="fn" rid="t3-fn2">$</xref></th><th align="center" valign="top" charoff="50">Genotype</th><th align="center" valign="top" charoff="50">Mutation segregation</th><th align="center" valign="top" charoff="50">T-DNA</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50"><italic>SlPIF4</italic></td><td align="center" valign="top" charoff="50">1</td><td align="center" valign="top" charoff="50">T0-22</td><td align="center" valign="top" charoff="50">Homozygote</td><td align="center" valign="top" charoff="50">d1d1</td><td align="center" valign="top" charoff="50">15d1d1</td><td align="center" valign="top" charoff="50">12+;3−</td></tr><tr><td align="left" valign="top" charoff="50"><italic>SlPIF4</italic></td><td align="center" valign="top" charoff="50">2</td><td align="center" valign="top" charoff="50">T0-19</td><td align="center" valign="top" charoff="50">Homozygote</td><td align="center" valign="top" charoff="50">d1d1</td><td align="center" valign="top" charoff="50">14d1d1</td><td align="center" valign="top" charoff="50">10+:4−</td></tr><tr><td align="left" valign="top" charoff="50"><italic>SlPIF4</italic></td><td align="center" valign="top" charoff="50">2</td><td align="center" valign="top" charoff="50">T0-8</td><td align="center" valign="top" charoff="50">Biallele</td><td align="center" valign="top" charoff="50">d2,d9</td><td align="center" valign="top" charoff="50">3d2d2,6e<xref ref-type="fn" rid="t3-fn3">*</xref>,5d9d9</td><td align="center" valign="top" charoff="50">10+:4−</td></tr><tr><td align="left" valign="top" charoff="50"><italic>SlPIF4</italic></td><td align="center" valign="top" charoff="50">2</td><td align="center" valign="top" charoff="50">T0-10</td><td align="center" valign="top" charoff="50">Biallele</td><td align="center" valign="top" charoff="50">d3,i1</td><td align="center" valign="top" charoff="50">1d3d3,7e,5i1i1</td><td align="center" valign="top" charoff="50">8+:5−</td></tr><tr><td align="left" valign="top" charoff="50"><italic>SlPDS</italic></td><td align="center" valign="top" charoff="50">1</td><td align="center" valign="top" charoff="50">T0-20</td><td align="center" valign="top" charoff="50">Chimera</td><td align="center" valign="top" charoff="50">d5,d12,c102,WT</td><td align="center" valign="top" charoff="50">8e</td><td align="center" valign="top" charoff="50">5+;1−</td></tr><tr><td align="left" valign="top" charoff="50"><italic>SlPIF4</italic></td><td align="center" valign="top" charoff="50">1</td><td align="center" valign="top" charoff="50">T0-3</td><td align="center" valign="top" charoff="50">Chimera</td><td align="center" valign="top" charoff="50">d3,d4,i1</td><td align="center" valign="top" charoff="50">13e,2i1</td><td align="center" valign="top" charoff="50">11+:4−</td></tr><tr><td align="left" valign="top" charoff="50"><italic>SlPIF4</italic></td><td align="center" valign="top" charoff="50">2</td><td align="center" valign="top" charoff="50">T0-12</td><td align="center" valign="top" charoff="50">Chimera</td><td align="center" valign="top" charoff="50">d2,d6,d17,c7</td><td align="center" valign="top" charoff="50">5d6,2e,5d2</td><td align="center" valign="top" charoff="50">All+</td></tr><tr><td align="left" valign="top" charoff="50"><italic>SlPIF4</italic></td><td align="center" valign="top" charoff="50">2</td><td align="center" valign="top" charoff="50">T0-16</td><td align="center" valign="top" charoff="50">Chimera</td><td align="center" valign="top" charoff="50">d1,d2,d3</td><td align="center" valign="top" charoff="50">8d2,11e,3d1</td><td align="center" valign="top" charoff="50">14+:8−</td></tr><tr><td align="left" valign="top" charoff="50"><italic>SlPIF4</italic></td><td align="center" valign="top" charoff="50">1</td><td align="center" valign="top" charoff="50">T0-6</td><td align="center" valign="top" charoff="50">WT</td><td align="center" valign="top" charoff="50">WT</td><td align="center" valign="top" charoff="50">13WT</td><td align="center" valign="top" charoff="50">12+:1−</td></tr><tr><td align="left" valign="top" charoff="50"><italic>SlPDS</italic></td><td align="center" valign="top" charoff="50">1</td><td align="center" valign="top" charoff="50">T0-18</td><td align="center" valign="top" charoff="50">WT</td><td align="center" valign="top" charoff="50">WT</td><td align="center" valign="top" charoff="50">16WT</td><td align="center" valign="top" charoff="50">9+:7−</td></tr></tbody></table><table-wrap-foot><fn id="t3-fn1"><p><sup>#</sup>Line name is in the format of T0-#.</p></fn><fn id="t3-fn2"><p><sup>$</sup>The zygosoty of homozygote, biallele and chimera in T0 plant lines were putative. d#, # of bp deleted at the target sites; i#, # number of bases insertion at target sites; c#, combined mutation; WT, wild-type sequence without mutations detected at target sites.</p></fn><fn id="t3-fn3"><p><sup>*</sup>e, heterogeneous, more than one sequence detected in the sample; +, T-DNA was detected; −, T-DNA was not detected.</p></fn></table-wrap-foot></table-wrap><table-wrap position="float" id="t4"><label>Table 4</label><caption><title>Mutation analyzed of potential off-target sites.</title></caption><graphic xlink:href="srep24765-t4"></graphic><table-wrap-foot><fn id="t4-fn1"><p>PAM sequence (NGG) is indicated in orange, the analogue NAG is also used for testing. Mismatch nucleotides are marked in red. <sup>$</sup>Examined plants were randomly selected from the T0 and T1 generations of sgRNA1/2-<italic>SlPIF4</italic> and sgRNA1-<italic>SlPDS</italic>, with the T1 plants were Cas9 positive or negative.</p></fn></table-wrap-foot></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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