Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule

Handle URI:
http://hdl.handle.net/10754/625405
Title:
Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule
Authors:
Butt, Haroon; Eid, Ayman; Ali, Zahir ( 0000-0002-7814-8908 ) ; Atia, Mohamed A. M.; Mokhtar, Morad M.; Hassan, Norhan; Lee, Ciaran M.; Bao, Gang; Mahfouz, Magdy M. ( 0000-0002-0616-6365 )
Abstract:
The CRISPR/Cas9 system has been applied in diverse eukaryotic organisms for targeted mutagenesis. However, targeted gene editing is inefficient and requires the simultaneous delivery of a DNA template for homology-directed repair (HDR). Here, we used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate the double-strand breaks) and repair template sequences (to direct HDR), flanked by regions of homology to the target. Gene editing was more efficient in rice protoplasts using repair templates complementary to the non-target DNA strand, rather than the target strand. We applied this cgRNA repair method to generate herbicide resistance in rice, which showed that this cgRNA repair method can be used for targeted gene editing in plants. Our findings will facilitate applications in functional genomics and targeted improvement of crop traits.
KAUST Department:
Laboratory for Genome Engineering, Division of Biological Sciences, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
Citation:
Butt H, Eid A, Ali Z, Atia MAM, Mokhtar MM, et al. (2017) Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule. Frontiers in Plant Science 8. Available: http://dx.doi.org/10.3389/fpls.2017.01441.
Publisher:
Frontiers Media SA
Journal:
Frontiers in Plant Science
Issue Date:
24-Aug-2017
DOI:
10.3389/fpls.2017.01441
Type:
Article
ISSN:
1664-462X
Sponsors:
This study was supported by King Abdullah University of Science and Technology.
Additional Links:
http://journal.frontiersin.org/article/10.3389/fpls.2017.01441/full
Appears in Collections:
Articles

Full metadata record

DC FieldValue Language
dc.contributor.authorButt, Haroonen
dc.contributor.authorEid, Aymanen
dc.contributor.authorAli, Zahiren
dc.contributor.authorAtia, Mohamed A. M.en
dc.contributor.authorMokhtar, Morad M.en
dc.contributor.authorHassan, Norhanen
dc.contributor.authorLee, Ciaran M.en
dc.contributor.authorBao, Gangen
dc.contributor.authorMahfouz, Magdy M.en
dc.date.accessioned2017-08-28T10:28:00Z-
dc.date.available2017-08-28T10:28:00Z-
dc.date.issued2017-08-24en
dc.identifier.citationButt H, Eid A, Ali Z, Atia MAM, Mokhtar MM, et al. (2017) Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule. Frontiers in Plant Science 8. Available: http://dx.doi.org/10.3389/fpls.2017.01441.en
dc.identifier.issn1664-462Xen
dc.identifier.doi10.3389/fpls.2017.01441en
dc.identifier.urihttp://hdl.handle.net/10754/625405-
dc.description.abstractThe CRISPR/Cas9 system has been applied in diverse eukaryotic organisms for targeted mutagenesis. However, targeted gene editing is inefficient and requires the simultaneous delivery of a DNA template for homology-directed repair (HDR). Here, we used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate the double-strand breaks) and repair template sequences (to direct HDR), flanked by regions of homology to the target. Gene editing was more efficient in rice protoplasts using repair templates complementary to the non-target DNA strand, rather than the target strand. We applied this cgRNA repair method to generate herbicide resistance in rice, which showed that this cgRNA repair method can be used for targeted gene editing in plants. Our findings will facilitate applications in functional genomics and targeted improvement of crop traits.en
dc.description.sponsorshipThis study was supported by King Abdullah University of Science and Technology.en
dc.publisherFrontiers Media SAen
dc.relation.urlhttp://journal.frontiersin.org/article/10.3389/fpls.2017.01441/fullen
dc.rightsThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.titleEfficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Moleculeen
dc.typeArticleen
dc.contributor.departmentLaboratory for Genome Engineering, Division of Biological Sciences, King Abdullah University of Science and Technology, Thuwal, Saudi Arabiaen
dc.identifier.journalFrontiers in Plant Scienceen
dc.eprint.versionPublisher's Version/PDFen
dc.contributor.institutionMolecular Genetics and Genome Mapping Laboratory, Agricultural Genetic Engineering Research Institute, Agricultural Research Center, Giza, Egypten
dc.contributor.institutionDepartment of Bioengineering, Rice University, Houston, TX, United Statesen
kaust.authorButt, Haroonen
kaust.authorEid, Aymanen
kaust.authorAli, Zahiren
kaust.authorHassan, Norhanen
kaust.authorMahfouz, Magdy M.en
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