Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

Handle URI:
http://hdl.handle.net/10754/579491
Title:
Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering
Authors:
Aouida, Mustapha ( 0000-0001-7945-5320 ) ; Eid, Ayman; Ali, Zahir ( 0000-0002-7814-8908 ) ; Cradick, Thomas; Lee, Ciaran; Deshmukh, Harshavardhan; Atef, Ahmed; Abu Samra, Dina Bashir Kamil ( 0000-0002-0974-5362 ) ; Gadhoum, Samah Zeineb; Merzaban, Jasmeen S. ( 0000-0002-7276-2907 ) ; Bao, Gang; Mahfouz, Magdy M. ( 0000-0002-0616-6365 )
Abstract:
The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.
KAUST Department:
Laboratory for Genome Engineering; Laboratory of Cell Signaling and Migration; Biological and Environmental Sciences and Engineering (BESE) Division
Citation:
Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering 2015, 10 (7):e0133373 PLOS ONE
Publisher:
Public Library of Science (PLoS)
Journal:
PLoS ONE
Issue Date:
30-Jul-2015
DOI:
10.1371/journal.pone.0133373
Type:
Article
ISSN:
1932-6203
Additional Links:
http://dx.plos.org/10.1371/journal.pone.0133373
Appears in Collections:
Articles; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorAouida, Mustaphaen
dc.contributor.authorEid, Aymanen
dc.contributor.authorAli, Zahiren
dc.contributor.authorCradick, Thomasen
dc.contributor.authorLee, Ciaranen
dc.contributor.authorDeshmukh, Harshavardhanen
dc.contributor.authorAtef, Ahmeden
dc.contributor.authorAbu Samra, Dina Bashir Kamilen
dc.contributor.authorGadhoum, Samah Zeineben
dc.contributor.authorMerzaban, Jasmeen S.en
dc.contributor.authorBao, Gangen
dc.contributor.authorMahfouz, Magdy M.en
dc.date.accessioned2015-10-08T07:42:29Zen
dc.date.available2015-10-08T07:42:29Zen
dc.date.issued2015-07-30en
dc.identifier.citationEfficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering 2015, 10 (7):e0133373 PLOS ONEen
dc.identifier.issn1932-6203en
dc.identifier.doi10.1371/journal.pone.0133373en
dc.identifier.urihttp://hdl.handle.net/10754/579491en
dc.description.abstractThe Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.en
dc.language.isoenen
dc.publisherPublic Library of Science (PLoS)en
dc.relation.urlhttp://dx.plos.org/10.1371/journal.pone.0133373en
dc.rightsThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are crediteden
dc.titleEfficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineeringen
dc.typeArticleen
dc.contributor.departmentLaboratory for Genome Engineeringen
dc.contributor.departmentLaboratory of Cell Signaling and Migrationen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.identifier.journalPLoS ONEen
dc.eprint.versionPublisher's Version/PDFen
dc.contributor.institutionDepartment of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, 30332, United States of Americaen
dc.contributor.affiliationKing Abdullah University of Science and Technology (KAUST)en
kaust.authorAouida, Mustaphaen
kaust.authorAli, Zahiren
kaust.authorGadhoum, Samahen
kaust.authorMerzaban, Jasmeen S.en
kaust.authorMahfouz, Magdy M.en
kaust.authorEid, Aymanen
kaust.authorAtef, Ahmeden
kaust.authorAbu Samra, Dina Bashir Kamilen
All Items in KAUST are protected by copyright, with all rights reserved, unless otherwise indicated.