Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering
Type
ArticleAuthors
Aouida, Mustapha
Eid, Ayman
Ali, Zahir

Cradick, Thomas
Lee, Ciaran
Deshmukh, Harshavardhan
Ahmed, Atef
Abu Samra, Dina Bashir Kamil

Gadhoum, Samah
Merzaban, Jasmeen

Bao, Gang
Mahfouz, Magdy M.

KAUST Department
Biological and Environmental Sciences and Engineering (BESE) DivisionBioscience Program
Desert Agriculture Initiative
KAUST Supercomputing Laboratory (KSL)
Laboratory for Genome Engineering
Laboratory of Cell Signaling and Migration
Plant Science
Plant Science Program
Date
2015-07-30Permanent link to this record
http://hdl.handle.net/10754/579491
Metadata
Show full item recordAbstract
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.Citation
Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering 2015, 10 (7):e0133373 PLOS ONEPublisher
Public Library of Science (PLoS)Journal
PLoS ONEPubMed ID
26225561Additional Links
http://dx.plos.org/10.1371/journal.pone.0133373ae974a485f413a2113503eed53cd6c53
10.1371/journal.pone.0133373
Scopus Count
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