Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering
Abu Samra, Dina Bashir Kamil
Gadhoum, Samah Zeineb
Mahfouz, Magdy M.
KAUST DepartmentDesert Agriculture Initiative
Biological and Environmental Sciences and Engineering (BESE) Division
Plant Science Program
Laboratory for Genome Engineering
Laboratory of Cell Signaling and Migration
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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.
CitationEfficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering 2015, 10 (7):e0133373 PLOS ONE
PublisherPublic Library of Science (PLoS)
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