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Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering.
oleh: Mustapha Aouida, Ayman Eid, Zahir Ali, Thomas Cradick, Ciaran Lee, Harshavardhan Deshmukh, Ahmed Atef, Dina AbuSamra, Samah Zeineb Gadhoum, Jasmeen Merzaban, Gang Bao, Magdy Mahfouz
Format: | Article |
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Diterbitkan: | Public Library of Science (PLoS) 2015-01-01 |
Deskripsi
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.