Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

Mustapha Aouida, Ayman Eid, Zahir Ali, Thomas Cradick, Ciaran Lee, Harshavardhan Deshmukh, Atef Ahmed, Dina Bashir Kamil Abu Samra, Samah Gadhoum, Jasmeen Merzaban, Gang Bao, Magdy M. Mahfouz

Research output: Contribution to journalArticlepeer-review

44 Scopus citations


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.
Original languageEnglish (US)
Pages (from-to)e0133373
JournalPLoS ONE
Issue number7
StatePublished - Jul 30 2015


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