title: Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering.
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.
authors.affiliation: Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Kingdom of Saudi Arabia.
authors.forename: Mustapha
authors.initials: M
authors.lastname: Aouida
authors.affiliation: Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Kingdom of Saudi Arabia.
authors.forename: Ayman
authors.initials: A
authors.lastname: Eid
authors.affiliation: Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Kingdom of Saudi Arabia.
authors.forename: Zahir
authors.initials: Z
authors.lastname: Ali
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, 30332, United States of America.
authors.forename: Thomas
authors.initials: T
authors.lastname: Cradick
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, 30332, United States of America.
authors.forename: Ciaran
authors.initials: C
authors.lastname: Lee
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, 30332, United States of America.
authors.forename: Harshavardhan
authors.initials: H
authors.lastname: Deshmukh
authors.affiliation: Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Kingdom of Saudi Arabia.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Atef
authors.affiliation: Laboratory of Cell Signaling and Migration, Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal, 23955, Saudi Arabia.
authors.forename: Dina
authors.initials: D
authors.lastname: AbuSamra
authors.affiliation: Laboratory of Cell Signaling and Migration, Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal, 23955, Saudi Arabia.
authors.forename: Samah Zeineb
authors.initials: SZ
authors.lastname: Gadhoum
authors.affiliation: Laboratory of Cell Signaling and Migration, Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal, 23955, Saudi Arabia.
authors.forename: Jasmeen
authors.initials: J
authors.lastname: Merzaban
authors.affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, 30332, United States of America.
authors.forename: Gang
authors.initials: G
authors.lastname: Bao
authors.affiliation: Laboratory for Genome Engineering, Division of Biological Sciences & Center for Desert Agriculture, 4700 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Kingdom of Saudi Arabia.
authors.forename: Magdy
authors.initials: M
authors.lastname: Mahfouz
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems; D059766:DNA End-Joining Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D017393:RNA Editing; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D015252:Deoxyribonucleases, Type II Site-Specific; D004720:Endonucleases; D011993:Recombinant Fusion Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; C035042:endodeoxyribonuclease FokI