title: Monomeric site-specific nucleases for genome editing.
abstract: Targeted manipulation of complex genomes often requires the introduction of a double-strand break at defined locations by site-specific DNA endonucleases. Here, we describe a monomeric nuclease domain derived from GIY-YIG homing endonucleases for genome-editing applications. Fusion of the GIY-YIG nuclease domain to three-member zinc-finger DNA binding domains generated chimeric GIY-zinc finger endonucleases (GIY-ZFEs). Significantly, the I-TevI-derived fusions (Tev-ZFEs) function in vitro as monomers to introduce a double-strand break, and discriminate in vitro and in bacterial and yeast assays against substrates lacking a preferred 5'-CNNNG-3' cleavage motif. The Tev-ZFEs function to induce recombination in a yeast-based assay with activity on par with a homodimeric Zif268 zinc-finger nuclease. We also fused the I-TevI nuclease domain to a catalytically inactive LADGLIDADG homing endonuclease (LHE) scaffold. The monomeric Tev-LHEs are active in vivo and similarly discriminate against substrates lacking the 5'-CNNNG-3' motif. The monomeric Tev-ZFEs and Tev-LHEs are distinct from the FokI-derived zinc-finger nuclease and TAL effector nuclease platforms as the GIY-YIG domain alleviates the requirement to design two nuclease fusions to target a given sequence, highlighting the diversity of nuclease domains with distinctive biochemical properties suitable for genome-editing applications.
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry,  University of Western Ontario, London, ON,  Canada N6A 5C1.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Kleinstiver
authors.forename: Jason M
authors.initials: JM
authors.lastname: Wolfs
authors.forename: Tomasz
authors.initials: T
authors.lastname: Kolaczyk
authors.forename: Alanna K
authors.initials: AK
authors.lastname: Roberts
authors.forename: Sherry X
authors.initials: SX
authors.lastname: Hu
authors.forename: David R
authors.initials: DR
authors.lastname: Edgell
mesh_terms: D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D059767:Recombinational DNA Repair / Q000502:physiology; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D004720:Endonucleases; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific