title: Using defined finger-finger interfaces as units of assembly for constructing zinc-finger nucleases.
abstract: Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger-finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the validation of a new set of two-finger modules that can be used for building ZFAs via conventional assembly methods or a new strategy-finger stitching-that increases the diversity of genomic sequences targetable by ZFNs. Instead of assembling ZFAs based on units of the zinc-finger structural domain, our finger stitching method uses units that span the finger-finger interface to ensure compatibility of neighbouring recognition helices. We tested this approach by generating and characterizing eight ZFAs, and we found their DNA-binding specificities reflected the specificities of the component modules used in their construction. Four pairs of ZFNs incorporating these ZFAs generated targeted lesions in vivo, demonstrating that stitching yields ZFAs with robust recognition properties.
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Cong
authors.initials: C
authors.lastname: Zhu
authors.forename: Ankit
authors.initials: A
authors.lastname: Gupta
authors.forename: Victoria L
authors.initials: VL
authors.lastname: Hall
authors.forename: Amy L
authors.initials: AL
authors.lastname: Rayla
authors.forename: Ryan G
authors.initials: RG
authors.lastname: Christensen
authors.forename: Benjamin
authors.initials: B
authors.lastname: Dake
authors.forename: Abirami
authors.initials: A
authors.lastname: Lakshmanan
authors.forename: Charlotte
authors.initials: C
authors.lastname: Kuperwasser
authors.forename: Gary D
authors.initials: GD
authors.lastname: Stormo
authors.forename: Scot A
authors.initials: SA
authors.lastname: Wolfe
mesh_terms: D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D003851:Deoxyribonucleases / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D009711:Nucleotides / Q000737:chemistry; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015027:Zebrafish; D016335:Zinc Fingers
chemical_list: D009711:Nucleotides; D011993:Recombinant Fusion Proteins; D004247:DNA; D003851:Deoxyribonucleases; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific