title: Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme.
abstract: Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome. Subsequent repair of this break via the nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene addition, respectively. Here, we report that ZFNs can be engineered to induce a site-specific DNA single-strand break (SSB) or nick. Using the CCR5-specific ZFNs as a model system, we show that introduction of a nick at this target site stimulates gene addition using a homologous donor template but fails to induce significant levels of the small insertions and deletions (indels) characteristic of repair via NHEJ. Gene addition by these CCR5-targeted zinc finger nickases (ZFNickases) occurs in both transformed and primary human cells at efficiencies of up to ∼1%-8%. Interestingly, ZFNickases targeting the AAVS1 "safe harbor" locus revealed similar in vitro nicking activity, a marked reduction of indels characteristic of NHEJ, but stimulated far lower levels of gene addition-suggesting that other, yet to be identified mediators of nick-induced gene targeting exist. Introduction of site-specific nicks at distinct endogenous loci provide an important tool for the study of DNA repair. Moreover, the potential for a SSB to direct repair pathway choice (i.e., HDR but not NHEJ) may prove advantageous for certain therapeutic applications such as the targeted correction of human disease-causing mutations.
authors.affiliation: Sangamo Biosciences Inc., Richmond, California 94804, USA.
authors.forename: Jianbin
authors.initials: J
authors.lastname: Wang
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Friedman
authors.forename: Yannick
authors.initials: Y
authors.lastname: Doyon
authors.forename: Nathaniel S
authors.initials: NS
authors.lastname: Wang
authors.forename: Carrie Jiaxin
authors.initials: CJ
authors.lastname: Li
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Miller
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Hua
authors.forename: Jenny Jiacheng
authors.initials: JJ
authors.lastname: Yan
authors.forename: Joshua E
authors.initials: JE
authors.lastname: Babiarz
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D053903:DNA Breaks, Double-Stranded; D053904:DNA Breaks, Single-Stranded; D059766:DNA End-Joining Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D015894:Genome, Human; D006657:Histones / Q000378:metabolism; D006801:Humans; D054643:INDEL Mutation; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D019713:Receptors, CCR5 / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D016335:Zinc Fingers
chemical_list: D006657:Histones; D019713:Receptors, CCR5; D011993:Recombinant Fusion Proteins; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific