title: Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases.
abstract: Efficient incorporation of novel DNA sequences into a specific site in the genome of living human cells remains a challenge despite its potential utility to genetic medicine, biotechnology, and basic research. We find that a precisely placed double-strand break induced by engineered zinc finger nucleases (ZFNs) can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in high-efficiency site-specific gene addition. Using an extrachromosomal DNA donor carrying a 12-bp tag, a 900-bp ORF, or a 1.5-kb promoter-transcription unit flanked by locus-specific homology arms, we find targeted integration frequencies of 15%, 6%, and 5%, respectively, within 72 h of treatment, and with no selection for the desired event. Importantly, we find that the integration event occurs in a homology-directed manner and leads to the accurate reconstruction of the donor-specified genotype at the endogenous chromosomal locus, and hence presumably results from synthesis-dependent strand annealing repair of the break using the donor DNA as a template. This site-specific gene addition occurs with no measurable increase in the rate of random integration. Remarkably, we also find that ZFNs can drive the addition of an 8-kb sequence carrying three distinct promoter-transcription units into an endogenous locus at a frequency of 6%, also in the absence of any selection. These data reveal the surprising versatility of the specialized polymerase machinery involved in double-strand break repair, illuminate a powerful approach to mammalian cell engineering, and open the possibility of ZFN-driven gene addition therapy for human genetic disease.
authors.affiliation: Sangamo BioSciences, Inc., Point Richmond Technology Center, 501 Canal Boulevard, Suite A100, Richmond, CA 94804, USA.
authors.forename: Erica A
authors.initials: EA
authors.lastname: Moehle
authors.forename: E A
authors.initials: EA
authors.lastname: Moehle
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Rock
authors.forename: J M
authors.initials: JM
authors.lastname: Rock
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Lee
authors.forename: Y L
authors.initials: YL
authors.lastname: Lee
authors.forename: Yann
authors.initials: Y
authors.lastname: Jouvenot
authors.forename: Y
authors.initials: Y
authors.lastname: Jouvenot
authors.forename: Russell C
authors.initials: RC
authors.lastname: DeKelver
authors.forename: R C
authors.initials: RC
authors.lastname: Dekelver
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: P D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Fyodor D
authors.initials: FD
authors.lastname: Urnov
authors.forename: F D
authors.initials: FD
authors.lastname: Urnov
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.forename: M C
authors.initials: MC
authors.lastname: Holmes
mesh_terms: D001483:Base Sequence; D003851:Deoxyribonucleases / Q000235:genetics*; D005069:Evaluation Studies as Topic; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D003851:Deoxyribonucleases