title: Multiple copies of a linear donor fragment released in situ from a vector improve the efficiency of zinc-finger nuclease-mediated genome editing.
abstract: Homologous recombination (HR) is a strategy for genetic correction. The efficiency of HR can be increased by creating a targeted double-strand break (DSB) via zinc-finger nucleases (ZFNs) and/or by introducing linear donor DNA intracellularly. Some studies have suggested that increased copy numbers of linear donor DNA may further improve HR efficiency. However, the introduction of multiple copies of a linear donor fragment remains a challenge, particularly in cell types with low transfection efficiency. In this study, we developed a vector that carries tandem repeats of a donor fragment, with each repeat flanked by ZFN target sequence fragments (TSFs). The cleavage of the flanking TSF sequence by ZFN would lead to the release of multiple linear fragment. We demonstrated that this novel vector carrying five copies of a linearizable donor fragment, when co-transfected with a ZFN-expressing vector in 293 cells, showed improved HR efficiency about 30 times, as compared with vector carry nonlinearizable donor. For the application of gene therapy, we then introduced this system into an adenoviral vector, which also revealed markedly improved ZFN-mediated HR efficiency in cells. The novel strategies presented here have the potential to promote the application of ZFNs in both basic research and disease therapy.
authors.affiliation: Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi'an, China.
authors.forename: W
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi'an, China.
authors.forename: D
authors.initials: D
authors.lastname: Wang
authors.affiliation: Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi'an, China.
authors.forename: S
authors.initials: S
authors.lastname: Liu
authors.affiliation: Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi'an, China.
authors.forename: X
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi'an, China.
authors.forename: H
authors.initials: H
authors.lastname: Ji
authors.affiliation: Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi'an, China.
authors.forename: H
authors.initials: H
authors.lastname: Xia
authors.affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Mao
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D015894:Genome, Human; D057809:HEK293 Cells; D059765:Homologous Recombination; D006801:Humans; D016335:Zinc Fingers
chemical_list: D003850:Deoxyribonuclease I