title: Targeted manipulation of mammalian genomes using designed zinc finger nucleases.
abstract: Targeted introduction of a double-stranded break (DSB) using designer zinc finger nucleases (ZFNs) in mammalian cells greatly enhances gene targeting - homologous recombination (HR) at a chosen endogenous target gene, which otherwise is limited by low spontaneous rate of HR. Here, we report that efficient ZFN-mediated gene correction occurs at a transduced, transcriptionally active, mutant GFP locus by homology-directed repair, and that efficient mutagenesis by non-homologous end joining (NHEJ) occurs at the endogenous, transcriptionally silent, CCR5 locus in HEK293 Flp-In cells, using designed 3- and 4-finger ZFNs. No mutagenesis by NHEJ was observed at the CCR2 locus, which has ZFN sites that are distantly related to the targeted CCR5 sites. We also observed efficient ZFN-mediated correction of a point mutation at the endogenous mutant tyrosinase chromosomal locus in albino mouse melanocytes, using designed 3-finger ZFNs. Furthermore, re-engineered obligate heterodimer FokI nuclease domain variants appear to completely eliminate or greatly reduce the toxicity of ZFNs to mammalian cells, including human cells.
authors.affiliation: Department of Environmental Health Sciences, Johns Hopkins School of Public Health, Baltimore, MD 21205, USA.
authors.forename: Karthikeyan
authors.initials: K
authors.lastname: Kandavelou
authors.forename: Sivaprakash
authors.initials: S
authors.lastname: Ramalingam
authors.forename: Viktoriya
authors.initials: V
authors.lastname: London
authors.forename: Mala
authors.initials: M
authors.lastname: Mani
authors.forename: Joy
authors.initials: J
authors.lastname: Wu
authors.forename: Vitali
authors.initials: V
authors.lastname: Alexeev
authors.forename: Curt I
authors.initials: CI
authors.lastname: Civin
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Chandrasegaran
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D016678:Genome / Q000235:genetics*; D006801:Humans; D008544:Melanocytes / Q000378:metabolism; D051379:Mice; D014442:Monophenol Monooxygenase / Q000235:genetics; D016296:Mutagenesis; D015202:Protein Engineering; D019713:Receptors, CCR5 / Q000235:genetics; D011995:Recombination, Genetic; D014161:Transduction, Genetic; D016335:Zinc Fingers
chemical_list: D019713:Receptors, CCR5; D014442:Monophenol Monooxygenase; D004720:Endonucleases