title: Identification of "safe harbor" loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair.
abstract: Zinc-finger nucleases (ZFNs) have proved to be successful tools for targeted genome manipulation in several organisms. Their main property is the induction of double-strand breaks (DSBs) at specific sites, which are further repaired through homologous recombination (HR) or non-homologous end joining (NHEJ). However, for the appropriate integration of genes at specific chromosomal locations, proper sites for gene integration need to be identified. These regions, hereby named safe harbor loci, must be localized in non-coding regions and possess high gene expression. In the present study, three different ZFN constructs (pZFN1, pZFN2, pZFN3), harboring β-glucuronidase (GUS) as a reporter gene, were used to identify safe harbor loci on rice chromosomes. The constructs were delivered into IR64 rice by using an improved Agrobacterium-mediated transformation protocol, based on the use of immature embryos. Gene expression was measured by histochemical GUS activity and the flanking regions were determined through thermal-asymmetric interlaced polymerase chain reaction (TAIL PCR). Following sequencing, 28 regions were identified as putative sites for safe integration, but only one was localized in a non-coding region and also possessed high GUS expression. These findings have significant applicability to create crops with new and valuable traits, since the site can be subsequently used to stably introduce one or more genes in a targeted manner.
authors.affiliation: Gene Transformation Lab, Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute Metro Manila, Philippines.
authors.forename: Christian
authors.initials: C
authors.lastname: Cantos
authors.affiliation: Gene Transformation Lab, Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute Metro Manila, Philippines.
authors.forename: Perigio
authors.initials: P
authors.lastname: Francisco
authors.affiliation: Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development Bogor, Indonesia.
authors.forename: Kurniawan R
authors.initials: KR
authors.lastname: Trijatmiko
authors.affiliation: Gene Transformation Lab, Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute Metro Manila, Philippines.
authors.forename: Inez
authors.initials: I
authors.lastname: Slamet-Loedin
authors.affiliation: Gene Transformation Lab, Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute Metro Manila, Philippines.
authors.forename: Prabhjit K
authors.initials: PK
authors.lastname: Chadha-Mohanty
keywords: double-strand breaks (DSBs); homologous recombination (HR); rice (Oryza sativa L.); safe harbor loci; zinc-finger nucleases (ZFNs)