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.
Keywords: zinc-finger nucleases (ZFNs), safe harbor loci, rice (Oryza sativa L.), homologous recombination (HR), double-strand breaks (DSBs)
Citation: Cantos C, Francisco P, Trijatmiko KR, Slamet-Loedin I and Chadha-Mohanty PK (2014) Identification of “safe harbor” loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair. Front. Plant Sci. 5:302. doi: 10.3389/fpls.2014.00302
Received: 03 March 2014; Paper pending published: 22 May 2014;
Accepted: 09 June 2014; Published online: 26 June 2014.
Edited by:Alma Balestrazzi, University of Pavia, Italy
Reviewed by:Alma Balestrazzi, University of Pavia, Italy
Copyright © 2014 Cantos, Francisco, Trijatmiko, Slamet-Loedin and Chadha-Mohanty. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Prabhjit K. Chadha-Mohanty, International Rice Research Institute, DAPO Box 7777, Metro Manila 1301, Philippines e-mail: firstname.lastname@example.org