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.

Keywords: double-strand breaks (DSBs); homologous recombination (HR); rice (Oryza sativa L.); safe harbor loci; zinc-finger nucleases (ZFNs).

Figure 1

Structure and key features of the pZFN1, pZFN2, and pZFN3 constructs. The vector system is based on the assembly of ZFN expression cassettes (pSAT4.hspP.ZFN and pSAT5.hspP.ZFN), a plant selection expression cassette (pSAT1.hpt), and GUS reporter cassette (pSAT6A.ZFN-TS^*::GUS) onto the plant binary vector pRCS2. Asterisk stands for the modification generated in the cassette. The plasmid carries a plant expression cassette engineered for constitutive expression of a mutated uidA (GUS) gene. A stop (TGA) codon was engineered within the 6-bp spacer of the ZFN target site, leading to premature termination of uidA translation in plant cells. The ZFN QQR recognition site is shown.

Figure 2

Generation of transgenic rice plants by Agrobacterium-mediated transformation. (A) Growth of A. tumefaciens LBA4404 on AB medium; (B) co-cultivation of immature embryos with Agrobacterium suspension; (C) selection of resistant calli; (D) regeneration of plantlets; (E) rice plantlets on MS0 rooting media.

Figure 3

Histochemical GUS staining; (A) Wild type; (B) Transgenic rice exhibiting high GUS expression; (C) Transgenic rice exhibiting medium GUS expression; (D) Transgenic rice exhibiting low GUS expression.

Figure 4

Agarose gel analysis of TAIL PCR products amplified from GUS-positive insertion lines. Bands shown in boxes were cut and sequenced. AD1, AD2, AD3, and AD4 = non-specific primers.