Figure 1.

Experimental approach and constructs for analyzing NHEJ-mediated genome modification in plants. A, A target DNA molecule was engineered to carry a functional expression cassette in which the target gene (gene A) is flanked by ZFN recognition sites. Gene A loss of function can facilitate the detection of site-specific mutagenesis, gene deletion, donor gene insertion (gene B), and target gene replacement (gene C). B, A GFP- and kanamycin-expressing target DNA molecule in which the GFP coding sequence is flanked by two quasipalindromic QQR ZFN recognition sites. This construct was used to produce GFP-overexpressing transgenic plants. C, A donor T-DNA molecule that was engineered to carry a promoterless hygromycin resistance-encoding gene flanked by two quasipalindromic QQR ZFN recognition sites and a constitutive QQR ZFN expression cassette. D, A dual-donor T-DNA molecule in which the promoterless hygromycin resistance-encoding gene and the QQR ZFN-expressing cassette are launched from two separate T-DNA molecules. gfp, GFP-encoding gene; hpt, hygromycin resistance-encoding gene; LB, left border; nptII, kanamycin resistance-encoding gene; P, generic promoter; P₁ and P₃, dual CaMV 35S promoter; P₂, octopine synthase promoter; QQR, QQR ZFN recognition site; RB, right border; T, generic terminator; T1 and T₃, CaMV 35S terminators; T2, octopine synthase terminator; ZFN, ZFN recognition site; ZFN*, altered ZFN recognition site. [See online article for color version of this figure.]