Split-Cre mediated deletion of DNA no longer needed after site-specific integration in rice.

Abstract

N-cre and C-cre added in separate lines reassemble functional Cre in F1 progeny to excise unnecessary DNA, includingcre DNA, thereby eliminating generations needed to cross in and outcre. Crop improvement via transgenesis can benefit through efficient DNA integration strategies. As new traits are developed, new transgenes can be stacked by in planta site-specific integration near previous transgenes, thereby facilitating their introgression to field cultivars as a single segregation locus. However, as each round of integration often requires use of selectable markers, it is more convenient to reuse the selection scheme. The Cre recombinase can be used to delete away previously used selection genes, and other DNA no longer needed after transformation, but the constitutive production of this DNA scanning protein can also affect plant growth. We had previously described in Arabidopsis a split Cre protein fragment complement scheme to reassemble a functional Cre recombinase. As our goal for developing this system was to deploy its use in major crop plants, here we show that Cre protein fragment complementation works in rice with precise recombination structures confirmed by DNA sequencing. As each N-terminal and C-terminal fragment is also flanked by lox recombination sites, they can also self-excise to avoid the need to segregate away the cre DNA. Options to form F1 hybrids homozygous for one transgene, or hemizygous for two different transgenes at the same chromosome location, are discussed.