Targeted Integration of Genes into the Maize Genome Facilitated By Cas9-gRNA System

Maize is one of the most important crops in the world. A growing global population requires a continuous effort to increase maize productivity and yield stability. Transgenic approach plays an important role in crop improvement. Currently, transgenes are randomly inserted into maize genome. When more than one gene are used to create a favorable trait or a group of traits, the transgenes reside in various parts of chromosomes, making the subsequent breeding costly and prolonging the time to bring products to farmers. Although molecular stacking in a transformation construct can combine multiple transgenes into one locus, this strategy is not always desirable because breeders often want to have the option to segregate out some of the transgenes to meet their trait goals. A preferred design would be placing multiple closely linked transgenes in designated chromosome regions. Previously, transgenes can be precisely inserted at a small number of locations in the genome with help of protein-based double strand break reagents like zinc-finger nucleases, homing endonucleases (meganucleases) and TALENs. Recently, the Cas9-gRNA system makes it possible to target transgenes into almost any location in the genome which would allow multiple transgenes to be precisely inserted at pre-selected, characterized, and closely linked genomic regions. Targeted integration of transgenes in maize via Cas9-gRNA will be presented.