Multiplex genome editing and precision breeding with CRISPR/Cas9

The bacterial cluster regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system has recently emerged as an efficient and versatile genome editing tool for basic biological research, medicine and agriculture. Recently, we have demonstrated precise genome editing and targeted mutagenesis in both monocot and dicot plants with the CRISPR/Cas9 system. To assess potential off-target effects and increase the editing specificity of CRISPR/Cas9, we have performed genome-wide prediction of highly specific gRNA spacer sequences in model plants and major crops and developed bioinformatic database and web tool for gRNA design. To facilitate multiplex genome editing, a novel polycistronic tRNA-gRNA strategy has been developed for simultaneous mutation of multiple genes, chromosomal fragment deletion, and other more sophisticated CRISPR/Cas9 applications. In addition, various strategies are being utilized to produce transgene-free, genetically modified crops such as rice, potato and mushroom. With improved genome editing strategies and bioinformatics tools, CRISPR/Cas9 has become a powerful technology for plant genome engineering and genetic improvement of agricultural crops.