CRISPR/Cas9 targeting systems have been applied to a variety of species. However, most of current CRISPR/Cas9 systems for plants can only modify one or a few target sites.Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon-optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with average 85.4% of mutation rate, mostly in biallelic and homozygous statuses. We reasoned that about 16% of the homozygous mutations in rice were generated through the nonhomologous end joining mechanism followed by homologous recombination-based repairing. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to 8) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.
Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H,Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, and Liu Y.G.(2015). A robustCRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicotplants. Mol. Plant.doi:10.1016/j.molp.2015.04.007.