Jinman Luo¹, Shaoya Li¹, Jiajing Xu¹, Lei Yan¹, Youzhi Ma^{1, *} and Lanqin Xia^{1, *}
　　Molecular Plant, IF: 12.084
　　https://doi.org/10.1016/j.molp.2021.03.024
　　Abstract
　　Pyramiding favorable alleles in an elite wheat variety by CRISPR/Cas9-mediated multiplex editing will greatly accelerate the breeding process. However, the hexaploidy nature of wheat makes pyramiding several agronomic important traits which are conferred by genes located at multiple genomic sites in an elite wheat variety, very challenging. Besides, most elite wheat varieties are recalcitrant to genetic transformation and usually have lower transformation efficiency. In this study, we developed a robust and efficient CRISPR/Cas9 multiplexing system by using a polycistronic tRNA strategy to simultaneously produce multiple sgRNAs. We demonstrated the simultaneous editing of two, three, four and five genes at up to 15 genomic loci in an elite wheat variety by multiplex gene editing for the first time. The simultaneous editing efficiency of multiple genes could reach as higher as 50%. Following embryo rescue and segregation, we successfully recovered transgene-free plants with targeted mutagenesis at up to 15 genomic loci in one generation and pyramided the favorable alleles in this elite wheat variety within one year. The established multiplexing system here would greatly facilitate fundamental biological research and translational breeding process in hexaploidy wheat as well as in other agriculturally important polyploid crop species for sustainable agriculture development.