A key step in plant gene editing is to deliver Cas9 protein and sgRNA to plant cells to play a role.
At present, the delivery methods in plants mainly include Agrobacterium and gene gun transformation, both of which require a long period of tissue culture and regeneration.
Obtain complete mutant plants
However, tissue culture and regeneration are still the rate-limiting steps of plant gene editing.
For some monocots, especially common wheat containing a large and complex hexaploid genome, genome editing is particularly difficult
The development of a delivery system that does not require tissue culture and is not restricted by genotype is a key technical problem that needs to be solved in plant genome editing
The Gao Caixia team of the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences and the Li Dawei team of China Agricultural University have jointly developed a barley stripe mosaic virus (BSMV)-based sgRNA delivery vector system-BSMV-sg, which has successfully established an efficient and heritable sgRNA in wheat.
, Does not require tissue culture genome editing delivery system
This research carried sgRNA to BSMVγ to construct the BSMVγ-sg system, which used mobile RNA elements (mAtFT, mTaFT, and tRNA) to modify sgRNA in order to promote the ability of BSMV virus to move to meristem cells to improve heritable editing Efficiency
The results show that the unmodified BSMVγ-sg has higher infection ability and editing activity in wheat leaves
The BSMV-sg system was further used to edit the three genes of the three wheat Cas9 expressing varieties, and it was found that BSMV-sg can obtain highly efficient heritable mutations in the progeny of virus-infected plants, with an efficiency of 12.
9%-100%, and The efficiency of pure and mutant is 1.
In addition, it was found that more than 53.
8% of the mutant offspring were virus-free
Multi-gene editing can be achieved by mixing BSMV-sg Agrobacterium containing multiple targets and infecting wheat
Finally, cross the BSMV-sg-infected Cas9 transgenic wheat pollen with wild-type wheat to obtain Cas9-free (Cas9-free) mutant offspring in the F2 generation
The BSMV-sg-mediated wheat genome editing delivery system has the characteristics of high efficiency, low cost, and no need for tissue culture and regeneration.
It can be applied to large-scale and high-throughput wheat genome editing, and is important for wheat functional gene research and molecular design breeding.
The research results will be published online in Molecular Plant magazine (DOI:10.
010) on July 14, 2021
Li Tingdong, a PhD student in the Gao Caixia research group of the Institute of Genetics and Development of the Chinese Academy of Sciences, and Hu Jia, a doctoral student in the Zhang Yongliang research group of the School of Biology, China Agricultural University, became the co-first authors of the paper.
The co-corresponding author of the paper
This research was funded by the Chinese Academy of Sciences Strategic Pilot Project A, the National Natural Science Foundation of China, the Open Project of the State Key Laboratory of Agricultural Biotechnology, and the funding of the Youth Innovation Promotion Association of the Chinese Academy of Sciences
Figure: BSMV- mediated high-efficiency wheat gene editing delivery system