TRV Vectors Deliver SgRNA-mediated Gene Editing Research

The third-generation gene editing technology CRISPR/Cas9 system has led the upsurge of gene function research in the field of animals and plants due to its simplicity and efficiency.Common delivery methods of CRISPR components are Agrobacterium-mediated Ti plasmid leaf injection,gene gun injection,protoplast transfection,and nanoparticle spraying.These methods can accurately deliver Cas9 protein and sgRNAs into nucleus of target cells.However,in order to obtain stable inherited mutant offspring;we have to use difficult regeneration of tissue embryos or controversial transgenic methods.The use of plant viruses to deliver CRISPR editing components have significant advantages: viruses can be rapidly amplified without transgenes in infecting plants;viruses can be systematically transferred in plants,and some RNA viruse vectors can also infect apical meristems and germplasm of plants.In this study,the TRV vector was used to deliver sgRNA to the overexpressing Sp Cas9 tobacco and cotton.By modifying the structure of TRV vectors,they greatly improve the efficiency of gene editing and the movement in tobacco and cotton.In addition,combining the virus induced gene silencing(VIGS)and virus induced gene editing(VIGE)technology,we simultaneously delivered the two components of VIGS and VIGE to the cotyledons of cotton through the TRV vector and gained the phenomenons of gene silencing and gene editing.The main results were as follows:1.Adding protective sequences at the 5'end and 3'end of sgRNA can improve the efficiency of gene editing.Taking Phytoenedesaturase(Phytoenedesaturase,PDS)gene and cotton Gh BASS5 gene as target genes,we constructed the TRV2 vectors with different lengths of protectived sequences at the 5' end and 3' end of sgRNA,respectively.The constructed vectors were infiltrated into overexpressing tobacco and cotton by Agrobacterium inoculation method.Through gene editing efficiency analysis,it was found that adding protective sequence could improve the efficiency of gene editing,and the effect of adding protective sequence with a length of 720 bp at the 3' end of sgRNA was the best.2.Adding viral subgenomic promoter before sgRNA improves the efficiency of gene editing.We added a PEBV promoter to the TRV2 virus vector,and at the same time,GFP fragment was added to the 5' end and 3' end of sgRNA as protective sequence respectively,and the constructed vectors were transfected into overexpressing tobacco and cotton by Agrobacterium inoculation method.The analysis of the experimental results showed that compared with the vector without additional promoter,the gene editing efficiency was indeed improved.Meanwhile,the analysis of the results also showed that when sgRNA contains a protective sequence,it could achieve better editing effect without the need of additional promoter sequence.3.The mobile RNA sequences can promote the systematic movement of sgRNA in plants.According to the idea that mobile RNA sequence can be fused with sgRNA to enhance virus mobility,protective sequence and mobile RNA sequence were added to the 3' end of sgRNA to ensure its mobility and editing effect.The result is that in tobacco and cotton,a good systematic editing effect was obtained,indicating that mobile RNA enhances the efficiency of systematic editing effect by promoting the movement of sgRNA..4.The silencing suppressor can promote the expression of sgRNA in plant.In the process of cotton gene editing research,the fusion of sgRNA and silencing suppressor improved the editing efficiency of infiltrated leaves and promoted the transmission of virus in cotton.5.Simultaneously delivering VIGS fragment and sgRNA sequence to shorten the flowering time of plants and promote gene editing of germ cells.Using VIGS technology to silence the cotton flowering negative regulation gene SELF PRUNING can shorten the growth cycle of cotton.By building a VIGS and VIGE fusion vector,TRV can deliver the two components together,not only making gene silencing effect and effectively promoting the cotton dwarf and flowering,but also enhancing the spread of the virus in the cotton,better realizing the effect of gene editing.By comparing the efficiency of targeted gene editing mediated by TRV based sgRNA delivery system,this study found that the addition of a protective sequence at the 3'end of sgRNA can greatly promote the effect of gene editing,while the fusion of mobile RNA sequences significantly improves viral vector-mediated systematic editing effect.This study also initially explored the possibility of combining VIGS and VIGE technologies to induce early flowering of plants to promote viral vectors carrying sgRNA to edit germ cells.This research provides a new strategy for the application of non-transgenic delivery of gene editing reagents using viral vectors and targeted editing of cotton and other crop genes,and lays a good foundation for the better realization of viral components infecting reproductive tissues.