CRISPR/dCas9 Associated Transcriptional Activation And Repression Study In Wheat

Wheat as a major staple by 40% of the world population,is a cultivated gramineous plant worldwide and its production is only second to corn.The CRISPR/Cas9 system has been applied to many species since it was applied in the field of gene editing since 2012.The inactivation of catalytical site in Cas9(dCas9)has been reprogrammed as an effective approach to regulate the transcriptional level of target genes,especially for the functionally essential genes and redundant genes.Studies have found that combining the transcriptional activation or repression domains with the C-terminus of dCas9 can more effectively achieve the corresponding transcriptional regulation,that is,up-regulation or down-regulationof the expression of the target gene.Although the CRISPR/Cas9 system has been used in wheat,it is still unknown whether the CRISPR/dCas9-derived CRISPR activation(CRISPRa)and CRISPR inhibition(CRISPRi)systems are amenable to wheat.In this project,based on the functional conservation of transcriptional elements between differentspecies of animals and plants,we constructed the CRISPRa and CRISPRi system in wheat,targeting the phytoene dehydrogenase encoding gene(PDS)gene,to manipulate its transcriptional expression level in common wheat.In order to improve target gene's expression,we generated transcription activators by fusing the activation domain of VP64,EDLL,and 6×TAL-2×VP64 to the C-terminus of dCas9 in the expression frame.With the purpose to inhibit the expression of transcription target genes,3×SRDX inhibitiondomain was conjugated to the C-terminus of dCas9 in frame.At the same time,in order tocompare the effects of using different g RNA processing methods on editing efficiency,wecompared the effects of the tandem single U3 promoter and endogenous tRNA processing methods on the CRISPR/dCas9 transcriptional regulatory system in wheat.These dCas9-based transcriptional regulators were guided to the promoter region of PDS in vivo and the gene expression levels were measured to evaluate the effect for each regulation.Our results showed that in stable transformed wheat variety,the transcriptional activation or repression domain of dCas9 fusion can effectively increase or decrease the transcription level of target genes,and its corresponding transcriptional regulation can be transferred to the next generation to some extent.Based on the study of the efficiency of the tRNA processing system in the transcription regulation system of CRISPR/dCas9,we found its advantages in some cases,which provide a theoretical basis for the rational application of this processing system.The accommodation of tRNA-processing system didn't reveal any advantage in T0 transgenic lines.However,as far as the T1 generation is concerned,the application of the tRNA processing system performs better to some extent.In summary,our work extends the application of the CRISPR/dCas9-based transcriptional activation and suppression system to an important crop-wheat,broadens its application range,and provides strong theoretical support for the functional research of wheat essential genes.At the same time,in order to develop more effective applications of transcription activation and suppression elements in wheat,we optimized the wheat protoplast preparation and transformation system.By analyzing the regulation of target gene expression by relatively new transcriptional activation and inhibitory elements in the wheat transient transformation and detection system,we will explore more effective transcriptional regulatory elements.