Application Of CRISPR/dCas-Based Transcription Activation System In Rice

The discovery of CRISPR/Cas system has promoted the rapid development of many research fields in biology.In recent years,the CRISPR/d Cas system has been improved,which plays a huge role in the regulation of gene expression levels.At present,several highefficiency transcriptional activation systems based on CRISPR/d Cas9 have been developed for animal and human cells.However,studies on d Cas9-mediated transcriptional activation in plants are relatively few and mainly focused on dicotyledonous Arabidopsis,a lot of work focused on identifying and optimizing the fusion of d Cas9 and activation domain,little is known about the effect of g RNA target sites on transcriptional activation efficiency.In addition,as a new type of CRISPR/Cas system,the CRISPR/Cpf1 system further expands the selection range of gene editing target sites.Because the extremely low offtarget efficiency and the simplicity of cr RNA structure,the CRISPR/d Cpf1 system has greater advantages in multiple gene regulation,but the application of CRISPR/d Cpf1 transcriptional activation system is difficult,and many studies are also in the exploration stage.Our study starts with development a CRISPR/d Cas9 transcriptional activation in rice.First,we constructed the second generation d Cas9-SAM system and analyzed its transcriptional activation effect in rice,we found that its activation efficiency was very low.So we chose the d Cas9–6TAL–VP128(d Cas9-TV)system,which has been shown to have a high transcriptional activation efficiency in plant protoplasts.In order to improve transcriptional activation efficiency in rice,we choose rice-specific Os U6 and Os U3 promoters to optimize the d Cas9-TV system.In order to investigate the effect of g RNA target sites on transcriptional activation efficiency,we designed a series of target sites at different positions upstream of the Transcription Start Site(TSS)in the promoter regions of the target genes Os WOX11 and Os YUC1,and also combined g RNAs of different target positions.Then we transformed the single target and multi-target transcriptional activation vectors into rice Zhonghua 11 by Agrobacterium-mediated stable transformation,respectively.We explored the transcriptional activation efficiency of different transgene events in rice according to the expression levels of target genes.In addition,we also tried to design and develop a d Cpf1-based transcriptional activation system.We fused d Cpf1 with the transcriptional activation elements Sun Tag and TV,and selected the same target genes in previous study to transform rice.We hope that these two methods can be used to quickly and easily overexpress rice genes,making it easier to study the function of genes and help to apply the transcriptional activation systems in rice.Our results show that the d Cas9-TV transcription activation system can strongly upregulate the expression levels of auxin-related genes Os WOX11 and Os YUC1 in rice,During the tissue culture differentiation stage,it showed obvious root phenotype,and the results of quantitative real-time PCR(q RT-PCR)also showed that the expression levels of the two target genes increased significantly.However,the d Cas9-SAM system has only a weak activation effect.Similarly,we also analyzed the target effect and found that the best targeted region choosen for transcriptional activation system in rice is within 350 bp upstream of the TSS.It will be more better if two targets are selected to target the same promoter at the same time.In conclusion,we have demonstrated a feasible and applicable transcriptional activation system in the stable transformation of rice,providing a useful tool for future research on rice gene function.