The CRISPR-Cas System Mediated Transcription Activation In Arabidopsis Thaliana

CRISPR-Cas system is a immune system aiming at exogenous gene.This system can use a small piece of RNA(sgRNA)to determine the specificity,implementation targeted sites knockdown.Because the operation is simple,high success rate,so many researchers turn them into gene editing tools.This topic used dcas9 which was the mutant of cas9 trasformed CRISPR-Cas system as a kind of specific transcriptional activation tool.First experiment we cloned a min35 s promoter lacking transcription factors to Pcambia 1380 vector,here we called it MP 1380.Then cloned ?-glucoside acid enzyme(GUS)gene to MP 1380 vector which was opened by min35 s promoter,called MGP 1380.Turned MGP 1380 into arabidopsis thaliana,obtained the stable expression plant,dyeing with X-gluc found that the plants didn't turn blue,indicated that the GUS gene haven't expressed.And then we would have dcas9-Vp64 fusion protein and a sgRNA-guid started by a U6 promoter cloned to a Pcambia1380 vector,We called the recombinant DVP 1380.Transferred DVP1380 to the above arabidopsis thaliana which had been transferred MGP 1380,got the stable expression one and then applied the same dyeing methods,the leaves turned blue.The experiment indicated that dcas9 protein togetherwith transcription activation domain Vp64 and sgRNA-guid targeting the promoter sit could open the expression of GUS gene.The reseacher proved that the CRISPR-Cas system can be used as an effective specific transcription activation or inhibit the tools,for the discovery of noncoding RNA(ncRNA)has a very important significance.