01979nam a2200217Ia 4500003001000000005001700010040000900027245010900036490006400145520122700209650001101436650002501447650001101472650001301483650003101496700001101527700001301538700001301551856015601564008004101720MX-MdCICY20260521091610.0  cCICY10aTargeted Transcriptional Activation in Plants Using a Potent Dead Cas9-Derived Synthetic Gene Activator.0 vCurrent Protocols in Molecular Biology, 127(1), p.e89, 20193 aGenetic tools for specific perturbation of endogenous gene expression are highly desirable for interrogation of plant gene functions and improvement of crop traits. Synthetic transcriptional activators derived from the CRISPR/Cas9 system are emerging as powerful new tools for activating the endogenous expression of genes of interest in plants. These synthetic constructs, generated by tethering transcriptional activation domains to a nuclease?dead Cas9 (dCas9), can be directed to the promoters of endogenous target genes by single guide RNAs (sgRNAs)to activate transcription. Here, we provide a detailed protocol for targeted transcriptional activation in plants using a recently developed, highly potent dCas9 gene activator construct referred to as dCas9?TV. This protocol covers selection of sgRNA targets, construction of sgRNA expression cassettes, and screening for an optimal sgRNA using a protoplast?based promoter?luciferase assay. Finally, the dCas9?TV gene activator coupled with the optimal sgRNA is delivered into plants via Agrobacterium?mediated transformation, thereby enabling robust upregulation of target gene expression in transgenic Arabidopsis and rice plants. © 2019 by John Wiley & Sons, Inc.14aCRISPR14aDCAS9 GENE ACTIVATOR14aPLANTS14aPROMOTER14aTRANSCRIPTIONAL ACTIVATION12aLi, Z.12aWang, F.12aLi, J.F.40uhttps://drive.google.com/file/d/1zVYRnP4LWFwTtuzgJu8U8wt8bZMcf-nC/view?usp=drivesdkzPara ver el documento ingresa a Google con tu cuenta: @cicy.edu.mx250602s9999    xx |||||s2   |||| ||und|d