摘要
Synthetic gene activators consisting of nucleasedead Cas9(dCas9)for single-guide RNA(sgRNA)-directed promoter binding and a transcriptional activation domain(TAD)represent new tools for gene activation from endogenous genomic locus in basic and applied plant research.However,multiplex gene coactivation by d Cas9-TADs has not been demonstrated in whole plants.There is also room to optimize the performance of these tools.Here,we report that our previously developed gene activator,dCas9-TV,could simultaneously upregulate OsGW7 and OsER1 in rice by up to 3,738 fold,with one sg RNA targeting to each promoter.The gene coactivation could persist to at least the fourth generation.Astonishingly,thepolycistronictRNA-sgRNAexpression under the maize ubiquitin promoter,a Pol II promoter,could cause enormous activation of these genes by up to>40,000-fold in rice.Moreover,the yeast GCN4 coiled coil-mediated dCas9-TV dimerization appeared to be promising for enhancing gene activation.Finally,we successfully introduced a self-amplification loop for dCas9-TV expression in Arabidopsis to promote the transcriptional upregulation of AtFLS2,a previously characterized dCas9-TV-refractory gene with considerable basal expression.Collectively,this work illustrates the robustness of dCas9-TV in multigene coactivation and provides broadly useful strategies for boosting transcriptional activation efficacy of dCas9-TADs in plants.
基金
supported by a grant from the National Transgenic Science and Technology Major Program of China(2019ZX08010003-001-009)。
作者简介
Xiangyu Xiong,contributed equally to this work;Jieping Liang,contributed equally to this work;Correspondence:Jian-Feng Li,lijfeng3@mail.sysu.edu.cn。
