RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins

摘要

Developing targeted genome regulation approaches holds much promise foraccelerating trait discovery and development in agricultural biotechnology. ClusteredRegularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) systemprovides bacteria and archaea with an adaptive molecular immunity mechanism againstinvading nucleic acids through phages and conjugative plasmids. The type IICRISPR/Cas system has been adapted for genome editing purposes across a variety ofcell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) proteincombined with guide RNAs (gRNAs) were used as a DNA-targeting platform tomodulate the expression patterns in bacterial, yeast and human cells. Here, we employedthis DNA-targeting system for targeted transcriptional regulation in planta by developingchimeric dCas9-based activators and repressors. For example, we fused to the C-terminusof dCas9 with the activation domains of EDLL and TAL effectors, respectively, togenerate transcriptional activators, and the SRDX repression domain to generatetranscriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TADactivators, guided by gRNAs complementary to promoter elements, induce strongtranscriptional activation on episomal targets in plant cells. Moreover, our data suggestthat the dCas9:SRDX repressor and the dCas9:EDLL and dCas9:TAD activators arecapable of markedly repressing or activating, respectively, the transcription of anendogenous genomic target. Our data indicate that the CRISPR/dCas9:TFs DNAtargeting system can be used in plants as a functional genomic tool and forbiotechnological applications.