The engineering of zinc-finger arrays capable of binding to a user-selected sequence is a difficult, expensive, laborious process and suffers from low reproducibility (Durai et al., 2005). These synthetic transcriptional regulators selectively activate or repress target genes (Bartsevich et al., 2003 Li et al., 2013b Mahfouz et al., 2012 Sanchez et al., 2002).
#SRDX RECOGNITION DOMAIN ACTIVATOR#
Synthetic transcriptional regulators have been generated using programmable DNA-binding modules such as zinc-finger domains and transcriptional activator (TA)-like (TAL) effectors, which are fused to a variety of activation or repression domains. This technology could discover novel traits and accelerate trait development in important crop species (Mojica et al., 2009). Developing these synthetic-biology approaches will enable highly efficient and precise interrogation of gene functions. This goal is feasible using synthetic transcriptional regulators, which are generated as chimeric proteins containing a DNA-binding domain fused to functional domain(s) that regulate transcriptional machinery (Mahfouz et al., 2012). Therefore, the development of new molecular tools to regulate gene function could also open new bioengineering strategies to express highly beneficial traits in agricultural crop species (Juillerat et al., 2014). The ability to specifically and simultaneously activate or repress single or multiple genes will enable the molecular investigation of gene and genome functions in their natural environment in response to different growth or stress signals (Hiratsu et al., 2003 Streubel et al., 2012). The observed CHS silencing induced by cosuppression in the early petunia study (Napoli et al., 1990) was reminiscent of RNAi-mediated gene silencing in C. elegans. During the late 1990s, pioneering experiments using short double-stranded RNA (dsRNA) introduced into Caenorhabditis elegans led to gene silencing this approach was designated as RNA interference (RNAi) (Fire et al., 1998). In the early 1990s, the phenomenon of cosuppression was observed when attempts to overexpress the chalcone synthase ( CHS) gene in petunia flowers unexpectedly resulted in down-regulation of gene expression (Napoli et al., 1990 Vanderkrol et al., 1990), and subsequent approaches utilized overexpression of antisense genes to down-regulate gene expression (Di Serio et al., 2001). Several methods have been developed to regulate gene transcription patterns in transformed plants, including generation of overexpression lines using constitutive or inducible promoters (Potenza et al., 2004). These responses are spatiotemporally controlled at the transcriptional level by transcription factors, activators or repressors, which modulate gene transcript levels, coordinate the transcriptional levels of genes within a single genetic pathway and orchestrate genomic expression at the cellular and system levels (Stam et al., 1997). Plants have evolved sophisticated molecular mechanisms that control their adaptive responses to dynamically changing environments. Our data indicate that the CRISPR/dCas9 DNA-targeting platform can be used in plants as a functional genomics tool and for biotechnological applications. Thus, our results suggest that the synthetic transcriptional repressor (dCas9:SRDX) and activators (dCas9:EDLL and dCas9:TAD) can be used as endogenous transcription factors to repress or activate transcription of an endogenous genomic target. Further, the dCas9:SRDX-mediated transcriptional repression of an endogenous gene.
#SRDX RECOGNITION DOMAIN .DLL#
Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3::uidA targets in plant cells. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology.
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