RNA‐guided transcriptional regulation in planta via synthetic dCas9‐based transcription factors

Abstract

SummaryTargeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. 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. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. 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. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 Cterminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. 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. Further, the dCas9:SRDX-mediated transcriptional repression of an endogenous gene. 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. Our data indicate that the CRISPR/dCas9 DNA-targeting platform can be used in plants as a functional genomics tool and for biotechnological applications.

Synthetic Botany

“…Other useful circuits comprise the Cre-Lox recombinase system that can be used for induction of transcription (Hoff et al 2001) or transgene excision (Chakraborti et al 2008), and a catalytically inactive version of Cas9 fused to regulatory domains. The latter has been shown to perform effective transcriptional activation and repression of an endogenous gene in Nicotiana (Piatek et al 2015) and Arabidopsis (Lowder et al 2015). Posttranscriptional control over gene expression in plants has been enabled by a variety of RNA-based technologies.…”

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation

“…However, when multiple gRNAs were coexpressed, a synergistic or additive transcriptional activation was observed. Repression of endogenous genes has also been demonstrated by expression of dCas9 or dCas9-repressor fusion proteins in human cells (Gilbert et al, 2013;Qi et al, 2013a) and tobacco (Piatek et al, 2014). Complex gene expression programs can be engineered with modified gRNA scaffolds that can simultaneously induce transcriptional activation or repression (Zalatan et al, 2015).…”

Genome-Editing Technologies: Principles and Applications

“…For example, TALEs and CRISPR-Cas9 have enabled rapid construction of custom genetic circuits and logic gates (Gaber et al 2014;Lebar et al 2014;Liu et al 2014b), complex gene regulation networks (Nielsen and Voigt 2014;Nissim et al 2014), and even facilitated cellular reprogramming (Gao et al 2013) and the differentiation of mouse embryonic fibroblasts to skeletal myocytes (Chakraborty et al 2014). dCas9 transcriptional effectors have even been used to efficiently mediate repression and activation of endogenous genes in Drosophila (Lin et al 2015b) and in plant cells (Piatek et al 2015). Both TALE and Cas9 activators have also been configured to stimulate transcription of latent HIV Ji et al 2016;Limsirichai et al 2016;Perdigao et al 2016;Saayman et al 2016), indicating their potential to work in concert with antiretroviral therapy for eradicating HIV infection.…”