RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors.
Targeted 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 C-terminus 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.
Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
Agnieszka
A
Piatek
Zahir
Z
Ali
Hatoon
H
Baazim
Lixin
L
Li
Aala
A
Abulfaraj
Sahar
S
Al-Shareef
Mustapha
M
Aouida
Magdy M
MM
Mahfouz
D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D018506:Gene Expression Regulation, Plant; D010944:Plants / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D014157:Transcription Factors
EDLL activation domain; SRDX repressor domain; chimeric dCas9 transcriptional activators and repressors; synthetic transcriptional regulators; targeted genomic regulation