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

Bibliography:

Agnieszka Piatek, Zahir Ali, Hatoon Baazim, Lixin Li, Aala Abulfaraj, Sahar Al-Shareef, Mustapha Aouida, Magdy M. Mahfouz. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnology Journal. DOI: 10.1111/pbi.12284

Authors:

Agnieszka Piatek, Zahir Ali, Hatoon Baazim, Lixin Li, Aala Abulfaraj, Sahar Al-Shareef, Mustapha Aouida, Magdy M. Mahfouz

Keywords:

Targeted genomic regulation, synthetic transcriptional regulators, chimeric dCas9transcriptional activators andrepressors, EDLL activation domain, SRDX repressor domain

Year:

2014

Abstract:

‚ÄčTargeted 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.

ISSN:

1467-7652