RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis

PLoS One. 2017 Jun 16;12(6):e0179410. doi: 10.1371/journal.pone.0179410. eCollection 2017.

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated protein 9 (Cas9) system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9) offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF) activation domain to dCas9 bound with the VP64 (tetramer of VP16) activation domain for application in plants. The redesigned CRISPR/Cas9 activation system was tested in Arabidopsis to increase endogenous transcriptional levels of production of anthocyanin pigment 1 (PAP1) and Arabidopsis thaliana vacuolar H+-pyrophosphatase (AVP1). The expression of PAP1 was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of PAP1 overexpressors. The AVP1 gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, AVP1 activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The AVP1 activated plants showed similar phenotypes to AVP1 overexpressors. Therefore, the redesigned CRISPR/Cas9 activation system containing modified p65-HSF provides a simple approach for producing activated plants by upregulating endogenous transcriptional levels.

MeSH terms

  • Arabidopsis Proteins* / biosynthesis
  • Arabidopsis Proteins* / genetics
  • Arabidopsis* / genetics
  • Arabidopsis* / metabolism
  • CRISPR-Cas Systems*
  • Gene Expression Regulation, Plant*
  • Inorganic Pyrophosphatase* / biosynthesis
  • Inorganic Pyrophosphatase* / genetics
  • Pancreatitis-Associated Proteins
  • Plant Leaves / genetics
  • Plant Leaves / metabolism
  • Transcription Factors* / biosynthesis
  • Transcription Factors* / genetics
  • Transcription, Genetic*
  • Transcriptional Activation*

Substances

  • Arabidopsis Proteins
  • PAP1 protein, Arabidopsis
  • Pancreatitis-Associated Proteins
  • REG3A protein, human
  • Transcription Factors
  • AVP1 protein, Arabidopsis
  • Inorganic Pyrophosphatase

Grants and funding

This work was supported by BioEnergy Science Center and The Samuel Roberts Noble Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.