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

• Published in: PloS one Vol. 12; no. 6; p. e0179410
• Main Authors: Park, Jong-Jin, Dempewolf, Emma, Zhang, Wenzheng, Wang, Zeng-Yu
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 16.06.2017; Public Library of Science (PLoS)
• Subjects: Acidification; Amino acids; Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis Proteins - biosynthesis; Arabidopsis Proteins - genetics; Arabidopsis thaliana; BASIC BIOLOGICAL SCIENCES; Biology and Life Sciences; Cloning; CRISPR; CRISPR-Cas Systems; Deactivation; Deoxyribonucleic acid; DNA; Drought; Endonuclease; Engineering; Enzymes; Functional anatomy; Gene expression; Gene Expression Regulation, Plant; Genes; Genetic aspects; Genetic modification; Genomes; Heat; Hydrogen; Inorganic pyrophosphatase; Inorganic Pyrophosphatase - biosynthesis; Inorganic Pyrophosphatase - genetics; Laboratories; Leaves; Mutation; NF-κB protein; Pancreatitis-Associated Proteins; Physiological aspects; Plant Leaves - genetics; Plant Leaves - metabolism; Proteins; Pyrophosphatase; Research and Analysis Methods; Ribonucleic acid; RNA; RNA modification; Science & Technology - Other Topics; Series (mathematics); Stresses; Transcription activation; Transcription factors; Transcription Factors - biosynthesis; Transcription Factors - genetics; Transcription, Genetic; Transcriptional Activation; Transgenic plants; VP16 protein; United States; United States > US; Tennessee; Oklahoma
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0179410

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.