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

Abstract

<div><p>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 <u><i>p</i></u><i>roduction of</i> <u><i>a</i></u><i>nthocyanin</i> <u><i>p</i></u><i>igment 1</i> (<i>PAP1</i>) and <u><i>A</i></u><i>rabidopsis thaliana</i> <u><i>v</i></u><i>acuolar H</i>^<i>+</i>-<u><i>p</i></u><i>yrophosphatase</i> (<i>AVP1</i>). The expression of <i>PAP1</i> was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of <i>PAP1</i> overexpressors. The <i>AVP1</i> gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, <i>AVP1</i> activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The <i>AVP1</i> activated plants showed similar phenotypes to <i>AVP1</i> 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.</p></div