A CRISPR/dCas9 toolkit for functional analysis of maize genes
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(2020) 16:133 Gentzel et al. Plant Methods https://doi.org/10.1186/s13007-020-00675-5
Open Access
RESEARCH
A CRISPR/dCas9 toolkit for functional analysis of maize genes Irene N. Gentzel1†, Chan Ho Park1†, Maria Bellizzi1, Guiqing Xiao1, Kiran R. Gadhave2, Colin Murphree2, Qin Yang2, Jonathan LaMantia3, Margaret G. Redinbaugh1,3, Peter Balint‑Kurti2, Tim L. Sit2 and Guo‑Liang Wang1*
Abstract Background: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9). Results: In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression. Conclusions: We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant. Keywords: Maize, Protoplasts, CRISPR/Cas9, Transcription activation, Transcription suppression Background The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is the method of choice for plant genome editing projects, as it combines simplicity with efficiency and precision [1, 2]. It relies on the nuclease activity of the Cas9 protein, a component of adaptive bacterial defense against bacteriophages or other nucleic acid threats [3]. The specificity of this system is conferred by the interaction of the Cas9 nuclease with an RNA composed of a scaffold RNA joined to a guide RNA (gRNA), which directs the protein to a specific target DNA sequence for cleavage [4]. The site of mutagenesis can therefore be programmed simply by adjusting the sequence of the gRNA, provided a protospacer adjacent motif (PAM) sequence is present in the *Correspondence: [email protected] † Irene N. Gentzel and Chan Ho Park contributed equally 1 Department of Plant Pathology, The Ohio State University, 483B Kottman Hall, 2021 Coffey Road, Columbus, OH 43210, USA Full list of author information is available at the end of the article
target DNA [4, 5]. The CRISPR/Cas9 system is also useful for other genetic manipulations beyond genomic sequence editing, such as regulation of gene expression and epigenetic modification [2, 4]. CRISPR-mediated transcription inhibition (CRISPRi) or activation (CRISPRa) is achieved by utilizing a nuclease-deactivated form of Cas9 (dCas9), a non-cutting variant which maintains its DNA-binding specificity [6, 7]. While the interaction of dCas9 itself with a specific promoter can reduce gene expression levels, fusion with a repression domain can enhance this effect [6]. One such repressor used in plant studies is the 12 amino acid SRDX domai
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