A CRISPR/dCas9 toolkit for functional analysis of maize genes

Irene N Gentzel,Margaret G Redinbaugh,Peter Balint-Kurti,Chan Ho Park,Jonathan Lamantia,Guiqing Xiao,Kiran R Gadhave,Colin Murphree,Tim L Sit,Maria Bellizzi,Qin Yang,Guo-Liang Wang

doi:10.1186/s13007-020-00675-5

Irene N Gentzel, Margaret G Redinbaugh + Show 10 more

Open Access

https://doi.org/10.1186/s13007-020-00675-5

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Abstract

BackgroundThe 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).ResultsIn 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.ConclusionsWe 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.

Highlights

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants
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, which directs the protein to a specific target DNA sequence for cleavage [4]
The CRISPR-mediated transcription inhibition (CRISPRi) and CRISPRa toolkits we describe offer a simple and time-efficient approach to facilitate screening guide RNA (gRNA) candidates in maize protoplasts prior to the generation of stable transgenic lines, for example, which streamlines the gRNA selection process and reduces the cost and time burdens associated with repeated maize transgenic production

Summary

Introduction

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to generate precise genomic mutations, and to manipulate gene expression when present as a deactivated protein (dCas). The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas 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 Cas protein, a component of adaptive bacterial defense against bacteriophages or other nucleic acid threats [3]. Alternative techniques to quickly manipulate maize gene expression in the desired genetic background would be highly beneficial

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