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Updated: Dec 6, 2025

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
Published on: September 2, 2021
A CRISPR/dCas9 toolkit for functional analysis of maize genes
Irene N Gentzel1, Chan Ho Park1, Maria Bellizzi1
1Department of Plant Pathology, The Ohio State University, 483B Kottman Hall, 2021 Coffey Road, Columbus, OH 43210 USA.
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.
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