A CRISPR/dCas9 toolkit for functional analysis of maize genes

Abstract

Background: The C lustered R egularly I nterspaced S hort P alindromic R epeats (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. Additionally, we describe the utility of Foxtail Mosaic Virus (FoMV), a positive-sense RNA monocot virus, as a vector for delivering guide RNAs (gRNAs) to maize protoplasts in addition to whole plants. 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.